JPH0995495A - Antisense oligonucleotide, nucleoside and intermediate for producing the same, its synthesis, oligonucleotide synthesizing unit and its synthesis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new nucleic acid having nuclease resistance and suppressing manifestation of a gene to cause a disease, containing a deoxyuridine having a carboxylic acid ester, etc., at its 5-position, and an N,N- dimethylaminohexyl-carbamoyl group. SOLUTION: This antisense oligonucleotide comprises a nucleoside containing a carboxylic acid ester or an aminohexylcarbamoyl group at its 5-position of deoxyuridine or its derivative and a nucleoside of 5-(N,N-dimethylaminohexyl) carbamoyl-2'-deoxyuridine or its derivative as constituent components. The oligonucleotide shows resistance to hydrolysis with nuclease, forms a complementary bond which is specific to a target mRNA and thermodynamically stable, controls the manifestation of a gene to cause a disease and is useful for treating the disease such as viral infectious disease, a cancer, etc. The nucleoside is obtained by reacting 5-iodo-2'-deoxyuridine with trifluoroethanol and carbon monoxide in the presence of a catalyst.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a second-generation antisense oligonucleotide, an intermediate nucleoside for producing the same, a method for synthesizing the same, an oligonucleotide synthesis unit, and a method for synthesizing the same. The present invention relates to an antisense oligonucleotide containing a deoxynucleoside whose base is modified, a nucleoside which is an intermediate for producing the same, a method for synthesizing the same, an oligonucleotide synthesis unit, and a method for synthesizing the same.

[0002]

2. Description of the Related Art When the cause of a disease is due to the regulation of gene expression and the gene product (protein) causes an infectious disease, cancer, etc., to suppress the production of these causative substances. In recent years, the concept of a method of suppressing the action of messenger RNA (mRNA) that transmits genetic information of a causative substance has been born. That is, mRNA is referred to as sense RNA, and DNA or RNA complementary thereto is referred to as antisense DNA or antisense RNA. By the antisense DNA or antisense RNA forming a double strand with mRNA, the action of mRNA is achieved. The idea is to control and cure the disease. Based on this concept
Various proposals have been made regarding antisense oligonucleotides for treating diseases such as viral infections such as AIDS (AIDS) and cancer (for example, JP-A-3-99).
093, Japanese Patent Application Laid-Open No. 4-154794, Japanese Patent Application Laid-Open No. 6-41185, Japanese Patent Application Laid-Open No. 6-179694, etc.), and methods for synthesizing antisense oligonucleotides and biological activities thereof have been actively studied. It has been reported at academic societies.

[0003] Antisense oligonucleotides are said to be effective in studies in vitro and at the cell level, but have problems with stability against the decomposition reaction of antisense oligonucleotides by nucleases and permeability of cell membranes. ing. To solve those problems, D
Conventionally, the natural phosphoric acid diester of NA or RNA is converted into a phosphonic acid ester, a thiophosphoric acid ester, or an amino acid bond.

[0004]

However, as has been hitherto studied, the phosphoric acid ester of an antisense oligonucleotide converted to a phosphonic acid ester or a thiophosphoric acid ester produces an unnatural isomer. Therefore, it is estimated that their physiological effects are low. Further, an antisense oligonucleotide in which a phosphoric acid ester is converted into a thiophosphoric acid ester type or an amino acid binding type exhibits a nonspecific antisense effect, which is a problem.

[0005] It should be noted that researches for enhancing the effect of antisense oligonucleotides by converting the base portion of the oligonucleotides have been reported in a few cases using natural nucleotides, but they are extremely rare.

The present invention has been made under the above circumstances and has a natural phosphodiester bond,
Useful to form duplexes that are biologically stable, that is, resistant to nuclease hydrolysis and that have specific and thermodynamically stable complementary bonds to the target mRNA An antisense oligonucleotide, a nucleoside that is an intermediate in the production of the antisense oligonucleotide, and a method for synthesizing the nucleoside, and the oligos required for synthesizing the antisense oligonucleotide from the nucleoside. It is an object to provide a nucleotide synthesis unit and a method for synthesizing the same.

