JPH07112997A - Oligonucleotide derivative, its production synthetic intermediate therefor and use thereof - Google Patents

Abstract

PURPOSE:To obtain the subject novel compound which is useful as an antitumor agent, antiviral agent and antiinflammatory agent, because it is excellent in resistance to hydrolytic enzymes, chemical stability and cell membrane permeability, and inhibits expression of malignant tumor gene, viral gene or the like. CONSTITUTION:A compound having the nucleotide sequence of formula I {B<1>, B<2>, B<3> each is nucleic acid residue; X<2>, X<3> are OH, SH, a 1 to 5 C alkyl, 1 to 5 C alkoxy, 1 to 5 C monoalkylamino, (substituted)aromatic ring; W is H, or a protecting group, when at least one of X<2> and X<3> is a (substituted)aromatic ring, and when both of X<2>, X<3> are groups other than (substituted)aromatic ring, W is the group of formula II (Y is a saccharide residue and X<1> is a group of X<2>, the group of formula III; R<1>, R<2>, R<3> each is H, OH, a halogen, 1 to 5C alkoxy (alkyloxy); (n) is 1 to 98}. For example galactose-bonding phosphorothioate oligonucleotide: (GAL)-TsGsGsAsGsCsCsAsTsAsGsCsGsAsGsGsC.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene that inhibits the expression of malignant tumor genes in malignant tumor cells, suppresses the expression of viral genes derived from viruses, and produces proteins that cause inflammation. Oligonucleotides or PTCA (Percutaneous Transluminal Coronary A) that ensure the effect of chemotherapy by suppressing the expression of chemotherapeutic drugs or controlling the expression of drug-resistant genes that cause problems during chemotherapy for malignant tumors.
ngioplasty; oligonucleotide derivative that inhibits the production of cell growth factor involved in vascular restenosis after percutaneous coronary artery dilatation), method for producing the oligonucleotide derivative, malignant tumor containing the oligonucleotide derivative, and viral disease Further, it relates to a drug that suppresses the expression of a specific gene such as a gene that causes inflammation, a screening method for various compounds using the oligonucleotide derivative, and the like.

[0002]

2. Description of the Related Art Many diseases caused by the expression of a specific gene and caused by the gene product are known, and examples thereof include the following. Virulence genes or oncogenes that are caused by oncogenes are those that are internal to the cell (for example, conversion of proto-oncogene to oncogene) and the result of infection with extracellular virus called exogenous. It has been clarified that the expression of these genes is one of the factors that cause the onset of viral diseases or the abnormal growth of normal cells. In addition, the expression of genes such as ICAM-1 causes the produced adhesion protein to cause inflammation. Furthermore, although the expression of these genes is not directly responsible for the etiology, the body acquires drug resistance to the antitumor drug that is always a problem during chemotherapy for malignant tumors, As a result, it is considered that the expression of the drug resistance gene is an indirect cause, such as making complete cure difficult. Therefore, a drug that controls activation or expression of a gene that causes such a disease is expected to be effective as an antiviral agent, an antitumor agent, an anti-inflammatory agent, or the like. Against this background, the technique of controlling the expression of a gene with an oligonucleotide derivative having a sequence in a region unique to the gene or a sequence complementary to the mRNA transcribed from the gene, that is, antisense The method is widely known (G. Zon, Pharmaceutical Re
s., vol. 5, No. 9, 539 (1988) .CA Steinetal., Ca
ncer Res., vol. 48, 2659 (1988) .E. Uhlman et al., C
hemical Reviews, vol.90, No.4, 543 (1990) .J. Goodch
ild, Bioconjugate Chem., vol.1, No.3, 165 (1990).
). In particular, attempts to control the expression of viral genes, oncogenes, etc. using these antisense oligonucleotides have been studied in detail. The antisense oligonucleotide used at this time is easily decomposed by a hydrolase such as nuclease in its natural form. Therefore, in order to increase the stability against these degrading enzymes, various chemically modified nucleotide derivatives have been developed. For example, phosphorothioate (F. Eckstein, Angew. Chem., Vol. 6,431, (198
3). F. Eckstein et al., Biochemistry, vol. 23, 3443
(1984). JW Stec et al., J. Am, Chem. Soc., Vol.
106, 6077 (1984). F. Eckstein et al., Annu Rev. Bi
ochem., vol. 54, 367, (1985).), methylphosphonate (PS Millar et al., Biochemistry, vol.18, 513)
4 (1979) .PS Millar et al., Biochimie, vol. 67,
769 (1985) .POP Ts'O et al., Ann NY Acad Sc
i., vol. 507 220 (1988).), phosphorodithioate (MH Caruthers et al., Tetrahedron Lett., vol. 2
9, 2911, (1988). MH Caruthers et al., J. Am. Che
m. Soc., vol. 111, 2321, (1989).). Alternatively, the 2'- of the ribose ring, which is a structural unit of nucleotides
Methylation of the 2'-sugar hydroxyl group is one of the attempts to improve stability by modifying the position (Y. Furukawa et a
l., Chem. Pharm. Bull., vol. 13, 1273 (1965). E. O.
htsuka et al., Nucleic Acids Res., vol. 15, 6131 (1
987).) And so on. Furthermore, in order to enhance the cell membrane permeability to such modified nucleotides, cholesterol (RL Letsinge
r et al., Proc. Natl. Acad. Sci. USA, vol. 86, 6553
(1989).) And poly (L-lysine) (M. Lemaitre et a
l., Proc. Natl. Acde. Sci. USA, vol. 84, 648 (198
Derivatives such as adding 7).) Have also been synthesized. Attempts have also been made to introduce antisense molecules into cells by encapsulating them in liposomes (PL Felgner et al., Proc. Natl. Acad. Sci. USA,
vol. 84, 7143 (1987).). Among these various derivatives, methylphosphonate, for example, can be expected to have higher chemical stability against hydrolases, and since it is a neutral molecule, it can be expected to have improved membrane permeability, but aqueous solutions It has the drawback of extremely poor solubility in. Phosphorodithioate loses the chirality of the phosphate group, but since there are two sulfur atoms in the phosphate group, it is considerably distorted compared to the natural type, and therefore double strand formation as an antisense Noh will fall. In addition, since it is difficult to synthesize it in comparison with other derivatives, there is a problem in performing large-scale synthesis. Attempts to introduce poly (L-lysine) etc. will result in considerably strong cytotoxicity (JP Leonetti
et al., Gene, vol. 72,323 (1988).), it is not a practical modification reaction for antisense. Under these circumstances, phosphorothioate is currently used as the most excellent antisense oligonucleotide, with an emphasis on its ability to exhibit antisense effects, stability against hydrolases, and low cytotoxicity. . However, this phosphorothioate inevitably reduces the ability to form double chains due to the chirality of the phosphate group, and the target sulfur compound and the trace amount of oxides are isolated and purified during the synthesis stage. There is a problem that it must be. In addition, phosphorothioate's low cell permeability and nuclear translocation become problems, causing problems in drug efficacy and persistence when developing as an antitumor agent, antiviral agent, antiinflammatory agent, etc. It is believed that.

[0003]

DISCLOSURE OF THE INVENTION The present invention maximizes the antisense effect by exhibiting resistance to hydrolases, having a chemically stable structure, and having excellent cell membrane permeability. A novel oligonucleotide derivative that can be exerted and does not have the above problems
Provided are a phosphorylated sugar derivative, a branched sugar derivative, and an antitumor agent, an antiviral agent, an anti-inflammatory agent, etc. containing the oligonucleotide derivative and the oligonucleotide derivative, which are also useful as intermediates for producing the oligonucleotide derivative. The purpose is to

[0004]

Means for Solving the Problems The inventors of the present invention have made earnest studies to solve the above problems, and as a result,

[0005]

[Chemical formula 26]

[Wherein W represents a hydrogen atom or a protective group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ²
May be the same or different in repeating n, and X ² and X ³ are respectively OH group, SH group, C
_1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, X ³ may be the same or different in each repeating n, R ¹ , R ² and R ³ each represent a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxy group or a C _1-5 alkoxyalkyloxy group, and R ² is the same or different in repeating n. Also, n represents an integer of 1 to 98. However, (1) X ² and X ³ are each an OH group, an SH group, a C _1-5 alkyl group,
A C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, wherein at least one of X ² and X ³ is an optionally substituted aromatic ring group, W represents a hydrogen atom or a protective group, and (2) X ² and X ³ are respectively an OH group, an SH group, a C _1-5 alkyl group,
When it is a C _1-5 alkoxy group or a C _1-5 monoalkylamino group, W is of the formula

[0007]

[Chemical 27]

Or the expression

[0009]

[Chemical 28]

(Wherein Y represents a sugar residue and X ¹ represents OH
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_{1-5 represents a} monoalkylamino group or an optionally substituted aromatic ring group. ) Represents a group represented by. ] It succeeded in synthesize | combining the oligonucleotide derivative (I) which has the nucleotide sequence represented by this, or its salt.

More specifically, the formula

[0012]

[Chemical 29]

[Wherein Q is Y or an equation

[0014]

[Chemical 30]

Represents a group represented by, Y represents a sugar residue,
X ¹ represents an OH group, a SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, and B ¹ , B ² and B ³ each represents a nucleic acid residue, B ² may be the same or different in repeating n, and X ⁴ and X ⁵ are OH group, SH group, C _1-5 alkyl group, C _{1-5, respectively.} An alkoxy group, a C _1-5 monoalkylamino group, X ⁵ may be the same or different in repeating n, R ¹ , R ² and R ³ are each a hydrogen atom, an OH group,
It represents a halogen atom, a C _1-5 alkoxyl group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in repeating n, and n is 1 to 1
An integer of 98 is shown. ] The oligonucleotide derivative (Ia) which has a nucleotide sequence represented by these, or its salt, and formula.

[0016]

[Chemical 31]

[Wherein W represents a hydrogen atom or a protecting group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ²
May be the same or different in repeating n, and X ⁶ and X ⁷ are respectively OH group, SH group, C
_1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, X ⁷ may be the same or different in each repeating n, At least one of X ⁶ and X ^{7 represents} an optionally substituted aromatic ring group, and R ¹ , R ² and R
³ each represents a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxyl group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in repeating n, and n is 1 to 1 An integer of 98 is shown. ]
We succeeded in synthesizing an oligonucleotide derivative (Ib) having a nucleotide sequence represented by or a salt thereof.

Then, the present inventors unexpectedly prepared the oligonucleotide derivative (Ia) by substituting the 2′-sugar hydroxyl group with a halogen atom, or by alkylating or alkoxyalkylating the oligonucleotide derivative (Ia). Also shows resistance to hydrolases,
We have found that it is chemically stable. In addition, since the ribose ring of the oligonucleotide derivative can have a 3'-endo conformation, target DNA, RNA
It has been found that it has a stronger complementary strand forming ability with respect to. Furthermore, by introducing various sugar derivatives into the 5'-end of the antisense molecule via a phosphate group,
(1) By using a branched oligosaccharide chemically synthesized in consideration of the cluster effect which is a characteristic of sugar receptor, a receptor that recognizes a sugar derivative such as glucose receptor, galactose receptor and mannose receptor existing in the living body is utilized. Then, the antisense molecule can be introduced into the cell, and (2) the affinity of the antisense nucleotide with the cell membrane can be increased by introducing a substituent having an increased hydrophobicity into the hydroxyl group of the sugar derivative,
(3) For neutral antisense such as methylphosphonate, a sugar derivative as a polyhydroxyl compound has an effect of increasing the solubility in an aqueous solution, and as a result, the effectiveness of these antisense molecules can be increased. Found the advantage of.

On the other hand, an oligonucleotide derivative (Ib)
Is at least one phenylphosphonate-type nucleotide in the molecule (preferably at the 5'end), preferably two (preferably at the 5'and 3'ends)
It is characterized by containing. The present inventors have found that this type of oligonucleotide has an excellent gene expression-suppressing activity regardless of whether or not it has a sugar derivative mediated by a phosphate group at the 5'-end. That is, by introducing a phenyl group into the phosphate group to form a phosphonate bond, (1) it is expected that the resistance of this oligonucleotide to a nucleolytic enzyme is increased, and (2) the lipophilicity of the antisense molecule is also increased. Since it increases, it is expected that the affinity with the cell membrane is increased, and as a result, the membrane permeability of this molecule is improved. (3) Furthermore, the presence of the phenyl group enhances the stacking effect between each base, resulting in high oligomericity. We have found many advantages such as possible stabilization of substructure. Furthermore, the present inventors succeeded in synthesizing a novel sugar derivative and nucleotide derivative which are useful intermediates for the production of the above-mentioned oligonucleotide derivative (I) or a salt thereof. The present inventors have completed the present invention as a result of further research based on these findings. That is, the present invention provides a novel oligonucleotide derivative or a salt thereof, a method for producing the same, a sugar derivative and a nucleotide derivative useful as an intermediate for producing the oligonucleotide derivative, and an antitumor agent or antivirus containing the oligonucleotide derivative. Agents, anti-inflammatory agents, etc. More specifically, the present invention provides the following [A]
Oligonucleotide derivative (I) or salt thereof, production method and use thereof, [B] oligonucleotide derivative (I
a) or a salt thereof, a production method and use thereof, [C] oligonucleotide derivative (Ib) or a salt thereof, a production method and use thereof, [D-1] sugar derivative (A), [D-2]
Provided are a sugar derivative (B), a [D-3] sugar derivative (C), and an [E] nucleotide derivative.

[A] Oligonucleotide derivative (I) or salt thereof shown in the following items (1) to (8), production method and use thereof (1) Formula

[0021]

[Chemical 32]

[In the formula, W represents a hydrogen atom or a protective group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ²
May be the same or different in repeating n, and X ² and X ³ are respectively OH group, SH group, C
_1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, X ³ may be the same or different in each repeating n, R ¹ , R ² and R ³ each represent a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxy group or a C _1-5 alkoxyalkyloxy group, and R ² is the same or different in repeating n. Also, n represents an integer of 1 to 98.

However, (i) X ² and X ³ are each O
H group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group,
When a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group and at least one of X ² and X ³ is an optionally substituted aromatic ring group, W is a hydrogen atom or a protecting group. (Ii) X ² and X ³ are OH
A group, an SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group or a C _1-5 monoalkylamino group, W is of the formula

[0024]

[Chemical 33]

Or the expression

[0026]

[Chemical 34]

(In the formula, Y represents a sugar residue, and X ¹ represents OH.
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_{1-5 represents a} monoalkylamino group or an optionally substituted aromatic ring group. ) Represents a group represented by. ] The oligonucleotide derivative or its salt which has a nucleotide sequence represented by these.

Equation (2)

[0029]

[Chemical 35]

[Wherein W, B ¹ , B ² , B ³ , R ¹ , R ² ,
R ³ and n have the same meanings as in claim 1, X ²¹ and X ³¹ each represent O or S, and X ³¹ may be the same or different in repeating n, and C
PG represents a solid phase carrier. ] Or a compound represented by the formula

[0031]

[Chemical 36]

[Wherein, W, B ¹ , B ² , B ³ , R ¹ , R ² ,
R ³ and n have the same meanings as in claim 1, and X ²² and X ³² are each a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or a substituent. It represents a good aromatic ring group, X ³² may be the same or different in each repeating n, and CPG represents a solid phase carrier. ] The protecting group of the compound represented by these is removed, The manufacturing method of the oligonucleotide derivative (I) or its salt as described in (1). (3) A gene expression inhibitor comprising the oligonucleotide derivative (I) or the salt thereof according to item (1). (4) The gene expression inhibitor according to item (3), which is an expression inhibitor of a gene causing a malignant tumor, a viral disease or inflammation. (5) Suppressing gene expression is (i) DNA-premRN
Repression of transcription to A, (ii) premRNA to mature mRN
Suppression of splicing to A or (iii) mature mRN
The gene expression inhibitor according to item (3) or (4), which suppresses translation of A into protein. (6) The gene causing the malignant tumor is sr
c, fps, yes, ros, myb, myc, er
b, rel, mos, abl, ras, fos, fe
A gene selected from the group consisting of s, fms, sis, raf, neu, and p53, and the genes causing the viral disease are AIDS virus, influenza virus, herpes virus, papilloma virus, polio virus, picornavirus, and adenovirus. Genes selected from the group consisting of viruses, which are genes that cause inflammation are ICAM-1, ELAM-
The gene expression inhibitor according to item (4), which is a gene selected from the group consisting of 1 and VLAM.

(7) The gene expression inhibitor according to item (3), wherein the gene is the ICAM-1 gene. (8) A PTC containing the oligonucleotide derivative (I) or the salt thereof according to item (1).
A post-A restenosis inhibitor.

[B] The oligonucleotide derivative (Ia) or a salt thereof shown in the following (1) to (47):
Production method and use thereof (1) An oligonucleotide derivative or a salt thereof in which a phosphate group bound to a sugar residue is bound to a phosphodiester at a 5'-terminal hydroxyl group.

Equation (2)

[0036]

[Chemical 37]

[Wherein Q is Y or an expression

[0038]

[Chemical 38]

Represents a group represented by, Y represents a sugar residue,
X ¹ represents an OH group, a SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, and B ¹ , B ² and B ³ each represents a nucleic acid residue, B ² may be the same or different in repeating n, and X ⁴ and X ⁵ are OH group, SH group, C _1-5 alkyl group, C _{1-5, respectively.} An alkoxy group, a C _1-5 monoalkylamino group, X ⁵ may be the same or different in repeating n, R ¹ , R ² and R ³ are each a hydrogen atom, an OH group,
It represents a halogen atom, a C _1-5 alkoxyl group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in repeating n, and n is 1 to 1
An integer of 98 is shown. ] The oligonucleotide derivative or a salt thereof according to item (1), which has a nucleotide sequence represented by:

(3) The group in which the sugar of the sugar residue represented by Y is composed of an optionally substituted monosaccharide, an optionally substituted oligosaccharide, and the above monosaccharide or a glycoside derivative of the above oligosaccharide. The oligonucleotide derivative according to item (2), which is selected from the following.

(4) The sugar of the sugar residue represented by Y is (i)
A hydroxyl group (preferably a hydroxyl group other than the hemiacetal hydroxyl group) is a halogen atom, a C _1-10 alkylcarbonyl group,
An acyl group optionally substituted by about 1 to 5 substituents selected from the group consisting of nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group, halogen atom, C _It may be substituted with about 1 to 5 substituents selected from the group consisting of _1-10 alkylcarbonyl group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group. Monosaccharides optionally substituted with about 1 to 4 substituents selected from the group consisting of alkyl groups and optionally substituted amino groups, (ii) hydroxyl groups (preferably hydroxyl groups other than hemiacetal hydroxyl groups) Is a halogen atom, C _1-10 alkylcarbonyl group, nitro group, C _1-10 alkyl group, C
_1-10 alkoxy group, a phenyl group and 1 to 5 or so substituents optionally substituted acyl group selected from the group consisting of naphthyl, halogen atom, Cl-IO alkyl group, a nitro group, C _{1 -10} alkyl group, C _1-10
1 to 4 selected from the group consisting of an alkyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of an alkoxy group, a phenyl group and a naphthyl group and an amino group which may be substituted. An oligosaccharide which may be substituted with a certain degree of substituent or (iii) a hydroxyl group of the above monosaccharide or oligosaccharide is a non-sugar component, -O-
A glycoside derivative substituted with R ⁷ , -S-R ⁸ or -N-R ⁹ (wherein R ⁷ , R ⁸ and R ⁹ each represent an optionally substituted hydrocarbon group); The oligonucleotide derivative according to the item (2). (5) The oligonucleotide derivative according to item (4), wherein the acyl group is a C _1-10 alkylcarbonyl group, a phenylcarbonyl group or a benzylcarbonyl group. (6) The oligonucleotide derivative according to item (4), wherein the alkyl group is a C _1-20 alkyl group. (7) The amino group has the formula —NR ⁴ R ⁵ (wherein R ⁴ and R ^4
5 is (i) hydrogen atom, (ii) halogen atom,
It may be substituted with about 1 to 5 substituents selected from the group consisting of C _1-10 alkylcarbonyl group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group. Good acyl group or (iii) halogen atom, C _1-10 alkylcarbonyl group, nitro group, C _1-10
The alkyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of an alkyl group, a C _1-10 alkoxy group, a phenyl group and a naphthyl group is shown. ) The oligonucleotide derivative according to item (4), which is an amino group represented by (8) Monosaccharide is 5-carbon sugar aldose, 5-carbon sugar ketose, 6
The oligonucleotide derivative according to item (3) or (4), which is a carbon sugar aldose or a hexacarbon sugar ketose. (9) 5-carbon sugar aldose is ribose, arabinose, xylose or lyxose, 5-carbon sugar ketose is ribulose or xylulose, and 6-carbon sugar aldose is allose, altrose, glucose, mannose,
The oligonucleotide derivative according to item (8), which is gulose, idose, galactose or talose, and the 6-carbon sugar ketose is psicose, fructose, sorbose or tagatose. (10) Oligosaccharides are lactose, α, α-trehalose, N-acetylneuraminyl lactose, difucosyl lactose, fucosyl lactose, lactulose, gentianose, isoliknoses, licnose, planteose, sucrose, biflucose, 1,6-di Glucosyl mannitol, galactosyl sucrose 6-β-
Glucosyl mannitol, laminaribiose, inulobiose, lycopoose, maltose, isomaltotriose, melibiose, neobifucose, neokestose, erulose, 6-α-galactosylgalactose, kojibiose, maltulose, melezitose, turanose, sophorose, gentio-oligosaccharide. Item 3, which is selected from the group consisting of, primeverose, sambubioose, lycobiose, lycobiose, lycotriose, sorabiose, strohantobiose, jigilandbiose, odrotriose, fungitetraose and β-1,3 xylo-oligo. The oligonucleotide derivative according to the item (4). (11) glycoside derivatives, -O-R ⁷ hydroxyl group of monosaccharides or oligosaccharides are non-sugar components, -S-R ⁸ or -
Item (3) or (4), which is substituted with NR ⁹ (wherein R ⁷ , R ⁸ and R ⁹ each represent an optionally substituted hydrocarbon group). Oligonucleotide derivative. (12) R ⁷ , R ⁸ and R ⁹ are each 1 to 5 selected from the group consisting of (i) a halogen atom, a C _1-10 alkylcarbonyl group, a nitro group and a C _1-10 alkoxy group.
1 to 1 selected from the group consisting of a C _1-30 alkyl group which may be substituted with about 1 substituent, (ii) a halogen atom, a C _1-10 alkylcarbonyl group, a nitro group and a C _1-10 alkoxy group. C _2-4 alkenyl group _optionally substituted with about 5 substituents, (iii) halogen atom, C _1-10
A C _2-4 alkynyl group optionally substituted with about 1 to 5 substituents selected from the group consisting of an alkylcarbonyl group, a nitro group and a C _1-10 alkoxy group, (iv) a halogen atom, C _{1- 10} alkylcarbonyl group, nitro group,
A C _6-12 aryl group which may be substituted with about 1 to 5 substituents selected from the group consisting of a C _1-10 alkyl group and a C _1-10 alkoxy group, or (v) a halogen atom, C _{1 A} C _7-14 aralkyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of a _-10 alkylcarbonyl group, a nitro group, a C _1-10 alkyl group and a C _1-10 alkoxy group. An oligonucleotide derivative according to the item (11). (13) The glycoside derivative is n-octyl glucoside,
n-octyl galactoside, n-octyl mannoside,
n-octyl thioglucoside, n-octyl thiogalactoside, n-octyl thiomannoside, stearyl glycoside, stearyl galactoside, stearyl mannoside, stearyl thioglycoside, stearyl thiogalactoside, stearyl thiomannoside, phenyl glucoside, phenyl galactoside The oligonucleotide derivative according to item (3) or (4), which is selected from the group consisting of and phenylmannoside. (14) Q is Y, and the sugar of the sugar residue represented by Y is
The oligonucleotide derivative according to item (2), which is a monosaccharide, oligosaccharide or glycoside derivative in which a hydroxyl group may be substituted with a C _1-10 alkylcarbonyl group, a phenylcarbonyl group or a C _1-20 alkyl group. (15) The oligonucleotide derivative according to item (14), wherein the monosaccharide is galactose, mannose, galactosamine or glucosamine. (16) The oligonucleotide derivative according to item (14), wherein the oligosaccharide is melibiose, gentiobiose or isomaltotriose. (17) The glycoside derivative is n-octyl glucoside,
The oligonucleotide derivative according to item (14), which is n-octylthioglucoside or phenylglucoside. (18) A monosaccharide in which a hydroxyl group is substituted with an amino group which may be substituted is glucosamine or galactosamine in which a 2-amino group may be substituted with a C _1-6 alkylcarbonyl group or a phenylcarbonyl group. (4)
The oligonucleotide derivative according to the item. (19) Q is Y, and the sugar of the sugar residue represented by Y is
Galactose or mannose whose hydroxyl group may be substituted with a C _1-6 alkylcarbonyl group, melibiose, gentiobiose or isomaltotriose whose hydroxyl group may be substituted with a phenylcarbonyl group,
The oligonucleotide derivative according to item (2), which is n-octylglucoside, n-octylthioglucoside or phenylglucoside, glucosamine or galactosamine optionally substituted with a C _1-10 alkylcarbonyl group or a phenylcarbonyl group. (20) Q is Y, and the sugar of the sugar residue represented by Y is galactose, mannose, melibiose, gentiobiose, isomaltotriose, n-octylglucoside, n-octylthioglucoside, phenylglucoside, galactosamine, N. -Benzoylgalactosamine,
The oligonucleotide derivative according to item (2), which is N-acetylgalactosamine, glucosamine, N-benzoylglucosamine or N-acetylglucosamine. (21) Q is the formula

[0042]

[Chemical Formula 39]

The oligonucleotide derivative according to item (2), wherein the sugar of the sugar residue represented by Y is a monosaccharide. (22) The oligonucleotide derivative according to the item (21), wherein the monosaccharide is galactose, mannose, or glucose. (23) The nucleic acid residue is hypoxanthine-9-yl, guanine-9-yl, adenine-9-yl, uracil-1-.
(2) to (22) selected from the group consisting of yl, thymin-1-yl and cytosyn-1-yl.
The oligonucleotide derivative according to the item. (24) X ¹ is OH group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, halogen atom, C _1-10 alkylcarbonyl group, O
C optionally substituted with about 1 to 5 substituents selected from the group consisting of H group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group.
_6-11 aryl group, C _1-6 alkyl group, C _2-6 alkenyl group, C _2-6 alkynyl group, C _3-6 cycloalkyl group, C _5-7 cycloalkenyl group, C _7-11 aralkyl group ,
C _6-14 aryl group, C _1-6 alkoxy group, C _6-14 aryloxy group, C _1-6 alkylcarbonyl group, C _6-14 aryl-carbonyl group, C _1-6 alkanoyloxy group, C
_6-14 aryl-carbonyloxy group, carboxyl group,
C _1-6 alkoxy-carbonyl group, carbamoyl group, N-
Mono-C _1-4 alkylcarbamoyl group, N, N-di-C
_1-4 alkylcarbamoyl group, halogen atom, mono-,
Di- or tri-halogeno-C _1-4 alkyl group, oxo group, amidino group, imino group, amino group, mono- or di-C
_1-4 1 to 4 selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom which may be substituted with about 1 to 5 substituents selected from the group consisting of an alkylamino group, an OH group and a nitro group. A 5- to 13-membered aromatic heterocycle containing about 12 heteroatoms and about 1 to 12 carbon atoms, wherein X ⁴ and X ⁵ are OH and SH, respectively.
Group, C _1-5 alkyl group, C _1-5 alkoxy group or C _1-5
The oligonucleotide derivative according to items (2) to (23), which is a monoalkylamino group. (25) The C _6-11 aryl group is a phenyl group or a naphthyl group, and contains 1 to 4 hetero atoms and 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom. The 5- to 13-membered aromatic heterocycle contained is 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 3- or 4-.
Pyridyl, 2-, 4- or 5-oxazolyl, 2-,
4- or 5-thiazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-imidazolyl, 3-, 4
-Or 5-isoxazolyl, 3-, 4- or 5-
Isothiazolyl, 3- or 5- (1,2,4-oxadiazolyl), 1,3,4-oxadiazolyl, 3- or 5- (1,2,4-thiadiazolyl), 1,3,4-thiadiazolyl, 4- Or 5- (1,2,3-thiadiazolyl),
1,2,5-thiadiazolyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1H- or 2H-tetrazolyl, N-oxide-2-, 3- or 4-pyridyl,
2-, 4- or 5-pyrimidinyl, N-oxide-2
-, 4- or 5-pyrimidinyl, thiomorpholinyl,
Morpholinyl, oxoimidazolinyl, dioxotriazinyl, pyrrolidinyl, piperidinyl, pyranyl, thiopyranyl, 1,4-oxazinyl, 1,4-thiazinyl,
1,3-thiazinyl, piperazinyl, triazinyl, oxotriazinyl, 3- or 4-pyridazinyl, pyrazinyl, N-oxide-3- or 4-pyridazinyl,
Benzofuryl, benzothiazolyl, benzoxazolyl, tetrazolo [1,5-b] pyridazinyl, triazolo [4,5-b] pyridazinyl, benzimidazolyl,
Quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, indolizinyl,
The oligonucleotide derivative according to paragraph (24), which is quinolidinyl, 1,8-naphthyridinyl, purinyl, pteridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenanthridinyl, chromanyl, benzoxazinyl, phenazinyl, phenothiazinyl or phenoxazinyl. . (26) X ¹ is OH group, SH group, C _1-5 alkyl group, C
_1-5 alkoxy group, C _1-5 monoalkylamino group, or halogen atom, C _1-10 alkylcarbonyl group, OH group,
A phenyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of a nitro group, a C _1-10 alkyl group, a C _1-10 alkoxy group, a phenyl group and a naphthyl group, and X ⁴ And X ⁵ are OH group, SH group, C
The oligonucleotide derivative according to item (2) to (23), which is a _1-5 alkyl group, a C _1-5 alkoxy group, or a C _1-5 monoalkylamino group. (27) X ¹ is OH group, SH group, methyl group, ethyl group,
Propyl group, phenyl group, (o, m, p) -alkoxyphenyl group, (o, m, p) -alkylphenyl group,
A group selected from the group consisting of a methoxy group and an ethoxy group, wherein X ⁴ and X ⁵ are each selected from the group consisting of an OH group, an SH group, a methyl group, an ethyl group, a propyl group, a methoxy group and an ethoxy group. Item (2) that is
The oligonucleotide derivative according to the item (23). (28) X ¹ is OH group, SH group, methyl group, ethyl group,
Selected from the group consisting of propyl group, phenyl group, (o, m, p) -C _1-10 alkoxyphenyl group, (o, m, p) -C _1-10 alkylphenyl group, methoxy group and ethoxy group. X ⁴ and X ⁵ are OH groups,
The oligonucleotide derivative according to item (2) to (23), which is selected from the group consisting of SH group, methyl group, ethyl group, propyl group, methoxy group and ethoxy group. (29) X ¹ is an OH group, an SH group, a methyl group or a phenyl group, and X ⁴ and X ⁵ are an OH group, an SH group,
The oligonucleotide derivative according to items (2) to (23), which is a methyl group. (30) R ¹ , R ² and R ³ are each a hydrogen atom, OH
The oligonucleotide derivative according to items (2) to (29), which is selected from the group consisting of a group, fluoro, chloro, bromo, methoxy group, ethoxy group and methoxymethyloxy group. (31) A case where R ¹ , R ² and R ³ are hydrogen atoms (2)
Item-The oligonucleotide derivative according to Item (29). (32) n is an integer of 1 to 38. (2) to (3)
1) The oligonucleotide derivative described in the item 1). (33) The oligonucleotide derivative according to any one of (2) to (32), which contains the nucleotide sequence represented by CAT. Formula (34)

[0044]

[Chemical 40]

[Wherein Y represents a sugar residue and X ¹ represents OH
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_{1-5 represents a} monoalkylamino group or an optionally substituted aromatic ring group, and the oligonucleotide a ₁ has the sequence number:
1 shows a nucleotide sequence selected from 1 to SEQ ID NO: 31 and SEQ ID NO: 34. ] The oligonucleotide derivative as described in (2) represented by these. (35) The sugar of the sugar residue represented by Y is galactose, mannose, melibiose, gentiobiose, isomalttriose, n-octylglucoside, n-octylthioglucoside, phenylglucoside, galactosamine,
N-benzoylgalactosamine, N-acetylgalactosamine, glucosamine, N-benzoylglucosamine or N-acetylglucosamine, X ¹ is an OH group,
The oligonucleotide derivative according to (34), which is an SH group, a methyl group or a phenyl group. (36)

[0046]

[Chemical 41]

[Wherein Y represents a sugar residue and X ¹ represents OH
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_{1-5 represents a} monoalkylamino group or an optionally substituted aromatic ring group, and the oligonucleotide a ₂ has a sequence number of
1 shows a nucleotide sequence selected from 1 to SEQ ID NO: 31 and SEQ ID NO: 34. ] The oligonucleotide derivative as described in (2) represented by these. (37) The sugar of the sugar residue represented by Y is galactose, mannose or glucose, X ¹ is an OH group, SH
The oligonucleotide derivative according to item (36), which is a group, a methyl group or a phenyl group. (38) Item (34) to (3), wherein the oligonucleotide a ₁ and the oligonucleotide a ₂ are represented by SEQ ID NO: 1, SEQ ID NO: 31 or SEQ ID NO: 34.
The oligonucleotide derivative according to the item 7). (39) Item (1) to (38), wherein the oligonucleotide derivative is an antisense oligonucleotide derivative
The oligonucleotide derivative according to the item. (40) The oligonucleotide derivative according to any one of items (1) to (39), which is encapsulated in a liposome.

Equation (41)

[0049]

[Chemical 42]

[Wherein Q, B ¹ , B ² , B ³ , R ¹ , R ² ,
R ³ and n have the same meanings as described in item (2), and X ¹¹ ,
X ⁴¹ and X ⁵¹ each represent O or S, and X ⁵¹ is n
May be the same or different, and CPG represents a solid phase carrier. ] The compound represented by
Or expression

[0051]

[Chemical 43]

[Wherein Q, B ¹ , B ² , B ³ , R ¹ , R ² ,
R ³ and n have the same meanings as described in item (2), and X ¹² ,
X ⁴² and X ⁵² each represent a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an aromatic ring group which may be substituted, and X ⁵² represents a repeating n group. CPG, which may be the same or different, represents a solid phase carrier. ] The protecting group of the compound represented by these is removed, The manufacturing method of the oligonucleotide derivative as described in (2) characterized by the above-mentioned. (42) A gene expression inhibitor comprising the oligonucleotide derivative according to any one of (1) to (40) or a salt thereof. (43) The gene expression inhibitor according to (42), which is an expression inhibitor of a gene causing a malignant tumor, a viral disease or inflammation. (44) Suppressing gene expression is (i) DNA-premR
Inhibition of transcription to NA, (ii) pre-mRNA to mature mR
Suppression of splicing to NA or (iii) mature mR
The gene expression inhibitor according to item (42) or (43), which suppresses translation of NA into protein. (45) The gene that causes the malignant tumor is sr
c, fps, yes, ros, myb, myc, er
b, rel, mos, abl, ras, fos, fe
A gene selected from the group consisting of s, fms, sis, raf, neu, and p53, and the genes causing the viral disease are AIDS virus, influenza virus, herpes virus, papilloma virus, polio virus, picornavirus, and adenovirus. Genes selected from the group consisting of viruses, which are genes that cause inflammation are ICAM-1, ELAM-
1. The gene expression inhibitor according to item (43), which is a gene selected from the group consisting of 1 and VLAM. (46) The gene wherein the gene is the ICAM-1 gene (42)
The gene expression inhibitor according to the item. (47) An agent for suppressing vascular restenosis after PTCA, which comprises the oligonucleotide derivative according to any one of items (1) to (40) or a salt thereof.

[C] The oligonucleotide derivative (Ib) or a salt thereof shown in the following (1) to (57):
Its manufacturing method and application (1) Formula

[0054]

[Chemical 44]

[Wherein W represents a hydrogen atom or a protecting group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ²
May be the same or different in repeating n, and X ⁶ and X ⁷ are respectively OH group, SH group, C
_1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, X ⁷ may be the same or different in each repeating n, At least one of X ⁶ and X ^{7 represents} an optionally substituted aromatic ring group, and R ¹ , R ² and R
³ each represents a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxyl group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in repeating n, and n is 1 to 1 An integer of 98 is shown. ]
An oligonucleotide derivative (Ib) having a nucleotide sequence represented by: or a salt thereof. (2) The protective group represented by W has the formula (i)

[0056]

[Chemical formula 45]

(Wherein Y represents a sugar residue), a group of formula (ii)

[0058]

[Chemical formula 46]

(Wherein Y represents a sugar residue), (iii) C _{1-6 optionally} substituted with a halogen atom.
Alkyl group, (iv) a halogen atom or C _1-6 alkyl optionally substituted C _6-11 also be an aryl group group, (v) a halogen atom or C _1-6 alkyl optionally substituted with a group C _{7 -12} aralkyl group, (vi) C _1-6 alkylcarbonyl group optionally substituted with a halogen atom, (vii) phenyloxycarbonyl group, (viii) C _7-12 aralkyl-carbonyl group, (ix) pyranyl group , (X) a furanyl group, (xi) a silyl group, (xii) a trityl group optionally substituted with 1 to 3 methoxy groups, or (xiii) a phenylxanthenyl group optionally substituted with a methoxy group. The oligonucleotide derivative or the salt thereof according to the certain item (1). (3) W is a hydrogen atom or formula

[0060]

[Chemical 47]

Or

[0062]

[Chemical 48]

An oligonucleotide derivative or a salt thereof according to item (1), which is a group represented by the formula (wherein Y represents a sugar residue). (4) The sugar of the sugar residue represented by Y is selected from the group consisting of an optionally substituted monosaccharide, an optionally substituted oligosaccharide, and the above monosaccharide or a glycoside derivative of the above oligosaccharide. The oligonucleotide derivative according to item (2) or (3), which is (5) In the sugar of the sugar residue represented by Y, (i) the hydroxyl group (preferably a hydroxyl group other than the hemiacetal hydroxyl group) is a halogen atom, a C _1-10 alkylcarbonyl group, a nitro group, C
_1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group, optionally substituted acyl group, halogen atom, C _1-10 alkyl selected from the group consisting of about 1 to 5 substituents. Carbonyl group, nitro group, C _1-10
A group consisting of an alkyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of an alkyl group, a C _1-10 alkoxy group, a phenyl group and a naphthyl group and an amino group which may be substituted. A monosaccharide which may be substituted with about 1 to 4 substituents selected from
(Ii) a hydroxyl group (preferably a hydroxyl group other than the hemiacetal hydroxyl group) is selected from a halogen atom, a C _1-10 alkylcarbonyl group, a nitro group, a C _1-10 alkyl group, a C _1-10 alkoxy group, a phenyl group and a naphthyl group. An acyl group, a halogen atom, a C _1-10 alkylcarbonyl group, a nitro group, a C _1-10 alkyl group, a C _1-10 alkoxy group which may be substituted with about 1 to 5 substituents selected from the group consisting of 1 to 4 selected from the group consisting of an alkyl group optionally substituted with about 1 to 5 substituents selected from the group consisting of a phenyl group and a naphthyl group and an amino group optionally substituted. -O-R ⁷ hydroxyl good oligosaccharides be substituted with a substituent or (iii) above monosaccharide or oligosaccharide is non-sugar components, -S
-R ⁸ or (wherein, R ^7, R ⁸ and R ⁹ represent, respectively, an optionally substituted hydrocarbon group.) -N-R ⁹ the a-substituted glycoside derivatives (2) term or The oligonucleotide derivative according to the item (3). (6) Monosaccharides are 5-carbon sugar aldose, 5-carbon sugar ketose, 6
The oligonucleotide derivative according to item (4) or (5), which is a saccharide sugar aldose or a 6-carbon sugar ketose. (7) 5-carbon sugar aldose is ribose, arabinose, xylose or lyxose, 5-carbon sugar ketose is ribulose or xylulose, and 6-carbon sugar aldose is allose, altrose, glucose, mannose,
The oligonucleotide derivative according to item (6), which is gulose, idose, galactose or talose, and the 6-carbon sugar ketose is psicose, fructose, sorbose or tagatose. (8) The oligosaccharide is lactose, α, α-trehalose,
N-acetylneuraminyl lactose, difucosyllactose, fucosyllactose, lactulose, gentianose, isoliknoses, licnose, planteose, sucrose, biflucose, 1,6-diglucosylmannitol, galactosylsucrose 6-β-glucosylmannitol, laminaribio. Su, inulobiose, lycopoose, maltose, isomaltotriose, melibiose, neobifucose, neokestose,
Ellulose, 6-α-galactosylgalactose, kojibiose, maltulose, melezitose, turanose, sophorose, gentiooligosaccharides, vicyanose,
Item (4) or item (4) or item (a) selected from the group consisting of primeverose, sambiose, lymbiose, lycobiose, lycotriose, sorabiose, strohantobiose, jigillandbiose, odrotriose, fungitetraose and β-1,3 xylo-oligo. The oligonucleotide derivative according to the item (5). (9) glycoside derivatives, -O-R ⁷ hydroxyl group of monosaccharides or oligosaccharides are non-sugar components, -S-R ⁸ or -N
-R ⁹ (wherein, R ^7, R ⁸ and R ⁹ represents. Each optionally substituted hydrocarbon group) first (4) are those substituted with claim or the (5) according to claim Oligonucleotide derivative. (10) 1 to 5 each of R ⁷ , R ⁸ and R ⁹ selected from the group consisting of (i) a halogen atom, a C _1-10 alkylcarbonyl group, a nitro group and a C _1-10 alkoxy group.
1 to 1 selected from the group consisting of a C _1-30 alkyl group which may be substituted with about 1 substituent, (ii) a halogen atom, a C _1-10 alkylcarbonyl group, a nitro group and a C _1-10 alkoxy group. C _2-4 alkenyl group _optionally substituted with about 5 substituents, (iii) halogen atom, C _1-10
A C _2-4 alkynyl group optionally substituted with about 1 to 5 substituents selected from the group consisting of an alkylcarbonyl group, a nitro group and a C _1-10 alkoxy group, (iv) a halogen atom, C _{1- 10} alkylcarbonyl group, nitro group,
A C _6-12 aryl group optionally substituted with about 1 to 5 substituents selected from the group consisting of a C _1-10 alkyl group and a C _1-10 alkoxy group, or (v) a halogen atom, C _{1 A} C _7-14 aralkyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of a _-10 alkylcarbonyl group, a nitro group, a C _1-10 alkyl group and a C _1-10 alkoxy group. The oligonucleotide derivative according to the item (9). (11) The glycoside derivative is n-octyl glucoside,
n-octyl galactoside, n-octyl mannoside,
n-octyl thioglucoside, n-octyl thiogalactoside, n-octyl thiomannoside, stearyl glycoside, stearyl galactoside, stearyl mannoside, stearyl thioglycoside, stearyl thiogalactoside, stearyl thiomannoside, phenyl glucoside, phenyl galactoside The oligonucleotide derivative according to item (4) or (5), which is selected from the group consisting of and phenylmannoside. (12) A monosaccharide in which a hydroxyl group is optionally substituted with an amino group is glucosamine or galactosamine in which a 2-amino group is optionally substituted with a C _1-6 alkylcarbonyl group or a phenylcarbonyl group. (5)
The oligonucleotide derivative according to the item. (13) W is a hydrogen atom or formula

[0064]

[Chemical 49]

The sugar of the sugar residue represented by Y is a monosaccharide or oligosaccharide whose hydroxyl group may be substituted with a C _1-10 alkylcarbonyl group, a phenylcarbonyl group or a C _1-20 alkyl group. Or a glycoside derivative (1)
The oligonucleotide derivative according to the item. (14) The oligonucleotide derivative according to item (13), wherein the monosaccharide is galactose, mannose, galactosamine or glucosamine. (15) The oligonucleotide derivative according to (13), wherein the oligosaccharide is melibiose, gentiobiose or isomaltotriose. (16) The glycoside derivative is n-octyl glucoside,
The oligonucleotide derivative according to item (13), which is n-octylthioglucoside or phenylglucoside. (17) W is a hydrogen atom or a formula

[0066]

[Chemical 50]

The sugar of the sugar residue represented by Y is
Galactose or mannose whose hydroxyl group may be substituted with a C _1-6 alkylcarbonyl group, melibiose, gentiobiose or isomaltotriose whose hydroxyl group may be substituted with a phenylcarbonyl group,
The oligonucleotide derivative according to item (1), which is n-octyl glucoside, n-octyl thioglucoside or phenyl glucoside, glucosamine or galactosamine optionally substituted with a C _1-10 alkylcarbonyl group or a phenylcarbonyl group. (18) W is a hydrogen atom or a formula

[0068]

[Chemical 51]

Wherein the sugar of the sugar residue represented by Y is galactose, mannose, melibiose, gentiobiose, isomalttriose, n-octylglucoside,
The oligonucleotide derivative according to item (1), which is n-octylthioglucoside, phenylglucoside, galactosamine, N-benzoylgalactosamine, N-acetylgalactosamine, glucosamine, N-benzoylglucosamine or N-acetylglucosamine. (19) W is a hydrogen atom or a formula

[0070]

[Chemical 52]

The oligonucleotide derivative according to item (1), wherein the sugar of the sugar residue represented by Y is a monosaccharide. (20) The oligonucleotide derivative according to item (19), wherein the monosaccharide is galactose, mannose, or glucose. (21) The nucleic acid residue is hypoxanthine-9-yl, guanine-9-yl, adenine-9-yl, uracil-1-
(1) to (20) selected from the group consisting of yl, thymin-1-yl and cytosyn-1-yl.
The oligonucleotide derivative according to the item. (22) X ⁶ and X ⁷ are an OH group, an SH group,
C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, halogen atom, C _1-10 alkylcarbonyl group, OH group, nitro group, C _1-10 alkyl group,
A C _6-11 aryl group which may be substituted with about 1 to 5 substituents selected from the group consisting of a C _1-10 alkoxy group, a phenyl group and a naphthyl group, or a C _1-6 alkyl group, C _{2 -6} alkenyl group, C _2-6 alkynyl group, C
_3-6 cycloalkyl group, C _5-7 cycloalkenyl group, C
_7-11 aralkyl group, C _6-14 aryl group, C _1-6 alkoxy group, C _6-14 aryloxy group, C _1-6 alkylcarbonyl group, C _6-14 aryl-carbonyl group, C _1-6 alkanoyl An oxy group, a C _6-14 aryl-carbonyloxy group,
Carboxyl group, C _1-6 alkoxy-carbonyl group, carbamoyl group, N-mono-C _1-4 alkylcarbamoyl group, N, N-di-C _1-4 alkylcarbamoyl group, halogen atom, mono-, di- or 1 to 5 selected from the group consisting of tri-halogeno-C _1-4 alkyl group, oxo group, amidino group, imino group, amino group, mono- or di-C _1-4 alkylamino group, OH group and nitro group 5 to 13 membered carbon atoms containing about 1 to about 4 heteroatoms and about 1 to about 12 carbon atoms selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom which may be substituted with a certain degree of substituents. Item (1) to (2) which are aromatic heterocycles
1) The oligonucleotide derivative described in the item 1). (23) The C _6-11 aryl group is a phenyl group or a naphthyl group, and contains 1 to 4 hetero atoms and 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom. The 5- to 13-membered aromatic heterocycle contained is 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 3- or 4-.
Pyridyl, 2-, 4- or 5-oxazolyl, 2-,
4- or 5-thiazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-imidazolyl, 3-, 4
-Or 5-isoxazolyl, 3-, 4- or 5-
Isothiazolyl, 3- or 5- (1,2,4-oxadiazolyl), 1,3,4-oxadiazolyl, 3- or 5- (1,2,4-thiadiazolyl), 1,3,4-thiadiazolyl, 4- Or 5- (1,2,3-thiadiazolyl),
1,2,5-thiadiazolyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1H- or 2H-tetrazolyl, N-oxide-2-, 3- or 4-pyridyl,
2-, 4- or 5-pyrimidinyl, N-oxide-2
-, 4- or 5-pyrimidinyl, thiomorpholinyl,
Morpholinyl, oxoimidazolinyl, dioxotriazinyl, pyrrolidinyl, piperidinyl, pyranyl, thiopyranyl, 1,4-oxazinyl, 1,4-thiazinyl,
1,3-thiazinyl, piperazinyl, triazinyl, oxotriazinyl, 3- or 4-pyridazinyl, pyrazinyl, N-oxide-3- or 4-pyridazinyl,
Benzofuryl, benzothiazolyl, benzoxazolyl, tetrazolo [1,5-b] pyridazinyl, triazolo [4,5-b] pyridazinyl, benzimidazolyl,
Quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, indolizinyl,
The oligonucleotide derivative according to item (22), which is quinolidinyl, 1,8-naphthyridinyl, purinyl, pteridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenanthridinyl, chromanyl, benzoxazinyl, phenazinyl, phenothiazinyl or phenoxazinyl. . (24) X ⁶ and X ⁷ are OH group, SH group, C
_1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, or halogen atom, C _1-10 alkylcarbonyl group, OH group, nitro group, C _1-10 alkyl group, C
_1-10 Item (1) to (23) which is a phenyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of an alkoxy group, a phenyl group and a naphthyl group.
The oligonucleotide derivative according to the item. (25) X ⁶ and X ⁷ are OH group, SH group, methyl group, ethyl group, propyl group, phenyl group, (o,
The oligo according to item (1) to (23), which is selected from the group consisting of (m, p) -alkoxyphenyl group, (o, m, p) -alkylphenyl group, methoxy group and ethoxy group. Nucleotide derivative. (26) X ⁶ and X ⁷ are OH group, SH group, methyl group, ethyl group, propyl group, phenyl group, (o, m,
p) -C _1-10 alkoxyphenyl group, (o, m, p)-
The oligonucleotide derivative according to item (1) to (23), which is selected from the group consisting of a C _1-10 alkylphenyl group, a methoxy group and an ethoxy group. (27) The oligonucleotide derivative according to any one of (1) to (23), wherein X ⁶ and X ⁷ are each an OH group, SH group, methyl group or phenyl group. (28) R ¹ , R ² and R ³ are each a hydrogen atom, OH
The oligonucleotide derivative according to item (1) to (27), which is selected from the group consisting of a group, fluoro, chloro, bromo, methoxy group, ethoxy group and methoxymethyloxy group. (29) A case where R ¹ , R ² and R ³ are hydrogen atoms (1)
Item-The oligonucleotide derivative according to Item (27). (30) n is an integer of 1 to 38, and the items (1) to (2)
The oligonucleotide derivative according to the item 9). Equation (31)

[0072]

[Chemical 53]

[Wherein W represents a hydrogen atom or a protecting group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ²
May be the same or different in repeating n, and X ⁸ is OH group, SH group, C _1-5 alkyl group, C
_1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, and R ¹ , R ² and R
³ each represents a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxy group or a C _1-5 alkoxyalkyloxy group, R ^{2 s} may be the same or different in repeating n, and n is 1 to 1 An integer of 98 is shown. ] The oligonucleotide derivative or a salt thereof according to item (1), which has a nucleotide sequence represented by: Formula (32)

[0074]

[Chemical 54]

[Wherein, W represents a hydrogen atom or a protecting group, B ¹ , B ²¹ , B ²² and B ³ each represent a nucleic acid residue, and B ²¹ may be the same or different in repeating n. Often, X ⁸ represents an OH group, an SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, and R ¹ and R ²
And R ³ each represent a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxy group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in the repetition of n, and n is An integer of 1 to 98 is shown. ] The oligonucleotide derivative or a salt thereof according to item (1), which has a nucleotide sequence represented by: (33) The nucleic acid residues represented by B ¹ , B ² and B ³ are hypoxanthine-9-yl, guanine-9-yl, adenine-9-yl, uracil-1-yl, thymine-1-yl. The oligonucleotide derivative according to item (31) or (32), which is selected from the group consisting of: and cytosyn-1-yl. (34) X ⁸ is OH group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, halogen atom, C _1-10 alkylcarbonyl group, O
C optionally substituted with about 1 to 5 substituents selected from the group consisting of H group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group.
_6-11 aryl group, C _1-6 alkyl group, C _2-6 alkenyl group, C _2-6 alkynyl group, C _3-6 cycloalkyl group, C _5-7 cycloalkenyl group, C _7-11 aralkyl group ,
C _6-14 aryl group, C _1-6 alkoxy group, C _6-14 aryloxy group, C _1-6 alkylcarbonyl group, C _6-14 aryl-carbonyl group, C _1-6 alkanoyloxy group, C
_6-14 aryl-carbonyloxy group, carboxyl group,
C _1-6 alkoxy-carbonyl group, carbamoyl group, N-
Mono-C _1-4 alkylcarbamoyl group, N, N-di-C
_1-4 alkylcarbamoyl group, halogen atom, mono-,
Di- or tri-halogeno-C _1-4 alkyl group, oxo group, amidino group, imino group, amino group, mono- or di-C
_1-4 1 to 4 selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom which may be substituted with about 1 to 5 substituents selected from the group consisting of an alkylamino group, an OH group and a nitro group. A 5- to 13-membered aromatic heterocycle containing about 3 heteroatoms and carbon atoms (3
The oligonucleotide derivative according to Item 1) or (32). (35) The C _6-11 aryl group is a phenyl group or a naphthyl group, and contains 1 to 4 hetero atoms and 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom. The 5- to 13-membered aromatic heterocycle contained is 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 3- or 4-.
Pyridyl, 2-, 4- or 5-oxazolyl, 2-,
4- or 5-thiazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-imidazolyl, 3-, 4
-Or 5-isoxazolyl, 3-, 4- or 5-
Isothiazolyl, 3- or 5- (1,2,4-oxadiazolyl), 1,3,4-oxadiazolyl, 3- or 5- (1,2,4-thiadiazolyl), 1,3,4-thiadiazolyl, 4- Or 5- (1,2,3-thiadiazolyl),
1,2,5-thiadiazolyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1H- or 2H-tetrazolyl, N-oxide-2-, 3- or 4-pyridyl,
2-, 4- or 5-pyrimidinyl, N-oxide-2
-, 4- or 5-pyrimidinyl, thiomorpholinyl,
Morpholinyl, oxoimidazolinyl, dioxotriazinyl, pyrrolidinyl, piperidinyl, pyranyl, thiopyranyl, 1,4-oxazinyl, 1,4-thiazinyl,
1,3-thiazinyl, piperazinyl, triazinyl, oxotriazinyl, 3- or 4-pyridazinyl, pyrazinyl, N-oxide-3- or 4-pyridazinyl,
Benzofuryl, benzothiazolyl, benzoxazolyl, tetrazolo [1,5-b] pyridazinyl, triazolo [4,5-b] pyridazinyl, benzimidazolyl,
Quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, indolizinyl,
The oligonucleotide derivative according to paragraph (34), which is quinolidinyl, 1,8-naphthyridinyl, purinyl, pteridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenanthridinyl, chromanyl, benzoxazinyl, phenazinyl, phenothiazinyl or phenoxazinyl. . (36) X ⁸ is an OH group, an SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group, or a halogen atom, a C _1-10 alkylcarbonyl group, an OH group, A phenyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group (31 ) To the oligonucleotide derivative according to (35). (37) X ⁸ is OH group, SH group, methyl group, ethyl group,
Propyl group, phenyl group, (o, m, p) -alkoxyphenyl group, (o, m, p) -alkylphenyl group,
The oligonucleotide derivative according to items (31) to (35), which is selected from the group consisting of a methoxy group and an ethoxy group. (38) X ⁸ is OH group, SH group, methyl group, ethyl group,
Selected from the group consisting of propyl group, phenyl group, (o, m, p) -C _1-10 alkoxyphenyl group, (o, m, p) -C _1-10 alkylphenyl group, methoxy group and ethoxy group. The oligonucleotide derivative according to the above (31) to (35). (39) The oligonucleotide derivative according to the item (31) to (35), wherein X ⁸ is an OH group, an SH group, a methyl group or a phenyl group. (40) R ¹ , R ² and R ³ are each a hydrogen atom, OH
The oligonucleotide derivative according to item (31) to (39), which is selected from the group consisting of a group, fluoro, chloro, bromo, methoxy group, ethoxy group and methoxymethyloxy group. (41) R ¹ , R ² and R ³ are hydrogen atoms (3
The oligonucleotide derivative according to items 1) to 39). (42) The oligonucleotide derivative according to any one of (31) to (41), wherein n is an integer of 1 to 38. (43) The oligonucleotide derivative according to any one of (31) to (41), wherein n is an integer of 1-28. (44) n is an integer of 4 to 38, in the repetition of n-1, the first X ⁸ is a phenyl group, and the n-th
The oligonucleotide derivative according to item (32), wherein the first X ⁸ is a phenyl group. Equation (45)

[0076]

[Chemical 55]

[In the formula, Y represents a sugar residue, and oligonucleotide b1 is SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, sequence A nucleotide sequence selected from the group consisting of NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48 is shown. ] (1) represented by
The oligonucleotide derivative according to the item. (46) The sugar of the sugar residue represented by Y is galactose, mannose, melibiose, gentiobiose, isomaltotriose, n-octylglucoside, n-octylthioglucoside, phenylglucoside, galactosamine,
The oligonucleotide derivative according to Item (45), which is N-benzoylgalactosamine, N-acetylgalactosamine, glucosamine, N-benzoylglucosamine or N-acetylglucosamine. (47) The oligonucleotide derivative according to (45), wherein the sugar of the sugar residue represented by Y is melibiose, gentiobiose or N-benzoylgalactosamine. Formula (48)

[0078]

[Chemical 56]

[In the formula, Y represents a sugar residue, and oligonucleotide b2 is SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, sequence A nucleotide sequence selected from the group consisting of NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48 is shown. ] (1) represented by
The oligonucleotide derivative according to the item. (49) The oligonucleotide derivative according to the item (48), wherein the sugar of the sugar residue represented by Y is galactose, mannose or glucose. (50) The oligonucleotide derivative according to the item (48), wherein the sugar of the sugar residue represented by Y is galactose.

Equation (51)

[0081]

[Chemical 57]

[Wherein Q, B ¹ , B ² , B ³ , R ¹ , R ² ,
R ³ and n have the same meanings as described in item (2), and X ¹¹ ,
X ⁴¹ and X ⁵¹ each represent O or S, and X ⁵¹ is n
May be the same or different, and CPG represents a solid phase carrier. ] The compound represented by
Or expression

[0083]

[Chemical 58]

[Wherein, Q, B ¹ , B ² , B ³ , R ¹ , R ² ,
R ³ and n have the same meanings as described in item (2), and X ¹² ,
X ⁴² and X ⁵² each represent a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an aromatic ring group which may be substituted, and X ⁵² represents a repeating n group. CPG, which may be the same or different, represents a solid phase carrier. ] The protecting group of the compound represented by these is removed, The manufacturing method of the oligonucleotide derivative as described in (1) characterized by the above-mentioned. (52) A gene expression inhibitor comprising the oligonucleotide derivative according to any one of (1) to (50) or a salt thereof. (53) The gene expression inhibitor according to the item (52), which is an expression inhibitor of a gene causing a malignant tumor, a viral disease or inflammation. (54) Suppressing gene expression is (i) premR from DNA
Inhibition of transcription to NA, (ii) pre-mRNA to mature mR
Suppression of splicing to NA or (iii) mature mR
The gene expression inhibitor according to item (52) or (53), which suppresses translation of NA into protein. (55) The gene that causes the malignant tumor is sr
c, fps, yes, ros, myb, myc, er
b, rel, mos, abl, ras, fos, fe
A gene selected from the group consisting of s, fms, sis, raf, neu, and p53, and the genes causing the viral disease are AIDS virus, influenza virus, herpes virus, papilloma virus, polio virus, picornavirus, and adenovirus. Genes selected from the group consisting of viruses, which are genes that cause inflammation are ICAM-1, ELAM-
The gene expression inhibitor according to item (53), which is a gene selected from the group consisting of 1 and VLAM. (56) The (52) wherein the gene is the ICAM-1 gene
The gene expression inhibitor according to the item. (57) An agent for suppressing vascular restenosis after PTCA, which comprises the oligonucleotide derivative according to any one of items (1) to (50) or a salt thereof. [D-1] Sugar derivative (1) represented by the following items (1) to (5):

[0085]

[Chemical 59]

[Wherein Q is Y or

[0087]

[Chemical 60]

Represents a group represented by and Y represents a sugar residue. ] The sugar derivative represented by these. (2) Q is Y, and the sugar of the sugar residue represented by Y is galactose, mannose, melibiose, gentiobiose and isomaltotriose, or these hydroxyl groups are substituted with a C _1-5 alkylcarbonyl group or a benzoyl group. Glucoside and thioglucoside or their hydroxyl groups substituted with a C _1-5 alkylcarbonyl group, benzoyl group or C _1-20 alkyl group, or glucosamine and galactosamine or their hydroxyl groups substituted with a halogen atom The sugar derivative according to item (1), which is substituted with an optionally substituted C _1-5 alkylcarbonyl group or a benzoyl group. (3) Q is Y, and the sugar of the sugar residue represented by Y is 1,2,3,
4-0-tetraacetylgalactose, 1,2,3,4-0-tetraacetylmannose, 1-On-octyl-2,3,4-O-tribenzoylglucoside, 1-On-octyl-2,3, 4-O-tribenzoylthioglucoside, 1-O-phenyl-2,3,4-O-tribenzoylglucoside, 1,3,4-O-tribenzoyl, 2-N-
Benzoylgalactosamine, 1,3,4-O-triacetyl, 2-N
-Acetylgalactosamine, 1,3,4-O-triacetyl, 2-N-
Acetylglucosamine, 1,3,4-O-tribenzoyl, 2-N-
Item (1) which is trifluoroacetylgalactosamine, 1,3,4-O-tribenzoyl, 2-N-trifluoroacetylglucosamine, heptabenzoylmelibiose, heptabenzoylgentiobiose or octabenzoylisomaltotriose The sugar derivative described. (4) Q is the formula

[0089]

[Chemical formula 61]

[In the formula, Y represents a sugar residue. ] The sugar derivative of the item (1), wherein the sugar of the sugar residue represented by Y is a monosaccharide in which the hydroxyl group may be substituted with a C _1-5 alkylcarbonyl group. (5) Q is the formula

[0091]

[Chemical formula 62]

[In the formula, Y represents a sugar residue. ] A sugar represented by the formula (1), wherein the sugar of the sugar residue represented by Y is galactose, mannose or glucose whose hydroxyl group may be substituted with a C _1-5 alkylcarbonyl group. Derivative.

[D-2] Sugar derivative (1) represented by the following items (1) to (7):

[0094]

[Chemical formula 63]

[Wherein Q is Y or an expression

[0096]

[Chemical 64]

Represents a group represented by, Y represents a sugar residue,
X ¹² represents a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an aromatic ring group which may be substituted. ] The sugar derivative represented by these. (2) Q is Y, and the sugar of the sugar residue represented by Y is galactose, mannose, melibiose, gentiobiose and isomaltotriose, or these hydroxyl groups are substituted with a C _1-5 alkylcarbonyl group or a benzoyl group. Glucoside and thioglucoside or their hydroxyl groups substituted with a C _1-5 alkylcarbonyl group, benzoyl group or C _1-20 alkyl group, or glucosamine and galactosamine or their hydroxyl groups substituted with a halogen atom The sugar derivative according to item (1), which is substituted with an optionally C _1-5 alkylcarbonyl group or a benzoyl group. (3) Q is Y, and the sugar of the sugar residue represented by Y is 1,2,3,
4-0-tetraacetylgalactose, 1,2,3,4-0-tetraacetylmannose, 1-On-octyl-2,3,4-O-tribenzoylglucoside, 1-On-octyl-2,3, 4-O-tribenzoylthioglucoside, 1-O-phenyl-2,3,4-O-tribenzoylglucoside, 1,3,4-O-tribenzoyl, 2-N-
Benzoylgalactosamine, 1,3,4-O-triacetyl, 2-N
-Acetylgalactosamine, 1,3,4-O-triacetyl, 2-N-
Acetylglucosamine, 1,3,4-O-tribenzoyl, 2-N-
Item (1) which is trifluoroacetylgalactosamine, 1,3,4-O-tribenzoyl, 2-N-trifluoroacetylglucosamine, heptabenzoylmelibiose, heptabenzoylgentiobiose or octabenzoylisomaltotriose The sugar derivative described. (4) Q is Y, and the sugar of the sugar residue represented by Y is 1-On-
Octyl-2,3,4-O-tribenzoylglucoside or 1-O
The sugar derivative according to item (1), which is -n-octyl-2,3,4-O-tribenzoylthioglucoside. (5) Q is the formula

[0098]

[Chemical 65]

(Wherein Y represents a sugar residue), and the sugar of the sugar residue represented by Y may have a hydroxyl group substituted with a C _1-5 alkylcarbonyl group. The sugar derivative according to item (1), which is a monosaccharide. (6) Q is the formula

[0100]

[Chemical formula 66]

(Wherein Y represents a sugar residue), and the sugar of the sugar residue represented by Y may have a hydroxyl group substituted with a C _1-5 alkylcarbonyl group. The sugar derivative according to item (1), which is galactose, mannose or glucose. (7) X ¹² is a C _1-5 alkyl group, a C _1-5 alkoxy group, C
_1-5 monoalkylamino group, or halogen atom, C
Substituted with about 1 to 5 substituents selected from the group consisting of _1-10 alkylcarbonyl group, OH, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group. The sugar derivative according to any one of items (1) to (6), which is an optionally substituted phenyl group.

[D-3] Sugar derivative (1) represented by the following items (1) to (4)

[0103]

[Chemical formula 67]

[In the formula, Y represents a sugar residue. ] The sugar derivative represented by these. (2) The sugar of the sugar residue represented by Y is a monosaccharide (1)
The sugar derivative according to the item. (3) Galactose in which the sugar of the sugar residue represented by Y may have a hydroxyl group substituted with a C _1-5 alkylcarbonyl group,
The sugar derivative according to item (1), which is mannose or glucose. (4) The sugar derivative according to item (1), wherein the sugar of the sugar residue represented by Y is galactose whose hydroxyl group may be substituted with a C _1-5 alkylcarbonyl group. [E] Nucleotide derivative shown in the following items (1) to (8) (1) Formula

[0105]

[Chemical 68]

[0106] wherein, W is a hydrogen atom or a protecting group, B ¹ represents a nucleic acid residues, R ¹ represents a hydrogen atom, OH group, a halogen atom, C _1-5 alkoxy group or C _1-5 alkoxy An alkyloxy group is shown. ] The nucleotide derivative represented by these. (2) W is a hydrogen atom, formula

[0107]

[Chemical 69]

Or

[0109]

[Chemical 70]

[In the formula, Y represents a sugar residue, and X ¹ represents OH.
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_{1-5 represents a} monoalkylamino group or an optionally substituted aromatic ring group. ] The nucleotide derivative as described in (1) represented by these. (3) W is a hydrogen atom or formula

[0111]

[Chemical 71]

Wherein the sugar of the sugar residue represented by Y is galactose, mannose, melibiose, gentiobiose and isomaltotriose, or the hydroxyl group thereof is substituted with a C _1-5 alkylcarbonyl group or a benzoyl group, Glucosides and thioglucosides or their hydroxyl groups substituted with C _1-5 alkylcarbonyl groups, benzoyl groups or C _1-20 alkyl groups, or glucosamine and galactosamine or their hydroxyl groups substituted with halogen atoms The nucleotide derivative according to item (1), which is substituted with a good C _1-5 alkylcarbonyl group or benzoyl group. (4) W is a hydrogen atom or formula

[0113]

[Chemical 72]

And the sugar of the sugar residue represented by Y is 1,2,3,
4-0-tetraacetylgalactose, 1,2,3,4-0-tetraacetylmannose, 1-On-octyl-2,3,4-O-tribenzoylglucoside, 1-On-octyl-2,3, 4-O-tribenzoylthioglucoside, 1-O-phenyl-2,3,4-O-tribenzoylglucoside, 1,3,4-O-tribenzoyl, 2-N-
Benzoylgalactosamine, 1,3,4-O-triacetyl, 2-N
-Acetylgalactosamine, 1,3,4-O-triacetyl, 2-N-
Acetylglucosamine, 1,3,4-O-tribenzoyl, 2-N-
Item (1) which is trifluoroacetylgalactosamine, 1,3,4-O-tribenzoyl, 2-N-trifluoroacetylglucosamine, heptabenzoylmelibiose, heptabenzoylgentiobiose or octabenzoylisomaltotriose The described nucleotide derivative. (5) W is a hydrogen atom or formula

[0115]

[Chemical formula 73]

The nucleotide derivative according to item (1), wherein the sugar of the sugar residue represented by Y is a monosaccharide whose hydroxyl group may be substituted with a C _1-5 alkylcarbonyl group. (6) W is a hydrogen atom or a formula

[0117]

[Chemical 74]

The nucleotide derivative according to item (1), wherein the sugar of the sugar residue represented by Y has a hydroxyl group of galactose, mannose or glucose. (7) X ¹ is OH group, SH group, C _1-5 alkyl group,
A C _1-5 alkoxy group, a C _1-5 monoalkylamino group,
Or a halogen atom, a C _1-10 alkylcarbonyl group,
A phenyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of an OH group, a nitro group, a C _1-10 alkyl group, a C _1-10 alkoxy group, a phenyl group and a naphthyl group. The nucleotide derivative according to items (1) to (6). (8) The nucleotide derivative according to any one of (1) to (6), wherein X ¹ is an OH group, an SH group, a methyl group or a phenyl group.

In the present specification, W represents a hydrogen atom or a protecting group. The protecting group represented by W may be any group as long as it can protect the OH group. For example,
Formula (i)

[0120]

[Chemical 75]

(In the formula, Y represents a sugar residue, and X ¹ represents OH.
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_{1-5 represents a} monoalkylamino group or an optionally substituted aromatic ring group. Group represented by), formula (ii)

[0122]

[Chemical 76]

(In the formula, Y represents a sugar residue, and X ¹ represents OH.
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_{1-5 represents a} monoalkylamino group or an optionally substituted aromatic ring group. ), A group represented by (iii), (iii) a halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), a C _1-6 alkyl group which may be substituted with a nitro group (eg, methyl, ethyl, n-). Propyl, i-
Propyl, n-butyl, tert-butyl etc.), (iv) halogen atom (eg fluoro, chloro, bromo, iodo etc.), C _1-6 alkyl group (eg methyl, ethyl, n-propyl, i-propyl) , N-butyl, tert-
Butyl, etc.), a phenyl group, a C _6-11 aryl group _optionally substituted with a nitro group (eg, phenyl, naphthyl, etc.), (v) a halogen atom (eg, fluoro,
Chloro, bromo, iodo, etc.), C _1-6 alkyl group (e.g., methyl, ethyl, n- propyl, i- propyl,
n-butyl, tert-butyl, etc.), a phenyl group, a C _7-12 aralkyl group _optionally substituted by a nitro group (eg, benzyl), (vi) a halogen atom (eg,
Fluoro, chloro, bromo, iodo, etc.) and the like, which may be substituted with C _1-6 alkylcarbonyl group (eg,
Formyl, methylcarbonyl, ethylcarbonyl, etc.), (vii) phenyloxycarbonyl group (eg,
Benzoxycarbonyl etc.), (viii) C _7-12 aralkyl-carbonyl group (eg benzyloxycarbonyl etc.), (ix) pyranyl group, (x) furanyl group, (x
i) a silyl group, (xii) a trityl group optionally substituted with 1 to 3 methoxy groups (eg, trityl,
(Methoxytrityl, dimethoxytrityl, trimethoxytrityl, etc.), (xiii) phenylxanthenyl group optionally substituted with methoxy group (eg, phenylxanthenyl, methoxyphenylxanthenyl, etc.) and the like are used.

In the present specification, Y represents a sugar residue. Y
As the sugar of the sugar residue represented by, those known per se are used, and may be chemically synthesized or naturally occurring. Specifically, an optionally substituted monosaccharide or a condensate thereof is used. Examples of the monosaccharide include an optionally substituted 5-carbon sugar aldose (eg, ribose, arabinose, xylose,
Lyxose, etc.), 5-carbon sugar ketose (for example, ribulose, xylulose, etc.), 6-carbon sugar aldose (for example,
Allose, altrose, glucose, mannose, gulose, idose, galactose, talose, etc.), 6
Carbon sugar ketose (for example, psicose, fructose, sorbose, tagatose, etc.) and the like are used. The monosaccharide may have, for example, a hydroxyl group generally substituted with about 1 to 4 substituents. As this substituent, for example,
Examples thereof include an optionally substituted acyl group, an optionally substituted alkyl group, and an optionally substituted amino group. As the acyl group of the optionally substituted acyl group, a C _1-10 alkylcarbonyl group (for example, C _1-6 alkylcarbonyl group such as formyl, acetyl and ethylcarbonyl), a phenylcarbonyl group, a C _7-10 Aralkylcarbonyl group (eg, benzylcarbonyl group), C _1-10 alkoxycarbonyl group (eg, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, etc.), phenyloxycarbonyl group (eg, benzoxycarbonyl group, etc.), naphthyl An oxycarbonyl group, a C _7-10 aralkyloxycarbonyl group (eg, benzyloxycarbonyl group, etc.), an allylsulfenyl group (eg, phenylsulfenyl, naphthylsulfenyl, etc.) and the like are used. As the acyl group, for example, a C _1-10 acyl group and the like are preferable, and examples thereof include a C _1-10 alkylcarbonyl group (eg, C _1-6 alkylcarbonyl group such as formyl, acetyl, ethylcarbonyl), phenylcarbonyl group, benzyl. A carbonyl group and the like are preferable. Examples of the substituent of the acyl group include a halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), a C _1-10 alkylcarbonyl group (eg, formyl, methylcarbonyl, ethylcarbonyl, etc.), a nitro group, C _1-10 alkyl groups (eg methyl,
Ethyl, etc.), C _1-10 alkoxy group (eg, methoxy, ethoxy, etc.), phenyl group, naphthyl group, etc. are used, and the number of substituents is usually about 1 to 5, preferably about 1 to 3. . Examples of the alkyl group which may be substituted include C _1-20 alkyl groups (eg, methyl, ethyl, n-propyl, i-propyl, n-butyl, n-pentyl, n-hexyl, n −
Heptyl, n-octyl, etc.), preferably a C _1-6 alkyl group (eg methyl, ethyl, n-propyl, i-
Propyl, n-butyl, n-pentyl, n-hexyl, etc.) are used. As a substituent of this alkyl group,
For example, halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), C _1-10 alkylcarbonyl group (eg, formyl, acetyl, ethylcarbonyl, etc., C
_1-6 alkylcarbonyl group), nitro group, C _1-10 alkyl group (eg methyl, ethyl etc.), C _1-10 alkoxy group (eg methoxy, ethoxy etc.), phenyl group, naphthyl group etc. are used. The number of substituents is usually about 1 to 5, preferably about 1 to 3. Examples of the optionally substituted amino group include, for example, a compound represented by the formula-
A group represented by NR ⁴ R ⁵ (in the formula, R ⁴ and R ⁵ each represent a hydrogen atom, an optionally substituted acyl group or an optionally substituted alkyl group) and the like are used. Examples of the optionally substituted acyl group represented by R ⁴ and R ⁵ and the optionally substituted alkyl group are:
The same as the above is used. The position of substitution of this amino group is preferably the 2-position of the monosaccharide. Thus, the monosaccharide in which the hydroxyl group is substituted with the amino group is a so-called amino sugar. Specific examples of such an amino sugar include, for example, glucosamine, galactosamine, N-methylglucosamine, 3-amino-3-deoxy whose amino group may be substituted with R ⁴ or (and) R ⁵ described above. Glucose, grosamine, neosamine B, neosamine C, fucosamine, rhamnosamine, aminomannuronic acid, quinovosamine and the like are preferable. Specifically, for example, glucosamine or galactosamine in which a 2-amino group may be substituted with a C _1-6 alkylcarbonyl group, a phenylcarbonyl group or the like (eg, glucosamine, galactosamine, N-
Acetylglucosamine, N-acetylgalactosamine,
N-benzoylglucosamine, N-benzoylgalactosamine, etc.) are preferable, and glucosamine and N- are particularly preferable.
Acetylglucosamine, N-benzoylglucosamine,
N-benzoylgalactosamine and the like are preferable. Among the above monosaccharides, for example, 5-carbon sugar aldose (for example,
Ribose, arabinose and the like), hexacarbon sugar aldose (eg, glucose, mannose, galactose and the like) and the like are more preferable. Especially galactose, mannose,
Glucose and the like are preferred. Substituents for the hydroxyl groups of monosaccharides include, for example, C _1-10 alkylcarbonyl groups (eg C such as formyl, acetyl, ethylcarbonyl, etc.
_1-6 alkylcarbonyl group), an acyl group such as a phenylcarbonyl group, and a C _1-20 alkyl group (eg, methyl, ethyl, n-propyl, i-propyl, n-butyl, n-).
Pentyl, n-hexyl, n-heptyl, n-octyl, etc.) are preferred. As the monosaccharide of Y in the formula (I), specifically, a 6-carbon sugar aldose in which a hydroxyl group may be substituted with a C _1-6 alkylcarbonyl group is particularly preferable. More specifically, for example, galactose, mannose, 1,2,3,4-0-tetraacetylgalactose, 1,2,3,4
-0-tetraacetylmannose, glucosamine, N-acetylglucosamine, N-benzoylglucosamine, N
-Benzoylgalactosamine and the like are preferred.