[0007]

The invention according to claim 1 is
Nucleoside having 5-carboxylic acid ester or aminohexylcarbamoyl group of deoxyuridine or its derivative and 5- (N, N-dimethylaminohexyl)
The antisense oligonucleotide was formed by including the nucleoside of carbamoyl-2'-deoxyuridine or its derivative as a constituent.

According to the second aspect of the invention, an antisense oligonucleotide is formed by including nucleoside of 5- (N, N-dimethylaminohexyl) carbamoyl-2'-deoxyuridine or its derivative as a constituent.

The antisense oligonucleotides of the inventions according to claim 1 and claim 2 are resistant to hydrolysis by nucleases, and are thermostable when they form a complementary strand and a double strand. Indicates.

The invention according to claim 3 is the antisense oligonucleotide according to claim 1 or 2, wherein a fatty acid is introduced via an amino linker of a uracil base derivative located at the 5'end or 3'end of the nucleoside. It is characterized in that a functional molecule selected from the group consisting of compounds, steroids, lipids and fluorescent chromophores is introduced.

The nucleoside of the invention according to claim 4 is 5
-Trifluoroethoxycarbonyl-2'-deoxyuridine.

The nucleoside of the invention according to claim 5 is 5
-(N, N-dimethylaminohexyl) carbamoyl-
It consists of 2'-deoxyuridine.

The invention according to claim 6 is 5-iodo-2 '.
-Deoxyuridine is used as a starting material to synthesize a nucleoside composed of 5-trifluoroethoxycarbonyl-2'-deoxyuridine.

The invention according to claim 7 is 5-iodo-2 '.
-Deoxyuridine as a starting material, 5-trifluoroethoxycarbonyl-2'-deoxyuridine and then 5-
(N, N-Dimethylaminohexyl) carbamoyl-
It is characterized in that a nucleoside composed of 2'-deoxyuridine is synthesized.

The oligonucleotide synthesis unit of the invention according to claim 8 is 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-3'-O- (cyanoethoxy) (N, N-diisopropylamino). ) Consists of phosphinyl-2'-deoxyuridine.

The oligonucleotide synthesis unit of the invention according to claim 9 is 5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-
It consists of 3'-O- (cyanoethoxy) (N, N-diisopropylamino) phosphinyl-2'-deoxyuridine.

The oligonucleotide synthesis unit of the invention according to claim 10 comprises a 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-deoxyuridine CPG unit.

The oligonucleotide synthesis unit of the invention according to claim 11 is 5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-
It consists of a 2'-deoxyuridine CPG unit.

The twelfth aspect of the present invention relates to 5-trifluoroethoxycarbonyl-2'-deoxyuridine to 5
-Trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-deoxyuridine to 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-3'-O- (cyanoethoxy) (N, N- It is characterized by synthesizing an oligonucleotide synthesis unit consisting of diisopropylamino) phosphinyl-2′-deoxyuridine.

The present invention according to claim 13 provides 5-trifluoroethoxycarbonyl-2'-deoxyuridine to 5
-Trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-deoxyuridine and 5- (N,
N-dimethylaminohexyl) carbamoyl-5′-O
-(N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-3'-O- (cyanoethoxy) (N, N-diisopurple) through dimethoxytrityl-2'-deoxyuridine. It is characterized in that an oligonucleotide synthesis unit consisting of pyramino) phosphinyl-2'-deoxyuridine is synthesized.

The present invention according to claim 14 provides 5-trifluoroethoxycarbonyl-2'-deoxyuridine to 5
-Trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-deoxyuridine and 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-3'-O-succinyl-2'-deoxyuridine in order. 5-trifluoroethoxycarbonyl-5 '
-O-dimethoxytrityl-2'-deoxyuridine C
It is characterized in that an oligonucleotide synthesis unit comprising a PG unit is synthesized.