As the condensate of the monosaccharides, for example, optionally substituted oligosaccharides, glycoside derivatives and the like are used. As the oligosaccharides, for example, those obtained by condensing 2 to 15 of the above-mentioned monosaccharides, preferably 2 to 8 are used, and for example, 2 to 5 of the above-mentioned monosaccharides, preferably 2 to 3 are used. Those in which individual pieces are condensed are also used. Specifically, those which are naturally present as free or as constituents of glycosides are used. For example, (1) lactose existing in the animal kingdom, α, α-
Trehalose, N-acetylneuraminyl lactose,
(2) Genthianose, isoliknoses, lycnose, plantateose, sucrose, biflucose, 1,6-diglucosylmannitol, galactosylsucrose 6-β-glucosyl existing in the plant kingdom, such as difucosyllactose, fucosyllactose, and lactulose. Mannitol, laminaribiose, inulobiose, lycopoose, maltose, isomalttriose,
(3) Sophorose, gentiooligosaccharides, vicyanose, primeverose, sambubiose, which are present as constituents of glycosides, such as melibiose, neobifucose, neokestose, erulose, 6-α-galactosylgalactose, kojibiose, maltulose, melezitose, and turanose. , Lycobiose, lycotriose, sorabiose, strohantobiose, gigilandbiose, odrotriose, fungitetraose, β
-1,3 Xylooligosaccharides and the like are used. Of these, for example, gentiobiose, melibiose, isomalttriose and the like are preferable. In the above oligosaccharide, for example, the hydroxyl group may be substituted. As this substituent,
The same substituents as the above monosaccharides, for example, an optionally substituted acyl group, an optionally substituted alkyl group, an optionally substituted amino group and the like are used.
Examples of the glycoside derivative include a glycoside derivative in which a hydroxyl group of the monosaccharide or oligosaccharide is substituted with a non-sugar aglycone (for example, an O-glycoside derivative,
S-glycoside derivative or N-glycoside derivative)
Are used. Examples of the aglycone include -O
^{-R 7, -S-R 8,} ( wherein, R ^7, R ⁸ and R ⁹ represent, respectively, an optionally substituted hydrocarbon group.) -N-R ⁹ and the like are used. Examples of the hydrocarbon group of the optionally substituted hydrocarbon group represented by R ⁷ , R ⁸ and R ⁹ include an alkyl group, an alkenyl group, an alkynyl group,
An aryl group and an aralkyl group are used. Examples of the alkyl group include C _1-30 alkyl groups (eg, methyl, ethyl, n-propyl, i-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-).
Octyl, stearyl, etc.), especially C _1-20 alkyl groups (eg methyl, ethyl, n-propyl, i-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, stearyl, etc. ) And the like are preferable. As the alkenyl group, for example, a C _2-4 alkenyl group (eg, allyl, propenyl, etc.) is used. As the alkynyl group, for example, a C _2-4 alkynyl group (eg, ethynyl, propynyl etc.) and the like are used. As the aryl group, for example, a C _6-12 aryl group such as a phenyl group or a naphthyl group is used. As the aralkyl group, for example, a C _7-14 aralkyl group (eg, benzyl, phenylethyl, naphthylmethyl, naphthylethyl, etc.) and the like are used. Examples of the substituent of the above alkyl group, alkenyl group and alkynyl group include a halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), a C _1-10 alkylcarbonyl group (eg, formyl, acetyl, ethylcarbonyl, etc.), Nitro group,
A C _1-10 alkoxy group (eg, methoxy, ethoxy, etc.) and the like are used. The number of substituents is usually about 1 to 5, preferably about 1 to 3. Examples of the substituent of the aryl group and aralkyl group include a halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), a C _1-10 alkylcarbonyl group (eg, formyl, acetyl, ethylcarbonyl, etc.), a nitro group, C
_1-10 alkyl group (eg, methyl, ethyl, etc.), C
_1-10 alkoxy groups (eg methoxy, ethoxy, etc.)
Are used. The number of substituents is usually about 1 to 5, preferably about 1 to 3. R ⁷ , R ⁸ and R ⁹ are each a C _1-30 alkyl group (for example,
Methyl, ethyl, n-propyl, i-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n
-Octyl, stearyl, etc.), a C _6-12 aryl group (eg, phenyl group, naphthyl group, etc.) and the like are preferable.
Specific examples of the glycoside derivative include n-
Octyl glucoside, n-octyl galactoside, n-
Octylmannoside, n-octylthioglucoside, n
-Octyl thiogalactoside, n-octyl thiomannoside, stearyl glycoside, stearyl galactoside, stearyl mannoside, stearyl thioglycoside, stearyl thiogalactoside, stearyl thiomannoside, phenyl glucoside, phenyl galactoside,
Phenyl mannoside and the like are preferable. Among the above, the sugar of the sugar residue represented by Y is, for example, Gal.
(Galactose), Man (mannose), Glu (glucose), n-oct-Glc (n-octylglucoside), n-oct-SGlc (n, -octyl-thioglucoside), PhGlc (phenylglucoside), P
hGal (phenylgalactoside), GalN (galactosamine), N-AcGal (N-acetylgalactosamine), N-BzGal (N-benzoylgalactosamine), GulN (glucosamine), N-AcGul (N).
-Acetylglucosamine), Mel (melibiose),
Gen (gentiobiose), Iso (isomaltotriose) and the like are preferable. Particularly, in the oligonucleotide derivative (Ia) of the present invention, Gal (galactose), Man (mannose), n-oct-Glc (n
-Octyl glucoside), n-oct-SGlc (n,
-Octyl-thioglucoside), PhGlc (phenylglucoside), PhGal (phenylgalactoside),
GalN (galactosamine), N-AcGal (N-acetylgalactosamine), N-BzGal (N-benzoylgalactosamine), GulN (glucosamine), N
-AcGul (N-acetylglucosamine), Mel
(Meribiose), Gen (Gentiobiose), Is
o (isomaltotriose) and the like are preferable. In the oligonucleotide derivative (Ib) of the present invention, Me
l (Meribiose), Gen (Gentiobiose), N
-BzGal (N-benzoylgalactosamine) and the like are preferable.

In the present specification, X ¹ is an OH group, SH
Group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or optionally substituted aromatic ring group. Examples of the C _1-5 alkyl group include methyl,
Ethyl, n-propyl, i-propyl, n-butyl, i
-Butyl, tert-butyl and the like are used. The C
_{As the 1-5} alkoxy group, for example, methoxy, ethoxy, propoxy and the like are used. Examples of the C _1-5 monoalkylamino group include methylamino, ethylamino, n
-Propylamino and the like are used. Examples of the substituent of the optionally substituted phenyl group include a halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), C _1-10 alkylcarbonyl group (eg, C such as formyl, acetyl, ethylcarbonyl, etc. _1-6 alkylcarbonyl group), OH, nitro group, C _1-10 alkyl group (eg, methyl, ethyl, etc.), C _1-10 alkoxy group (eg, methoxy, ethoxy, n-propyl, i-propyl, n -Butyl, i-butyl, tert-butyl, etc.), a phenyl group, a naphthyl group, etc. are used, and the number of substituents is usually about 1 to 5, preferably about 1 to 3. Examples of the aromatic ring group include an aryl group,
An aromatic heterocyclic group or the like is used.

As the aryl group, for example, a C _6-11 aryl group (eg, phenyl group, naphthyl group, etc.) is used, and a phenyl group is particularly preferable. Examples of the substituent of the aryl group include a halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), a C _1-10 alkylcarbonyl group (eg, formyl, acetyl, propionyl, n-butyryl, iso-butyryl, etc.), OH group, nitro group, C _1-10
Alkyl group (eg, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, etc.), C _1-10 alkoxy group (eg, methoxy, ethoxy, propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, etc.), phenyl group, naphthyl group and the like. The number of substituents is about 1 to 5, preferably about 1 to 3. The aromatic heterocyclic group is, for example, a 5- to 13-membered aromatic group containing about 1 to 4 hetero atoms and about 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom. Group heterocyclic groups and the like are used, and 5- to 9-membered aromatic heterocyclic groups are particularly preferable. For example, 2- or 3-thienyl, 2- or 3-furyl,
2- or 3-pyrrolyl, 2-, 3- or 4-pyridyl, 2-, 4- or 5-oxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-imidazolyl, 3-, 4- or 5-isoxazolyl, 3-, 4- or 5-isothiazolyl, 3- or 5- (1,2,4-oxadiazolyl), 1,3,4-oxadiazolyl, 3-or 5-
(1,2,4-thiadiazolyl), 1,3,4-thiadiazolyl, 4- or 5- (1,2,3-thiadiazolyl), 1,
2,5-thiadiazolyl, 1,2,3-triazolyl, 1,
2,4-triazolyl, 1H- or 2H-tetrazolyl or other 1 to about 4 carbon atoms, as well as an oxygen atom,
5-membered aromatic heterocyclic group containing 1 to 4 heteroatoms selected from sulfur atom, nitrogen atom and the like, for example, N-oxide-2-, 3- or 4-pyridyl, 2-, 4- or 5- Pyrimidinyl, N-oxide-2-, 4- or 5
-Pyrimidinyl, thiomorpholinyl, morpholinyl, oxoimidazolinyl, dioxotriazinyl, pyrrolidinyl, piperidinyl, pyranyl, thiopyranyl, 1,4-
2 to 5 such as oxazinyl, 1,4-thiazinyl, 1,3-thiazinyl, piperazinyl, triazinyl, oxotriazinyl, 3- or 4-pyridazinyl, pyrazinyl, N-oxide-3- or 4-pyridazinyl
6-membered aromatic heterocyclic group containing 1 to 4 heteroatoms selected from oxygen atom, sulfur atom, nitrogen atom, etc. in addition to about 4 carbon atoms, for example, benzofuryl, benzothiazolyl, benzoxazolyl, tetrazolo [1 , 5-b] pyridazinyl, triazolo [4,5-b] pyridazinyl,
Benzimidazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, indolizinyl, quinolidinyl, 1,8-naphthyridinyl, purinyl, pteridinyl, dibenzofuranyl,
In addition to 1 to 8 carbon atoms such as carbazolyl, acridinyl, phenanthridinyl, chromanyl, benzoxazinyl, phenazinyl, phenothiazinyl, and phenoxazinyl, a hetero atom selected from oxygen atom, sulfur atom, nitrogen atom, etc. Examples thereof include 5- to 9-membered aromatic heterocyclic groups containing 1 to 4 groups. The substituent which the aromatic heterocyclic group may have is, for example, C _1-6 alkyl (eg, methyl, ethyl, propyl, isopropyl,
Butyl, isobutyl, sec-butyl, tert-butyl, etc., C _2-6 alkenyl (eg, vinyl, 1-methylvinyl, 1-propenyl, allyl, etc.), C _2-6 alkynyl (eg, ethynyl, 1-propynyl) , Propargil, etc.),
C _3-6 cycloalkyl (eg, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.), C _5-7
Cycloalkenyl (eg, cyclopentenyl, cyclohexenyl, etc.), C _7-11 aralkyl (eg, benzyl, α-
Methylbenzyl, phenethyl etc.), C _6-14 aryl (eg phenyl, naphthyl etc.), C _1-6 alkoxy (eg methoxy, ethoxy, propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy). , Tert-
Butoxy), C _6-14 aryloxy (eg, phenoxy etc.), C _1-6 alkylcarbonyl (eg, formyl,
Acetyl, propionyl, n-butyryl, iso-butyryl etc.), C _6-14 aryl-carbonyl (eg benzoyl etc.), C _1-6 alkanoyloxy (eg formyloxy, acetyloxy, propionyloxy, n-butyryloxy, iso-butyryloxy, etc.), C _6-14 aryl-
Carbonyloxy (eg, benzoyloxy etc.), carboxyl, C _1-6 alkoxy-carbonyl (eg, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, iso-propoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, tert-butoxy Carbonyl), carbamoyl group, N-mono-C _1-4 alkylcarbamoyl (eg, N-methylcarbamoyl, N-ethylcarbamoyl, N-propylcarbamoyl, N-isopropylcarbamoyl, N-butylcarbamoyl, etc.), N, N −
Di-C _1-4 alkylcarbamoyl (eg, N, N-dimethylcarbamoyl, N, N-diethylcarbamoyl, N, N-dipropylcarbamoyl, N, N-dibutylcarbamoyl, etc.), halogen atom (eg, fluoro, Chloro, bromo, iodo, etc.), mono-, di- or tri-halogeno-C _1-4 alkyl (eg, chloromethyl, dichloromethyl, trifluoromethyl, trifluoroethyl, etc.), oxo group, amidino group, imino group , An amino group, a mono- or di-C _1-4 alkylamino group (eg, methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, etc.), Examples thereof include OH group and nitro group. The number of substitutions is about 1 to 6, preferably about 1 to 3. Examples of X ¹ include OH group, SH group, methyl group, ethyl group, n-
Propyl group, phenyl group, (o, m, p) -C _1-10 alkoxyphenyl group, (o, m, p) -C _1-10 alkylphenyl group, methoxy, ethoxy and the like are preferable. In particular,
OH group, SH group, methyl group, phenyl group and the like are preferable.

In the present specification, Q is Y (Y represents a sugar residue) or a formula

[0129]

[Chemical 77]

[In the formula, Y represents a sugar residue. ] The group represented by is shown. As the sugar residue represented by Y, the same ones as described above are used. In the present specification, B ¹ ,
B ² , B ²¹ , B ²² and B ³ each represent a nucleic acid residue. Examples of the nucleic acid residue include hypoxanthine-9-
Ilyl, guanin-9-yl, adenin-9-yl, uracil-1-yl, thymine-1-yl, cytosin-1-yl and the like are used. B ² may be the same or different in each repeating n. B ¹ , B ² , B ²¹ ,
B ²² and B ³ can be appropriately selected depending on the oligonucleotide sequence to be produced. X ² , X ³ ,
X ⁶ , X ⁷ and X ⁸ respectively represent an OH group, an SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group. ,
X ³ , X ⁷ and X ⁸ may be the same or different in each repeating n. X ⁴ and X ⁵ are OH
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group or C _1-5 monoalkylamino group, and X ⁵ may be the same or different in repeating n. X
C _1-5 represented by ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁸
Examples of the alkyl group include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and ter.
t-Butyl or the like is used. X ² , X ³ , X ⁴ , X ⁵ , X
Examples of the C _1-5 alkoxy group represented by ⁶ , X ⁷ and X ⁸ include methoxy, ethoxy, propoxy and the like. Examples of the C _1-5 monoalkylamino group represented by X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁸ include methylamino, ethylamino, n-propylamino and the like. Examples of the substituent of the optionally substituted phenyl group include a halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), a C _1-10 alkylcarbonyl group (eg, C such as formyl, acetyl, ethylcarbonyl, etc.). _1-6 alkylcarbonyl group), OH group, nitro group, C _1-10 alkyl group (eg, methyl, ethyl, etc.), C _1-10 alkoxy group (eg, methoxy, ethoxy, n-propyl, i-propyl, (n-butyl, i-butyl, tert-butyl, etc.), phenyl group, naphthyl group and the like are used, and the number of substituents is usually about 1 to 5, preferably about 1 to 3. X ² , X ³ , X
Examples of the aromatic ring group of the optionally substituted aromatic ring group represented by ⁶ , X ⁷ and X ⁸ include an aryl group and an aromatic heterocyclic group. As the aryl group, for example, a C _6-11 aryl group (eg, phenyl group, naphthyl group, etc.) is used, and a phenyl group is particularly preferable.
Examples of the substituent of the aryl group include a halogen atom (eg, fluoro, chloro, bromo, iodo, etc.), C
_1-10 alkylcarbonyl group (eg, formyl, acetyl,
Propionyl, n-butyryl, iso-butyryl, etc.), OH
Group, nitro group, C _1-10 alkyl group (eg, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, se
c-butyl, tert-butyl, etc.), C _1-10 alkoxy group (eg, methoxy, ethoxy, propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-
Butoxy), phenyl group, naphthyl group and the like. The number of substituents is about 1 to 5, preferably about 1 to 3. The aromatic heterocyclic group is, for example, a 5- to 13-membered group containing about 1 to 4 hetero atoms and about 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom. An aromatic heterocyclic group or the like is used, and a 5- to 9-membered aromatic heterocyclic group or the like is particularly preferable. For example, 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl,
2-, 3- or 4-pyridyl, 2-, 4- or 5-
Oxazolyl, 2-, 4- or 5-thiazolyl, 3
-, 4- or 5-pyrazolyl, 2-, 4- or 5-
Imidazolyl, 3-, 4- or 5-isoxazolyl, 3-, 4- or 5-isothiazolyl, 3- or 5- (1,2,4-oxadiazolyl), 1,3,4-oxadiazolyl, 3- or 5- (1,2,4-thiadiazolyl), 1,3,4-thiadiazolyl, 4- or 5- (1,
2,3-thiadiazolyl), 1,2,5-thiadiazolyl,
1,2,3-triazolyl, 1,2,4-triazolyl, 1
5-membered aromatic heterocyclic group containing 1 to 4 heteroatoms selected from oxygen atom, sulfur atom, nitrogen atom, etc. in addition to 1 to 4 carbon atoms such as H- or 2H-tetrazolyl, for example, N -Oxide-2-, 3- or 4-
Pyridyl, 2-, 4- or 5-pyrimidinyl, N-oxide-2-, 4- or 5-pyrimidinyl, thiomorpholinyl, morpholinyl, oxoimidazolinyl, dioxotriazinyl, pyrrolidinyl, piperidinyl, pyranyl, thiopyranyl, 2 to 1, such as 1,4-oxazinyl, 1,4-thiazinyl, 1,3-thiazinyl, piperazinyl, triazinyl, oxotriazinyl, 3- or 4-pyridazinyl, pyrazinyl, N-oxide-3- or 4-pyridazinyl. A 6-membered aromatic heterocyclic group containing 1 to 4 heteroatoms selected from oxygen atom, sulfur atom, nitrogen atom, etc. in addition to about 5 carbon atoms, for example, benzofuryl, benzothiazolyl, benzoxazolyl, tetrazolo [ 1,5-b] pyridazinyl, triazolo [4,5]
-B] pyridazinyl, benzimidazolyl, quinolyl,
Isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, indolizinyl, quinolidinyl, 1,8-naphthyridinyl, purinyl, pteridinyl, dibenzofuranyl, carbazolyl, acridinyl,
An oxygen atom other than about 1 to 8 carbon atoms such as phenanthridinyl, chromanyl, benzoxazinyl, phenazinyl, phenothiazinyl, and phenoxazinyl.
Examples thereof include 5- to 9-membered aromatic heterocyclic groups containing 1 to 4 heteroatoms selected from sulfur atom, nitrogen atom and the like. The substituent that the aromatic heterocyclic group may have is, for example, C _1-6 alkyl (eg, methyl, ethyl,
Propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, etc.), C _2-6 alkenyl (eg, vinyl, 1-methylvinyl, 1-propenyl, allyl, etc.), C
_2-6 alkynyl (eg, ethynyl, 1-propynyl, propargyl, etc.), C _3-6 cycloalkyl (eg, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.), C _5-7 cycloalkenyl (eg, cyclopentenyl, cyclo) Hexenyl, etc.), C _7-11 aralkyl (eg,
Benzyl, α-methylbenzyl, phenethyl, etc.), C
_6-14 aryl (eg, phenyl, naphthyl, etc.), C _1-6
Alkoxy (eg methoxy, ethoxy, propoxy, is
o-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, etc.), C _6-14 aryloxy (eg, phenoxy, etc.), C _1-6 alkylcarbonyl (eg, formyl, acetyl, propionyl, n-butyryl, iso-butyryl etc.), C _6-14 aryl-carbonyl (eg benzoyl etc.), C _1-6 alkanoyloxy (eg formyloxy, acetyloxy, propionyloxy, n-butyryloxy, iso-butyryloxy etc. ), C _6-14 aryl-carbonyloxy (eg, benzoyloxy, etc.), carboxyl, C _1-6 alkoxy-carbonyl (eg, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, iso-propoxycarbonyl, n-butoxycarbonyl) , Isobutoxycarbonyl,
tert-butoxycarbonyl etc.), carbamoyl group, N
-Mono-C _1-4 alkylcarbamoyl (eg, N-methylcarbamoyl, N-ethylcarbamoyl, N-propylcarbamoyl, N-isopropylcarbamoyl, N-butylcarbamoyl, etc.), N, N-di-C _1-4 alkyl Carbamoyl (eg, N, N-dimethylcarbamoyl, N, N-diethylcarbamoyl, N, N-dipropylcarbamoyl, N, N-dibutylcarbamoyl, etc.), halogen atom (eg, fluoro, chloro, bromo, iodo, etc.) , Mono-, di- or tri-halogeno-C _1-4 alkyl (eg, chloromethyl, dichloromethyl, trifluoromethyl, trifluoroethyl, etc.), oxo group, amidino group, imino group, amino group,
Mono- or di-C _1-4 alkylamino group (eg, methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino,
Dipropylamino, diisopropylamino, dibutylamino and the like), OH group, nitro group and the like. The number of substitutions is about 1 to 6, preferably about 1 to 3. Examples of X ² , X ³ , X ⁶ , X ⁷ and X ⁸ include OH group, SH group, methyl group, ethyl group, n-propyl group, phenyl group, (o, m, p) -C _1-10 Alkoxyphenyl group, (o, m, p) -C _1-10 alkylphenyl group, methoxy, ethoxy and the like are preferable. Especially OH
A group, SH group, methyl group, phenyl group and the like are preferable.
As X ⁴ and X ⁵ , for example, OH group, SH group, methyl group, ethyl group, n-propyl group, methoxy group, ethoxy group and the like are preferable. Particularly, OH group, SH group, methyl group and the like are preferable.

In the present specification, R ¹ , R ² , R ²¹ , R ²²
And R ³ each represent a hydrogen atom, OH, a halogen atom, a C _1-5 alkoxyl group, a C _1-5 alkoxyalkyloxy group, and R ² and R ²¹ may be the same or different in repeating n. . Examples of the halogen atom include fluoro, chloro, bromo and iodo. Examples of the C _1-5 alkoxy group include methoxy, ethoxy, n-propoxy, i-
Propoxy, n-butoxy and the like are used. The C _1-5
Examples of the alkoxyalkyloxy group include C _1-5 alkoxy-, such as methoxymethyloxy, methoxyethyloxy, methoxypropyloxy, ethoxymethyloxy, ethoxyethyloxy and ethoxypropyloxy.
C _1-6 alkyl-oxy or the like is used. R ¹ , R ² ,
R ²¹ , R ²² and R ³ are each a hydrogen atom or O.
H, a halogen atom, a methoxy group, an ethoxy group, a methoxymethyloxy group and the like are preferable, and a hydrogen atom is particularly preferable. n shows the integer of 1-98. Among them, 1-38
Is an integer of 1 to 28, more preferably an integer of 1 to 28, especially 5 to 18
Is preferably an integer. That is, the oligonucleotide derivative of the present invention is usually about 3 to 100, preferably about 3 to 40, and more preferably 3 to 3.
It is an oligonucleotide derivative consisting of about 0 nucleotides, particularly preferably about 7 to 20 nucleotides.

Hereinafter, when the oligonucleotide chain portion is referred to in the present specification, for example, in the oligonucleotide derivative (Ia), B ¹ , B ² and B ³ are thymin-1-yl and R ¹ , R ² and When R ³ is a hydrogen atom and an oligonucleotide chain TTT in which n is 1 is represented,
When X ¹ , X ² and X ³ are OH groups, they are simply expressed as TTT, and when X ¹ is an OH group and X ² and X ³ are SH groups, T _S T
_S T, when X ¹ is an OH group and X ² and X ³ are methyl groups, it is expressed as T _Me T _Me T, X ¹ is an OH group and X ² and X
^{When 3} is a phenyl group, it is expressed as T _Ph T _Ph T, and X ¹ is OH.
In the case where X ² is an SH group and X ³ is a phenyl group, T _S T _Ph T
It may be written as. In the oligonucleotide derivative (Ia) of the present invention, Q is, for example, Y or a formula

[0133]

[Chemical 78]

A group represented by and the like are preferable. When Q is Y, the sugar of the sugar residue represented by Y is, for example, a C _1-10 alkylcarbonyl group (eg, C _1-6 alkylcarbonyl group such as formyl, acetyl, ethylcarbonyl),
An acyl group such as a phenylcarbonyl group or a C _1-20 alkyl group (eg, methyl, ethyl, n-propyl, i-propyl, n-butyl, n-pentyl, n-hexyl, n-
Preferred are monosaccharides, oligosaccharides, glycoside derivatives and the like which may be substituted with heptyl, n-octyl and the like). As the monosaccharide, for example, pentose aldose (eg, ribose, arabinose, etc.), hexacarbonate aldose (eg, glucose, mannose, galactose, etc.) and the like are preferable, and galactose, mannose, galactosamine and the like are particularly preferable. As the oligosaccharide, melibiose, gentiobiose, isomaltotriose and the like are preferable. As the glycoside derivative,
N-octyl glucoside, n-octyl thioglucoside, phenyl glucoside and the like are preferable. Also, C _1-6
2 with an alkylcarbonyl group or a phenylcarbonyl group
-Glucosamine or galactosamine optionally substituted with an amino group and the like are also preferable. Among these,
Galactose or mannose whose hydroxyl group may be substituted with a C _1-6 alkylcarbonyl group (eg, formyl, acetyl, ethylcarbonyl, etc.), melibiose, gentiobiose or isomalt which may be substituted with a phenylcarbonyl group. Substituted with triose, n-octyl glucoside, n-octyl thioglucoside or phenyl glucoside, C _1-10 alkylcarbonyl group (eg C _1-6 alkylcarbonyl group such as formyl, acetyl, ethylcarbonyl) or phenylcarbonyl group Glucosamine, galactosamine and the like which may be present are preferred. In particular,
For example, Gal (galactose), Man (mannose), n-oct-Glc (n-octylglucoside), n-oct-SGlc (n-octyl-thioglucoside), PhGlc (phenylglucoside), PhG.
al (phenylgalactoside), GalN (galactosamine), N-AcGal (N-acetylgalactosamine), N-BzGal (N-benzoylgalactosamine), GulN (glucosamine), N-AcGul (N).
-Acetylglucosamine), Mel (melibiose),
Gen (gentiobiose), Iso (isomaltotriose) and the like are preferable. Preference is also given to hexose aldose, which may have a hydroxyl group substituted with a C1-6 alkylcarbonyl group (eg, formyl, acetyl, ethylcarbonyl, etc.), and specific examples include galactose, mannose, 1,2. Preferred are 3,3,4-0-tetraacetylgalactose, 1,2,3,4-0-tetraacetylmannose, glucosamine, N-acetylglucosamine, N-benzoylglucosamine and N-benzoylgalactosamine.

Q is an expression

[0136]

[Chemical 79]

In the case of the group represented by, the sugar of the sugar residue represented by Y is preferably, for example, a monosaccharide. As the monosaccharide, for example, galactose, mannose, glucose and the like are preferable, and galactose is particularly preferable. X
¹ is OH group, SH group, C _1-5 alkyl group,
A C _1-5 alkoxy group, a C _1-5 monoalkylamino group,
Halogen atom, C _1-10 alkylcarbonyl group, OH
C, which may be substituted with about 1 to 5 substituents selected from the group consisting of a group, a nitro group, a C _1-10 alkyl group, a C _1-10 alkoxy group, a phenyl group and a naphthyl group.
_6-11 aryl group (eg, phenyl group, naphthyl group, etc.), or C _1-6 alkyl group, C _2-6 alkenyl group,
C _2-6 alkynyl group, C _3-6 cycloalkyl group, C _5-7 cycloalkenyl group, C _7-11 aralkyl group, C _6-14 aryl group, C _1-6 alkoxy group, C _6-14 aryloxy group Base,
C _1-6 alkylcarbonyl group, C _6-14 aryl-carbonyl group, C _1-6 alkanoyloxy group, C _6-14 aryl-carbonyloxy group, carboxyl group, C _1-6 alkoxy-
Carbonyl group, carbamoyl group, N-mono-C _1-4 alkylcarbamoyl group, N, N-di-C _1-4 alkylcarbamoyl group, halogen atom, mono-, di- or tri-halogeno-C _1-4 alkyl Substituted with about 1 to 5 substituents selected from the group consisting of a group, an oxo group, an amidino group, an imino group, an amino group, a mono- or di-C _1-4 alkylamino group, an OH group and a nitro group. 1 selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom
A 5- to 13-membered aromatic heterocyclic group containing about 4 to about 4 hetero atoms and about 1 to 12 carbon atoms is preferable. The 5- to 13-membered aromatic heterocyclic group containing 1 to 4 hetero atoms and 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom is 2- or 3 -Thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 3-
Or 4-pyridyl, 2-, 4- or 5-oxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-imidazolyl, 3-, 4- Or 5-isoxazolyl, 3-, 4
-Or 5-isothiazolyl, 3-or 5- (1,2,
4-oxadiazolyl), 1,3,4-oxadiazolyl, 3- or 5- (1,2,4-thiadiazolyl), 1,
3,4-thiadiazolyl, 4- or 5- (1,2,3-thiadiazolyl), 1,2,5-thiadiazolyl, 1,2,3
-Triazolyl, 1,2,4-triazolyl, 1H- or 2H-tetrazolyl, N-oxide-2-, 3- or 4-pyridyl, 2-, 4- or 5-pyrimidinyl,
N-oxide-2-, 4- or 5-pyrimidinyl, thiomorpholinyl, morpholinyl, oxoimidazolinyl,
Dioxotriazinyl, pyrrolidinyl, piperidinyl,
Pyranyl, thiopyranyl, 1,4-oxazinyl, 1,4
-Thiazinyl, 1,3-thiazinyl, piperazinyl, triazinyl, oxotriazinyl, 3- or 4-pyridazinyl, pyrazinyl, N-oxide-3- or 4-
Pyridazinyl, benzofuryl, benzothiazolyl, benzoxazolyl, tetrazolo [1,5-b] pyridazinyl, triazolo [4,5-b] pyridazinyl, benzimidazolyl, quinolyl, isoquinolyl, cinnolinyl,
Phthalazinyl, quinazolinyl, quinoxalinyl, indolizinyl, quinolidinyl, 1,8-naphthyridinyl, purinyl, pteridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenanthridinyl, chromanyl,
Preferred are benzoxazinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like. As X ¹ ,
In particular, OH group, SH group, methyl group, ethyl group, n-propyl group, phenyl group, (o, m, p) -C _1-10 alkoxyphenyl group, (o, m, p) -C _1-10 alkyl Phenyl, methoxy, ethoxy and the like are preferred. Above all, O
H group, SH group, methyl group, phenyl group and the like are preferable. Examples of X ⁴ and X ⁵ are OH group, SH
Group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5
A monoalkylamino group and the like are preferable. Above all, OH
Group, SH group, methyl group, ethyl group, n-propyl group, methoxy, ethoxy and the like are preferable, and particularly OH group, SH
A group and a methyl group are preferable.

As the combination of X ¹ , X ⁴ and X ⁵ , (i) X ¹ is an OH group, an SH group, a C _1-5 alkyl group,
A C _1-5 alkoxy group, a C _1-5 monoalkylamino group,
Or a halogen atom, a C _1-10 alkylcarbonyl group,
A phenyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of OH group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group. , X ⁴ and X ⁵ are an OH group and S, respectively.
H group, C _1-5 alkyl group, C _1-5 alkoxy group or C
_{In the case of 1-5} monoalkylamino group, (ii) X ¹ is OH group, SH group, methyl group, ethyl group, propyl group, phenyl group, (o, m, p) -alkoxyphenyl group, (o , M, p) -alkylphenyl group, methoxy group and ethoxy group, wherein X ⁴ and X ⁵ are OH group, SH group, methyl group, ethyl group, propyl group and methoxy group, respectively. Or when it is an ethoxy group, (iii) X ¹ is OH group, SH group, methyl group, ethyl group, propyl group, phenyl group, (o, m, p) -C _1-10 alkoxyphenyl group, (o, m, p) -C _1-10 alkylphenyl group, methoxy group or ethoxy group, and X ⁴ and X ⁵ are OH group, SH group, methyl group, ethyl group, propyl group, methoxy group and ethoxy group, respectively. In this case, (iv) X ¹ is an OH group, S It is preferably a H group, a methyl group or a phenyl group, and when X ⁴ and X ⁵ are an OH group, an SH group or a methyl group, and the like. Examples of the nucleic acid residues represented by B ¹ , B ² and B ³ include hypoxanthin-9-yl, guanine-9-yl, adenin-9-yl, uracil-1-yl, thymine-1-yl, cytosine. -1-yl and the like are preferable, and can be appropriately selected depending on the oligonucleotide sequence to be produced. R ¹ ,
As R ² and R ³ , a hydrogen atom, OH, a halogen atom, a methoxy group, an ethoxy group, a methoxymethyloxy group and the like are preferable, and a hydrogen atom is particularly preferable. n
Represents an integer of 1 to 98. Among them, integers from 1 to 38,
More preferably, it is an integer of 1-28, particularly preferably an integer of 5-18. Oligonucleotide derivative (Ia) of the present invention
As, any derivative in which the preferable ones of the above-mentioned symbols are appropriately combined is preferable. Of these, an oligonucleotide derivative containing a nucleotide sequence represented by CAT is preferable. Further, the oligonucleotide derivative (Ia) of the present invention is more preferably a so-called antisense oligonucleotide derivative. Furthermore, it is preferable that they are entrapped in liposomes (for example, lipofectin). In the oligonucleotide derivative (Ib) of the present invention, W is, for example, a hydrogen atom, a formula

[0139]

[Chemical 80]

A group represented by the formula:

[0141]

[Chemical 81]

A group represented by and the like are preferable.

W is an expression

[0144]

[Chemical formula 82]

In the case of the group represented by, the sugar of the sugar residue represented by Y is, for example, a C _1-10 alkylcarbonyl group (for example, C such as formyl, acetyl or ethylcarbonyl).
_1-6 alkylcarbonyl group), an acyl group such as a phenylcarbonyl group, and a C _1-20 alkyl group (eg, methyl, ethyl, n-propyl, i-propyl, n-butyl, n-).
(Pentyl, n-hexyl, n-heptyl, n-octyl etc.) and the like, monosaccharides, oligosaccharides or glycoside derivatives which may be substituted are preferable. As the monosaccharide, for example, 5-carbon sugar aldose (for example, ribose, arabinose, etc.), 6-carbon sugar aldose (for example, glucose, mannose, galactose, etc.) and the like are preferable,
Galactose, mannose, galactosamine and the like are particularly preferable. As the oligosaccharide, melibiose, gentiobiose, isomaltotriose and the like are preferable. Examples of the glycoside derivative include n-octyl glucoside,
N-octylthioglucoside, phenylglucoside and the like are preferable. In addition, glucosamine or galactosamine in which the 2-amino group may be substituted with a C _1-6 alkylcarbonyl group or a phenylcarbonyl group is also preferable. Among these, galactose or mannose whose hydroxyl group may be substituted with a C _1-6 alkylcarbonyl group (eg, formyl, acetyl, ethylcarbonyl, etc.) and melibiose whose hydroxyl group may be substituted with a phenylcarbonyl group , Gentiobiose or isomaltotriose, n-octyl glucoside, n-octyl thioglucoside or phenyl glucoside, C _1-10 alkylcarbonyl group (for example, C _1-6 alkylcarbonyl group such as formyl, acetyl, ethylcarbonyl) or phenyl Glucosamine, galactosamine and the like, which may be substituted with a carbonyl group, are suitable. Specifically, for example, Gal (galactose), Man (mannose), n-oct-Glc (n
-Octyl glucoside), n-oct-SGlc (n-
Octyl-thioglucoside), PhGlc (phenylglucoside), PhGal (phenylgalactoside), G
alN (galactosamine), N-AcGal (N-acetylgalactosamine), N-BzGal (N-benzoylgalactosamine), GulN (glucosamine), N-
AcGul (N-acetylglucosamine), Mel (Meribiose), Gen (Gentiobiose), Iso
(Isomaltotriose) and the like are preferable. Above all,
Mel (melibiose), Gen (gentiobiose), N-BzGal (N-benzoylgalactosamine) and the like are preferable. Preference is also given to hexose aldose, which may have a hydroxyl group substituted with a C _1-6 alkylcarbonyl group (eg, formyl, acetyl, ethylcarbonyl, etc.), and specific examples include galactose, mannose, 1, 2,3,4-0-tetraacetylgalactose, 1,2,3,4-0-tetraacetylmannose, glucosamine, N-acetylglucosamine, N-benzoylglucosamine, N-benzoylgalactosamine and the like are preferable.