The present invention according to claim 15 provides 5-trifluoroethoxycarbonyl-2'-deoxyuridine to 5
-Trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-deoxyuridine, 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-3'-O-succinyl-2'-deoxyuridine and 5- (N, N-Dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-3'-O-succinyl-2'-deoxyuridine followed by 5- (N, N)
-Dimethylaminohexyl) carbamoyl-5'-O-
It is characterized by synthesizing an oligonucleotide synthesis unit comprising a dimethoxytrityl-2'-deoxyuridine CPG unit.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The best embodiment of the present invention will be described below.

First, the nucleoside synthesis, which is the first step in chemically synthesizing an antisense oligonucleotide, will be described. As shown in FIG. 1, 5-trifluoroethoxycarbonyl-2′-deoxyuridine (compound 2) is synthesized from 5-iodo-2′-deoxyuridine (compound 1). Further, the carboxylic acid ester of Compound 2 is converted into an N, N-dimethylaminohexylcarbamoyl group to synthesize 5- (N, N-dimethylaminohexyl) carbamoyl-2′-deoxyuridine (Compound 7). By thus synthesizing compound 7 via compound 2, compound 7 can be synthesized under extremely mild conditions.

Next, the synthesis of the oligonucleotide synthesis unit, which is the second step in chemically synthesizing the antisense oligonucleotide, will be described. When compound 2 or compound 7 is incorporated into a nucleotide sequence excluding the 3'end of an oligonucleotide, it is used as a synthetic unit.
-Trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-3'-O- (cyanoethoxy) (N, N
-Diisopropylamino) phosphinyl-2'-deoxyuridine (compound 4) and 5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-3'-O- (cyanoethoxy) ( N, N-Diisopropylamino) phosphinyl-2′-deoxyuridine (Compound 6) is synthesized by applying a known method.
That is, compound 4 is synthesized from compound 2 via 5-trifluoroethoxycarbonyl-5′-O-dimethoxytrityl-2′-deoxyuridine (compound 3) as shown in FIG. In addition, the compound 6 includes compounds 2 to 3 and 5- (N, N-dimethylaminohexyl) carbamoyl-5′-O-dimethoxytrityl-.
It is synthesized via 2'-deoxyuridine (Compound 5).

When compound 2 or compound 7 is located at the 3'end of the oligonucleotide sequence, compound 2 and compound 7 are used as synthetic units by applying known methods to CPG (Controlled-P), respectively.
ore Glass) carrier-bound compound, 5-trifluoroethoxycarbonyl-5′-O-dimethoxytrityl-2′-deoxyuridine CPG unit (compound 9) and 5- (N, N-dimethylaminohexyl) carbamoyl-5. Induced to'-O-dimethoxytrityl-2'-deoxyuridine CPG unit (Compound 11). That is, the compound 9 is converted from the compound 2 to the compound 3 and then from the compound 3 to 5-trifluoroethoxycarbonyl-5′-O-dimethoxytrityl-3′-O-succinyl-2′-, as shown in FIG. Derived via deoxyuridine (Compound 8). In addition, compound 11 is
From compound 2 to compound 3, further from compound 3 to compound 8 and 5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-3'-O-
Derived via succinyl-2'-deoxyuridine (compound 10).

Then, as a third step, an oligonucleotide is synthesized from the above oligonucleotide synthesis unit. Oligonucleotides, by the known solid phase synthesis method,
It can be synthesized using a DNA synthesizer.

The basic structure of the DNA oligonucleotides (ON-1 to ON-5) synthesized as described above is shown below.
~ Chemical formula 5 are shown respectively.

[0029]

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In the formulas 1 to 5, Cm is 5-methyl-
2'-deoxycytidine, T is thymidine, E is 5-trifluoroethoxycarbonyl-2'-deoxyuridine, M is 5- (N, N-dimethylaminohexyl) carbamoyl-2'-deoxyuridine, N is 5- Aminohexylcarbamoyl-2'-deoxyuridine is shown respectively.

The oligonucleotides ON-1 and ON-2 shown in Chemical formula 1 and Chemical formula 2 respectively locate the compound 2 at the 3'end or the 5'end, and respectively form the 5'end or the 3'end on the other side. It is an oligonucleotide containing compound 7 within the sequence. Each of these oligonucleotides ON-1 and ON-2 is treated with 1,6-diaminohexane to give a compound located at the 3'end or 5'end, as shown in Chemical formula 3 and Chemical formula 4, respectively. Oligonucleotides ON-3 and ON-4 in which 2 is converted to 5-aminohexylcarbamoyl-2′-deoxyuridine, whose structural formula is shown in Chemical formula 6, are obtained. In addition, the oligonucleotide ON-5 is
It is an oligonucleotide containing compound 7 at both 5 ′ and 3 ′ ends and inside the sequence.

[0032]

[Chemical 6]

It is assumed that the nucleoside derivative contained in the oligonucleotide obtained as described above forms a hydrogen bond pair with adenine nucleoside in the same manner as uracil nucleoside or thymine nucleoside. Therefore, assuming that the newly obtained oligonucleotide will be applied as an antisense oligonucleotide, its thermal stability and resistance to nuclease were examined.

First, a complementary oligonucleotide DNA-0: 5'-d (TGGAGA, in which an adenine nucleoside is located at a position complementary to the obtained oligonucleotide.
GAGAGAGAGAGAGAGAGGT) -3 ′ and RNA-0: 5′-r (UGGAGAGAGAGAGAGA
GAGAGAGAGGU) -3 ′ are synthesized by known methods, and their complementary oligonucleotides DN
A-0 and RNA-0 and the oligonucleotide (control) shown in Chemical formula 7 and the oligonucleotide ON-
Double strands of 3, ON-4 and ON-5 were respectively formed, and the thermal stability of the double strands in each case was measured. Table 1 shows the results.

[0035]

[Chemical 7]

[0036]

[Table 1]

From the results shown in Table 1, N, at the 5-position of the uracil base of the nucleoside contained in the oligonucleotide,
When an N-dimethylaminohexylcarbamoyl group or aminohexylcarbamoyl group is introduced, DNA-
It can be seen that the thermal stability of the double strand of DNA or DNA-RNA is enhanced. This is the oligonucleotide ON
It shows that specificity and effectiveness can be expected when -3, ON-4, and ON-5 are applied to antisense oligonucleotides.

Next, in the study on the resistance of the antisense oligonucleotide to the hydrolysis of the oligonucleotide by nuclease, which shows the stability in vivo, the degradation by exonuclease was hardly observed within 2 hours. . This suggests that the presence of the substituent at the 5-position of the uracil base of the nucleoside contained in the oligonucleotide enhances the resistance to nuclease.

Further, the oligonucleotides ON-3 and O
N-4 contains an oligonucleoside having an aminoalkyl group (aminohexyl group) at the 3'end or the 5'end, respectively, so that it serves as an amino linker to introduce various functional groups. You can For example, an oligonucleotide to which a fatty acid, a steroid, a lipid, a fluorescent chromophore, or the like is bound can be obtained. This improves the cell membrane permeability of the oligonucleotide, or when the oligonucleotide binds to mRNA,
It can be used as a fluorescent label that emits fluorescence.

[0040]

EXAMPLES Hereinafter, specific synthesis examples of each of the above substances will be described.

[5-trifluoroethoxycarbonyl-
Synthesis Example of 2'-deoxyuridine (Compound 2)] 3.00 g of 5-iodo-2'-deoxyuridine (Compound 1)
(8.47 mmol) was dissolved in 150 ml of N, N-dimethylformamide, and 60.8 ml (847 mmol) of 2,2,2-trifluoroethanol, 1.18 ml (8.47 mmol) of triethylamine and bis (benzonitrile) were dissolved in this solution. ) Dichloropalladium (II) 65
mg (0.169 mmol) was added, and the mixture was stirred under a carbon monoxide stream at a temperature of 70 ° C. overnight.
The reaction solution was filtered through Celite, the Celite was washed with ethanol, and the solvent was evaporated. The residue was purified by silica gel column chromatography (solvent: chloroform containing 15% methanol), whereby 2.11 g of compound 2 was obtained.
(5.94 mmol, yield 70%), obtained as a single brown solid.