W is an expression

[0147]

[Chemical 83]

In the case of the group represented by, the sugar of the sugar residue represented by Y is preferably, for example, a monosaccharide. As the monosaccharide, for example, galactose, mannose, glucose and the like are preferable, and galactose is particularly preferable. X
⁶ and X ⁷ are OH group, SH group,
C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, halogen atom, C _1-10 alkylcarbonyl group, OH group, nitro group, C _1-10 alkyl group, C
_1-10 alkoxy group, C _6-11 aryl group which may be substituted with about 1 to 5 substituents selected from the group consisting of phenyl group and naphthyl group (for example, phenyl group,
Naphthyl group), or C _1-6 alkyl group, C _2-6 alkenyl group, C _2-6 alkynyl group, C _3-6 cycloalkyl group, C _5-7 cycloalkenyl group, C _7-11 aralkyl group,
C _6-14 aryl group, C _1-6 alkoxy group, C _6-14 aryloxy group, C _1-6 alkylcarbonyl group, C _6-14 aryl-carbonyl group, C _1-6 alkanoyloxy group, C
_6-14 aryl-carbonyloxy group, carboxyl group,
C _1-6 alkoxy-carbonyl group, carbamoyl group, N-
Mono-C _1-4 alkylcarbamoyl group, N, N-di-C
_1-4 alkylcarbamoyl group, halogen atom, mono-,
Di- or tri-halogeno-C _1-4 alkyl group, oxo group, amidino group, imino group, amino group, mono- or di-C
_1-4 1 to 4 selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom which may be substituted with about 1 to 5 substituents selected from the group consisting of an alkylamino group, an OH group and a nitro group. A 5- to 13-membered aromatic heterocyclic group containing about 1 to 12 carbon atoms and about 1 to 12 carbon atoms is preferable.

The 5- to 13-membered aromatic heterocyclic group containing 1 to 4 hetero atoms and 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom is 2 -Or 3-thienyl, 2-
Or 3-furyl, 2- or 3-pyrrolyl, 2,3
-Or 4-pyridyl, 2-, 4- or 5-oxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-imidazolyl, 3-, 4 -Or 5-isoxazolyl, 3-, 4
-Or 5-isothiazolyl, 3-or 5- (1,2,
4-oxadiazolyl), 1,3,4-oxadiazolyl, 3- or 5- (1,2,4-thiadiazolyl), 1,
3,4-thiadiazolyl, 4- or 5- (1,2,3-thiadiazolyl), 1,2,5-thiadiazolyl, 1,2,3
-Triazolyl, 1,2,4-triazolyl, 1H- or 2H-tetrazolyl, N-oxide-2-, 3- or 4-pyridyl, 2-, 4- or 5-pyrimidinyl,
N-oxide-2-, 4- or 5-pyrimidinyl, thiomorpholinyl, morpholinyl, oxoimidazolinyl,
Dioxotriazinyl, pyrrolidinyl, piperidinyl,
Pyranyl, thiopyranyl, 1,4-oxazinyl, 1,4
-Thiazinyl, 1,3-thiazinyl, piperazinyl, triazinyl, oxotriazinyl, 3- or 4-pyridazinyl, pyrazinyl, N-oxide-3- or 4-
Pyridazinyl, benzofuryl, benzothiazolyl, benzoxazolyl, tetrazolo [1,5-b] pyridazinyl, triazolo [4,5-b] pyridazinyl, benzimidazolyl, quinolyl, isoquinolyl, cinnolinyl,
Phthalazinyl, quinazolinyl, quinoxalinyl, indolizinyl, quinolidinyl, 1,8-naphthyridinyl, purinyl, pteridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenanthridinyl, chromanyl,
Preferred are benzoxazinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like. X ⁶ and X ⁷ are respectively OH group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, halogen atom, C _1-10 alkylcarbonyl group, Phenyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of OH group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group, and the like. OH group, S
H group, methyl group, ethyl group, n-propyl group, phenyl group, (o, m, p) -C _1-10 alkoxyphenyl group,
(O, m, p) -C _1-10 alkylphenyl group, methoxy group, ethoxy group and the like are preferable. Especially, OH group, SH
A group, a methyl group, a phenyl group and the like are preferable. B ¹ ,
Examples of the nucleic acid residues represented by B ² , B ²¹ , B ²² and B ³ include hypoxanthine-9-yl, guanine-9-yl,
Adenine-9-yl, uracil-1-yl, thymine-1
-Yl, cytosyn-1-yl and the like are preferable and can be appropriately selected depending on the oligonucleotide sequence to be produced. As R ¹ , R ² , R ²¹ , R ²² and R ³ ,
Hydrogen atom, OH, halogen atom, methoxy group,
Ethoxy group, methoxymethyloxy group and the like are preferable,
A hydrogen atom is particularly preferable. n shows the integer of 1-98. Among them, an integer of 1 to 38, more preferably 1 to 2
An integer of 8 is preferable, and an integer of 5 to 18 is particularly preferable.

The oligonucleotide derivative of the present invention (I
Among b), those containing a phenylphosphonate type nucleotide at the 5'-terminal are particularly preferable. That is, the formula

[0151]

[Chemical 84]

An oligonucleotide derivative (Ib-1) having a nucleotide sequence represented by: is preferred. W,
Preferable ranges of B ¹ , B ² , B ³ , R ¹ , R ² , R ³ and n are the same as those described above as the preferable range of the oligonucleotide derivative (Ib).
X ⁸ is OH group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, halogen atom, C _1-10 alkylcarbonyl group, O
C optionally substituted with about 1 to 5 substituents selected from the group consisting of H group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group.
_6-11 aryl group (eg, phenyl group, naphthyl group, etc.), or C _1-6 alkyl group, C _2-6 alkenyl group,
C _2-6 alkynyl group, C _3-6 cycloalkyl group, C _5-7 cycloalkenyl group, C _7-11 aralkyl group, C _6-14 aryl group, C _1-6 alkoxy group, C _6-14 aryloxy group Base,
C _1-6 alkylcarbonyl group, C _6-14 aryl-carbonyl group, C _1-6 alkanoyloxy group, C _6-14 aryl-carbonyloxy group, carboxyl group, C _1-6 alkoxy-
Carbonyl group, carbamoyl group, N-mono-C _1-4 alkylcarbamoyl group, N, N-di-C _1-4 alkylcarbamoyl group, halogen atom, mono-, di- or tri-halogeno-C _1-4 alkyl Substituted with about 1 to 5 substituents selected from the group consisting of a group, an oxo group, an amidino group, an imino group, an amino group, a mono- or di-C _1-4 alkylamino group, an OH group and a nitro group. 1 selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom
A 5- to 13-membered aromatic heterocyclic group containing about 1 to about 4 hetero atoms and about 1 to about 12 carbon atoms is preferable. The 5- to 13-membered aromatic heterocyclic group containing 1 to 4 hetero atoms and 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom is 2- or 3 -Thienyl, 2-
Or 3-furyl, 2- or 3-pyrrolyl, 2,3
-Or 4-pyridyl, 2-, 4- or 5-oxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-imidazolyl, 3-, 4 -Or 5-isoxazolyl, 3-, 4
-Or 5-isothiazolyl, 3-or 5- (1,2,
4-oxadiazolyl), 1,3,4-oxadiazolyl, 3- or 5- (1,2,4-thiadiazolyl), 1,
3,4-thiadiazolyl, 4- or 5- (1,2,3-thiadiazolyl), 1,2,5-thiadiazolyl, 1,2,3
-Triazolyl, 1,2,4-triazolyl, 1H- or 2H-tetrazolyl, N-oxide-2-, 3- or 4-pyridyl, 2-, 4- or 5-pyrimidinyl,
N-oxide-2-, 4- or 5-pyrimidinyl, thiomorpholinyl, morpholinyl, oxoimidazolinyl,
Dioxotriazinyl, pyrrolidinyl, piperidinyl,
Pyranyl, thiopyranyl, 1,4-oxazinyl, 1,4
-Thiazinyl, 1,3-thiazinyl, piperazinyl, triazinyl, oxotriazinyl, 3- or 4-pyridazinyl, pyrazinyl, N-oxide-3- or 4-
Pyridazinyl, benzofuryl, benzothiazolyl, benzoxazolyl, tetrazolo [1,5-b] pyridazinyl, triazolo [4,5-b] pyridazinyl, benzimidazolyl, quinolyl, isoquinolyl, cinnolinyl,
Phthalazinyl, quinazolinyl, quinoxalinyl, indolizinyl, quinolidinyl, 1,8-naphthyridinyl, purinyl, pteridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenanthridinyl, chromanyl,
Preferred are benzoxazinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like. For X ⁸ ,
In particular, OH group, SH group, methyl group, ethyl group, n-propyl group, phenyl group, (o, m, p) -C _1-10 alkoxyphenyl group, (o, m, p) -C _1-10 alkyl Phenyl, methoxy, ethoxy and the like are preferred. Above all, O
H group, SH group, methyl group, phenyl group and the like are preferable. More preferred oligonucleotide derivatives (Ib) include those containing a phenylphosphonate-type nucleotide at the second position from the 5'-end and the 3'-end. That is, the formula

[0153]

[Chemical 85]

An oligonucleotide derivative (Ib-2) having a nucleotide sequence represented by is preferable.

Preferable ranges of W, B ¹ , B ³ , R ¹ , R ³ and n are the same as those mentioned above as the preferable range of the oligonucleotide derivative (Ib). More preferred oligonucleotide derivative (I
Examples of b) include those containing two phenylphosphonate-type nucleotides at the 5'-end and phenylphosphonate-type nucleotides at the second and third positions from the 3'-end. That is, n-1 in the above-mentioned oligonucleotide derivative (Ib-2)
(N is an integer of 4 to 38, preferably an integer of 4 to 28, and more preferably an integer of 4 to 18) is repeated, and the first X ⁸ is a phenyl group, and the n-1th An oligonucleotide derivative in which X ⁸ is a phenyl group is preferred. Examples of the preferable range of B ²¹ and B ²² include the same range as described above as the preferable range of B ² of the oligonucleotide derivative (Ib).
Preferable range of R ²¹ and R ²² is the same range as described above as a preferable range of R ² of the oligonucleotide derivative (Ib). The preferred range of X ^8, include the same range as those described above as the preferred range of X ⁸ oligonucleotide derivative (Ib-1). The oligonucleotide derivative of the present invention (I
b) may be a so-called antisense oligonucleotide derivative, or may be encapsulated in liposomes (eg lipofectin etc.).

The oligonucleotide derivatives (I), (Ia) and (Ib) of the present invention are the target DNAs.
Any one may be used as long as it suppresses the expression of. That is, (1) whether it is complementary to the sequence of the target DNA and suppresses transcription of DNA into pre-mRNA,
(2) Complementary to the sequence of pre-mRNA,
Any one may be used as long as it suppresses the splicing from NA to mature mRNA or (3) suppresses the translation from mature mRNA to protein. If the subject is DNA, it may be complementary to any of the exon site, the intron site, and the site containing both exons and introns. Specifically, it suppresses the expression of genes that produce proteins that cause various diseases (eg, malignant tumors, viral diseases, inflammation, vascular restenosis after PTCA, etc.). In particular, the oligonucleotide derivative (Ia) of the present invention or a salt thereof can be used as a so-called antisense oligonucleotide derivative. Genes that cause malignant tumors include oncogenes such as src, fps, yes, and r.
os, myb, myc, erb, rel, mos, ab
l, ras, fos, fes, fms, sis, ra
Examples include f, neu and p53 genes. Examples of genes that cause viral diseases include AIDS virus (eg, env, gag, pol, n
ef, tat and rev), influenza virus, herpes virus, papilloma virus, polio virus, picorna virus, adenovirus and the like. Genes that produce proteins that cause inflammation (particularly adhesion proteins) include:
For example, ICAM-1, ELAM-1, VLAM and the like can be mentioned. Examples of genes that produce proteins that cause vascular restenosis after PTCA include PD
Examples include GF gene and FGF gene. The oligonucleotide derivative of the present invention may be complementary to a part of the DNA or mRNA sequence of these genes. For example, since the expression initiation codon, 5'-ATG-3 'or 5'-AUG-3', is contained in the DNA or mRNA sequence, the sequence containing the ATG, AUG, or sequences before and after them. Is complementary to the DNA or mR
The expression of NA can be suppressed. Therefore, examples of the oligonucleotide derivative of the present invention include 5'-ATG.
5'-CAT- which is complementary to 3'or 5'-AUG-3 '
An oligonucleotide derivative having a nucleotide sequence represented by 3 ′ is preferable.

The specific embodiments of the oligonucleotide derivative of the present invention are shown below, but the present invention is not limited thereto. Examples of the oligonucleotide derivative (Ia) complementary to ICAM-1, which is a gene that produces an adhesion protein that causes inflammation, include compounds of the formula:

[0158]

[Chemical 86]

[In the formula, Y and X 1 have the same meanings as described above. ] The oligonucleotide derivative (Ia-
1),

[0160]

[Chemical 87]

[In the formula, Y and X 1 have the same meanings as described above. ] The oligonucleotide derivative (Ia-
2) and the like. Oligonucleotide derivative (I
Examples of the oligonucleotide a ₁ and the oligonucleotide a ₂ in a-1) and (Ia-2) include, for example, TGGGAGCCATAGCGAGGC (SEQ ID NO: 1) TGGGAGCCATAGCGAGGCT (SEQ ID NO: 2) GGCTGCTGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 3) GGCTGCTGGGAGCCATAGCGAGGCTGAGG (sequence). No .: 4) GGCTGCTGGGAGCCATAGCGAGGCTGAG (SEQ ID NO: 5) GGCTGCTGGGAGCCATAGCGAGGCTGA (SEQ ID NO: 6) GGCTGCTGGGAGCCATAGCGAGGCTG (SEQ ID NO: 7) GGCTGCTGGGAGCCATAGCGACGGC: GC #: GC10: GGCTGCTGGGAGCCAGGCGGCAGCT: SEQ ID NO: 8) : 11) GGCTGCTGGGAGCCATAGCGA (SEQ ID NO: 12) GGCTGCTGGGAGCCATAGCG (SEQ ID NO: 13) GGCTGCTGGGAGCCATAGC (SEQ ID NO: 14) GGCTGCTGGGAGCCATAG (SEQ ID NO: 15) GGCTGCTGGGAGCCATA (SEQ ID NO: 16) GGCTGCTGGGAGCCAT (SEQ ID NO: 17) JishitijiCTGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 18) CTGCTGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 19) TGCTGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 20) GCTGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 21) CTGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 22) TGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 23) GGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 24) GGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 25) GAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 26) AGCCATAGCGAGGCTGAGGT (SEQ ID NO: 27) GCCATAGCGAGGCTGAGGT (SEQ ID NO: 28) CCATAGCGAGGCTGAGGT (SEQ ID NO: 29) CATAGCGAGGCTGAGGT (SEQ ID NO: 30). X ¹ , X ⁴ and X ⁵ in these oligonucleotides are OH groups, but X ⁴ and / or X ⁵ may be SH groups or methyl groups. Examples of such an oligonucleotide a ₁ and an oligonucleotide a ₂ include, for example, T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C (SEQ ID NO: 31) T _Me G _Me GGAGCCATAGCGAG _Me G _Me C (SEQ ID NO: 34) and the like. In particular, SEQ ID NO: 1, SEQ ID NO: 3
1, oligonucleotide a ₁ and oligonucleotide a ₂ represented by SEQ ID NO: 34 and the like are preferable.

Specific examples of Y in the oligonucleotide derivative (Ia-1) include Gal (galactose), Man (mannose) and n-oct-Glc (n.
-Octyl glucoside), n-oct-SGlc (n-
Octyl-thioglucoside), PhGlc (phenylglucoside), PhGal (phenylgalactoside), G
alN (galactosamine), N-AcGal (N-acetylgalactosamine), N-BzGal (N-benzoylgalactosamine), GulN (glucosamine), N-
AcGul (N-acetylglucosamine), Mel (Meribiose), Gen (Gentiobiose), Iso
(Isomaltotriose) and the like are preferable. As Y in the oligonucleotide derivative (Ia-2), specifically Gal (galactose), Man (mannose), Glu (glucose) and the like are preferable, and Ga is particularly preferable.
1 (galactose) is preferred. In addition, an oligonucleotide derivative (I
As b), for example, the formula

[0163]

[Chemical 88]

[In the formula, Y has the same meaning as described above. ] The oligonucleotide derivative (Ib-3) represented by

[0165]

[Chemical 89]

[In the formula, Y has the same meaning as described above. Such as an oligonucleotide derivative (Ib-4 and the like represented by]. Examples of oligonucleotides b ₁ and oligonucleotide b ₂ in an oligonucleotide derivative (Ib-3) and (Ib-4), for example TGGGAGCCATAGCGAGGC (SEQ No .: 1) TGGGAGCCATAGCGAGGCT (SEQ ID NO: 2) GGCTGCTGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 3) GGCTGCTGGGAGCCATAGCGAGGCTGAGG (SEQ ID NO: 4) GGCTGCTGGGAGCCATAGCGAGGCGC number: 5) GGCTGCTGGGAGCCGC: GC number: 5) GGCTGCTGGGAGCCTG: GC number: 5) : 8) GGCTGCTGGGAGCCATAGCGAGGC (SEQ ID NO: 9) GGCTGCTGGGAGCCATAGCGAGG (SEQ ID NO: 10) GGCTGCTGGGAGCCATAGCGAG (SEQ ID NO: 11) GGCTGCTGGGAGCCATAGCGA (SEQ ID NO: 12) GGCTGCTGGGAGCCATAGCG (SEQ ID NO: 13) GGCTGCTGGGAGAG GGCTGCTGGGAGCCATAG (SEQ ID NO: 15) GGCTGCTGGGAGCCATA (SEQ ID NO: 16) GGCTGCTGGGAGCCAT (SEQ ID NO: 17) GCTGCTGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 18) CTGCTGGGAGCCATAGCGAGGGAGCGA21GC (SEQ ID NO: 19) TGCTGGGAGCCAGTAG: GC20: SEQ ID NO: 19) (SEQ ID NO: 22) TGGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 23) GGGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 24) GGAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 25) GAGCCATAGCGAGGCTGAGGT (SEQ ID NO: 26) AGCCATAGCGAGGCTGAGGT (SEQ ID NO: 27) GCCATAGATAAGAGGC SEQ ID NO: 29) CATAGCGAGGCTGAGGT (SEQ ID NO: 30) CCCCCACCACTTCCCCT (SEQ ID NO: 49) CCCTGTCCCGGGATAGGT (SEQ ID NO: 50) GGAGCCATAGCGAGGC (SEQ ID NO: 51) AGCCATAGCGAG (SEQ ID NO: 52) AGCCA TAGC (SEQ ID NO: 53) GGTTTTTTTTTTTTTTTGGG (SEQ ID NO: 54) AATTTTTTTTTTTTTTTAAA (SEQ ID NO: 55) and other oligonucleotides in which at least one of X ⁶ and X ⁷ is a phenyl group (ie, at least one nucleotide is phenyl) A phosphonate type oligonucleotide) is used, for example, T _Ph G _Ph GGAGCCATAGCGAG _Ph G _Ph C (SEQ ID NO: 32) T _Ph GGGAGC _Ph C _Ph AT _Ph AGC _Ph GAGGC _PH T (SEQ ID NO: 33) T _Ph GGGAGCCATAGCGAGG _Ph C (SEQ ID NO: 35) C _Ph G _Ph GAGCGATACCGAGG _Ph G _Ph T (SEQ ID NO: 37) T _Ph G _Ph GGAGC _Ph C _Ph AT _Ph AGC _Ph GAG _Ph G _Ph C (SEQ ID NO: 39) C _Ph G _Ph GAGC _Ph GAT _Ph AC _Ph C _Ph GAGG _Ph G _Ph T (SEQ ID NO: 40) C _Ph C _Ph CCCACCACTTCCC _Ph C _Ph T (SEQ ID NO: 41) C _Ph C _Ph CTGTCCCGGGATAG _Ph G _Ph T (SEQ ID NO: 42) T _Ph G _Ph G _Ph G _Ph A _Ph G _Ph C _Ph C _Ph A _Ph T _Ph A _Ph G _Ph C _Ph G _Ph A _Ph G _Ph G _Ph C (SEQ ID NO: 43) G _Ph G _Ph AGCCATAGCGAG _Ph G _Ph C (SEQ ID NO: 44) A _Ph G _Ph CCATAGCG _Ph A _Ph G (SEQ ID NO: 45) A _Ph G _Ph CCATA _Ph G _Ph C (SEQ ID NO: 46) G _Ph G _Ph TTTTTTTTTTTTTG _Ph G _Ph G (SEQ ID NO: 47) A _Ph A _Ph TTTTTTTTTTTTTTTA _Ph A _Ph A (SEQ ID NO: 48) and the like are preferable.

Specific examples of Y in the oligonucleotide derivative (Ib-3) include Gal (galactose), Man (mannose) and n-oct-Glc (n.
-Octyl glucoside), n-oct-SGlc (n-
Octyl-thioglucoside), PhGlc (phenylglucoside), PhGal (phenylgalactoside), G
alN (galactosamine), N-AcGal (N-acetylgalactosamine), N-BzGal (N-benzoylgalactosamine), GulN (glucosamine), N-
AcGul (N-acetylglucosamine), Mel (Meribiose), Gen (Gentiobiose), Iso
(Isomaltotriose) and the like are preferable. In particular, Me
l (Meribiose), Gen (Gentiobiose), N
-BzGal (N-benzoylgalactosamine) and the like are preferable. As Y in the oligonucleotide derivative (Ib-4), specifically Gal (galactose), Man (mannose), Glu (glucose) and the like are preferable, and Gal (galactose) is particularly preferable. As the oligonucleotide derivative of the present invention, more specifically, Examples 3, 4, 6, 8, 10, 12, 1
4, 16, 18, 20, 22, 24, 26, 28, 3
Those produced in 0, 32 to 60 and 64 are preferable, and in particular, Examples 10, 12, 14, 16, 18,
20, 22, 24, 26, 32, 33, 34, 35, 4
More preferred are those manufactured by 4, 45, 46,
Those manufactured in Examples 18, 44, 45 and 46 are particularly preferable.

In the oligonucleotide derivative (I) of the present invention, first of all, regarding the oligonucleotide, the starting nucleoside is selectively and appropriately protected at the base site such as purine or pyrimidine and the sugar hydroxyl group according to a known method ( "Oligonucleotide Synthesis; a prac
tical approach "MJ Gait (ed), IRL PRESS, (198
4).), Leading 3'-hydroxyl group to phosphinic acid derivative or H-phosphonate derivative (MD Matteuci et al., Tet
rahedron Let., vol. 21, 719 (1980) .SL Beaucage
et al., Tetrahedron Lett., vol. 22, 1859 (1981).
LJ McBride et al., Tetrahedron Lett., Vol. 2
4, 245 (1983). BC Froehler et al., Nucleic Aci
ds Res., vol. 14, 5399 (1986). BC Froehler et a
l., Tetrahedron Lett., vol. 27, 469 (1986) .PJ G
aregg et al., Tetrahedron Lett., vol. 27, 4051 (198
6). E. Uhlman et al., Chemical Reviews, vol.90, N
o.4, 543 (1990).), they can be synthesized as a unit according to the known phosphoramidite method, H-phosphonate method, etc. described in the above-mentioned reference. The method for producing the oligonucleotide derivatives (Ia) and (Ib) of the present invention will be described in detail below. [Method for producing oligonucleotide derivative (Ia)] First,
The sugar residue to be introduced into the oligonucleotide derivative is
First, a sugar compound as a raw material is, for example, according to a known method,
If necessary, protect the secondary sugar hydroxyl groups (see "Reagent
s for Organic Synthesis ", vol. 1, 453, L. Fieser
(eds), John Wiley and Sons, INC. (1967). DD Reyn
olds et al., Org. Syn., Coll. vol. 3, 432 (1955).
RR Schmidt, Angew. Chem. Int. Ed. Engl., Vol.
25, 212 (1986).), Its primary hydroxyl group is introduced into a phosphinic acid derivative or an H-phosphonate derivative according to the known method described in the above-mentioned reference, and these are used as a unit in the known phosphoramidite method described in the above-mentioned reference, H
-It can be synthesized according to the phosphonate method or the like. Hereinafter, the oligonucleotide derivative of the present invention (I
The production method of a) will be described by taking as an example the production method of an oligonucleotide derivative in which Q is Y and the sugar of the sugar residue represented by Y is a galactose derivative, but Y is another group. Oligonucleotide derivatives (Ia) and Q have the formula

[0169]

[Chemical 90]

An oligonucleotide derivative (Ia) which is
Can be similarly obtained. The oligonucleotide derivative (Ia) of the present invention (eg, an oligonucleotide derivative having a galactose derivative introduced) has the following reaction (a):
To (c).
Phosphorylation of the 6-position hydroxyl group of a sugar derivative (eg, tetra-O-benzoylgalactose) protected by an appropriate protecting group has been known in the literature (MD Matt-euci et al., Tetrahe).
dron Let., vol. 21, 719 (1980). SLBeaucage et a
l., Tetrahedron Lett., vol. 22, 1859 (1981). LJ
McBride et al., Tetrahed-ron Lett., Vol. 24, 245
(1983). BC Froehler et al., Nucleic Acids Re
s., vol. 14, 5399 (1986). BC Froehler et al., Te
trahedron Lett., vol.27, 469 (1986) .PJ Garegg e
t al., Tetrahedron Lett., vol. 27, 4051 (1986). E.
Uhlman et al., Chemical Reviews, vol.90, No.4, 543
(1990).).

Formula used in the following production method

[0172]

[Chemical Formula 91]

[In the formula, Q has the same meaning as described above. ] The sugar derivative (A) represented by

[0174]

[Chemical Formula 92]

[Wherein, Q has the same meaning as defined above, and X ¹² represents a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring. Indicates a group. ] The sugar derivative (B) represented by] is useful as an intermediate for the production of the oligonucleotide derivative (I). In sugar derivatives (A) and (B), when Q is Y, Y
As the sugar of the sugar residue represented by, those similar to those described in the above-mentioned oligonucleotide derivative (I) are used. Of these, galactose, mannose, melibiose, gentiobiose and isomaltotriose, or those in which these hydroxyl groups are substituted with an appropriate group such as C _1-5 alkylcarbonyl group (eg, acetyl group), benzoyl group, glucoside And those in which thioglucoside or these hydroxyl groups are substituted with a suitable group such as C _1-5 alkylcarbonyl group (eg, acetyl group) benzoyl group, C _1-20 alkyl group (eg, octyl group), Glucosamine and galactosamine or their hydroxyl groups are substituted with an appropriate group such as C _1-5 alkylcarbonyl group (eg, acetyl group) optionally substituted with halogen atom (eg, fluoro), benzoyl group, etc. The thing etc. are preferable.
Specifically, for example, 1,2,3,4-0-tetraacetylgalactose, 1,2,3,4-0-tetraacetylmannose, 1-On
-Octyl-2,3,4-O-tribenzoylglucoside, 1-On-
Octyl-2,3,4-O-tribenzoylthioglucoside, 1-O
-Phenyl-2,3,4-O-tribenzoylglucoside, 1,3,4-
O-tribenzoyl, 2-N-benzoylgalactosamine, 1,
3,4-O-triacetyl, 2-N-acetylgalactosamine, 1,
3,4-O-triacetyl, 2-N-acetylglucosamine, 1,3,4
-O-tribenzoyl, 2-N-trifluoroacetylgalactosamine, 1,3,4-O-tribenzoyl, 2-N-trifluoroacetylglucosamine, heptabenzoyl melibiose,
Heptabenzoyl gentiobiose, octabenzoyl isomaltotriose and the like are preferable. In the sugar derivative (B), 1-On-octyl-2,3,4-O-tribenzoylglucoside, 1-On-octyl-2,3,4-O-tribenzoylthioglucoside and the like are particularly preferable. C _1-5 alkyl group represented by X ¹² in the sugar derivative (B), C
_{As the 1-5} alkoxy group, C _1-5 monoalkylamino group, and optionally substituted aromatic ring group, the same groups as those described above for X ¹ , X ² and X ³ can be used. Are preferred.

Sugar derivatives (A) and (B) in which Q is Y
Is bound to the 5′-terminal side of the oligonucleotide, for example, Gal (galactose), Man (mannose), n-oct-Gln (n-octylglucoside), n-oct-SGlc (n-octyl-thio). Glucoside), PhGlc (phenyl glucoside), PhG
al (phenylgalactoside), GalN (galactosamine), N-AcGal (N-acetylgalactosamine), N-BzGal (N-benzoylgalactosamine), GulN (glucosamine), N-AcGul (N).
-Acetylglucosamine), Mel (melibiose),
Those that can be introduced into Gen (gentiobiose), Iso (isomaltotriose) and the like are preferable. In particular, as the sugar derivative (B) in which Q is Y, for example, n-oct is attached to the 5'terminal side of the oligonucleotide.
-Gln (n-octyl glucoside), n-oct-S
Those that can be introduced into Glc (n-octyl-thioglucoside) and the like are preferable. Specific examples of the sugar derivatives (A) and (B) in which Q is Y are those in which the hydroxyl groups of these sugars are appropriately substituted, and examples thereof include Examples 1, 2, 5, 7, 9 , 11, 13, 15, 1
Those manufactured by 7, 19, 21, 23, 25, 27, 29 and 31 are preferable. Q is the formula

[0177]

[Chemical formula 93]

As the sugar derivatives (A) and (B) which are, for example, Gal (galactose), Man (mannose),
Glu (glucose) and the like, particularly those that can be introduced into Gal (galactose) and the like are preferable. For example,
Monosaccharides (eg, galactose, mannose, glucose, etc., preferably galactose, in which the sugar of the sugar residue represented by Y may have a hydroxyl group substituted with a C _1-5 alkylcarbonyl group (eg, acetyl group) and the like. And the like) and the like, and sugar derivatives (A) and (B) are preferable, and specifically, those produced in Example 63 are preferable. (1) X ¹ , X ⁴ and X ⁷ are each an OH group or SH
Method for producing oligonucleotide derivative (Ia) which is a group Reaction formula (a)

[0179]

[Chemical 94]

[In the formula, Bz represents a benzoyl group. In the reaction formula (a), the hydroxyl group of the compound (II) is reacted with the phosphorylating agent (III) in which O adjacent to P is protected with cyanoethyl to obtain the compound (IV). Compound (II)
About 1 to 2 mmol of phosphorylating agent (III) and about 2 to 5 mmol of ethyldiisopropylamine are used per 1 mmol, and halogenated hydrocarbons (eg,
Methylene chloride, chloroform), ethers (eg, diethyl ether, tetrahydrofuran), aromatic hydrocarbons (eg, benzene, toluene), acetonitrile, pyridine, dimethylformamide, dimethyl sulfoxide, alcohols (eg, methanol, ethanol) Among them, methylene chloride is preferably used, and the reaction temperature is usually from -20 ° C to 70 ° C, preferably from 15 ° C to 35 ° C, and the reaction time is usually from 0.5 hour to 5 hours, preferably. Is reacted for about 1 to 2 hours to obtain the compound (IV). The starting compounds (II) and (III) can be produced by a method known per se or a method analogous thereto. For example, the raw material compound (II) is known in the literature ("Reagents for Organic Synthesi"
s ", vol. 1, 453, L. Fieser (eds), John Wiley and
Sons, INC. (1967). DD Reynolds et al., Org. Sy
n., Coll. vol. 3, 432 (1955). RR Schmidt, Angew.
Chem. Int. Ed. Engl., Vol.25, 212 (1986).). Raw material compound (II
I) is a known document (MDMatteuci et al., Tetrahedron L
et., vol. 21, 719 (1980). SL Beaucage et al., T
etrahedron Lett., vol. 22, 1859 (1981). LJ McBr
ide et al. Te-trahedron Lett., vol. 24, 245 (198
3). BC Froehler et al., Nucleic Acids Res.,
vol. 14, 5399 (1986). BC Froehler et al., Tetrahe
dron Lett., vol.27, 469 (1986) .PJ Garegg et a
l., Tetrahedron Lett., vol. 27, 4051 (1986). E. Uhl
man et al., Chemical Reviews, vol.90, No.4, 543 (199
It can be manufactured according to the method of 0).).

Reaction formula (b)

[0182]

[Chemical 95]

[In the formula, Bz represents a benzoyl group, and X ⁴¹
And X ⁵¹ each represent O or S, X ⁴¹ may be the same or different in repeating n, CPG represents a solid-phase carrier (Controlled Pore Glass), and other symbols have the same meanings as described above. . The reaction formula (b) is the compound (IV) obtained in the reaction formula (a).
With an appropriately protected nucleotide derivative or oligonucleotide derivative (V) (eg, O adjacent to P is protected with cyanoethyl).
About 20 to 100 μmol of the phosphorous-phosphorylated sugar derivative compound (V) relative to 1 μmol of the compound (IV)
And 1 to 20 micromol of 1-H tetrazole are used as a solvent, halogenated hydrocarbons (eg methylene chloride, chloroform), ethers (eg diethyl ether, tetrahydrofuran), aromatic hydrocarbons (eg , Benzene, toluene), acetonitrile, pyridine, dimethylformamide, dimethylsulfoxide, alcohols (eg, methanol, ethanol) can be used, and among them, acetonitrile is preferably used, and the reaction temperature is usually from −20 ° C. to about 70 ° C. , Preferably 15 to 35 ° C, and usually a reaction time of 3 minutes to 3
The compound (VI) is obtained by reacting for about 0 minutes, preferably for about 5 to 20 minutes. The starting material compound, 1-H tetrazole, can be produced by a method known per se or a method analogous thereto. The optionally protected nucleotide derivative or oligonucleotide derivative (V), which is a raw material compound, can be produced by a method known per se or a method analogous thereto. That is, a method of synthesizing an oligonucleotide using a general nucleotide derivative ("Oligonucleotide S
ynthesis; a practical approach "MJ Gait (ed), IR
L PRESS, (1984).). Although a series of nucleotide chain extension reactions can be carried out by either the manual method or the method using an automatic synthesizer, the operation method can be simplified by performing the reaction on an automatic synthesizer,
In addition, a method using an automatic synthesizer is desirable from the viewpoint of synthesis accuracy.

Reaction formula (c)

[0185]

[Chemical 96]

[In the formula, Bz represents a benzoyl group,
X ¹¹ , X ⁴¹ and X ⁵¹ each represent O or S, and X ^11
4 1 may be the same or different in each repetition of n, and CPG is a solid phase carrier (Controlled Pore Glas
s), and other symbols have the same meanings as described above. The reaction formula (c) is the compound (VI) obtained in the reaction formula (b).
To obtain compound (VII). Compound (VI) with respect to 1 micromolar, oxidizing agent (e.g., mCPBA, NBA, NBS, I 2, DMSO-
DCC or the like, preferably I ₂ ) or a sulfiding agent (eg,
Di (phenylacetyl) disulfide, 3H-1,2-
Benzodithiol-3-one, tetraethylthiuram disulfide, dibenzoyl tetrasulfide, dibenzoyl trisulfide, dibenzoyl disulfide, etc.,
The reaction temperature is usually from -20 ° C to 70 ° C, preferably from 15 ° C to 35 ° C, preferably by adding about 20 µmol to 100 µmol of tetraethylthiuram disulfide). The reaction time is usually 30 seconds to 100 minutes, preferably 30 seconds to 5 minutes in the case of oxidation reaction, and 30 seconds to 100 minutes, preferably 30 in the case of sulfurization reaction. It is about 20 seconds to a second. Then, by removing the protecting group of the obtained compound (VII), X ¹ , X ⁴ and X ⁵ are respectively OH group or S
An oligonucleotide derivative (Ia) that is an H group can be produced. As a method for removing the protecting group, a method known per se or a method analogous thereto can be used. For example, first, an ammonia / pyridine (5: 1) solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier. Then, the protecting groups for the phosphate group, base moiety and sugar moiety are removed. After removing all protecting groups, reverse phase HP
It can be purified using LC etc. ("Oligonucleo
tide Synthesis; a prac-tical approach ", MJ Gai
t (ed), IRL PRESS, (1984).).