[Synthesis Example of 5- (N, N-Dimethylaminohexyl) carbamoyl-2'-deoxyuridine (Compound 7)] 0.200 g (0.564 mmol) of Compound 2 was added to 2,2,2-trifluoro. 16m ethanol
1 and dissolved in this solution, N, N-dimethyl-1,6-
Hexanediamine 0.807 ml (5.64 mmol)
Was added and the mixture was stirred at room temperature overnight. After distilling off the solvent of the reaction solution, the residue was dissolved in 5 ml of pyridine, and 1.06 ml (11.3 mmo of acetic anhydride was added to this solution.
1) was added and the mixture was stirred at room temperature for 4 hours. 1 ml of water was added to the reaction solution, the solution was stirred for 10 minutes, and then the solvent was distilled off. The residue was dissolved in chloroform, separated once with water and once with saturated aqueous sodium hydrogen carbonate, and washed once with saturated brine. The extracted organic layer is dried over anhydrous sodium sulfate, the solvent is distilled off, and the residue is subjected to silica gel column chromatography (solvent: 20%
Purified by methanol-containing chloroform). 0.19 g (0.393 mmol, yield 70%) of the obtained white foamy substance was dissolved in 5 ml of methanol, and then sodium methylate 0.0564 mmol of anhydrous methanol 1 was added.
The 29 ml solution was added dropwise under ice cooling, and the solution was stirred at room temperature for 10 minutes. The reaction solution is Dowex 50W (H
+) Was neutralized, the resin was filtered off, and the solution was evaporated. As a result, 0.07 g (0.177 m) of compound 7 was obtained.
mol, yield 47%), and obtained as a yellow candy-like substance.

[5-trifluoroethoxycarbonyl-
Synthesis example of 5'-O-dimethoxytrityl-2'-deoxyuridine (Compound 3)] 0.98 g (2.77 mmo
Compound 2 of 1) was azeotroped twice with pyridine, then it was dissolved in 20 ml of pyridine, 1.22 g (3.60 mmol) of dimethoxytrityl chloride was added to this solution, and the mixture was stirred at room temperature for 3 hours. It was stirred. Ethanol was added to the reaction solution, the solution was stirred for 10 minutes, the solvent was distilled off, chloroform was added to the residue, and the solution was partitioned twice with saturated aqueous sodium hydrogen carbonate and washed once with saturated brine. The extracted organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off, and the residue was purified by silica gel column chromatography (solvent: 4% methanol-chloroform containing 0.2% pyridine), whereby the compound 3 to 1.
Obtained as 69 g (2.57 mmol, 93% yield) as a white foam.

[5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-
Synthesis example of 2'-deoxyuridine (compound 5)] 50.
0 mg (0.0761 mmol) of compound 3 was dissolved in 3 ml of pyridine, 13.1 mg (0.0913 mmol) of N, N-dimethyl-1,6-hexanediamine was added, and the solution was stirred overnight at room temperature. The reaction solvent was distilled off, the residue was dissolved in chloroform, and the mixture was washed with saturated aqueous sodium hydrogencarbonate solution (2).
The solution was separated and washed once with saturated saline. The extracted organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by silica gel column chromatography (solvent: 10% methanol-chloroform containing 0.5% triethylamine). Accordingly, 0.050 g (0.0712 mmol, yield 94) of compound 5 was obtained.
%), And obtained as a colorless candy.