(2) X ¹ , X ⁴ and X ⁵ are each C _1-5.
Method for producing oligonucleotide derivative (Ia) having alkyl group, C _1-5 alkoxy group or C _1-5 monoalkylamino group Reaction formula (d)

[0188]

[Chemical 97]

Reaction formula (e)

[0190]

[Chemical 98]

Reaction formula (f)

[0192]

[Chemical 99]

The phosphorylating agent (VIII) which is a raw material compound is
It can be produced by a method known per se or a method analogous thereto. For example, publicly known literature (MDMatte
uciet al., Tetrahedron Let., vol. 21, 719 (1980).
SL Beaucage et al., Tetrahedron Lett., Vol. 22,
1859 (1981). LJ McBride et al. Te-trahedronLet
t., vol. 24, 245 (1983). BC Froehler et al., N
Nucleic Acids Res., vol. 14, 5399 (1986). BC F
roehler et al., Tetrahedron Lett., vol.27,469 (198
6) .PJ Garegg et al., Tetrahedron Lett., Vol. 2
7, 4051 (1986) .E. Uhlman et al., Chemical Reviews,
vol.90, No.4, 543 (1990).). The reaction formula (d) is the same as the above reaction formula (a),
The reaction formula (e) can be performed in the same manner as the above reaction formula (b), and the reaction formula (f) can be performed in the same manner as the above reaction formula (c). Then, by removing the protecting group of the obtained compound (XII), X ¹ , X ⁴ and X ⁵ are each a C _1-5 alkyl group, a C _1-5 alkoxy group or a C _1-5 monoalkylamino group. The oligonucleotide derivative (Ia) can be produced.

[Production Method of Oligonucleotide Derivative (Ib)] The formula used in the following production method

[0195]

[Chemical 100]

[In the formula, W, B ¹ and R ¹ have the same meanings as described above. ] The nucleotide derivative represented by the following is useful as an intermediate for the production of the oligonucleotide derivative (Ib). W is, for example, a hydrogen atom or a formula

[0197]

[Chemical 101]

Or

[0199]

[Chemical 102]

[In the formula, Y and X ¹ have the same meanings as described above. ] The group etc. which are represented by these are preferable. W is the formula

[0201]

[Chemical 103]

In the case of the group represented by, examples of the sugar of the sugar residue represented by Y include galactose, mannose, melibiose, gentiobiose and isomaltotriose, or a hydroxyl group other than these hemiacetal hydroxyl groups is C _{1- 5} those substituted with an alkylcarbonyl group or a benzoyl group, glucosides and thioglucosides or those whose hydroxyl groups are substituted with a C _1-5 alkylcarbonyl group, benzoyl group or C _1-20 alkyl group, or glucosamine and Galactosamine or a hydroxyl group other than these hemiacetal hydroxyl groups is preferably substituted with a C _1-5 alkylcarbonyl group which may be substituted with a halogen atom or a benzoyl group. Specifically, 1,2,3,4-0-tetraacetylgalactose, 1,2,3,4-0-tetraacetylmannose, 1-On-octyl-2,3,4-O-tribenzoylglucoside , 1-On-octyl-2,3,4-O-tribenzoylthioglucoside, 1-O-phenyl-2,3,4-O-tribenzoylglucoside, 1,3,4-O-tribenzoyl, 2 -N-benzoylgalactosamine, 1,3,4-O-triacetyl, 2-N-acetylgalactosamine, 1,3,4-O-triacetyl, 2-N-acetylglucosamine, 1,3,4-O- Tribenzoyl, 2-N-trifluoroacetylgalactosamine, 1,3,4-O-tribenzoyl, 2-N
-Trifluoroacetylglucosamine, heptabenzoyl melibiose, heptabenzoyl gentiobiose,
Octabenzoylisomaltotriose and the like are preferable. Examples of X ¹ include OH group, SH group, C _1-5
Alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, or halogen atom, C _1-10 alkylcarbonyl group, OH group, nitro group, C _1-10 alkyl group, C
_A phenyl group which may be substituted with 1 to 5 substituents selected from the group consisting of a _1-10 alkoxy group, a phenyl group and a naphthyl group is preferable, and an OH group is particularly preferable.
SH group, methyl group, phenyl group and the like are preferable. W is the formula

[0203]

[Chemical 104]

[In the formula, Y and X ¹ have the same meanings as described above. In the case of the group represented by the following, as the sugar of the sugar residue represented by Y, for example, a monosaccharide in which a hydroxyl group may be substituted with a C _1-5 alkylcarbonyl group (eg, acetyl group) and the like (eg, , Galactose, mannose, glucose, etc., preferably galactose, etc.) are suitable. X ¹ is, for example, OH group, SH group,
C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, or halogen atom, C _1-10 alkylcarbonyl group, OH group, nitro group, C _1-10 alkyl group, C _{1 -10} is preferably a phenyl group which may be substituted with about 1 to 5 substituents selected from the group consisting of an alkoxy group, a phenyl group and a naphthyl group, and particularly, an OH group, an SH group, a methyl group, a phenyl group and the like. Is preferred.

The method for producing the oligonucleotide derivative (Ib) of the present invention is described below, wherein W is a protecting group; Pix group (Ib-1) A method for producing an oligonucleotide derivative which is a phenylxanthenyl group) will be described by way of example.

[0206]

[Chemical 105]

Or expression

[0208]

[Chemical formula 106]

An oligonucleotide derivative (Ib) which is
Etc. can be similarly obtained. (1) An oligonucleotide derivative (Ib-1) in which X ⁸ is an OH group or an SH group (that is, X ⁶ is a phenyl group and X ⁷ is an OH group or S
Method for producing H-group oligonucleotide derivative (Ib))

[0210]

[Chemical formula 107]

[In the formula, Pix represents a phenylxanthenyl group, and EtN (iPr) 2 represents ethyldiisopropylamine. The reaction formula (a) is to obtain the compound (XV) by reacting the hydroxyl group of the compound (XIII) with the phosphorylating agent (XIV) in which the phenyl group is directly bonded to the phosphorus atom. Compound (XII
I) About 1 to 2 mmol of phosphorylating agent (XIV) and about 2 to 5 mmol of ethyldiisopropylamine are used as a solvent, and halogenated hydrocarbons (eg, methylene chloride, chloroform, carbon tetrachloride) as a solvent. ), Ethers (eg, diethyl ether, tetrahydrofuran), aromatic hydrocarbons (eg, benzene, toluene),
Acetonitrile, pyridine, dimethylformamide, dimethylsulfoxide, alcohols (eg, methanol,
Ethanol) can be used, and among them, methylene chloride is preferably used, and the reaction temperature is usually -20 to 70 ° C, preferably 15 to 35 ° C, and the reaction time is usually 0.5 to 5 hours. Desirably, the compound (IXV) is obtained by reacting for about 1 to 2 hours. The starting compounds (XIII) and (XIV) can be produced by a method known per se or a method analogous thereto,
Known literature (MD Matteuci et al., Tetrahedron Let
t., vol. 21, 719 (1980). SL Beaucage et al., Tet
rahedron Lett., vol. 22, 1859 (1981). LJ McBride
et al., Tetrahedron Lett., vol. 24, 245 (1983). B.
C. Froehler et al., Nucleic Acids Res., Vol. 14, 5
399 (1986). BC Froehler et al., Tetrahedron Let
t., vol. 27, 4051 (1986). E. Uhlman et al., Chemica
l Reviews, vol. 90, No. 4, 543 (1990).).

[0212]

[Chemical 108]

[Wherein, X ⁸¹ represents O or S, and may be the same or different in repeating n. The reaction formula (b) is the compound (XV) obtained in the reaction formula (a).
With a nucleotide derivative or oligonucleotide derivative (XVI) that is appropriately protected (eg, O adjacent to P is protected with a cyanoethyl group). 20 to 1 per 1 micromol of compound (XVI)
A nucleotide derivative (XV) having a phenylphosphonite structure of about 00 μmol and 1-H tetrazole of about 20 to 100 μmol are used, and a halogenated hydrocarbon (eg, methylene chloride, chloroform, etc.) is used as a solvent.
Carbon tetrachloride), ethers (eg diethyl ether, tetrahydrofuran), aromatic hydrocarbons (eg benzene, toluene), acetonitrile, pyridine, dimethylformamide, dimethyl sulfoxide, alcohols (eg methanol, ethanol) Of these, acetonitrile is preferably used, and the reaction temperature is usually −20 ° C. to 70 ° C., preferably 15 ° C. to 35 ° C., and the reaction time is usually 3 minutes to 30 minutes, preferably 5 minutes to 20 minutes. The compound (XVII) is obtained by reacting for about a minute. The starting material compound, 1-H tetrazole, can be produced by a method known per se or a method analogous thereto. The optionally protected nucleotide derivative or oligonucleotide derivative (XVI), which is a raw material compound, can be produced by a method known per se or a method analogous thereto. That is, a method of synthesizing an oligonucleotide using a general nucleotide derivative (“Oligonucleotide Sy
nthesis; a practical approach "MJ Gait (ed), IR
L PRESS, (1984).). Although a series of nucleotide chain extension reactions can be performed by either the manual method or the method using an automatic synthesizer, using an automatic synthesizer simplifies the operation method and, in terms of the accuracy of synthesis, an automatic synthesizer is used. The method used is preferred.

[0214]

[Chemical 109]

The reaction formula (c) is obtained by oxidizing or sulfiding the compound (XVII) obtained in the reaction formula (b).
Is what you get. An oxidizing agent (eg, mCPBA, NBS, NBA, I ₂ ,
DMSO-DCC and the like, preferably I ₂ ) or sulfiding agents (eg di (phenylacetyl) disulfide, 3-H,
1,2-benzodithiol-3-one, tetraethylthiuram disulfide, dibenzoyltetrasulfide, dibenzoyltrisulfide, dibenzoyldisulfide, etc., preferably tetraethylthiuram disulfide, etc.) is usually added in an amount of about 20 μmol to 100 μmol, Usually, the reaction temperature is about -20 ° C to 70 ° C, preferably about 15 ° C to 35 ° C. In the case of oxidation reaction, the reaction time is usually 30 seconds to 100 minutes,
It is preferably about 30 seconds to 5 minutes, and in the case of a sulfurization reaction, the reaction time is usually about 30 seconds to 100 minutes, preferably about 30 seconds to 20 minutes.

Then, the protecting group of the obtained compound (XVIII) is selectively removed to produce an oligonucleotide derivative (Ib) in which X ⁶ is a phenyl group and X ⁷ is an OH group or an SH group. be able to.
As a method for removing the protecting group, a method known per se or a method analogous thereto can be used. For example, first, an ammonia / pyridine (5: 1) solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier. Then, the protecting groups for the phosphate group, base moiety and sugar moiety are removed. After removing all the protecting groups, it can be purified using reverse phase HPLC or the like (“Oligonucleotide Synthesis;
acticalapproach "MJ Gait (ed), IRL PRESS, (198
Four).). Furthermore, after removing the Pix group in the above production method, another protecting group, for example, a compound of the formula

[0217]

[Chemical 110]

Or

[0219]

[Chemical 111]

[In the formula, Y and X ¹ have the same meanings as described above. ] A group represented by the above can be introduced by a method known per se or a method analogous thereto.

(2) An oligonucleotide derivative (Ib-1) in which X ⁸ is a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group. ) (Ie, X ⁶ is a phenyl group and X ⁷ is C
Method for producing oligonucleotide derivative (Ib)) having _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or optionally substituted aromatic ring group

[0222]

[Chemical 112]

[In the formula, Pix represents a phenylxanthenyl group, and EtN (iPr) 2 represents ethyldiisopropramine. ]

[0224]

[Chemical 113]

[In the formula, X ⁸² represents a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an aromatic ring group which may be substituted; It may be the same or different. ]

[0226]

[Chemical 114]

The optionally protected nucleotide derivative or oligonucleotide derivative (XIX), which is the starting material compound, can be produced by a method known per se or a method analogous thereto. That is, a method for synthesizing an oligonucleotide using a general nucleotide derivative (“Oligonucleotide Synthesis; a practi
cal approach "MJ Gait (ed), IRL PRESS, (1984).)
Can be done according to. Although a series of nucleotide chain extension reactions can be performed by either the manual method or the method using an automatic synthesizer, using an automatic synthesizer simplifies the operation method and, in terms of the accuracy of synthesis, an automatic synthesizer is used. The method used is preferred. The reaction formula (d) can be performed in the same manner as the above reaction formula (a), the reaction formula (e) can be performed in the same manner as the reaction formula (b), and the reaction formula (f) can be performed in the same manner as the reaction formula (c).
Then, by selectively removing the protective group of the obtained compound (XXI), X ⁶ is a phenyl group and X ⁷ is C _1-5.
The oligonucleotide derivative (Ib) which is an alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group can be produced. As the method for removing the protecting group, the same method as described above is used. Furthermore, after removing the Pix group in the above production method, another protecting group can be introduced according to a method known per se or a modification thereof. As described above, the sugar derivative (A) and the sugar derivative (B) of the present invention can be produced according to a method known per se or a method analogous thereto, but in the sugar derivative (A) of the present invention, Q is a formula

[0228]

[Chemical 115]

The method for producing a sugar derivative having a branched oligosaccharide represented by is specifically described below.

[0230]

[Chemical formula 116]

In the reaction formula (a), the hydroxyl group of the glycerol derivative (XXIII) protected with a benzyl group is reacted with the fluorinated galactose derivative (XXII) protected with an acetyl group,
The compound (XXIV) is obtained. Compound (XXIII) 1
The fluorinated galactose derivative protected with about 2 to 5 mmol of acetyl group is used as Lewis acid (BF _{3-).
Et 2 O, SnCl 4, Cp} 2 HfCl 2 -AgClO 4, C
p ₂ ZrCl ₂ -AgClO ₄ , among these, preferably BF
_3- Et ₂ O. Cp represents cyclopentadienyl. ) Is used as a solvent, and halogenated hydrocarbons (eg, methylene chloride, chloroform), ethers (eg, benzene, toluene), acetonitrile, pyridine, dimethylformamide, dimethylsulfoxide, alcohol are used as a solvent. Although it is possible to use a group (eg, methanol, ethanol), it is preferable to use methylene chloride among them, and the reaction temperature is usually −78 ° C.
The compound (XXIV) is obtained by reacting at about 80 ° C to about 15 ° C to about 35 ° C for a reaction time of usually 0.5 hours to 5 hours, preferably 1 hour to 2 hours.

Starting compounds (XXII) and (XXIII) are
It can be produced by a method known per se or a method analogous thereto. For example, the raw material compound (XXII) is a known document (K. Suzuki et al., Tetrahedron Lett., Vol. 2
9, 3571 (1988). K. Suzukiet al., Tetrahedron Lett.,
vol.29, 3575 (1988) .K. Suzuki et al., Tetrahedron
Lett., Vol.30, 6879 (1989). K. Suzuki et al., Tet
rahedron Lett., vol.30, 4853 (1989). T. Ogawa et
al., Tetrahedron Lett., vol.31, 1597 (1990). T. Og
awa et al., Tetrahedron Lett., vol.33, 6343 (199
2). T. Ogawaet al., Tetrahedron Lett., Vol.33, 7
907 (1992) .T. Ogawa et al., Tetrahedron Lett.,
vol.34, 1061 (1993) .KC Nicolaou et al., J. Am.
Chem. Soc., Vol.112, 3693 (1990). KC Nicolaou
et al., J. Am. Chem. Soc., vol.114, 8701 (1992).
ORGANIC SYNTHESIS "Second Edition, TW Greene a
nd PGM Wuts, John Willey & Sons, Inc. (199
It can be manufactured according to the method of 1).).

[0233]

[Chemical 117]

The reaction formula (b) is obtained by deprotecting the benzyl group of the compound (XXIV) by catalytic reduction in the presence of Pd.
V) is what you get. 30 mg to 60 mg of 10% Pd (OH) ₂ was added to 1 mmol of compound (XXIV), and halogenated hydrocarbons (eg, methylene chloride, chloroform), ethers (eg, benzene, toluene), acetonitrile as a solvent. , Pyridine, dimethylformamide, dimethylsulfoxide, ethyl acetate, alcohols (eg, methanol, ethanol) can be used. Among them, methanol-ethyl acetate (1: 1) is preferably used, and medium pressure hydrogenation (3. 5 kg / c
m ² ), the reaction temperature is usually −78 ° C. to 80 ° C., preferably 15 ° C. to 35 ° C., and the reaction time is usually 1 hour to
The compound (XXV) is obtained by reacting for about 24 hours, preferably for 3 to 15 hours.

[0235]

[Chemical 118]

In the reaction formula (c), the hydroxyl group of the compound (XXV) is reacted with the O adjacent to P to the phosphorylating agent (XXVI) protected with a cyanoethyl group to obtain the compound (XXVII). Using 1 to 2 mmol of the phosphorylating agent (XXVI) and 2 to 5 mmol of ethyldiisopropylamine per 1 mmol of the compound (XXV), halogenated hydrocarbons (eg, methylene chloride, chloroform) as a solvent,
Ethers (eg, benzene, toluene), acetonitrile, pyridine, dimethylformamide, dimethylsulfoxide, ethyl acetate, alcohols (eg, methanol, ethanol) can be used. Of these, methylene chloride is preferably used, and the reaction temperature is usually used. -20 ° C to 70
C., preferably about 15.degree. C. to 35.degree. C., usually for a reaction time of about 0.5 hours to 5 hours, preferably for about 1 to 2 hours to obtain compound (XXVII). The raw material compound (XXVI) is known in the literature (MD Matteuci et al., Tetrahed
ron Lett., vol.21, 719 (1980) .SL Beaucage et a
l., Tetrahedron Lett., vol.22, 1859, (1981). LJ
McBride et al., Tetrahedron Lett., Vol.24, 245,
(1983). BC Froehler et al., Nucleic Acids Res.,
vol.14, 5399 (1986) .BCFroehler et al., Tetrah
edron Lett., vol.27, 469 (1986). E. Uhlman et al.,
Chemical Reviews, vol.90, No.4, 543 (1990).). Formulas including the starting compound (XXV) used in the above production method

[0237]

[Chemical formula 119]

[In the formula, Y has the same meaning as described above, and R ¹⁰ represents a hydrogen atom or a benzyl group. ] The sugar derivative represented by

[0239]

[Chemical 120]

It is useful as an intermediate for the production of sugar derivatives (A) and (B). In the sugar derivative (C), Y
The sugar of the sugar residue represented by is a monosaccharide in which a hydroxyl group may be substituted with a C _1-5 alkylcarbonyl group (eg, acetyl group) and the like (eg, galactose, maltose glucose, etc., preferably galactose Such)
Is preferred. Specifically, those which can be produced in Example 61 or 62 are suitable.

When the thus obtained oligonucleotide derivatives (I), (Ia) and (Ib) are obtained in free form, they can be converted into salts by a method known per se or a method analogous thereto, and conversely. When obtained with a salt, by a method known per se or a method analogous thereto,
It can be converted into the educt or other salt. The oligonucleotide derivatives (I), (Ia) and (I of the present invention
As the salt of b), physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid),
Alternatively, organic acids (eg, acetic acid, formic acid, propionic acid,
Fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid,
Malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like salts are used. The oligonucleotide derivative (I), (Ia), (Ib) of the present invention (hereinafter, referred to as the oligonucleotide derivative (I) including the oligonucleotide derivative (Ia) and the oligonucleotide derivative (Ib)) or a salt thereof is , D of genes that cause various diseases (eg, malignant tumors, viral diseases, inflammation, vascular restenosis after PTCA)
By complementation to NA or mRNA, expression of a gene that produces a harmful protein in vivo is suppressed, and a protein involved in the expression of a gene that produces a harmful protein (for example, binding to a promoter to initiate transcription) The expression of a gene that produces a protein (for example, can be suppressed). For example, (1) inhibit the expression of malignant tumor gene in malignant tumor cells, (2)
Suppresses the expression of viral genes derived from viruses, (3) suppresses the expression of genes that produce proteins that cause inflammation, and (4) drug resistance genes that are problematic during chemotherapy for malignant tumors. (5) Since it is possible to inhibit the production of cell growth factors involved in vascular restenosis after PTCA, the expression of antitumor agents, antiviral agents, anti-inflammatory agents, drug resistance genes, etc. It can be used as a drug that controls the expression of a specific gene. Also, these compounds can be used in drug screening to screen for other compounds that affect the conditions being tested. For example, when looking at tumor cells where the tumor is derived from the expression of N-ras, oligonucleotide compounds targeting N-ras (i.e., antisense compounds directed against the limiting part of N-ras such as mRNA translation initiation site). ) Can be used, and other compounds that affect tumors can be screened. In this way, various agents can be selected and developed. In particular, the oligonucleotide derivative (Ia) of the present invention can be used as a so-called antisense oligonucleotide derivative. In vivo, as a result of the expression of a specific gene, many diseases caused by the gene product are known,
The examples are as follows. For example, conversion of proto-oncogene to oncogene (eg ras), overexpression as oncogene (eg neu / erb.B2)
and so on. There are two major types of pathogenic viral genes or oncogenes: those that are internal to the cell (for example, structural genes such as ras), and those that are the result of infection with a virus called extracellular from the outside. It has been clarified that the expression of these genes is one of the factors that cause the onset of viral diseases or the abnormal growth of normal cells. Also, I
The expression of genes such as CAM-1 causes the produced adhesion protein to cause inflammation. Furthermore, although the expression of these genes is not directly responsible for the etiology, the body acquires drug resistance to the antitumor drug that is always a problem during chemotherapy for malignant tumors, As a result, it is considered that the expression of the drug resistance gene is an indirect cause, such as making complete cure difficult. Therefore, the oligonucleotide derivative (I) of the present invention or a salt thereof regulates the activation or expression of a gene causing such a disease, thereby allowing warm-blooded mammals (eg, rat,
Mouse, rabbit, rat, pig, sheep, monkey, dog,
It is effective as a safe and low-toxicity therapeutic agent (for example, antitumor agent, antiviral agent, antiinflammatory agent, etc.) for cats, humans, etc.). In order to enhance the cell membrane permeability and lipophilicity of the oligonucleotide derivative of the present invention or a salt thereof, the oligonucleotide derivative (I) of the present invention or a salt thereof is used as a liposome (eg lipofectin).
It can be used inclusively. In particular, it is preferable to include the oligonucleotide derivative (Ia) of the present invention or a salt thereof in a liposome (eg, lipofectin).

Oligonucleotide derivative (I) of the present invention
Alternatively, when the salt thereof is used as the above-mentioned drug, it may be orally administered as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or water or other pharmaceutically acceptable. It can be used parenterally in the form of an injectable solution such as a sterile solution with a liquid or a suspension. For example, the oligonucleotide derivative (I) of the present invention or a salt thereof together with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc. is required for generally accepted pharmaceutical practice. It can be manufactured by mixing in a unit dose form. The amount of active ingredient in these preparations is such that a suitable amount within the indicated range can be obtained. Additives that can be mixed with tablets, capsules, etc. include, for example, gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Puffing agents, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. . Aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-
Mannitol, sodium chloride, etc.)
It may be used in combination with a suitable solubilizing agent such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant (eg polysorbate 80, HCO-50) and the like. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservation You may mix | blend with agents (for example, benzyl alcohol, phenol, etc.), antioxidants, etc. The adjusted injection solution is usually filled in a suitable ampoule. Although the dose varies depending on the type and symptoms of the target disease, when administered orally, it is generally for adults (60k
(as g) about 0.1 mg to 10 per day
It is 0 mg, preferably 1.0 to 50 mg, more preferably 1.0 to 20 mg. When administered parenterally,
The single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, in an adult (60 kg), it is usually about 0.0 per day.
It is convenient to administer about 1 mg to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg by intravenous injection. In the case of other mammals, the same dose is calculated in terms of kg.

Further, since the oligonucleotide derivative (I) of the present invention or its salt has excellent complementarity to DNA, it can be used as a probe for screening DNA. Generally, for DNA screening, an appropriate oligonucleotide is synthesized,
A method of screening the target DNA using it as a probe is used. However, if the synthetic oligonucleotide has low complementarity to DNA, it has a drawback that screening efficiency is lowered. Since the oligonucleotide derivative (I) of the present invention or its salt has excellent complementarity to DNA, it is also effective as a material for efficient screening. Furthermore, the oligonucleotide derivative (I) of the present invention or a salt thereof is
Since it has a sugar derivative etc. at the 5'end, the target D
It is also possible to detect the target DNA by performing a sugar analysis after binding to NA in a complementary manner. Further, the oligonucleotide derivative (I) of the present invention or a salt thereof can be used as a probe instead of other oligonucleotides or can be used for screening.

[0244]

[Reference Example 1]

ICAM-1 antisense phosphorothioate: T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C (SEQ ID NO: 3
Synthesis of 1) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
500 micrograms of CPG introduced with 0 micromol (1
0 micromole was used as an automatic synthesizer (ABI DNA sy
The reaction column installed in the nthesizer Model 381 A) is packed. Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind the guanosine derivative having the next base sequence, the same operation as described above is repeated. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically quantifying DMTr liberated by the operation of (1) was 99% or more. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide
Synthesis; a practical approach ", MJ Gait (e
d), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBo
Purification is carried out using ndasphere _C18 ). By the above operation, the target ICAM-1 antisense phosphorothioate: T _S G _S G _S G _{S AS} G _S C _S C _S A _S T _S A _S G _S C _S G _S
A _S G _S G _S C was synthesized. Yield; 893.3 OD λmax; 257.4 nm λmin; 230.2 nm

[0245]

[Reference Example 2] ICAM-1 antisense phosphorothioate reverse sequence phosphorothioate _{oligonucleotides: C S G S G S A} S G S C S G S A S T S A S C S C S G S A S G S G S G
_S T (SEQ ID NO: 36) thymidine derivative synthesized 5'hydroxyl group is protected with DMTr group was 20 micromoles introduced per gram CPG5
00 milligrams (10 micromoles) are packed in a reaction column installed in an automatic synthesizer (DNA synthesizer Model 381 A manufactured by ABI). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the thymidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind the guanosine derivative having the next base sequence, the same operation as described above is repeated. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically quantifying DMTr liberated by the operation of (1) was 99% or more. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide
Synthesis; a practical approach ", MJ Gait (e
d), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBo
Purification is carried out using ndasphere _C18 ). More of the object by operating ICAM-1 antisense phosphorothioate reverse sequence phosphorothioate _{oligonucleotides: C S G S G S A} S G S C S G S A S T S A S C S C S G S A S G S
G _S G _S T yield; 719.1 OD λmax; 257.1 nm λmin; 230.9 nm

[0246]

[Reference Example 3] N- benzo Irganox lactosamine phosphate bound phosphorothioate reverse sequence _{oligonucleotide: (N-BzGalPO) -C S} G S G S A S G S C S G S A S T
_{S A S C S C S G} S A S G S G S G S T ( SEQ ID NO: 36) thymidine derivative synthesized 5'hydroxyl group is protected with DMTr group was 20 micromoles introduced per gram CPG5
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the thymidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind the guanosine derivative having the next base sequence, the same operation as described above is repeated. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically quantifying DMTr liberated by the operation of (1) was 99% or more. After condensing the 18-mer cytidylic acid, Example 1 was added to the 5'end.
In order to bind the N-benzoylgalactosamine derivative synthesized in 7, after similarly dissolving it in a concentration of 0.2 M and mounting it on an automatic synthesizer, (1), (4) and (5) are as follows. The above operations (1) to (7) are repeated except that the above is set. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected cytidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2MN-benzoylgalactosamine amidite unit synthesized in Example 17 and 0.4M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. By the above operation, the N-benzoylgalactosamine derivative can be introduced at the 5'end of the reverse sequence oligonucleotide. The deprotection operation after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a practical approac
h ", MJ Gait (ed), IRL PRESS, (1984).) After removing the CPG from the reaction column,
This is treated with an ammonia / pyridine (5: 1) solution at 60 ° C.,
By reacting for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). By the above operation, a phosphorothioate reverse sequence oligonucleotide to which the desired N-benzoylgalactosamine phosphate is bound: (N-BzGalPO) -C
_S G _S G _S A _S G _S C _S G _S A _S T _S A _S C _S C _S G _S A _S G _S G _S G _S
T was synthesized. Yield: 70.5 OD λmax; 257.2 nm λmin; 229.6 nm

[0247]

Reference Example 4 Phosphate Reverse Sequence Oligonucleotide Partially Introduced Methylphosphonate Bond: Synthesis of C _Me G _Me GAGCGATACCCGAGG _Me G _Me T (SEQ ID NO: 38) 5′-hydroxyl protected thymidine derivative CPG5 with 20 micromoles introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the guanosine methylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine methylphosphonoamidite unit and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine methylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind the guanosine methylphosphonate derivative having the next base sequence, the same operation as described above is repeated. In order to bind guanosine phosphate of the next base sequence, guanosine phosphoramidite unit was added to 0.2M.
After being dissolved in the concentration of 1 and attached to an automatic synthesizer, the above operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine methylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasphere C ₁₈ )
Is used for purification. By the above operation, a phosphate reverse sequence oligonucleotide in which a methylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product: C _Me G _Me
GAGCGATACCCGAGG _Me G _Me T was synthesized. Yield: 71.0 OD λmax; 256.4 nm λmin; 234.0 nm

[0248]

Reference Example 5 Phosphate Antisense Oligonucleotide Partially Introduced Methylphosphonate Bond: T _Me G _Me GGAGCCATAGCGAG _Me G _Me C (SEQ ID NO: 34) Synthesis N-4 amino group with benzoyl group 5′- DM hydroxyl group
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine methylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine methylphosphonoamidite unit and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine methylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine methylphosphonate derivative having the next base sequence, the same operation as described above is repeated. To bind adenosine phosphate of the next base sequence, 0.2M of adenosine phosphoramidite unit was added.
After being dissolved in the concentration of 1 and attached to an automatic synthesizer, the above operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine methylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRLPRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasphere C ₁₈ )
Is used for purification. By the above operation, a phosphate antisense oligonucleotide in which a methylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product: T
_Me G _Me GGAGCCATAGCGAG _Me G _Me C was synthesized. Yield; 17.0 OD λmax; 255.6 nm λmin; 229.8 nm

[0249]

Example 1 1,2,3,4-O-tetraacetylgalactose-6-O-N, N-
Synthesis of diisopropylamino-O-cyanoethyl phosphoramidite 1,2,3,4-O-tetraacetylgalactose (0.87 g,
2.49 mmol) is azeotropically dehydrated with anhydrous pyridine 2 ml × 3 times, and then azeotropically distilled with anhydrous toluene 2 ml × 3 times and anhydrous methylene chloride 2 ml × 3 times. After that, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (2.17 ml, 12.45 mmol) and N, N- were added to this solution.
Diisopropylamino-O-cyanoethylphosphorochloride (1.11 ml, 4.98 mmol) was added, and the mixture was reacted at room temperature for 1 hour, then diluted with 50 ml of methylene chloride, and the solution was diluted with 50 ml of saturated aqueous sodium hydrogen carbonate solution.
After being washed 3 times with, it was dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (hexane / methylene chloride = 80:20, 2% triethylamine) to obtain the desired product, 1,2,3,4-O-tetraacetylgalactose-6-O. -N, N-diisopropylamino-O-cyanoethyl phosphoramidite (1.17
g, 2.14 mmol, 86%) was obtained. 1H-NMR (CDCl ₃ ) δ6.34 (m, 1H, H-1), 5.67-5.3
0 (m, 3H, H-2, H-3, H-4), 4.34-4.18 (m, 3H, H-5, H-6), 3.90-
3.56 (m, 2H, methine of isopropyl), 2.65 (m, 4H, methyle
ne of cyanoethyl), 2.14-1.99 (m, 12H, methyl of acety
l), 1.31-1.19 (m, 12H, methyl of isopropyl) ppm ³¹ P-NMR (CDCl ₃ ) δ 152.2, 152.3, 153.6, 15
3.7 ppm

[0250]

Example 2 Synthesis of 1,2,3,4-O-tetraacetylmannose-6-O-N, N-diisopropylamino-O-cyanoethyl phosphoramidite 1,2,3,4-O-tetraacetyl Mannose (0.97g,
2.78 mmol) is azeotropically dehydrated with 2 ml of anhydrous pyridine × 3 times, and then azeotropically distilled with 2 ml of anhydrous toluene × 3 times and with 2 ml of anhydrous methylene chloride × 3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (2.42 ml, 13.90 mmol) and N, N- were added to this solution.
Diisopropylamino-O-cyanoethyl phosphorochloride (1.24 ml, 5.56 mmol) was added, and the mixture was reacted at room temperature for 1 hour, then diluted with 50 ml of methylene chloride, and this solution was diluted with 50 ml of saturated aqueous sodium hydrogen carbonate solution.
After being washed 3 times with, it was dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (hexane / methylene chloride = 80: 20, 2% triethylamine) to obtain the desired product, 1,2,3,4-O-tetraacetylmannose-6-O. -N, N-diisopropylamino-O-cyanoethyl phosphoramidite (1.25
g, 2.28 mmol, 82%) was obtained. 1 H-NMR (CDCl ₃ ) δ 6.08 (d, 1H, H-1), 5.36-5.2
5 (m, 3H, H-2, H-3, H-4), 3.87-3.77 (m, 3H, H-5, H-6), 3.64
(m, 2H, methine of isopropyl), 2.66 (m, 4H, methylene o
f cyanoethyl), 2.19-2.01 (m, 12H, methyl of acetyl),
1.30-1.16 (m, 12H, methyl of isopropyl) ppm ³¹ P-NMR (CDCl ₃ ) δ 149.6, 149.7, 150.2, 15
0.3 ppm

[0251]

EXAMPLE 3 galactose bound phosphorothioate _{oligonucleotides: (GAL) -T S G S} G S G S A S G S C S C S A S T S A S G S C
_{S G S A S G S G} S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to bind the galactose derivative synthesized in the above Example 1 to the 5'end, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then (1) and (4)
Regarding the above, the above operations (1) to (7) are repeated except for the following. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) By the 0.2M galactose phosphoramidite unit synthesized in Example 1 and 0.4M tetrazole,
The condensation reaction is carried out for 5 minutes. By the above operation, the galactose derivative can be introduced into the 5'end of the oligonucleotide. The deprotection procedure after the completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthetic
thesis; a practical approach ", MJ Gait (ed), IR
L PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasph
Purification is carried out using ere _C18 ). Phosphorothioate oligonucleotides galactose desired product are bonded by the above _{procedure: (GAL) -T S G S} G S G S A S G S C
It was synthesized _{S C S A S T S A} S G S C S G S A S G S G S C. Yield: 43.65 OD λmax; 256.3 nm λmin; 229.3 nm

[0252]

Example 4 mannose is linked phosphorothioate _{oligonucleotides: (MAN) -T S G S} G S G S A S G S C S C S A S T S A S G S C
_{S G S A S G S G} S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to bind the mannose derivative previously synthesized in Example 2 to the 5'end, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. After that, with respect to (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is performed for 5 minutes using the 0.2M mannose phosphoramidite unit synthesized in Example 2 and 0.4M tetrazole. By the above operation, the mannose derivative can be introduced at the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthetic
thesis; a practical approach ", MJ Gait (ed), IR
L PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasph
Purification is carried out using ere _C18 ). Phosphorothioate oligonucleotides mannose of the target compound are bonded by the above _{procedure: (MAN) -T S G S} G S G S A S G S C S
C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C was synthesized. Yield: 33.47 OD λmax; 256.3 nm λmin; 229.3 nm

[0253]

Example 5 1-On-octyl-2,3.4-O-tribenzoylglucoside-6-
Synthesis of ON, N-diisopropylamino-O-cyanoethyl phosphoramidite 1-O-n-octyl-2,3.4-O-tribenzoylglucoside (1.22 g, 2.02 mmol) was added to anhydrous pyridine 2 m.
After azeotropic dehydration 1 × 3 times, anhydrous toluene 2 ml × 3
And azeotropically with 2 ml of anhydrous methylene chloride × 3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.76 ml, 10.10 mmo) was added to this solution.
1) and N, N-diisopropylamino-O-cyanoethyl phosphorochloride (0.90 ml, 4.04 mmo
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1-O-n-octyl-2,3.4-O-tribenzoylglucoside-
6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (1.42 g, 1.78 mmol, 88
%) Was obtained. ¹ H-NMR (CDCl ₃ ) δ 8.02-7.80 (m, 6H, oH of benz
oyl), 7.55-7.26 (m, 9H, m, pH of benzoyl), 5.82 (d, 1H,
H-1), 5.60-5.29,5.60-5.30 (m, 3H, H-2,3,4), 3.95-3.77
(m, 3H, H-5, H-6), 3.59-3.50 (m, 2H, CH of isopropyl),
2.58-2.55 (m, 2H, CH ₂ of cyanoethyl), 1.17-1.09 (m, 20
H, CH3 of isopropyl, CH ₂ of octyl), 0.83 (t, 3H, CH3 of
Octyl) ppm ³¹ P-NMR (CDCl ₃ ) δ149.57, 149.65, 149.68, 1
49.74 ppm

[0254]

EXAMPLE 6 n- octylglucoside bound phosphorothioate antisense oligonucleotides: (n-oct-Glc) -T S G S G S G S A S G S C S C S A S T S A
_{S G S C S G S A} S G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to attach the n-octyl glucoside derivative synthesized in Example 5 to the 5 ′ end, the n-octyl glucoside derivative was similarly dissolved in 0.2M to prepare an automatic synthesizer. After the attachment, the above-mentioned operations (1) to (7) are repeated except for (1) and (4) as described below. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes with the 0.2Mn-octylglucoside phosphoramidite unit synthesized in Example 5 and 0.4M tetrazole. By the above operation, the n-octyl glucoside derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection operation after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a practical approac
h ", MJ Gait (ed), IRL PRESS, (1984).) After removing the CPG from the reaction column,
This is treated with an ammonia / pyridine (5: 1) solution at 60 ° C.,
By reacting for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). The above operation by the desired product n- octyl glucoside bound phosphorothioate antisense oligonucleotides: (n-oct-Glc) -T S G S G S G S A S G S C
It was synthesized _{S C S A S T S A} S G S C S G S A S G S G S C. Yield: 38.7 OD λmax; 256.5 nm λmin; 229.1 nm

[0255]

Example 7 Synthesis of 1-On-octyl-2,3.4-O-tribenzoylthioglucoside-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite 1-O-n-octyl-2,3.4 -O-Tribenzoylthioglucoside (0.76 g, 1.22 mmol) was azeotropically dehydrated 3 times with 2 ml of anhydrous pyridine, and then 2 ml of anhydrous toluene.
Azeotropic distillation is performed 3 times and 2 ml of anhydrous methylene chloride 3 times.
After that, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.06 ml, 6.10 mmo) was added to this solution.
1) and N, N-diisopropylamino-O-cyanoethyl phosphorochloride (0.50 ml, 2.44 mmo
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1-O-n-octyl-2,3.4-O-tribenzoylthioglucoside-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (0.82 g, 1.00 mmol, 8
2%) was obtained. 1H-NMR (CDCl ₃ ) δ7.97-7.93 (m, 6H, oh of ben
zoyl), 7.52-7.26 (m, 9H, m, pH of benzoyl), 5.82 (d, 1
H, H-1), 5.61-5.45,4.82-4.75 (m, 3H, H-2,3,4), 3.85-3.7
8 (m, 3H, H-5, H-6), 3.68-3.50 (m, 2H, CH of isopropyl),
2.61-2.54 (m, 2H, CH ₂ of cyanoethyl), 1.29-1.10 (m, 20
H, CH3 of isopropyl, CH ₂ of octyl), 0.90-0.86 (t, 3H, C
H3 of octyl) ppm ³¹ P-NMR (CDCl ₃ ) δ 149.51, 149.91, 151.18 pp
m

[0256]

Example 8 n- octylthioglucoside bound phosphorothioate antisense oligonucleotides: (n-oct-SGlc) -T S G S G S G S A S G S C S C S A S T S
_{A S G S C S G S} A S G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. To bind the guanosine derivative of the following base sequence,
Similarly, after dissolving at a concentration of 0.2 M and attaching to an automatic synthesizer, the above operations (1) to (7) are repeated except that the steps (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylate, in order to attach the n-octylthioglucoside derivative synthesized in Example 7 to the 5 ′ end, 0.2M was similarly added.
After being dissolved in the concentration of 1 and attached to an automatic synthesizer, the above operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes with the 0.2Mn-octylthioglucoside phosphoramidite unit synthesized in Example 7 and 0.4M tetrazole. By the above operation, the n-octylthioglucoside derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthesis; a practical appr
oach ", MJGait (ed), IRL PRESS, (1984).). After removing the CPG in the reaction column, it was washed with an ammonia / pyridine (5: 1) solution at 60%.
By reacting at 6 ° C. for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). The phosphorothioate antisense oligonucleotide to which the target n-octylthioglucoside was bound by the above operation: (n-oct-SGlc) -T _S G _S G _S
G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C was synthesized. Yield: 14.7 OD λmax; 256.9 nm λmin; 229.6 nm

[0257]

Example 9 1-On-octyl-2,3,4-O-tribenzoylglucoside-6-
Synthesis of O- (N, N-diisopropylamino) phenylphosphonoamidite 1-On-octyl-2,3,4-O-tribenzoylglucoside (1.15g, 1.91mmol) was added to anhydrous pyridine 2m.
After azeotropic dehydration 1 × 3 times, anhydrous toluene 2 ml × 3
And azeotropically with 2 ml of anhydrous methylene chloride × 3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.66 ml, 9.54 mmo) was added to this solution.
1) and chloro-N, N-diisopropylaminophenylphosphonoamidite (0.93 g, 3.82 mmo
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1-On-octyl-2,3,4-O-tribenzoylglucoside-6-
O- (N, N-diisopropylamino) phenylphosphonoamidite (1.01 g, 1.24 mmol, 65%) was obtained. 1H-NMR (CDCl ₃ ) δ 7.98-7.82 (m, 11H, oh of ben
zoyl, phenyl), 7.53-7.24 (m, 9H, m-, pH of benzoyl),
5.93-5.83 (s, 1H, H-1), 4.84-4.80,5.54-5.45 (m, 3H, H-2,
3,4), 3.92-3.77 (m, 3H, H-5,6), 3.56-3.47 (m, 2H, CH of is
opropyl), 1.38-1.22 (m, 20H, CH3 of isopropyl, CH ₂ of
octyl), 0.86-0.80 (t, 3H, CH3 of octyl) ppm ³¹ P-NM
R (CDCl ₃ ) δ 120.86, 121.95 ppm

[0258]

Example 10 Phosphorothioate Antisense Oligonucleotide Conjugated with n-Octyl Glucoside Phenylphosphonate: (n-oct-GlcPh) -T _S G _S G _S G _S A _S G _S C _S C _{S
A S T S A S G S} C S G S A S G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to bond the n-octyl glucoside phenylphosphonate derivative synthesized in Example 9 to the 5'end, it was similarly dissolved at a concentration of 0.2 M and automatically synthesized. After mounting on the machine, the above-mentioned operations (1) to (7) are repeated except that (1), (4) and (5) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) 0.2M n-octylglucoside phenylphosphonoamidite unit synthesized in Example 9 and 0.4M
The condensation reaction is performed for 5 minutes with tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. By the above operation, the n-octyl glucoside phenylphosphonate derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection operation after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthesis;
a practical approach ", MJ Gait (ed), IRL PRESS,
(1984).). CP in reaction column
After G is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. After that, reverse-phase HPLC (μBondaspher
e C ₁₈ ) is used for purification. The phosphorothioate antisense oligonucleotide to which the target n-octyl glucoside phenylphosphonate was bound by the above operation: (n-oct-GlcPh) -T _S G _S G _S G G _S A _S G _S C
It was synthesized _{S C S A S T S A} S G S C S G S A S G S G S C. Yield: 52.2 OD λmax; 256.3 nm λmin; 229.3 nm

[0259]

Example 11 Synthesis of 1-On-octyl-2,3,4-O-tribenzoylthioglucoside-6-O- (N, N-diisopropylamino) phenylphosphonoamidite 1-On-octyl-2, 3,4-O-Tribenzoylthioglucoside (0.84 g, 1.35 mmol) was added to anhydrous pyridine 2
After azeotropic dehydration 3 times with 3 ml, dry toluene 2 ml 3
And azeotropically with 2 ml of anhydrous methylene chloride × 3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.17 ml, 6.75 mmo) was added to this solution.
l) and chloro-N, N-diisopropylaminophenylphosphonoamidite (0.66 g, 2.69 mmo
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1-On-octyl-2,3,4-O-tribenzoylthioglucoside-6-O- (N, N-diisopropylamino) phenylphosphonoamidite (0.51 g, 0.62 mmol, 46
%) Was obtained. 1H-NMR (CDCl ₃ ) δ 7.97-7.80 (m, 11H, oh of ben
zoyl, phenyl), 7.79-7.19 (m, 9H, m-, pH of benzoyl),
5.91 (d, 1H, H-1), 5.58-5.46, 4.85-4.79, 4.07-4.00 (m, 6
H, H-2,3,4,5,6), 3.33-3.28 (m, 2H, CH of isopropyl),
1.57-1.00 (m, 20H, CH3 of isopropyl, CH ₂ of octyl),
0.85 (t, 3H, CH3 of octyl) ppm ³¹ P-NMR (CDCl ₃ ) δ119.68, 122.60 ppm

[0260]

Example 12 Phosphorothioate Antisense Oligonucleotide Conjugated with n-Octylthioglucoside Phenylphosphonate: (n-oct-SGlcPh) -T _S G _S G _S G _S A _S G _S C _S C _{S S} A
_{S T S A S G S C} S G S A S G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to bond the n-octylthioglucoside phenylphosphonate derivative synthesized in Example 11 to the 5'end, it was similarly dissolved in 0.2M to automatically After mounting on the synthesizer, the above operations (1) to (7) are repeated except that the steps (1), (4) and (5) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2 M n-octylthioglucoside phenylphosphonoamidite unit synthesized in Example 11 and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. By the above operation, the n-octylthioglucoside phenylphosphonate derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after the completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthes
is; a practical approach ", MJ Gait (ed), IRL PR
ESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. After that, reverse-phase HPLC (μBondaspher
e C ₁₈ ) is used for purification. The above operation by the desired product n- octylthioglucoside phosphonate is bonded to phosphorothioate antisense oligonucleotides: (n-oct-SGlcPh) -T S G S G S G S A
It was synthesized _{S G S C S C S A} S T S A S G S C S G S A S G S G S C. Yield: 40.3 OD λmax; 256.8 nm λmin; 229.6 nm

[0261]

Example 13 1-O-phenyl-2,3,4-O-tribenzoylglucoside-6-O-
Synthesis of N, N-diisopropylamino-O-cyanoethyl phosphoramidite 1-O-phenyl-2,3,4-O-tribenzoylglucoside (1.14 g, 2.00 mmol) was added to anhydrous pyridine 2 m.
After azeotropic dehydration 1 × 3 times, anhydrous toluene 2 ml × 3
And azeotropically with 2 ml of anhydrous methylene chloride × 3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.74 ml, 10.00 mmo) was added to this solution.
1) and N, N-diisopropylamino-O-cyanoethylphosphorochloride (0.89 ml, 4.00 mmo
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1-O-phenyl-2,3.4-O-tribenzoylglucoside-6-O-
N, N-diisopropylamino-O-cyanoethyl phosphoramidite (1.31 g, 1.71 mmol, 85%) was obtained. ¹ H-NMR (CDCl ₃ ) δ7.97-7.80 (m, 6H, oH of benz
oyl), 7.51-7.25, 7.18-7.06 (m, 14H, m-, pH of benzoy
l, phenyl), 6.00-5.37 (m, 4H, H-1,2,3,4), 4.18-4.12,
3.92-3.72 (3H, H-5,6) 3.61-3.50 (m, 2H, CH of isopropy
l), 2.57-2.42 (4H, CH ₂ of cyanoethyl), 1.16-1.04 (12
H, CH ₃ of isopropyl) ppm ³¹ P-NMR (CDCl ₃ ) δ149.67, 149.
19 ppm

[0262]

Example 14 Phosphorothioate antisense oligonucleotides phenyl glucoside _{bound: (PhGlc) -T S G S} G S G S A S G S C S C S A S T S A S
_{G S C S G S A S} G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced on CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. As a result of colorimetrically quantifying DMTr liberated by the operation of (1), the calculated average yield of each condensation reaction was 9
It was 9% or more. After condensing the 18-mer thymidylic acid, in order to bind the phenylglucoside derivative synthesized in Example 13 to the 5'end, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. After that, with respect to (1), (4) and (5), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is performed for 5 minutes using the 0.2 M phenylglucoside phosphoramidite unit synthesized in Example 13 and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the phenylglucoside derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a practical
approach ", MJ Gait (ed), IRL PRESS, (1984).). After removing CPG from the reaction column, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C for 6 hours. Thus, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed.
After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). More phenyl glucoside of the object is bound by the operation phosphorothioate antisense _{oligonucleotides: (PhGlc) -T S G S} G S G S A S G S
C _S C _S A _S T _{S S} A _S G _S C _S G _S A _S G _S G _S C was synthesized. Yield: 56.6 OD λmax; 256.7 nm λmin; 228.9 nm

[0265]

Example 15 1-O-phenyl-2,3,4-O-tribenzoylgalactoside-6-
Synthesis of ON, N-diisopropylamino-O-cyanoethyl phosphoramidite 1-O-phenyl-2,3.4-O-tribenzoylgalactoside (1.14 g, 2.00 mmol) was added to 2 m of anhydrous pyridine.
After azeotropic dehydration 1 × 3 times, anhydrous toluene 2 ml × 3
And azeotropically with 2 ml of anhydrous methylene chloride × 3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.74 ml, 10.00 mmo) was added to this solution.
1) and N, N-diisopropylamino-O-cyanoethylphosphorochloride (0.89 ml, 4.00 mmo
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1-O-phenyl-2,3.4-O-tribenzoylgalactoside-6-
ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (1.37 g, 1.79 mmol, 90%)
Got ¹ H-NMR (CDCl ₃ ) δ8.12-7.80 (m, 6H, oH of benz
oyl), 7.50-7.05 (m, 14H, m-, pH of benzoyl, phenyl),
6.03-5.98,5.79-5.60,5.43-5.29 (m, 4H, H-1,2,3,4), 4.3
6-4.12 (3H, H-5,6) 3.60-3.47 (m, 2H, CH of isopropyl),
2.75-2.52 (4H, CH ₂ of cyanoethyl), 1.30-1.06 (12H, CH ₃
of isopropyl) ppm ³¹ P-NMR (CDCl ₃ ) δ149.98, 149.60ppm

[0266]

Example 16 Phosphorothioate phenyl galactoside bound antisense _{oligonucleotides: (PhGal) -T S G S} G S G S A S G S C S C S A S T S A S
_{G S C S G S A S} G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to bind the phenylgalactoside derivative synthesized in Example 15 to the 5'end, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. After that, with respect to (1), (4) and (5), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2Mn-phenylgalactoside phosphoramidite unit synthesized in Example 15 and 0.4M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the phenylgalactoside derivative can be introduced at the 5'end of the antisense oligonucleotide. The deprotection operation after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a practica
l approach ", MJ Gait (ed), IRL PRESS, (1984).)
Follow the instructions. After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). More phenyl glucoside of the object is bound by the operation phosphorothioate antisense _{oligonucleotides: (PhGal) -T S G S} G S G S A
It was synthesized _{S G S C S C S A} S T S A S G S C S G S A S G S G S C. Yield: 61.5 OD λmax; 257.1 nm λmin; 229.4 nm

[0269]

Example 17 Synthesis of 1,3,4-O-tribenzoyl, 2-N-benzoylgalactosamine-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite 1,3,4-O-tribenzoyl 2,2-N-benzoylgalactosamine (1.05 g, 1.76 mmol) was added to anhydrous pyridine 2
After azeotropic dehydration 3 times with 3 ml, dry toluene 2 ml 3
And azeotropically with 2 ml of anhydrous methylene chloride × 3 times. After that, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.53 ml, 8.79 mmo was added to this solution.
l) and N, N-diisopropylamino-O-cyanoethyl phosphorochloride (0.78 ml, 3.52 mmo
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1,3,4-O-tribenzoyl, 2-N-benzoylgalactosamine-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (0.97 g, 1.22 mmol, 70
%) Was obtained. 1H-NMR (CDCl ₃ ) δ8.50 (s, 1H, -NH), 8.22-7.95
(m, 8H, oH of benzoyl), 7.82-7.35 (m, 12H, m-, pH of be
nzoyl), 6.61-6.56, 6.32-6.18 (4H, H-1,2,3,4), 4.32-3.
91,3.87-3.64 (m, 3H, H-5,6), 3.45-3.22 (m, 2H, CH of iso
propyl), 2.73-2.45 (m, 4H, CH ₂ of cyanoethyl), 1.39-1.
02 (m, 12H, CH ₃ of isopropyl) ppm ³¹ P-NMR (CDCl ₃ ) δ149.84, 150.09, 150.43ppm

[0270]

Example 18 N-Benzoylgalactosamine Phosphate-Linked Phosphorothioate Antisense Oligonucleotide: (N-BzGalPO) -T _S G _S G _S G _S A _S G _S C _S C _S A
_{S T S A S G S C} S G S A S G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced on CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. DM released by operation (1)
The average yield of each condensation reaction calculated as a result of the colorimetric determination of Tr was 99% or more. After condensing the 18-mer thymidylic acid, in order to attach the N-benzoylgalactosamine derivative synthesized in Example 17 to the 5'end, it was dissolved in a concentration of 0.2 M in an automatic synthesizer in the same manner. After mounting, the above operations (1) to (7) are repeated except that the steps (1), (4) and (5) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2MN-benzoylgalactosamine amidite unit synthesized in Example 17 and 0.4M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the N-benzoylgalactosamine derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection operation after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a
practical approach ", MJ Gait (ed), IRL PRESS,
(1984).). CP in reaction column
After G is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. After that, reverse-phase HPLC (μBondaspher
e C ₁₈ ) is used for purification. The above operation by the desired product N- benzo Irganox lactosamine phosphate bound phosphorothioate antisense _{oligonucleotides: (N-BzGalPO) -T S} G S G S G S A S G S C
It was synthesized _{S C S A S T S A} S G S C S G S A S G S G S C. Yield: 51.6 OD λmax; 256.2 nm λmin; 228.9 nm

[0273]

Example 19 1,3,4-O-triacetyl, 2-N-acetylgalactosamine-6-
Synthesis of ON, N-diisopropylamino-O-cyanoethyl phosphoramidite 1,3,4-O-triacetyl, 2-N-acetylgalactosamine (0.38 g, 1.09 mmol) was added to anhydrous pyridine 2 m.
After azeotropic dehydration 1 × 3 times, anhydrous toluene 2 ml × 3
And azeotropically with 2 ml of anhydrous methylene chloride × 3 times. Then, dissolve in 5 ml of methylene chloride, and add diisopropylethylamine (0.87 ml, 5.00 mmo) to this solution.
1) and N, N-diisopropylamino-O-cyanoethyl phosphorochloride (0.45 ml, 2.00 mmo
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1,3,4-O-triacetyl, 2-N-acetylgalactosamine-6-
ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (0.40 g, 0.75 mmol, 69%)
Got ¹ H-NMR (CDCl ₃ ) δ8.50 (s, 1H, -NH), 6.20, 5.70
-5.56,5.32 (m, 4H, H-1,2,3,4), 4.22-4.10 (m, 3H, H-5,6),
3.90-3.77,3.61-3.47 (m, 12H, CH ₃ of acetyl), 2.80-2.5
0 (m, 4H, CH ₂ of cyanoethyl), 1.30-1.11 (m, 12H, CH ₃ of
isopropyl) ppm ³¹ P-NMR (CDCl ₃ ) δ149.50, 149.57, 150.81pp
m

[0274]

EXAMPLE 20 N- acetylgalactosamine phosphate bound phosphorothioate antisense _{oligonucleotides: (N-AcGal) -T S} G S G S G S A S G S C S C S A S T S
_{A S G S C S G S} A S G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced on CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylate, in order to attach the N-acetylgalactosamine derivative synthesized in Example 19 to the 5'end, 0.2
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
With respect to (4) and (5), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2MN-acetylgalactosamine amidite unit synthesized in Example 19 and 0.4M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the N-acetylgalactosamine derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a pr
actical approach ", MJGait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasphere C ₁₈ )
Is used for purification. With the above operation, the target N
-Acetylgalactosamine phosphate linked phosphorothioate antisense oligonucleotides: (N
-AcGal) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _{S S}
G _S C _S G _S A _S G _S G _S C was synthesized. Yield: 9.1 OD λmax; 257.4 nm λmin; 229.6 nm

[0277]

Example 21 1,3,4-O-triacetyl, 2-N-acetylglucosamine-6-O-
Synthesis of N, N-diisopropylamino-O-cyanoethyl phosphoramidite 1,3,4-O-triacetyl, 2-N-acetylglucosamine (0.82g, 1.39mmol) was added to anhydrous pyridine 2m.
After azeotropic dehydration 1 × 3 times, anhydrous toluene 2 ml × 3
And azeotropically with 2 ml of anhydrous methylene chloride × 3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.21 ml, 6.95 mmo) was added to this solution.
1) and N, N-diisopropylamino-O-cyanoethylphosphorochloride (0.62 ml, 2.78 mmo)
l) was added and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, silica gel column chromatography (hexane / methylene chloride = 8)
0:20, 2% triethylamine)
1,3,4-O-triacetyl, 2-N-acetylglucosamine-6-O-
N, N-diisopropylamino-O-cyanoethyl phosphoramidite (0.47 g, 0.59 mmol, 42%) was obtained. ¹ H-NMR (CDCl ₃ ) δ8.38 (s, 1H, -NH), 6.15-5.99,
5.80-5.52 (m, 4H, H-1,2,3,4), 4.28-4.08 (m, 3H, H-5,6),
3.96-3.57 (m, 12H, CH ₃ of acetyl), 2.78-2.53 (m, 4H, CH ₂
of cyanoethyl), 1.41-1.08 (m, 12H, CH ₃ of isopropyl)
ppm ³¹ P-NMR (CDCl ₃ ) δ149.76, 150.72 ppm

[0278]

EXAMPLE 22 N- acetylglucosamine phosphate bound phosphorothioate antisense _{oligonucleotides: (N-AcGul) -T S} G S G S G S A S G S C S C S A S T S
_{A S G S C S G S} A S G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to attach the N-acetylglucosamine derivative synthesized in Example 21 to the 5 ′ end, 0.2M was similarly added.
After being dissolved in the concentration of 1 and attached to an automatic synthesizer, the above-mentioned operations (1) to (7) are repeated except that (1), (4) and (5) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes with the 0.2MN-acetylglucosamine amidite unit synthesized in Example 21 and 0.4M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. By the above operation, the N-acetylglucosamine derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthesis; a practical approa").
ch ", MJ Gait (ed), IRL PRESS, (1984).) The CPG in the reaction column was taken out, and then it was washed with an ammonia / pyridine (5: 1) solution at 60%.
By reacting at 6 ° C. for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). The phosphorothioate antisense oligonucleotide bound with N-acetylglucosamine phosphate of the target product by the above operation: (N-AcGul)-
T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G
_S C was synthesized. Yield: 42.5 OD λmax; 257.3 nm λmin; 229.6 nm

[0279]

Example 23 Synthesis of 1,3,4-O-tribenzoyl, 2-N-trifluoroacetylgalactosamine-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite 1,3,4-O- Tribenzoyl, 2-N-trifluoroacetylgalactosamine (0.58 g, 0.99 mmol) was azeotropically dehydrated with 2 ml of anhydrous pyridine 3 times, and then azeotropically distilled with 2 ml of anhydrous toluene 3 times and 2 ml of anhydrous methylene chloride 3 times. Then, dissolve in 5 ml of methylene chloride, and add diisopropylethylamine (0.87 ml, 5.0
0 mmol) and N, N-diisopropylamino-O-
Cyanoethyl phosphorochloride (0.45 ml, 2.0
(0 mmol) was added, and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and then dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography (hexane / methylene chloride = 80: 20, 2% triethylamine),
1,3,4-O-tribenzoyl, 2-N-trifluoroacetylgalactosamine-6-ON, N-diisopropylamino-
O-cyanoethyl phosphoramidite (0.42 g, 0.
53 mmol, 53%) was obtained. ¹ H-NMR (CDCl ₃ ) δ8.70 (s, 1H, -NH), 8.18-7.79
(m, 6H, oH of benzoyl), 7.66-7.21 (m, m-, pH of benzoy
l), 6.66-6.15 (m, 4H, H-1,2,3,4), 4.04-3.70 (m, 3H, H-5,
6), 3.61-3.42 (m, 2H, CH of cyanoethyl), 2.68-2.41 (m, 4
H, CH ₂ of cyanoethyl), 1.31-0.92 (m, 12H, CH ₃ of cyano
ethyl) ppm ³¹ P-NMR (CDCl ₃ ) δ 148.96, 149.38, 150.10ppm

[0280]

EXAMPLE 24 galactosamine phosphate bound phosphorothioate antisense _{oligonucleotides: (GalN) -T S G S} G S G S A S G S C S C S A S T S A S G S
_{C S G S A S G S} G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to bind the galactosamine derivative synthesized in Example 23 to the 5'end, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. , (1) and (4)
Regarding (5), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2 M galactosamine amidite unit synthesized in Example 23 and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the galactosamine derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthesis; a practical appr
Oach ", MJ Gait (ed), IRL PRESS, (1984).). After removing the CPG in the reaction column, it was washed with an ammonia / pyridine (5: 1) solution at 60%.
By reacting at 6 ° C. for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). More of the object by operating galactosamine phosphate bound phosphorothioate antisense _{oligonucleotides: (GalN) -T S G S} G S G S A S
G _S C _S C _{S AS} T _{S AS} G _S C _S G _S A _S G _S G _S C was synthesized. Yield: 38.7 OD λmax; 256.4 nm λmin; 229.3 nm

[0283]

Example 25 Synthesis of 1,3,4-O-tribenzoyl, 2-N-trifluoroacetylglucosamine-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite 1,3,4-O- Tribenzoyl, 2-N-trifluoroacetylglucosamine (0.82 g, 1.39 mmol) was azeotropically dehydrated 3 times with 2 ml of anhydrous pyridine and then with anhydrous toluene 2
Azeotrope is performed 3 times with ml and 2 times with 2 ml of anhydrous methylene chloride. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (1.21 ml, 6.95) was added to this solution.
mmol) and N, N-diisopropylamino-O-cyanoethylphosphorochloride (0.62 ml, 2.78).
(mmol) and reacted at room temperature for 1 hour, 50 ml of methylene chloride was added to dilute the solution, and the solution was washed 3 times with 50 ml of a saturated aqueous solution of sodium hydrogen carbonate and then dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (hexane / methylene chloride = 80: 20, 2% triethylamine) to obtain the desired product, 1,3,4-O-tribenzoyl, 2-N-trifluoro. Acetylglucosamine-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (0.47 g, 0.59
mmol, 42%) was obtained. ¹ H-NMR (CDCl ₃ ) δ8.74 (s, 1H, -NH), 8.19-7.81
(m, 6H, oH of benzoyl), 7.62-7.26 (m, m-, pH of benzoy)
l), 6.63-6.32 (m, 4H, H-1,2,3,4), 4.22-3.84 (m, 3H, H-5,
6), 3.58-3.42 (m, 2H, CH of cyanoethyl), 2.70-2.56 (m, 4
H, CH ₂ of cyanoethyl), 1.26-1.07 (m, 12H, CH ₃ of cyano
ethyl) ppm ³¹ P-NMR (CDCl ₃ ) δ 150.33, 150.77, 151.39ppm

[0284]

EXAMPLE 26 glucosamine phosphate bound phosphorothioate antisense _{oligonucleotides: (GulN) -T S G S} G S G S A S G S C S C S A S T S A S G S
_{C S G S A S G S} G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to attach the glucosamine derivative synthesized in Example 25 to the 5 ′ end, after similarly dissolving it at a concentration of 0.2 M and attaching it to an automatic synthesizer. , (1) and (4)
Regarding (5), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2 M glucosamine amidite unit synthesized in Example 25 and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the glucosamine derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthesis; a practical approa").
ch ", MJ Gait (ed), IRLPRESS, (1984).) The CPG in the reaction column was taken out, and then it was washed with an ammonia / pyridine (5: 1) solution at 60%.
By reacting at 6 ° C. for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). More glucosamine phosphate of the target compound is bound by the operation phosphorothioate antisense _{oligonucleotides: (GulN) -T S G S} G S G S A S G S
C _S C _S A _S T _{S S} A _S G _S C _S G _S A _S G _S G _S C was synthesized. Yield: 33.4 OD λmax; 257.3 nm λmin; 229.4 nm

[0287]

Example 27 Synthesis of heptabenzoyl melibiose-N, N-diisopropylamino-O-cyanoethyl phosphoramidite Heptabenzoyl melibiose (1.07 g, 1.00
(mmol) was azeotropically dehydrated 2 ml × 3 times with anhydrous pyridine, and then 2 ml × 3 times with anhydrous toluene and 2 m of anhydrous methylene chloride.
Azeotropically perform 1x3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (0.
87 ml, 5.00 mmol) and N, N-diisopropylamino-O-cyanoethylphosphorochloride (0.
45 ml, 2.00 mmol) was added, and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous sodium hydrogen carbonate solution three times, and dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (hexane / methylene chloride = 80: 20, 2% triethylamine) to obtain the desired product, heptabenzoyl melibiose-N, N-diisopropylamino-O-cyanoethylphospho. Loamidite (1.91 g, 1.50 mmol, 75
%) Was obtained. ¹ H-NMR (CDCl ₃ ) δ8.13-8.09, 8.03-7.87, 7.84
-7.80 (m, 14H, oH of benzoyl), 7.56-7.24 (m, 21H, m-, p-
H of benzoyl), 5.99-5.80, 5.58-5.41 (m, 8H, H-1,2,3,
4,1 ', 2', 3'4 '), 4.61-4.40, 3.83-3.41 (m, 6H, H-5,6), 2.5
8-2.40 (m, 4H, CH ₂ of cyanoethyl), 1.12-0.96 (m, 12H, CH ₃
of isopropyl) ppm ³¹ P-NMR (CDCl ₃ ) δ 149.45, 149.72ppm

[0288]

Example 28 Mellibiose Phosphate-Linked Phosphorothioate Antisense Oligonucleotide: (Mel) -T _S G _S G _S G _{S AS} G _S C _S C _S A _S T _S A _S G _S C
_{S G S A S G S G} S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to bind the melibiose derivative synthesized in Example 27 to the 5'end, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. , (1) and (4)
Regarding (5), the above operations (1) to (7) are repeated except that the following is performed. (1) 3.0
% TCA / CH ₂ Cl ₂ treatment is carried out for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2 M melibiose amidite unit synthesized in Example 27 and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the melibiose derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthesis; a practical approa").
ch ", MJ Gait (ed), IRLPRESS, (1984).) The CPG in the reaction column was taken out, and then it was washed with an ammonia / pyridine (5: 1) solution at 60%.
By reacting at 6 ° C. for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). The above operation by the desired product melibiose phosphate bound phosphorothioate antisense _{oligonucleotides: (Mel) -T S G S} G S G S A S G S C
It was synthesized _{S C S A S T S A} S G S C S G S A S G S G S C. Yield: 78.0 OD λmax; 256.6 nm λmin; 229.2 nm

[0290]

Example 29 Synthesis of heptabenzoyl gentiobiose-N, N-diisopropylamino-O-cyanoethyl phosphoramidite Heptabenzoyl gentiobiose (1.24 g, 1.
16 mmol) was azeotropically dehydrated with anhydrous pyridine 2 ml × 3 times, and then azeotropically distilled with anhydrous toluene 2 ml × 3 times and anhydrous methylene chloride 2 ml × 3 times. Then methylene chloride 5m
It was dissolved in 1, and to this solution were added diisopropylethylamine (1.01 ml, 5.80 mmol) and N, N-diisopropylamino-O-cyanoethylphosphorochloride (0.50 ml, 2.32 mmol), and the mixture was stirred at room temperature for 1 hour.
After reacting for a period of time, 50 ml of methylene chloride was added to dilute, and this solution was diluted with 50 ml of a saturated aqueous solution of sodium hydrogen carbonate to 3 times.
After washing twice, it was dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (hexane / methylene chloride = 80: 20, 2% triethylamine) to obtain the desired product, heptabenzoylgentiobiose-N, N-diisopropylamino-O-cyanoethylphospho. Loamidite (1.36g, 1.07mmo
1, 93%). ¹ H-NMR (CDCl ₃ ) δ 8.10-7.92, 7.86-7.71 (m, 14
H, oH of benzoyl), 7.52-7.19 (m, 21H, m-, pH of benzo
yl), 6.01-5.83, 5.57-5.45 (m, 8H, H-1,2,3,4,1 ', 2', 3 '
4 '), 4.63-4.46, 3.81-3.43 (m, 6H, H-5,6), 2.55-2.43 (m,
4H, CH ₂ of cyanoethyl), 1.15-0.94 (m, 12H, CH ₃ of isopr
opyl) ppm ³¹ P-NMR (CDCl ₃ ) δ 150.12, 150.43ppm

[0291]

Example 30 gentian obi triose phosphate bound phosphorothioate antisense _{oligonucleotides: (Gen) -T S G S} G S G S A S G S C S C S A S T S A S G S C
_{S G S A S G S G} S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to attach the gentiobiose derivative synthesized in Example 29 to the 5'end, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. , (1) and (4) and (5), the above operations (1) to (7) are repeated except that the following is performed. (1)
3.0% TCA / CH ₂ Cl ₂ treatment for 60 seconds,
The 5'-DMTr group of the protected thymidine derivative is removed. (4) A condensation reaction is carried out for 5 minutes using the 0.2 M gentiobiose amidite unit synthesized in Example 29 and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the gentiobiose derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection operation after the completion of the synthesis reaction is performed by a conventional method ("Oligonucleotide Synthesis; a practical ap
proach ", MJ Gait (ed), IRL PRESS, (1984).) After removing the CPG in the reaction column, it was added to a solution of ammonia / pyridine (5: 1) 6 times.
By reacting at 0 ° C. for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). More of the object by operating gentian Obi triose phosphate bound phosphorothioate antisense _{oligonucleotides: (Gen) -T S G S} G S G S A
It was synthesized _{S G S C S C S A} S T S A S G S C S G S A S G S G S C. Yield: 37.9 OD λmax; 256.6 nm λmin; 229.0 nm

[0294]

Example 31 Synthesis of Octabenzoyl Isomaltotriose-N, N-Diisopropylamino-O-Cyanoethyl Phosphoramidite Octabenzoyl Isomaltotriose (1.56 g,
After azeotropic dehydration of anhydrous pyridine 2 ml × 3 times, anhydrous pyridine 2 ml × 3 times and anhydrous methylene chloride 2 ml × 3 times are azeotropically distilled. After that, it was dissolved in 5 ml of methylene chloride, diisopropylethylamine (0.87 ml, 5.00 mmol) and N, N-diisopropylamino-O-cyanoethylphosphorochloride (0.45 ml, 2.00 mmol) were added to this solution, and the mixture was stirred at room temperature. After reacting for 1 hour with 50 ml of methylene chloride for dilution, the solution was washed 3 times with 50 ml of a saturated aqueous solution of sodium hydrogen carbonate and then dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (hexane / methylene chloride = 80: 20, 2% triethylamine), and the desired product octabenzoylisomaltotriose-N, N-diisopropylamino-O-cyanoethyl was obtained. Phosphoramidite (1.21g, 0.69m
mol, 69%) was obtained. ³¹ P-NMR (CDCl ₃ ) δ 150.92, 151.23 ppm

[0295]

EXAMPLE 32 isomaltotriose phosphate bound phosphorothioate antisense _{oligonucleotides: (Iso) -T S G S} G S G S A S G S C S C S A S T S A S G S C
_{S G S A S G S G} S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced on CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated by the colorimetric quantification of DMTr liberated by the operation of (1) is 99.
% Or more. After condensing the 18-mer thymidylic acid, in order to bind the isomaltotriose derivative synthesized in Example 31 to the 5 ′ end, it was similarly dissolved at a concentration of 0.2M in an automatic synthesizer. After mounting, the above operations (1) to (7) are repeated except that the steps (1), (4) and (5) are as follows. (1)
3.0% TCA / CH ₂ Cl ₂ treatment for 60 seconds,
The 5'-DMTr group of the protected thymidine derivative is removed. (4) A condensation reaction is carried out for 5 minutes using the 0.2M isomaltotrioseamidite unit synthesized in Example 31 and 0.4M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF.

By the above operation, the isomalttriose derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection operation after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a practica
l approach ", MJ Gait (ed), IRL PRESS, (1984).)
Follow the instructions. After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). By the above operation, the phosphorothioate antisense oligonucleotide to which the desired product isomaltotriose phosphate is bound: (Iso) -T _S
G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C
Was synthesized. Yield: 51.8 OD λmax; 256.4 nm λmin; 228.9 nm

[0298]

Example 33 Phosphate Antisense Oligonucleotide Partially Introduced Phenylphosphonate Bond: T _Ph G _Ph GGAGCCATAGCGAG _Ph G _Ph C (SEQ ID NO: 32) Synthesis N-4 amino group with benzoyl group, 5′- DM hydroxyl group
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. To further bind adenosine phosphate having the following nucleotide sequence, adenosine phosphoramidite unit is dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, and then (1) and (4) are The above operations (1) to (7) are repeated except that the above is set. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product by the above operation:
T _Ph G _Ph GGAGCCATAGCGAG _Ph G _Ph C was synthesized. Yield: 35.4 OD λmax; 256.5 nm λmin; 229.1 nm

[0300]

Example 34 Phosphate Antisense Oligonucleotide with Partially Introduced Phenylphosphonate Bond and Gentiobiose Bonded to the 5 ′ End: (Gen) -T _Ph G _Ph GGAGCCATAGCGAG _Ph
Synthesis of G _Ph C (SEQ ID NO: 32) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected adenosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the adenosine phenylphosphonate derivative of the next base sequence, the same operation as described above is repeated. To further bind guanosine phosphate having the following nucleotide sequence, the guanosine phosphoramidite unit was dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, (1) and (4) were as follows. The above operations (1) to (7) are repeated except that the above is set. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, after similarly dissolving the gentiobiose amidite unit in a concentration of 0.2 M and attaching it to an automatic synthesizer , (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-P.
The ix group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole.

By the above operation, the gentiobiose derivative can be introduced into the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oli
gonucleotide Synthesis; a practical approach ", M.
J. Gait (ed), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HP
Purification is carried out using LC (μBondasphere C ₁₈ ).
Through the above operation, a phenylphosphonate bond was introduced into the 5'and 3'terminal 2 bases of the target product, and a phosphate antisense oligonucleotide having gentiobiose bonded to the 5'terminal: (Gen) -T _Ph G _Ph GGAGCCATA.
GCGAG _Ph G _Ph C was synthesized. Yield; 17.0 OD λmax; 255.6 nm λmin; 229.8 nm

[0303]

Example 35 Phosphate Antisense Oligonucleotide with Partially Introduced Phenylphosphonate Bond and Meribiose Bonded to 5 ′ End: (Mel) -T _Ph G _Ph GGAGCCATAGCGAG _Ph
Synthesis of G _Ph C (SEQ ID NO: 32) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH2Cl2 for 60 seconds to remove the 5'-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. In order to bind adenosine phosphate having the next base sequence, 0.2 units of adenosine phosphoramidite units are added.
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the melibiose derivative to the 5'end, the melibiose amidite unit was similarly dissolved at a concentration of 0.2M and attached to an automatic synthesizer. , (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-P.
The ix group is removed. (4) A condensation reaction is performed with 0.2 M melibiose phosphoramidite unit and 0.4 M tetrazole for 5 minutes.

By the above operation, the melibiose derivative can be introduced at the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligo
nucleotide Synthesis; a practical approach ", M.
J. Gait (ed), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HP
Purification is carried out using LC (μBondasphere C ₁₈ ).
Phosphoantisense oligonucleotide in which a phenylphosphonate bond was introduced into the 5'and 3'terminals 2 bases of the target product by the above-mentioned operation and melibiose was bonded to the 5'terminal: (Mel) -T _Ph G _Ph GGAGCACATAGC.
GAG _Ph G _Ph C was synthesized. Yield; 22.5 OD λmax; 255.2 nm λmin; 229.6 nm

[0305]

Example 36 Phosphate Antisense Oligonucleotide Partially Introduced Phenylphosphonate Bond: T _Ph GGGAGC _Ph C _Ph AT _Ph AGC _Ph GAGGC _Ph T
Synthesis of (SEQ ID NO: 33) CPG5 in which 20 μmol of a thymidine derivative having a 5′-hydroxyl group protected by a DMTr group was introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the cytidine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is carried out for 5 minutes with 0.2 M cytidine phenylphosphonoamidite unit and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected cytidine phenylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind guanosine phosphate having the following nucleotide sequence, the guanosine phosphoramidite unit was dissolved at a concentration of 0.2M and attached to an automatic synthesizer. The above steps (1) to (7) are repeated except that the above steps are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Mox group of the protected cytidine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide having a phenylphosphonate bond introduced into a pyrimidine nucleotide by the above operation: T _Ph GG
GAGC _Ph C _Ph AT _Ph AGC _Ph GAGGC _Ph T was synthesized. Yield: 44.4 OD λmax; 256.1 nm λmin; 228.2 nm

[0306]

Example 37 Phosphate antisense oligonucleotide in which a phenylphosphonate bond was partially introduced and gentiobiose was bonded to the 5 ′ end: (Gen) -T _Ph GGGAGC _Ph C _Ph AT _Ph AGC _Ph G
Synthesis of AGGC _Ph T (SEQ ID NO: 33) CPG5 in which 20 μmol of thymidine derivative having 5′-hydroxyl group protected by DMTr group was introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the cytidine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is carried out for 5 minutes with 0.2 M cytidine phenylphosphonoamidite unit and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected cytidine phenylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind guanosine phosphate having the following nucleotide sequence, the guanosine phosphoramidite unit was dissolved at a concentration of 0.2M and attached to an automatic synthesizer. The above steps (1) to (7) are repeated except that the above steps are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Mox group of the protected cytidine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 19th mer thymidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, after similarly dissolving the gentiobiose amidite unit at a concentration of 0.2M and attaching it to an automatic synthesizer. , (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-P.
The ix group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole.

By the above operation, the gentiobiose derivative can be introduced at the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oli
gonucleotide Synthesis; a practical approach ", M.
J. Gait (ed), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HP
Purification is carried out using LC (μBondasphere C ₁₈ ).
By the above operation, a phenylphosphonate bond was introduced into the target pyrimidine nucleotide, and a phosphate antisense oligonucleotide in which gentiobiose was bonded to the 5 ′ end: (Gen) -T _Ph GGGAGC _Ph C _Ph A
T _Ph AGC _Ph GAGGC _Ph T was synthesized. Yield: 32.4 OD λmax; 255.7 nm λmin; 229.2 nm

[0308]

Example 38 Phosphate Antisense Oligonucleotide Partially Introduced Methylphosphonate Bond and Gentiobiose Bonded to 5 ′ End: (Gen) -T _Me G _Me GGAGCCATAGCGAG _Me
Synthesis of G _Me C (SEQ ID NO: 34) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine methylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine methylphosphonoamidite unit and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine methylphosphonate derivative can be condensed with the cytidine derivative introduced on CPG. In order to bind the guanosine methylphosphonate derivative having the next base sequence, the same operation as described above is repeated.
To further bind adenosine phosphate having the following nucleotide sequence, adenosine phosphoramidite unit is dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, and then (1) and (4) are The above operations (1) to (7) are repeated except that the above is set. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine methylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, after similarly dissolving the gentiobiose amidite unit in a concentration of 0.2 M and attaching it to an automatic synthesizer , (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-D.
Remove the MTr group. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole.

By the above operation, the gentiobiose derivative can be introduced into the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oli
gonucleotide Synthesis; a practical approach ", M.
J. Gait (ed), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HP
Purification is carried out using LC (μBondasphere C ₁₈ ).
A phosphate antisense oligonucleotide in which a methylphosphonate bond was introduced into the 5 ′ and 3 ′ terminal 2 bases of the target product and gentiobiose was bonded to the 5 ′ end by the above operation: (Gen) -T _Me G _Me GGAGCCATAG
CGAG _Me G _Me C was synthesized. Yield: 65.7 OD λmax; 256.0 nm λmin; 229.8 nm

[0311]

Example 39 Phosphate Antisense Oligonucleotide Partially Introduced Methylphosphonate Bond and Melibiose Bonded to 5 ′ End: (Mel) -T _Me G _Me GGAGCCATAGCGAG _Me
Synthesis of G _Me C (SEQ ID NO: 34) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine methylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine methylphosphonoamidite unit and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine methylphosphonate derivative can be condensed with the cytidine derivative introduced on CPG. In order to bind the guanosine methylphosphonate derivative having the next base sequence, the same operation as described above is repeated.
To further bind adenosine phosphate having the following nucleotide sequence, adenosine phosphoramidite unit is dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, and then (1) and (4) are The above operations (1) to (7) are repeated except that the above is set. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine methylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the melibiose derivative to the 5'end, the melibiose amidite unit was similarly dissolved at a concentration of 0.2M and attached to an automatic synthesizer. For (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-D.
Remove the MTr group. (4) A condensation reaction is performed with 0.2 M melibiose phosphoramidite unit and 0.4 M tetrazole for 5 minutes. By the above operation, the melibiose derivative can be introduced into the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synth
esis; a practical approach ", MJ Gait (ed), IRL
PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. After that, reverse-phase HPLC (μBondaspher
e C ₁₈ ) is used for purification. By the above operation, a methylphosphonate bond was introduced into the 5'and 3'terminal 2 bases of the target product, and a phosphate antisense oligonucleotide in which melibiose was bonded to the 5'terminal: (Mel)-
T _Me G _Me GGAGCCATAGCGAG _Me G _Me C was synthesized. Yield: 59.3 OD λmax; 256.9 nm λmin; 227.8 nm

[0313]

Example 40 Phosphate antisense oligonucleotide in which a phenylphosphonate bond is partially introduced and N-benzoylgalactosamine is bonded to the 5 ′ end: (N-BzGal) -T _Ph G _Ph GGAGCCATAGC
Synthesis of GAG _Ph G _Ph C (SEQ ID NO: 32) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove 5′-Pix group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. In order to bind adenosine phosphate having the next base sequence, 0.2 units of adenosine phosphoramidite units are added.
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the N-benzoylgalactosamine derivative to the 5'end, similarly, the N-benzoylgalactosamine amidite unit was dissolved in a concentration of 0.2M to prepare an automatic synthesizer. After attaching to, the above-mentioned operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-P.
The ix group is removed. (4) A condensation reaction is performed for 5 minutes with 0.2M N-benzoylgalactosamine phosphoramidite unit and 0.4M tetrazole. By the above operation, N- is added to the 5'end of the oligonucleotide.
A benzoylgalactosamine derivative can be introduced. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligo
nucleotide Synthesis; a practical approach ", M.
J. Gait (ed), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HP
Purification is carried out using LC (μBondasphere C ₁₈ ).
Through the above operation, a phenylphosphonate bond was introduced into the 5'and 3'terminal 2 bases of the target product, and a phosphate antisense oligonucleotide having N-benzoylgalactosamine bonded to the 5'terminal: (N-BzGal) -T _Ph G
_Ph GGAGCCATAGCGAG _Ph G _Ph C was synthesized. Yield; 21.2 OD λmax; 256.5 nm λmin; 228.9 nm

[0314]

Example 41 Phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond: Synthesis of T _Ph GGGAGCCCATAGCGAGG _Ph C (SEQ ID NO: 35) N-4 amino group is benzoyl group and 5′-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind guanosine phosphate having the following nucleotide sequence, the guanosine phosphoramidite unit was dissolved at a concentration of 0.2M and attached to an automatic synthesizer. The above steps (1) to (7) are repeated except that the above steps are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5 ', 3'terminal 1 base of the target product by the above operation:
T _Ph GGGAGGCCATAGCGAGG _Ph was synthesized. Yield; 20.9 OD λmax; 256.0 nm λmin; 228.4 nm

[0315]

Example 42 Phosphate Antisense Oligonucleotide with Partially Introduced Phenylphosphonate Bond and Gentiobiose Bonded at the 5'End: (Gen) -T _Ph GGGAGCACATAGCGAGG _Ph
Synthesis of C (SEQ ID NO: 35) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind guanosine phosphate having the following nucleotide sequence, the guanosine phosphoramidite unit was dissolved at a concentration of 0.2M and attached to an automatic synthesizer. The above steps (1) to (7) are repeated except that the above steps are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, after similarly dissolving the gentiobiose amidite unit in a concentration of 0.2 M and attaching it to an automatic synthesizer , (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-P.
The ix group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole. The gentiobiose derivative can be introduced into the 5'end of the oligonucleotide by the above operation. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide S
ynthesis; a practical approach ", MJ Gait (ed),
IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondas
Purification is carried out using phere _C18 ). Phosphoantisense oligonucleotide in which a phenylphosphonate bond was introduced into the 5'and 3'ends 1 base of the target product and gentiobiose was bonded to the 5'end by the above operation:
(Gen) -T _Ph GGGAGCCCATAGCGAGG _Ph
C was synthesized. Yield; 21.1 OD λmax; 256.3 nm λmin; 228.4 nm

[0316]

Example 43 Phosphate Antisense Oligonucleotide with Partially Introduced Phenylphosphonate Bond and N-Benzoylgalactosamine at the 5'End: (N-BzGal) -T _Ph GGGAGCCCATAGCG
Synthesis of AGG _Ph C (SEQ ID NO: 35) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind guanosine phosphate having the following nucleotide sequence, the guanosine phosphoramidite unit was dissolved at a concentration of 0.2M and attached to an automatic synthesizer. The above steps (1) to (7) are repeated except that the above steps are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the N-benzoylgalactosamine derivative to the 5'end, similarly, the N-benzoylgalactosamine amidite unit was dissolved in a concentration of 0.2M to prepare an automatic synthesizer. After attaching to, the above-mentioned operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-P.
The ix group is removed. (4) A condensation reaction is performed for 5 minutes with 0.2M N-benzoylgalactosamine phosphoramidite unit and 0.4M tetrazole.

By the above operation, the N-benzoylgalactosamine derivative can be introduced into the 5'end of the oligonucleotide. The deprotection operation after the completion of the synthesis reaction is performed by a conventional method ("Oligonucleotide Synthesis; a practical ap
proach ", MJ Gait (ed), IRLPRESS, (1984).) The CPG in the reaction column was taken out, and then it was treated with an ammonia / pyridine (5: 1) solution at 60%.
By reacting at 6 ° C. for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). By the above operation, 5 ', 3'end 1 of the target product
Phosphoantisense oligonucleotide in which a phenylphosphonate bond is introduced into the base and N-benzoylgalactosamine is bonded to the 5'end thereof: (N-BzGa
1) -T _Ph GGGAGCCCATAGCGAGG _Ph C was synthesized. Yield; 25.8 OD λmax; 256.1 nm λmin; 228.6 nm

[0318]

Example 44 Phosphate Reverse Sequence Oligonucleotide Partially Introduced Phenylphosphonate Bond: Synthesis of C _Ph G _Ph GAGCGATACCCGAGG _Ph G _Ph T (SEQ ID NO: 37) 5′-hydroxyl protected thymidine derivative CPG5 with 20 micromoles introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. Further, in order to bind guanosine phosphate having the next base sequence, guanosine phosphoramidite unit was added to 0.2
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasphere C ₁₈ )
Is used for purification. By the above operation, a phosphate reverse sequence oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product: C _Ph G
_Ph GAGCGATACCCGAGG _Ph G _Ph T was synthesized. Yield: 14.8 OD λmax; 257.3 nm λmin; 229.9 nm

[0319]

Example 45 Phosphate Reverse Sequence Oligonucleotide with Partially Introduced Phenylphosphonate Bond and Gentiobiose at the 5'end: (Gen) -C _Ph G _Ph GAGCGATACCCGAGG _Ph
Synthesis of G _Ph T (SEQ ID NO: 37) CPG5 into which 20 μmol of thymidine derivative having 5′-hydroxyl group protected by DMTr group was introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. Further, in order to bind guanosine phosphate having the next base sequence, guanosine phosphoramidite unit was added to 0.2
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer cytidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, the gentiobiose amidite unit was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, Regarding (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to obtain a protected cytidylphosphonic acid derivative 5′-M.
The ox group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole.

By the above operation, the gentiobiose derivative can be introduced at the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oli
gonucleotide Synthesis; a practical approach ", M.
J. Gait (ed), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HP
Purification is carried out using LC (μBondasphere C ₁₈ ).
By the above operation, a phenylphosphonate bond was introduced into the 5'and 3'terminal 2 bases of the target product, and a phosphate reverse sequence oligonucleotide in which gentiobiose was bonded to the 5'terminal: (Gen) -C _Ph G _Ph GAGCGCATACCGA
GG _Ph G _Ph T was synthesized. Yield; 12.2 OD λmax; 257.2 nm λmin; 230.0 nm

[0321]

Example 46 Phosphoantisense Reverse Sequence Oligonucleotide with Partially Introduced Phenylphosphonate Bond and Mellibiose at the 5'end: (Mel) -C _Ph G _Ph GAGCGATACCCGAGG _Ph
Synthesis of G _Ph T (SEQ ID NO: 37) CPG5 into which 20 μmol of thymidine derivative having 5′-hydroxyl group protected by DMTr group was introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. Further, in order to bind guanosine phosphate having the next base sequence, guanosine phosphoramidite unit was added to 0.2
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer cytidylphosphonic acid, in order to bind the melibiose derivative to the 5'end, the melibiose amidite unit was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, With respect to (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to obtain a protected cytidylphosphonic acid derivative 5′-M.
The ox group is removed. (4) A condensation reaction is performed with 0.2 M melibiose phosphoramidite unit and 0.4 M tetrazole for 5 minutes. By the above operation, the melibiose derivative can be introduced into the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synth
esis; a practical approach ", MJ Gait (ed), IRL
PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. After that, reverse-phase HPLC (μBondaspher
e C ₁₈ ) is used for purification. By the above operation, a phenylphosphonate bond was introduced into the 5'and 3'terminal 2 bases of the desired product, and a phosphate antisense oligonucleotide in which melibiose was bonded to the 5'terminal: (Mel)
-C _Ph G _Ph GAGCGATACCCGAGG _Ph G _Ph T was synthesized. Yield; 15.3 OD λmax; 257.2 nm λmin; 230.3 nm

[0322]

Example 47 Phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond: T _Ph G _Ph GGAGC _Ph C _Ph AT _Ph AGC _Ph GAG _Ph G _Ph
Synthesis of C (SEQ ID NO: 39) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. In order to bind adenosine phosphate having the next base sequence, 0.2 units of adenosine phosphoramidite units are added.
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5 ', 3'terminal two bases of the target product and a phenylphosphonate bond is also introduced into a pyrimidine base by the above operation: T _Ph G _Ph GGAGC _Ph C _Ph AT _Ph AGC _Ph GA
G _Ph G _Ph C was synthesized. Yield; 43.1 OD λmax; 257.6 nm λmin; 229.4 nm

[0323]

Example 48 Phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond and bound with gentiobiose at the 5'end: (Gen) -T _Ph G _Ph GGAGC _Ph C _Ph AT _Ph AGC _Ph.
Synthesis of GAG _Ph G _Ph C (SEQ ID NO: 39) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. ． (1) 3.0% TCA
/ CH ₂ Cl ₂ treatment for 60 seconds, 5'-DMTr
Remove the group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. In order to bind adenosine phosphate having the next base sequence, 0.2 units of adenosine phosphoramidite units are added.
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, after similarly dissolving the gentiobiose amidite unit in a concentration of 0.2 M and attaching it to an automatic synthesizer , (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-P.
The ix group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole.

By the above operation, the gentiobiose derivative can be introduced at the 5'end of the oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oli
gonucleotide Synthesis; a practical approach ", M.
J. Gait (ed), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HP
Purification is carried out using LC (μBondasphere C ₁₈ ).
By the above operation, a phenylphosphonate bond was introduced into the 5'and 3'terminal 2 bases of the target product, and a phosphate antisense oligonucleotide having gentiobiose bonded to the 5'terminal, (Gen) -TpGpGGAGGCpCpA.
TpAGCpGAGpGpC was synthesized. Yield; 43.3 OD λmax; 257.9 nm λmin; 229.5 nm

[0325]

Example 49 Phosphate Antisense Oligonucleotide Partially Introduced Phenylphosphonate Bond: C _Ph G _Ph GAGC _Ph GAT _Ph AC _Ph C _Ph GAGG _Ph G _Ph
Synthesis of T (SEQ ID NO: 40) CPG5 in which 20 μmol of a thymidine derivative having a 5′-hydroxyl group protected by a DMTr group was introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. Further, in order to bind guanosine phosphate having the next base sequence, guanosine phosphoramidite unit was added to 0.2
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M guanosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide having a phenylphosphonate bond introduced into the 5 ', 3'terminal 2 bases of the target product and a phenylphosphonate bond introduced into the pyrimidine base by the above operation: C _Ph G _Ph GAGC _Ph GAT _Ph AC _Ph C _Ph GAG
G _Ph G _Ph T was synthesized. Yield: 38.0 OD λmax; 256.7 nm λmin; 229.0 nm

[0326]

Example 50 Phosphate Antisense Oligonucleotide Partially Introduced Phenylphosphonate Bond: Synthesis of C _Ph C _Ph CCCACCACTTCCC _Ph C _Ph T (SEQ ID NO: 41) Thymidine derivative in which 5′-hydroxyl group is protected with DMTr group CPG5 with 20 micromoles introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the cytidine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is carried out for 5 minutes with 0.2 M cytidine phenylphosphonoamidite unit and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected cytidine phenylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind the cytidine phenylphosphonate derivative having the following base sequence, the same operation as described above is repeated. To bind the cytidine phosphate of the next base sequence,
After dissolving the cytidine phosphoramidite unit at a concentration of 0.2M and attaching it to the automatic synthesizer, the above steps (1) to (7) are performed except that the steps (1) and (4) are as follows. Repeat. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Mox group of the protected cytidine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M cytidine amidite unit and 0.4M
The condensation reaction is performed for 5 minutes with tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer cytidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, the gentiobiose amidite unit was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, Regarding (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to obtain a protected cytidylphosphonic acid derivative 5′-M.
The ox group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole. The gentiobiose derivative can be introduced into the 5'end of the oligonucleotide by the above operation. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide S
ynthesis; a practical approach ", MJ Gait (ed),
IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondas
Purification is carried out using phere _C18 ). A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product by the above operation: C _Ph C _Ph CCCACCACTTCCC _Ph C _Ph
T was synthesized. Yield: 37.6 OD λmax; 256.1 nm λmin; 229.1 nm

[0327]

Example 51 Phosphate antisense oligonucleotide partially introduced with a phenylphosphonate bond and gentiobiose introduced at the 5 ′ end: (Gen) -C _Ph C _Ph CCCACCACTTCCC _Ph C
Synthesis of _Ph T (SEQ ID NO: 41) CPG5 in which 20 μmol of thymidine derivative having 5′-hydroxyl group protected by DMTr group was introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the cytidine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is carried out for 5 minutes with 0.2 M cytidine phenylphosphonoamidite unit and 0.4 M tetrazole. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected cytidine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the cytidine phenylphosphonate derivative having the following base sequence, the same operation as described above is repeated. To bind the cytidine phosphate of the next base sequence,
After dissolving the cytidine phosphoramidite unit at a concentration of 0.2M and attaching it to the automatic synthesizer, the above steps (1) to (7) are performed except that the steps (1) and (4) are as follows. Repeat. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Mox group of the protected cytidine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M cytidine amidite unit and 0.4M
The condensation reaction is performed for 5 minutes with tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer cytidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, the gentiobiose amidite unit was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, Regarding (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to obtain a protected cytidylphosphonic acid derivative 5′-M.
The ox group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole. The gentiobiose derivative can be introduced into the 5'end of the oligonucleotide by the above operation. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide S
ynthesis; a practical approach ", MJ Gait (ed),
IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondas
Purification is carried out using phere _C18 ). By the above operation, a phosphate antisense oligonucleotide in which a phenylphosphonate bond was introduced at the 5 ', 3'terminal 2 bases of the target product and gentiobiose was introduced at the 5'terminal: (G
en) -C _Ph C _Ph CCCACCACTTCCCC _Ph C _Ph T
Was synthesized. Yield: 55.7 OD λmax; 257.9 nm λmin; 234.5 nm

[0328]

Example 52 Phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond: Synthesis of C _Ph C _Ph CTGTCCCGGGATAG _Ph G _Ph T (SEQ ID NO: 42) 5′-hydroxyl protected thymidine derivative CPG5 with 20 micromoles introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. To further bind adenosine phosphate having the following nucleotide sequence, adenosine phosphoramidite unit is dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, and then (1) and (4) are The above operations (1) to (7) are repeated except that the above is set. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product by the above operation:
C _Ph C _Ph CTGTCCCGGGATAG _Ph G _Ph T was synthesized. Yield: 57.1 OD λmax; 259.1 nm λmin; 230.9 nm

[0330]

Example 53 Phosphate antisense oligonucleotide partially introduced with a phenylphosphonate bond and gentiobiose introduced at the 5'end: (Gen) -C _Ph C _Ph CTGTCCCGGGATAG _Ph
Synthesis of G _Ph T (SEQ ID NO: 42) CPG5 in which 20 μmol of thymidine derivative having 5′-hydroxyl group protected by DMTr group was introduced per gram
An automatic synthesizer (AB
It is packed in the reaction column installed in the DNA synthesizer Model 381 A) manufactured by Company I. Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas.

By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the thymidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. To further bind adenosine phosphate having the following nucleotide sequence, adenosine phosphoramidite unit is dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, and then (1) and (4) are The above operations (1) to (7) are repeated except that the above is set. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. After condensing the 18-mer cytidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, the gentiobiose amidite unit was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, Regarding (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to obtain a protected cytidylphosphonic acid derivative 5′-M.
The ox group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole. The gentiobiose derivative can be introduced into the 5'end of the oligonucleotide by the above operation. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide S
ynthesis; a practical approach ", MJ Gait (ed),
IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondas
Purification is carried out using phere _C18 ). By the above operation, a phosphate antisense oligonucleotide in which a phenylphosphonate bond was introduced at the 5 ', 3'terminal 2 bases of the target product and gentiobiose was introduced at the 5'terminal: (G
en) -C _Ph C _Ph CTGTCCCCGGGATAG _Ph G _Ph
T was synthesized. Yield: 52.4 OD λmax; 258.4 nm λmin; 230.7 nm

[0332]

Example 54 Phenylphosphonate Antisense Oligonucleotide: T _Ph G _Ph G _Ph G _Ph A _Ph G _Ph C _Ph C _Ph A _Ph T _Ph A _Ph G _Ph C
Synthesis of _Ph G _Ph A _Ph G _Ph G _Ph C (SEQ ID NO: 43) N-4 amino group is benzoyl group and 5'-hydroxyl group is DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99.
% Or more. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthesis; a practical appr
Oach ", MJ Gait (ed), IRL PRESS, (1984).). After removing the CPG in the reaction column, it was washed with an ammonia / pyridine (5: 1) solution at 60%.
By reacting at 6 ° C. for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. Then, purification is carried out using reverse phase HPLC (μBondasphere C ₁₈ ). Phenylphosphonate antisense oligonucleotide in which all the internucleotide bonds of the target compound are converted to phenylphosphonate bonds by the above operation:
T _Ph G _Ph G _Ph G _Ph A _Ph G _Ph C _Ph C _Ph A _Ph T _Ph A _Ph G _Ph C
_Ph G _Ph A _Ph G _Ph G _Ph C was synthesized. Yield: 35.6 OD λmax; 254.7 nm λmin; 227.5 nm

[0333]

Example 55 Phenylphosphonate antisense oligonucleotide having gentiobiose linked to the 5 ′ end: (Gen) -T _Ph G _Ph G _Ph G _Ph A _Ph G _Ph C _Ph C _Ph A _Ph T
_Ph A _Ph G _Ph C _Ph G _Ph A _Ph G _Ph G _Ph C (SEQ ID NO: 43)
Synthesis of N-4 amino group with benzoyl group and 5'-hydroxyl group with DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99.
% Or more. After condensing the 18-mer thymidylphosphonic acid, in order to bind the gentiobiose derivative to the 5'end, after similarly dissolving the gentiobiose amidite unit in a concentration of 0.2 M and attaching it to an automatic synthesizer , (1) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to give a protected thymidylphosphonic acid derivative 5′-P.
The ix group is removed. (4) A condensation reaction is carried out for 5 minutes with 0.2 M gentiobiose phosphoramidite unit and 0.4 M tetrazole. By the above operation, 5'of the oligonucleotide
A gentiobiose derivative can be introduced at the end. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligo
nucleotide Synthesis; a practical approach ", M.
J. Gait (ed), IRL PRESS, (1984).). After CPG in the reaction column is taken out, it is reacted with an ammonia / pyridine (5: 1) solution at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HP
Purification is carried out using LC (μBondasphere C ₁₈ ).
By the above-mentioned operation, all the internucleotide bonds of the target compound are converted into phenylphosphonate bonds, and a phenylphosphonate antisense oligonucleotide in which gentiobiose is introduced at the 5'end, (Gen) -T _Ph G _Ph G _Ph
G _Ph A _Ph G _Ph C _Ph C _Ph A _Ph T _Ph A _Ph G _Ph C _Ph G _Ph A _Ph G
_Ph G _Ph C was synthesized. Yield; 23.1 OD λmax; 253.4 nm λmin; 226.7 nm

[0334]

Example 56 Phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond: Synthesis of G _Ph G _Ph AGCCATAGCGAG _Ph G _Ph C (SEQ ID NO: 44) N-4 amino group with benzoyl group, 5′- DM hydroxyl group
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. In order to bind adenosine phosphate having the next base sequence, 0.2 units of adenosine phosphoramidite units are added.
After being dissolved in the concentration of M and attached to the automatic synthesizer, (1)
Regarding steps (4) and (4), the above operations (1) to (7) are repeated except that the following is performed. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M adenosine amidite unit and 0.4
The condensation reaction is carried out for 5 minutes with M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product by the above operation:
G _Ph G _Ph AGCCATAGCGAG G _Ph G _Ph C was synthesized. Yield: 38.3 OD λmax; 256.3 nm λmin; 231.9 nm

[0335]

Example 57 Phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond: A _Ph G _Ph CCATAGCG _Ph A _Ph G (SEQ ID NO: 45)
Synthesis of N-2 amino group with isobutyryl group and 5'-hydroxyl group with D
50 mg of CPG (1 μmol) into which 20 μmol of the guanosine derivative protected by the MTr group was introduced was added to an automatic synthesizer (DNA manufactured by ABI).
Pack in the reaction column installed in synthesizer Model 381 A). Then, the adenosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with 0.2M adenosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected adenosine phenylphosphonate derivative can be condensed with the guanosine derivative introduced on CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. Furthermore, in order to bind cytidine phosphate having the following base sequence, 0.2M of cytidine phosphoramidite unit was added.
After being dissolved in the concentration of 1 and attached to an automatic synthesizer, the above operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M cytidine amidite unit and 0.4M
The condensation reaction is performed for 5 minutes with tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product by the above operation:
A _Ph G _Ph CCATAGCG _Ph A _Ph G was synthesized. Yield: 34.6 OD λmax; 258.1 nm λmin; 230.4 nm

[0336]

Example 58 Phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond: Synthesis of A _Ph G _Ph CCAT A _Ph G _Ph C (SEQ ID NO: 46) N-4 amino group with benzoyl group, 5′- DM hydroxyl group
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the cytidine derivative introduced onto CPG. In order to bind the adenosine phenylphosphonate derivative of the next base sequence, the same operation as described above is repeated. To further bind thymidine phosphate having the following nucleotide sequence, a thymidine phosphoramidite unit was dissolved at a concentration of 0.2 M and attached to an automatic synthesizer, and then (1) and (4) were as follows The above operations (1) to (7) are repeated except that the above is set. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected adenosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M thymidine amidite unit and 0.4M
The condensation reaction is performed for 5 minutes with tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product by the above operation:
It was synthesized _{A Ph G Ph CCATA Ph G Ph} C. Yield: 33.0OD λmax; 258.6nm λmin; 229.8nm

[0337]

Example 59 Synthesis of phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond: G _Ph G _Ph TTTTTTTTTTTTTTG _Ph G _Ph G (SEQ ID NO: 47) Synthesis N-5 amino group with isobutyryl group at 5′-hydroxyl group DM
50 mg of CPG (1 μg of guanosine derivative protected by a Tr group) was introduced in an amount of 20 μmol / g.
Micromole is an automatic synthesizer (DNA synt manufactured by ABI).
Pack in the reaction column installed in hesizer Model381 A). Then, the guanosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) 3.0% TCA /
CH ₂ Cl ₂ treatment is performed for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with a 0.2M guanosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine phenylphosphonate derivative can be condensed with the guanosine derivative introduced onto CPG. In order to bind the guanosine phenylphosphonate derivative having the next nucleotide sequence, the same operation as described above is repeated. To bind thymidine phosphate of the next base sequence, a thymidine phosphoramidite unit was added to 0.2M.
After being dissolved in the concentration of 1 and attached to an automatic synthesizer, the above operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected guanosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M thymidine amidite unit and 0.4M
The condensation reaction is performed for 5 minutes with tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product by the above operation:
G _Ph G _Ph TTTTTTTTTTTTTTTG _Ph G _Ph G was synthesized. Yield; 37.0OD λmax; 263.7nm λmin; 232.5nm

[0338]

Example 60 Phosphate antisense oligonucleotide partially introduced with phenylphosphonate bond: A _Ph A _Ph TTTTTTTTTTTTTTA _Ph A _Ph A (SEQ ID NO: 48) Synthesis N-6 amino group to 5'-hydroxyl group with benzoyl group To DMT
2 g / g of adenosine derivative protected by r group
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the adenosine phenylphosphonoamidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction of 5 with 0.2M adenosine phenylphosphonoamidite unit and 0.4M tetrazole
Do it for a minute. (5) The oxidation reaction is carried out for 30 seconds with 0.1 M iodine / water / pyridine / THF. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected adenosine phenylphosphonate derivative can be condensed with the adenosine derivative introduced on CPG. In order to bind the adenosine phenylphosphonate derivative of the next base sequence, the same operation as described above is repeated. To bind thymidine phosphate of the next base sequence, a thymidine phosphoramidite unit was added to 0.2M.
After being dissolved in the concentration of 1 and attached to an automatic synthesizer, the above operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-Pix group of the protected adenosine phenylphosphonate derivative. Save this solution for calculating the condensation yield. (4) 0.2M thymidine amidite unit and 0.4M
The condensation reaction is performed for 5 minutes with tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. The average yield of each condensation reaction calculated as a result of colorimetrically determining the trityl alcohol liberated by the operation of (1) was 99% or more. The deprotection procedure after the completion of the synthesis reaction is carried out by a conventional method ("Oligonucleotide Synthesis; a prac
tical approach ", MJ Gait (ed), IRL PRESS, (198
4).) The CPG in the reaction column was taken out, and then it was mixed with ammonia / pyridine (5:
1) The solution is reacted at 60 ° C. for 6 hours to cut out the nucleotide chain from the solid phase carrier and remove the protecting group. Then reverse-phase HPLC (μBondasphere C ₁₈ )
Is used for purification. A phosphate antisense oligonucleotide in which a phenylphosphonate bond is introduced into the 5'and 3'terminal 2 bases of the target product by the above operation:
A _Ph A _Ph TTTTTTTTTTTTTTTA _Ph A _Ph A was synthesized. Yield: 77.5OD λmax; 263.6nm λmin; 233.8nm

[0339]

Example 61 Synthesis of 1,3-O-bis (2,3,4,6-tetraacetylgalactosyl) -2-O-benzylglycerol 2-O-benzylglycerol (182.2 mg,
1.00 mmol) and 1-O-fluoro-2,3,4,
6-O-tetraacetylgalactose (770.6m
g, 2.20 mmol) to anhydrous methylene chloride (5 m
l) and trifluoroborane etherate (BF ₃ -Et ₂ O) (425.8 mg, 3.00 m)
mol) and molecular sieves (1.0 g).
The reaction solution is stirred at room temperature for 3 hours and then filtered through Celite. 50 ml of methylene chloride was added to the filtrate to dilute it, and this solution was diluted with saturated aqueous sodium hydrogen carbonate solution 5
After washing 3 times with 0 ml, the solvent is removed under reduced pressure. After that, the reaction product was purified by silica gel column chromatography (hexane / ethyl acetate = 8: 2) to obtain the desired product 1,3.
-O-bis (2,3,4,6-tetraacetylgalactosyl) -2-O-benzylglycerol is obtained.

[0340]

Example 62 Synthesis of 1,3-O-bis (2,3,4,6-tetraacetylgalactosyl) glycerol 1,3-O-bis (2,3,4,6-tetraacetylgalactosyl) -2 -O-benzyl glycerol (84
2.8 mg, 1.00 mmol) was dissolved in methanol-ethyl acetate (1: 1) (10 ml), and 10% P was added thereto.
Add d (OH) ₂ (30 mg). This solution was subjected to medium pressure hydrogenation (3.5 kg / cm ² ) at room temperature.
React for hours. After completion of the reaction, filtration is carried out using Celite, 50 ml of methylene chloride is added to the filtrate for dilution, the solution is washed 3 times with 50 ml of a saturated sodium hydrogen carbonate aqueous solution, and then the solvent is removed under reduced pressure. After that, the reaction product is purified by silica gel column chromatography (hexane / ethyl acetate = 8: 2) to obtain 1,3-O-bis (2,3,4,6-tetraacetylgalactosyl) glycerol as a target product.

[0341]

Example 63 Synthesis of 1,3-O-bis (2,3,4,6-tetraacetylgalactosyl) -2-O- (N, N, diisopropylamino-O-cyanoethyl phosphoramidite) glycerol 1, 3-O-bis (2,3,4,6-tetraacetylgalactosyl) glycerol (752.7 mg, 1.00
(mmol) was azeotropically dehydrated 2 ml × 3 times with anhydrous pyridine, and then 2 ml × 3 times with anhydrous toluene and 2 m of anhydrous methylene chloride.
Azeotropically perform 1x3 times. Then, it was dissolved in 5 ml of methylene chloride, and diisopropylethylamine (0.
87 ml, 5.00 mmol) and N, N-diisopropylamino-O-cyanoethyl phosphorochloride (0.
45 ml, 2.00 mmol) was added, and the mixture was reacted at room temperature for 1 hour, diluted with 50 ml of methylene chloride, washed with 50 ml of a saturated aqueous solution of sodium hydrocarbon three times, and dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography (hexane / methylene chloride = 8: 2, 2% triethylamine) to obtain the desired product, 1,3-O-bis (2,3,3).
4,6-Tetraacetylgalactosyl) -2-O-
(N, N, Diisopropylamino-O-cyanoethyl phosphoramidite) glycerol is obtained.

[0342]

Example 64] 1, 3-O-bis galactosyl glycerol bound phosphorothioate antisense _{oligonucleotides: ((Gal) 2 Glyc)} -T S G S G S G S A S G S C S C S
_{A S T S A S G S} C S G S A S G S G S C ( SEQ ID NO: 31) with benzoyl group the synthetic N-4 amino group, a 5'-hydroxyl group DM
2 gram of cytidine derivative protected by Tr group per gram
50 micrograms (1 micromol) of CPG introduced with 0 micromol was used as an automatic synthesizer (DNA synthe manufactured by ABI).
Pack in the reaction column installed in sizer Model 381 A). Then, the guanosine phosphoramidite unit is dissolved in anhydrous acetonitrile to a concentration of 0.2 M, packed in a bottle for storing the unit, and attached to an automatic synthesizer. Then, the following operations are performed in the reaction column by an automatic synthesizer. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group. Save this solution for calculating the condensation yield. (2) Wash the CPG with acetonitrile. (3) Dry the CPG with argon gas. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. (5) Sulfurization reaction is carried out for 15 minutes with 0.5 M tetraethylthiuram disulfide / acetonitrile. (6) 0.25M acetic anhydride / 1-methylimidazole /
The unreacted 5'-hydroxyl group is blocked by reacting the lutidine / tetrahydrofuran solution for 30 seconds. (7) Wash with acetonitrile and dry with argon gas. By the above operation, the protected guanosine derivative can be condensed with the cytidine derivative introduced onto CPG by a phosphorothioate bond. In order to bind a guanosine derivative having the following nucleotide sequence, it was similarly dissolved at a concentration of 0.2 M and attached to an automatic synthesizer. Then, with respect to (1) and (4), The operations (1) to (7) are repeated. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected guanosine derivative. Save this solution for calculating the condensation yield. (4) A condensation reaction is performed for 5 minutes with 0.2 M guanosine phosphoramidite unit and 0.4 M tetrazole. By repeating the above operation, the nucleotide chain is sequentially extended. After condensing the 18-mer thymidylic acid, in order to attach the galactosylglycerol derivative synthesized in Example 63 to the 5 ′ end, 0.2M was similarly added.
After being dissolved in the concentration of 1 and attached to an automatic synthesizer, the above operations (1) to (7) are repeated except that (1) and (4) are as follows. (1) Treatment with 3.0% TCA / CH ₂ Cl ₂ for 60 seconds to remove the 5′-DMTr group of the protected thymidine derivative. (4) A condensation reaction is carried out for 5 minutes using the 0.2 M galactosylglycerol phosphoramidite unit synthesized in Example 63 and 0.4 M tetrazole. By the above operation, the galactosylglycerol derivative can be introduced into the 5'end of the antisense oligonucleotide. The deprotection procedure after completion of the synthesis reaction is a conventional method ("Oligonucleotide Synthesis; a practical approa").
ch ", MJ Gait (ed), IRL PRESS, (1984).) The CPG in the reaction column was taken out, and then it was washed with an ammonia / pyridine (5: 1) solution at 60%.
By reacting at 6 ° C for 6 hours, the nucleotide chain is cleaved from the solid phase carrier and the protecting group is removed. After that, purification is performed using reverse phase HPLC (μBondasphere C ₁₈ ). By the above operation, the desired galactoside glycerol-linked phosphorothioate antisense oligonucleotide: ((Gal) ₂ Glyc) -T _S G _S
G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C _S is synthesized.

A list of sugar derivatives and oligonucleotide derivatives produced in the above Reference Examples and Examples is shown below. Reference Example 1 T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C (SEQ ID NO: 31) (Reference Example 2 ] C _S G _S G _S A _S G _S C _S G _S A _S T _S A _S C _S C _S G _S A _S G _S G _S G _S T (SEQ ID NO: 36) (Reference Example 3) (N- BzGalPO) -C _S G _S G _S A _S G _S C _S G _S A _S T _S A _S C _S C _S G _S A _S G _S G _S G _S T ((N
-BzGalPO) -SEQ ID NO: 36) (Reference Example 4) C _Me G _Me GAGCGATACCGAGG _Me G _Me T (SEQ ID NO: 38) (Reference Example 5) T _Me G _Me GGAGCCATAGCGAG _Me G _Me C (SEQ ID NO: 34) Example 1] 1,2,3,4-O-tetraacetylgalactose-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite [Example 2] 1,2,3,4-O-tetraacetyl Mannose-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (Example 3) (GAL) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((GAL) -SEQ ID NO: 31) (Example 4) (MAN) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((MAN) -SEQ ID NO: 31) (Example 5) 1-On-octyl-2,3,4-O-tribenzoylglucoside-6-
ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (Example 6) (n-oct-Glc) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((n
-oct-Glc) -SEQ ID NO: 31) (Example 7) 1-On-octyl-2,3.4-O-tribenzoylthioglucoside-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite Example 8) (n-oct-SGlc) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C
((n-oct-SGlc) -SEQ ID NO: 31) [Example 9] 1-On-octyl-2,3,4-O-tribenzoylglucoside-6-
O- (N, N-diisopropylamino) phenylphosphonoamidite [Example 10] (n-oct-GlcPh) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C
((n-oct-GlcPh) -SEQ ID NO: 31) [Example 11] 1-On-octyl-2,3,4-O-tribenzoylthioglucoside-6-O- (N, N-diisopropylamino) Phenylphosphonoamidite (Example 12) (n-oct-SGlcPh) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C
((n-oct-SGlcPh) -SEQ ID NO: 31) [Example 13] 1-O-phenyl-2,3,4-O-tribenzoylglucoside-6-O-
N, N-Diisopropylamino-O-cyanoethyl phosphoramidite (Example 14) (PhGlc) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((PhGl
c) -SEQ ID NO: 31) [Example 15] 1-O-phenyl-2,3,4-O-tribenzoylgalactoside-6-
ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (Example 16) (PhGal) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((PhGa
l) -SEQ ID NO: 31) Example 17 1,3,4-O-tribenzoyl, 2-N-benzoylgalactosamine-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite Example 18 ] (N-BzGalPO) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((N
-BzGalPO) -SEQ ID NO: 31) Example 19 1,3,4-O-triacetyl, 2-N-acetylgalactosamine-6-
ON, N-diisopropylamino-O-cyanoethyl phosphoramidite (Example 20) (N-AcGal) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((NA
cGal) -SEQ ID NO: 31) Example 21 1,3,4-O-triacetyl, 2-N-acetylglucosamine-6-O-
N, N-Diisopropylamino-O-cyanoethyl phosphoramidite (Example 22) (N-AcGul) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((NA
cGul) -SEQ ID NO: 31) Example 23 1,3,4-O-tribenzoyl, 2-N-trifluoroacetylgalactosamine-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite Example 24) (GalN) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((GalN)-
SEQ ID NO: 31) [Example 25] 1,3,4-O-tribenzoyl, 2-N-trifluoroacetylglucosamine-6-ON, N-diisopropylamino-O-cyanoethyl phosphoramidite [Example 26] (GulN) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((GulN)-
SEQ ID NO: 31) [Example 27] Heptabenzoyl melibiose-N, N-diisopropylamino-O-cyanoethyl phosphoramidite [Example 28] (Mel) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((Mel) -SEQ ID NO: 31) [Example 29] Heptabenzoylgentiobiose-N, N-diisopropylamino -O-Cyanoethyl phosphoramidite (Example 30) (Gen) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((Gen) - SEQ ID NO: 31) example 31 oct benzoyl isomaltotriose -N, N-diisopropylamino -O- cyanoethyl phosphoramidite example 32] (iso) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C ((Iso) -SEQ ID NO: 31) (Example 33) T _Ph G _Ph GGAGCCATAGCGAGPHG _Ph C (SEQ ID NO: 32) (Example 34) (Gen) -T _Ph G _Ph GGAGCCATAGCGAGPHG _Ph C ((Gen) -SEQ ID NO: 3)
2) (Example 35) (Mel) -T _Ph G _Ph GGAGCCATAGCGAG _Ph G _Ph C ((Mel) -SEQ ID NO: 3
2) (Example 36) T _Ph GGGAGC _Ph C _Ph AT _Ph AGC _Ph GAGGC _Ph T (SEQ ID NO: 33) (Example 37) (Gen) -T _Ph GGGAGC _Ph C _Ph AT _Ph AGC _Ph GAGGC _Ph T (( Gen) -SEQ ID NO: 33) (Example 38) (Gen) -T _Me G _Me GGAGCCATAGCGAG _Me G _Me C ((Gen) -SEQ ID NO: 3
4) (Example 39) (Mel) -T _Me G _Me GGAGCCATAGCGAG _Me G _Me C ((Mel) -SEQ ID NO: 3
4) (Example 40) (N-BzGal) -T _Ph G _Ph GGAGCCATAGCGAG _Ph G _Ph C ((N-BzGal) -SEQ ID NO: 32) (Example 41) T _Ph GGGAGCCATAGCGAGG _Ph C (SEQ ID NO: 35) Example 42 (Gen) -T _Ph GGGAGCCATAGCGAGG _Ph C ((Gen) -SEQ ID NO: 35) Example 43 (N-BzGal) -T _Ph GGGAGCCATAGCGAGG _Ph C ((N-BzGal) -SEQ ID NO: 35) (Example 44) C _Ph G _Ph GAGCGATACCGAGG _Ph G _Ph T (SEQ ID NO: 37) (Example 45) (Gen) -C _Ph G _Ph GAGCGATACCGAGG _Ph G _Ph T ((Gen) -SEQ ID NO: 3)
7) (Example 46) (Mel) -C _Ph G _Ph GAGCGATACCGAGG _Ph G _Ph T ((Mel) -SEQ ID NO: 3
7) (Example 47) T _Ph G _Ph GGAGC _Ph C _Ph AT _Ph AGC _Ph GAG _Ph G _Ph C (SEQ ID NO: 39) (Example 48) (Gen) -T _Ph G _Ph GGAGC _Ph C _Ph AT _Ph AGC _Ph GAG _Ph G _Ph C ((Gen) -SEQ ID NO: 39) [Example 49] C _Ph G _Ph GAGC _Ph GAT _Ph AC _Ph C _Ph GAGG _Ph G _Ph T (SEQ ID NO: 4)
0) [Example 50] C _Ph C _Ph CCCACCACTTCCC _Ph C _Ph T (SEQ ID NO: 41) [Example 51] (Gen) -C _Ph C _Ph CCCACCACTTCCC _Ph C _Ph T ((Gen) -SEQ ID NO: 41) (Example 52) C _Ph C _Ph CTGTCCCGGGATAG _Ph G _Ph T (SEQ ID NO: 42) (Example 53) (Gen) -C _Ph C _Ph CTGTCCCGGGATAG _Ph G _Ph T ((Gen) -SEQ ID NO: 4)
2) Example 54 T _Ph G _Ph G _Ph G _Ph A _Ph G _Ph C _Ph C _Ph A _Ph T _Ph A _Ph G _Ph C _Ph G _Ph A _Ph G _Ph G
_Ph C (SEQ ID NO: 43) (Example 55) (Gen) -T _Ph G _Ph G _Ph G _Ph A _Ph G _Ph C _Ph C _Ph A _Ph T _Ph A _Ph G _Ph C _Ph G _Ph A
_Ph G _Ph G _Ph C ((Gen) -SEQ ID NO: 43) [Example 56] G _Ph G _Ph AGCCATAGCGAG _Ph G _Ph C (SEQ ID NO: 44) [Example 57] A _Ph G _Ph CCATAGCG _Ph A _Ph G (SEQ ID NO: 45) (Example 58) A _Ph G _Ph CCATA _Ph G _Ph C (SEQ ID NO: 46) (Example 59) G _Ph G _Ph TTTTTTTTTTTTTG _Ph G _Ph G (SEQ ID NO: 47) (Example 60) ] A _Ph A _Ph TTTTTTTTTTTTTA _Ph A _Ph A (SEQ ID NO: 48) (Example 61) 1,3-O-bis (2,3,4,6-tetraacetylgalactosyl) -2-O
-Benzylglycerol [Example 62] 1,3-O-bis (2,3,4,6-tetraacetylgalactosyl) glycerol [Example 63] 1,3-O-bis (2,3,4,6- Tetraacetylgalactosyl) -2-O
-(N, N, diisopropylamino-O-cyanoethyl phosphoramidite) glycerol Example 64 ((Gal) ₂ Glyc) -T _S G _S G _S G _S A _S G _S C _S C _S A _S T _S A _S G _S C _S G _S A _S G _S G _S C
((Gal) ₂ Glyc-) SEQ ID NO: 31)

[0344]

[Test Example 1] Cell adhesion protein ICAM-1 (Interce
llular Adhesion Molecule 1) Pre-mRN of expressed genes
A derivative in which the phosphorothioate antisense oligonucleotide of the present invention for the vicinity of the translation initiation site of A is entrapped with cationic lipofectin (DOTMA), and an unmodified phosphorothioate antisense oligonucleotide having a homologous base sequence also entrapped in DOTMA, and The ICAM-1 expression inhibitory activity of the corresponding reverse nucleotide sequence oligonucleotide was measured. ICAM-1
The expression inhibition measurement was performed according to a conventional method. (MY C
hiang et al., J. Biol. Chem., 266, 18162 (1991).)
That is, after washing A549 cells (human lung cancer-derived cells) three times with serum-free medium (High Glucose D-MEM), an appropriate amount
(1.00 μM, 0.33 μM) Phosphorothioate antisense oligonucleotide of the present invention and lipofectin (DOTMA) 10 μg / ml in serum-free medium 1
After adding 00 μl / well and culturing at 37 ° C. for 4 hours, the medium was replaced with 10% serum-containing medium containing the same amount of antisense, and further culturing at 37 ° C. for 4 hours. Then, the expression of ICAM-1 was induced by adding hIL1-β (final concentration 0.1 ng / ml) or hTNF-α (final concentration 1 ng / ml), and after culturing for 15 hours, ICAM-1 Cell
The expression level of ICAM-1 is measured by EIA,
The ICAM-1 expression inhibitory activity of the phosphorothioate antisense oligonucleotide of the present invention entrapped with lipofectin is measured. At the same time, the evaluation of cell toxicity by the crystal violet staining is also performed. [Table 1] As the ICAM-1 inhibitory activity, the comparative activity of the phosphorothioate antisense oligonucleotide of the present invention included in lipofectin when the activity of the unmodified phosphorothioate antisense oligonucleotide included in lipofectin was taken as 100 (ICAM -1 Relative inhibition (%)) and Cell i
Write the result of nhibition (%).

[0345]

[Table 1]

Each of the phosphorothioate antisense oligonucleotides of the present invention, when incorporated with lipofectin, was superior in suppressing ICAM-1 expression to the phosphorothioate antisense oligonucleotide having the same unmodified base sequence incorporated with lipofectin. It was confirmed to have an effect. An antisense oligonucleotide to which a compound in which the anomeric hydroxyl group is substituted with a phenyl group as in Example 16 is bound, and Example 1
It was found that the inhibitory effect was enhanced in antisense oligonucleotides into which 2-amino sugar derivatives such as 8, 20, 22, 24 and 26 were introduced. In addition,
The cytotoxicity of these antisense oligonucleotides is almost unchanged compared to unmodified ones,
Since the phosphorothioate oligonucleotide having the reverse sequence of Reference Example 2 showed almost no suppressive effect, it was revealed that the antisense effect was enhanced by modifying the 5'end of the antisense with a sugar derivative.

[0347]

[Test Example 2] Cell adhesion protein ICAM-1 (Interce
llular Adhesion Molecule 1) Pre-mRN of expressed genes
The ICAM-1 expression inhibitory activity of the phosphorothioate antisense oligonucleotide of the present invention in the vicinity of the translation initiation site of A, as well as the unmodified phosphorothioate antisense oligonucleotide and the corresponding reverse nucleotide sequence oligonucleotide was measured. ICAM-
1 The measurement of expression inhibition was performed according to a conventional method. (MY
Chiang et al., J. Biol. Chem., 266, 18162 (1991).)
That is, A549 cells (human lung cancer-derived cells) were washed three times with serum-free medium (High Glucose D-MEM), and appropriate amounts (20.0 μM, 30.0 μM) of the phosphorothioate antisense oligonucleotide of the present invention were added. 100 μl / well was added to the serum-free medium containing the cells, and the cells were cultured at 37 ° C. for 4 hours, replaced with 10% serum-containing medium containing the same amount of antisense, and further cultured at 37 ° C. for 4 hours. Then hIL1
-Β (final concentration 0.1 ng / ml) or hTNF-
By adding α (final concentration 1 ng / ml), ICAM-1
Expression was induced and after culturing for 15 hours, ICAM-1
The expression level of ICAM-1 was measured by Cell EIA to measure the phosphorothioate antisense of the present invention.
The ICAM-1 expression inhibitory activity of the oligonucleotide is measured. At the same time, the evaluation of cell toxicity by the crystal violet staining is also performed. [Table 2] shows the ICAM-1 expression inhibitory activity (ICAM-1 inhib) of the phosphorothioate antisense oligonucleotide of the present invention when lipofectin is not included.
ition (%)) and Cell inhibition (%).

[0348]

[Table 2]

Unmodified phosphorothioate antisense oligonucleotides show almost no inhibitory effect on ICAM-1 expression when not encapsulated with lipofectin. However, the N-benzoylgalactosamine-conjugated phosphorothioate antisense oligonucleotide prepared in Example 18 of the present invention had a remarkable ICAM.
-1 Expression suppression effect was observed. In addition, the cytotoxicity of this antisense showed almost no change as compared with the unmodified one, and almost all the expression-inhibiting effect was observed in the phosphorothioate oligonucleotide bound with N-benzoylgalactosamine having the reverse sequence of Reference Example 3. It was revealed that the antisense effect was enhanced by modifying the 5'end of the antisense with N-benzoylgalactosamine.

[0350]

[Test Example 3] Cell adhesion protein ICAM-1 (Interce
llular Adhesion Molecule 1) Pre-mRN of expressed genes
The phosphate antisense oligonucleotide of the present invention partially having a phosphonate bond, the unmodified phosphorothioate antisense oligonucleotide having a homologous base sequence, and the corresponding reverse type base sequence oligonucleotide with respect to the vicinity of the translation initiation site of A. IC
The AM-1 expression inhibitory activity was measured. The measurement of ICAM-1 expression inhibition was performed according to a conventional method. (MY Chiang
et al., J. Biol. Chem., 266, 18162 (1991). That is, A549 cells (human lung cancer-derived cells) are serum-free medium.
After washing with (High Glucose D-MEM) three times, 30.0 μM antisense of the present invention. 100 μl / well was added to a serum-free medium containing an oligonucleotide, and the mixture was cultured at 37 ° C. for 4 hours, replaced with a medium containing 10% serum containing the same amount of antisense, and further cultured at 37 ° C. for 4 hours. Then hIL1-
β (final concentration 0.1 ng / ml) or hTNF-α
(Final concentration 1 ng / ml) was added to induce the expression of ICAM-1, and after culturing for 15 hours, ICAM-1 C was added.
The expression level of ICAM-1 was measured by using cell EIA, and ICAM-of the phosphate antisense oligonucleotide partially having a phosphonate bond of the present invention was used.
1 Expression suppression activity is measured. At the same time, the cytotoxicity (cell inhibiti
on) together with the evaluation. In Table 4, ICAM-1 expression inhibitory activity (ICAM-1 inhi) of the antisense oligonucleotide of the present invention when lipofectin is not included
Write the results of bition (%)) and cell inhibition (%).

[0351]

[Table 3]

Unmodified phosphorothioate antisense oligonucleotides show almost no effect of suppressing ICAM-1 expression unless lipofectin is included.
However, Examples 33 to 35, 44 to 46, 5 of the present invention
A remarkable inhibitory effect on ICAM-1 expression was observed for the phosphate antisenses partially prepared with 4, 55, 56 and 59 and partially having a phenylphosphonate bond. At this time, since almost no cytotoxicity was observed, the partially introduced phenylphosphonate bond was bound to ICAM-
It is clear that this is the cause of the inhibitory effect on the expression of 1.

[0353]

INDUSTRIAL APPLICABILITY The oligonucleotide derivative (I) of the present invention or a salt thereof can be prepared by complementation of genes causing various diseases with DNA or mRNA.
(1) inhibit the expression of oncogenes in tumor cells,
(2) Suppress expression of viral genes derived from viruses, (3) Suppress expression of gene products that cause inflammation, (4) Drug resistance gene that becomes a problem during chemotherapy for malignant tumors (5) PTCA (Percutaneous Transluminal Coro)
nary Angioplasty (percutaneous coronary luminal dilatation) inhibits the production of cell growth factors involved in vascular restenosis, so expression of specific genes such as antitumor agents, antiviral agents, anti-inflammatory agents, and resistance genes It can be used as a controlling drug. Furthermore, the oligonucleotide derivative (I) of the present invention or its salt is a target DNA or mRNA.
In addition, since it binds very efficiently as compared with ordinary DNA fragments, it can be efficiently used as a probe. These can also be used for drug screening.

[0354]

[Sequence list]

[0355]

[SEQ ID NO: 1] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TGGGAGCCAT AGCGAGGC
18

[0356]

[SEQ ID NO: 2] Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TGGGAGCCAT AGCGAGGGCT
19

[0357]

[SEQ ID NO: 3] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGC GAGGCTGAGGT 30

[0358]

[SEQ ID NO: 4] Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG AGGCTGAGG
29

[0359]

[SEQ ID NO: 5] Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG AGGCTGAG
28

[0360]

[SEQ ID NO: 6] Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG AGGCTGA 27

[0361]

[SEQ ID NO: 7] Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG AGGCTG
26

[0362]

[SEQ ID NO: 8] Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG AGGCT
25

[0363]

[SEQ ID NO: 9] Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG AGGC 24

[0364]

[SEQ ID NO: 10] Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG AGG
23

[0365]

[SEQ ID NO: 11] Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG AG
22

[0366]

[SEQ ID NO: 12] Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG A 21

[0367]

[SEQ ID NO: 13] Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGCG
20

[0368]

[SEQ ID NO: 14] Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAGC
19

[0369]

[SEQ ID NO: 15] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATAG 18

[0370]

[SEQ ID NO: 16] Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCATA
17

[0371]

[SEQ ID NO: 17] Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGCTGCTGGG AGCCAT
16

[0372]

[SEQ ID NO: 18] Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GCTGCTGGGA GCCATAGCGA GGCTGAGGT 29

[0373]

[SEQ ID NO: 19] Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CTGCTGGGAG CCATAGCGAG GCTGAGGT
28

[0374]

[SEQ ID NO: 20] Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TGCTGGGAGC CATAGCGAGG CTGAGGGT
27

[0375]

[SEQ ID NO: 21] Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GCTGGGAGCC ATAGCGAGGC TGAGGT 26

[0376]

[SEQ ID NO: 22] Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CTGGGAGCCA TAGCGAGGCT GAGGT
25

[0377]

[SEQ ID NO: 23] Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TGGGAGCCAT AGCGAGGCTG AGGT
24

[0378]

[SEQ ID NO: 24] Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGGAGCCATA GCGAGGCTGA GGT 23

[0379]

[SEQ ID NO: 25] Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGAGCCATAG CGAGGCTGAG GT
22

[0380]

[SEQ ID NO: 26] Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GAGCCATAGC GAGGCTGAGG T
21

[0380]

[SEQ ID NO: 27] Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AGCCATAGCG AGGCTGAGGT 20

[0382]

[SEQ ID NO: 28] Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GCCATAGCGA GGCTGAGGT
19

[0383]

[SEQ ID NO: 29] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCATAGCGAG GCTGAGGT
18

[0384]

[SEQ ID NO: 30] Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CATAGCGAGG CTGAGGT 17

[0385]

[SEQ ID NO: 31] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNNNNNNNNNNNNNNNNC
18

[0386]

[SEQ ID NO: 32] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNGGAGCCAT AGCGANNNC
18

[0387]

[SEQ ID NO: 33] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NGGGAGNNAN AGNGAGGNT 19

[0388]

[SEQ ID NO: 34] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNGGAGCCAT AGCGANNNC
18

[0389]

[SEQ ID NO: 35] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NGGGAGCCAT AGCGAGNC
18

[0390]

[SEQ ID NO: 36] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNNNNNNNNN NNNNNNNT 18

[0391]

[SEQ ID NO: 37] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNGAGCGATA CCGAGNNT
18

[0390]

[SEQ ID NO: 38] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNGAGCGATA CCGAGNNT
18

[0393]

[SEQ ID NO: 39] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNGGAGNNAN AGNGANNC 18

[0394]

[SEQ ID NO: 40] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNGAGNGANA NNGAGNNT
18

[0395]

[SEQ ID NO: 41] Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNCCCACCAC TTCCNNT
17

[0396]

[SEQ ID NO: 42] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNCTGTCCCG GGATANNT 18

[0397]

[SEQ ID NO: 43] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNNNNNNNNNNNNNNNNC
18

[0398]

[SEQ ID NO: 44] Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNAGCCATAG CGANNNC
16

[0399]

[SEQ ID NO: 45] Sequence length: 12 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNCCATAGCN NG 12

[0400]

[SEQ ID NO: 46] Sequence length: 9 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNCCATNNC
9

[0401]

[SEQ ID NO: 47] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNTTTTTTTTTTTTTNNG
18

[0402]

[SEQ ID NO: 48] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence NNTTTTTTTT TTTTTNNA 18

[0403]

[SEQ ID NO: 49] Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCCCCACCAC TTCCCCT
17

[0404]

[SEQ ID NO: 50] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCCTGTCCCG GGATAGGT
18

[0405]

[SEQ ID NO: 51] Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGAGCCATAG CGAGGC 16

[0406]

[SEQ ID NO: 52] Sequence length: 12 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AGCCATAGCG AG
12

[0407]

[SEQ ID NO: 53] Sequence length: 9 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AGCCATAGC
9

[0408]

[SEQ ID NO: 54] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGTTTTTTTT TTTTTGGG 18

[0409]

[SEQ ID NO: 55] Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AATTTTTTTT TTTTTAAA
18

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location C07H 21/00 (72) Inventor Susumu Iwasa 21-21 Ozumigaoka, Tanabe-cho, Tsuzuki-gun, Kyoto Prefecture

Claims (26)

[Claims] 1. The formula: [In the formula, W represents a hydrogen atom or a protecting group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ² may be the same or different in repeating n, and X ²
And X ³ each represent an OH group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or optionally substituted aromatic ring group, and X ³ is n May be the same or different, and R ¹ , R ² and R ³ are each a hydrogen atom, an OH group,
It represents a halogen atom, a C _1-5 alkoxy group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in repeating n, and n is 1-9
Indicates an integer of 8. However, (1) X ² and X ³ are each an OH group, an SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an aromatic ring group which may be substituted. And when at least one of X ² and X ³ is an optionally substituted aromatic ring group, W represents a hydrogen atom or a protecting group, and (2) X ² and X ³ are respectively an OH group and an SH group. , A C _1-5 alkyl group, a C _1-5 alkoxy group or a C _1-5 monoalkylamino group, W has the formula: Or the formula (In the formula, Y represents a sugar residue, X ¹ represents an OH group, an SH group, a C
_1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group is shown. ) Represents a group represented by. ] The oligonucleotide derivative or its salt which has a nucleotide sequence represented by these. 2. The formula: [In the formula, Q is Y or the formula: Represents a group represented by, Y represents a sugar residue, X ¹ represents OH
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_{1-5 represents a} monoalkylamino group or an optionally substituted aromatic ring group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ² may be the same or different in repeating n. Well, X ⁴ and X ⁵ are OH
Group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_1-5 monoalkylamino group, X ⁵ may be the same or different in repeating n, R ¹ ,
R ² and R ³ each represent a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxyl group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in each repeating n. n shows the integer of 1-98. ] The oligonucleotide derivative or salt thereof according to claim 1, which has a nucleotide sequence represented by: 3. Q is Y, the sugar of the sugar residue represented by Y is galactose or mannose whose hydroxyl group may be substituted with a C _1-6 alkylcarbonyl group, and the hydroxyl group is a phenylcarbonyl group. -Melibiose, which may be substituted with an amino group, gentiobiose or isomaltotriose, n-octylglucoside, n-octylthioglucoside or phenylglucoside, C
The oligonucleotide derivative according to claim 2, which is glucosamine or galactosamine optionally substituted with a _1-10 alkylcarbonyl group or a phenylcarbonyl group. 4. Q is of the formula: The oligonucleotide derivative according to claim 2, wherein the sugar of the sugar residue represented by Y is a monosaccharide. 5. The nucleic acid residues represented by B ¹ , B ² and B ³ are hypoxanthine-9-yl, guanine-9-yl,
Adenine-9-yl, uracil-1-yl, thymine-1
5. The oligonucleotide derivative according to claims 2 to 4, which is selected from the group consisting of -yl and cytosyn-1-yl. 6. X ¹ is an OH group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group, halogen atom, C _1-10 alkylcarbonyl group,
C _6-11 optionally substituted with about 1 to 5 substituents selected from the group consisting of OH group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group. Aryl group, or C _1-6 alkyl group, C _2-6 alkenyl group, C _2-6 alkynyl group, C _3-6 cycloalkyl group, C _5-7 cycloalkenyl group, C _7-11 aralkyl group,
C _6-14 aryl group, C _1-6 alkoxy group, C _6-14 aryloxy group, C _1-6 alkylcarbonyl group, C _6-14 aryl-carbonyl group, C _1-6 alkanoyloxy group,
C _6-14 aryl-carbonyloxy group, carboxyl group, C _1-6 alkoxy-carbonyl group, carbamoyl group, N-mono-C _1-4 alkylcarbamoyl group, N, N-
A di-C _1-4 alkylcarbamoyl group, a halogen atom, a mono-, di- or tri-halogeno-C _1-4 alkyl group,
It may be substituted with about 1 to 5 substituents selected from the group consisting of an oxo group, an amidino group, an imino group, an amino group, a mono- or di-C _1-4 alkylamino group, an OH group and a nitro group. 5 to 13 containing 1 to 4 hetero atoms and 1 to 12 carbon atoms selected from the group consisting of nitrogen atom, oxygen atom and sulfur atom
Member aromatic heterocycle, wherein X ⁴ and X ⁵ are each OH
The oligonucleotide derivative according to claim 2, which is a group, an SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group or a C _1-5 monoalkylamino group. 7. R ¹ , R ² and R ³ are each a hydrogen atom,
The oligonucleotide derivative according to claims 2 to 6, which is selected from the group consisting of an OH group, fluoro, chloro, bromo, methoxy group, ethoxy group and methoxymethyloxy group. 8. The oligonucleotide derivative according to claim 2, which is an antisense oligonucleotide derivative. 9. The formula: [In the formula, W represents a hydrogen atom or a protective group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ² may be the same or different in repeating n, and X ⁶
And X ⁷ each represent an OH group, an SH group, a C _1-5 alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, and X ⁷ is n Which may be the same or different, and at least one of X ⁶ and X ^{7 represents} an optionally substituted aromatic ring group, R ¹ , R ² and R ³ are each a hydrogen atom, an OH group, It represents a halogen atom, a C _1-5 alkoxyl group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in repeating n, and n represents an integer of 1 to 98. ] The oligonucleotide derivative or salt thereof according to claim 1, which has a nucleotide sequence represented by: 10. A protective group represented by W has the formula (i): (Wherein Y represents a sugar residue), a group represented by the formula (ii): (In the formula, Y represents a sugar residue.), (Iii)
C _1-6 alkyl group optionally substituted with halogen atom, (iv) halogen atom or C _6-11 aryl group optionally substituted with C _1-6 alkyl group, (v) halogen atom or C _{1 -6} C _optionally substituted with an alkyl group
_7-12 aralkyl group, (vi) C _1-6 alkylcarbonyl group optionally substituted with a halogen atom, (vii) phenyloxycarbonyl group, (viii) C _7-12 aralkyl-carbonyl group, (ix) pyranyl Group, (x) furanyl group, (x
The oligonucleotide according to claim 9, which is i) a silyl group, (xii) a trityl group optionally substituted with 1 to 3 methoxy groups, or (xiii) a phenylxanthenyl group optionally substituted with a methoxy group. Derivative or salt thereof. 11. W is a hydrogen atom or a compound of the formula: And the sugar of the sugar residue represented by Y has a hydroxyl group of C
_1-6 Galactose or mannose optionally substituted with an alkylcarbonyl group, melibiose, gentiobiose or isomaltotriose whose hydroxyl group may be substituted with a phenylcarbonyl group, n-octylglucoside, n-octylthioglucoside or phenyl The oligonucleotide derivative according to claim 9, which is glucosamine, glucosamine or galactosamine optionally substituted with a C _1-10 alkylcarbonyl group or a phenylcarbonyl group. 12. W is a hydrogen atom or a compound of the formula: 10. The oligonucleotide derivative according to claim 9, wherein the sugar of the sugar residue represented by Y is a monosaccharide. 13. The nucleic acid residues represented by B ¹ , B ² and B ³ are hypoxanthine-9-yl, guanine-9-yl,
Adenine-9-yl, uracil-1-yl, thymine-1
13. The oligonucleotide derivative according to claims 9 to 12, which is selected from the group consisting of -yl and cytosyn-1-yl. 14. X ⁶ and X ⁷ are respectively an OH group and S
H group, C _1-5 alkyl group, C _1-5 alkoxy group, C
_1-5 monoalkylamino group, halogen atom, C _1-10 alkylcarbonyl group, OH group, nitro group, C _1-10 alkyl group, C _1-10 alkoxy group, phenyl group and naphthyl group. A C _6-11 aryl group which may be substituted with about 1 to 5 substituents, or C
_1-6 alkyl group, C _2-6 alkenyl group, C _2-6 alkynyl group, C _3-6 cycloalkyl group, C _5-7 cycloalkenyl group, C _7-11 aralkyl group, C _6-14 aryl group, C _1-6 alkoxy group, C _6-14 aryloxy group, C _1-6 alkylcarbonyl group, C _6-14 aryl-carbonyl group, C _1-6 alkanoyloxy group, C _6-14 aryl-carbonyloxy group, Carboxyl group, C _1-6 alkoxy-carbonyl group, carbamoyl group, N-mono-C _1-4 alkylcarbamoyl group, N, N-di-C _1-4 alkylcarbamoyl group,
Halogen atom, mono-, di- or tri-halogeno-C
1 to 5 substituents selected from the group consisting of _1-4 alkyl group, oxo group, amidino group, imino group, amino group, mono- or di-C _1-4 alkylamino group, OH group and nitro group. It is a 5- to 13-membered aromatic heterocycle containing 1 to 4 hetero atoms and 1 to 12 carbon atoms selected from the group consisting of an optionally substituted nitrogen atom, oxygen atom and sulfur atom. Claims 9 to 13
The described oligonucleotide derivative. 15. R ¹ , R ² and R ³ are each a hydrogen atom, OH group, fluoro, chloro, bromo, methoxy group,
The oligonucleotide derivative according to claim 9, which is selected from the group consisting of an ethoxy group and a methoxymethyloxy group. 16. The formula: [In the formula, W represents a hydrogen atom or a protective group, B ¹ , B ² and B ³ each represent a nucleic acid residue, and B ² may be the same or different in each repeating n, X ⁸
Represents an OH group, SH group, C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or optionally substituted aromatic ring group, and R ¹ , R ² and R ³ Each represents a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxy group or a C _1-5 alkoxyalkyloxy group, and R ² may be the same or different in repeating n, and n is 1 to 98. Indicates an integer. ] The oligonucleotide derivative or its salt of Claim 9 which has a nucleotide sequence represented by these. 17. The formula: [In the formula, W represents a hydrogen atom or a protective group, and B ¹ ,
B ²¹ , B ²² and B ³ each represent a nucleic acid residue, and B ²¹
May be the same or different in repeating n, and X ⁸ is OH group, SH group, C _1-5 alkyl group, C
_1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, and R ¹ , R ² and R
³ each represents a hydrogen atom, an OH group, a halogen atom, a C _1-5 alkoxy group or a C _1-5 alkoxyalkyloxy group, R ^{2 s} may be the same or different in repeating n, and n is 1 to 1 An integer of 98 is shown. ] The oligonucleotide derivative or its salt of Claim 9 which has a nucleotide sequence represented by these. 18. The formula: [In the formula, W, B ¹ , B ² , B ³ , R ¹ , R ² , R ³ and n have the same meanings as in claim 1, X ²¹ and X ³¹ each represent O or S, and X ³¹ may be the same or different in each repetition of n, and CPG represents a solid phase carrier. ] Or a compound represented by the formula: [In the formula, W, B ¹ , B ² , B ³ , R ¹ , R ² , R ³ and n have the same meanings as in claim 1, X ²² and X ³² are each a C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, X ³² may be the same or different in repeating n, and CPG represents a solid phase carrier. Show. ] The protecting group of the compound represented by these is removed, The manufacturing method of the oligonucleotide derivative or its salt of Claim 1 characterized by the above-mentioned. 19. The formula: [In the formula, Q, B ¹ , B ² , B ³ , R ¹ , R ² , R ³ and n have the same meanings as in claim 2, and X ¹¹ , X ⁴¹ and X ⁵¹ represent O or S respectively. As shown, X ⁵¹ may be the same or different in each repetition of n, and CPG represents a solid phase carrier. ] Or a compound represented by the formula: [In the formula, Q, B ¹ , B ² , B ³ , R ¹ , R ² , R ³ and n have the same meanings as in claim 2, and X ¹² , X ⁴² and X ⁵² are each C _1-5. Alkyl group, C _1-5 alkoxy group, C _1-5
It represents a monoalkylamino group or an optionally substituted aromatic ring group, X ⁵² may be the same or different in each repeating n, and CPG represents a solid phase carrier. ] The protective group of the compound represented by these is removed, The manufacturing method of the oligonucleotide derivative of Claim 2 characterized by the above-mentioned. 20. The formula: [In the formula, W, B ¹ , B ² , B ³ , R ¹ , R ² , R ³ and n have the same meanings as in claim 9, X ⁶¹ and X ⁷¹ each represent O or S, and X ⁷¹ may be the same or different in each repetition of n, and CPG represents a solid phase carrier. ] Or a compound represented by the formula: [In the formula, W, B ¹ , B ² , B ³ , R ¹ , R ² , R ³ and n have the same meanings as in claim 9, X ⁶² and X ⁷² are each a C _1-5 alkyl group, C _1-5 alkoxy group, C _1-5 monoalkylamino group or an optionally substituted aromatic ring group, X ⁷² may be the same or different in repeating n, and CPG represents a solid phase carrier. Show. ] The protecting group of the compound represented by these is removed, The manufacturing method of the oligonucleotide derivative of Claim 9 characterized by the above-mentioned. 21. A gene expression inhibitor comprising the oligonucleotide derivative according to any one of claims 1 to 17 or a salt thereof. 22. A PTCA (Percutaneous Transluminal Coronary) containing the oligonucleotide derivative according to any one of claims 1 to 17 or a salt thereof.
Angioplasty; Percutaneous coronary lumen dilatation) inhibitor for vascular restenosis. 23. The formula: [Wherein Q is Y or the formula: And Y represents a sugar residue. ] The sugar derivative represented by these. 24. The formula: [Wherein Q is Y or the formula: Represents a group, Y represents a sugar residue, and X ¹² represents C _1-5.
An alkyl group, a C _1-5 alkoxy group, a C _1-5 monoalkylamino group or an optionally substituted aromatic ring group is shown. ]
A sugar derivative represented by. 25. The formula: [In the formula, Y represents a sugar residue, and R ¹⁰ represents a hydrogen atom or a benzyl group. ] The sugar derivative represented by these. 26. The formula: [In the formula, W represents a hydrogen atom or a protecting group, B ¹ represents a nucleic acid residue, R ¹ represents a hydrogen atom, an OH group, a halogen atom,
A C _1-5 alkoxy group or a C _1-5 alkoxyalkyloxy group is shown. ] The nucleotide derivative represented by these.