[5-trifluoroethoxycarbonyl-
Synthesis example of 5′-O-dimethoxytrityl-3′-O- (cyanoethoxy) (N, N-diisopropylamino) phosphinyl-2′-deoxyuridine (Compound 4)]
After azeotropically distilling 0.584 g (0.889 mmol) of compound 3 with pyridine, it is dissolved in 10 ml of methylene chloride and 0.2 ml of diisopropylethylamine is added to this solution.
3 ml (1.33 mmol) and 2-cyanoethyl-N,
N-diisopropylchlorophosphoramidite 0.29
7 ml (1.33 mmol) was added and the mixture was stirred at room temperature for 20 minutes. After distilling off the reaction solvent, chloroform was added to the residue, and the mixture was partitioned twice with saturated aqueous sodium hydrogen carbonate and washed once with saturated brine. The extracted organic layer is dried over anhydrous sodium sulfate, the solvent is evaporated, and the residue is subjected to neutral silica gel column chromatography (solvent:
Hexane-ethyl acetate (hexane: ethyl acetate = 1:
2)) and 0.502 g (0.5
86 mmol, 66% yield) was obtained as a white foam.

[5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-
Synthesis Example of 3'-O- (cyanoethoxy) (N, N-diisopropylamino) phosphinyl-2'-deoxyuridine (Compound 6)] 1.99 g (2.83 mmol) of Compound 5 was co-used with pyridine. After boiling, it was dissolved in 30 ml of methylene chloride and 0.986 ml (5.66 mmol) of diisopropylethylamine and 1.42 ml (5.66 mmol) of 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite were added to this solution. Add
The mixture was stirred at room temperature for 20 minutes. After distilling off the reaction solvent, chloroform was added to the residue, and the mixture was partitioned 3 times with saturated aqueous sodium hydrogen carbonate and washed once with saturated brine. The extracted organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by neutral silica gel column chromatography (solvent: acetone) to give compound 6 (1.63 g, 1.78 mmol, yield). Rate 63%), and obtained as a white foamy substance.

[5-trifluoroethoxycarbonyl-
Example of Synthesis of 5'-O-Dimethoxytrityl-3'-O-succinyl-2'-deoxyuridine (Compound 8)] 1.
54 g (2.35 mmol) of compound 3 was added to pyridine 35
0.470 g of succinic anhydride
(4.70 mmol) and 4-dimethylaminopyridine 4
7 mg (0.385 mmol) was added and the mixture was stirred at room temperature for 2 days. Water was added to the reaction solution, the solution was stirred for 10 minutes, the solvent was distilled off, the residue was dissolved in chloroform, and the solution was separated once with water and three times with a saturated aqueous solution of potassium dihydrogen phosphate, and then saturated. It was washed once with brine. The extracted organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (solvent: chloroform containing 20% methanol), whereby 0.79 of compound 3 was obtained.
6 g (1.21 mmol, yield 51%) and Compound 8 (0.644 g, 0.851 mmol, yield 36%) were obtained as white foamy substances.

[5-trifluoroethoxycarbonyl-
Synthesis example of 5'-O-dimethoxytrityl-2'-deoxyuridine CPG unit (Compound 9)] 0.104 m
g (0.137 mmol) of compound 8 and 27.4 mg
(0.137 mmol) 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added to N,
It was dissolved in 3 ml of N-dimethylformamide, and this solution was mixed with aminopropyl / CPG (85.7 mmol / g, 4
00 mg, 34.3 μmol) and reacted at room temperature for 2 days. The reaction solution was washed with pyridine and added with 0.1M
3 ml of a mixed solution of 4-dimethylaminopyridine with pyridine-acetic anhydride (9: 1) was added, and the mixture was reacted at room temperature for 12 hours. The reaction product was washed with ethanol and acetone and then dried under reduced pressure, whereby Compound 9 was added to 21.
Obtained with an activity of 9 μmol / g.

[5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-
Synthesis Example of 3'-O-succinyl-2'-deoxyuridine (Compound 10)] 0.470 g (0.621 mmo
Compound 8 of 1) was dissolved in 7 ml of pyridine, and N, N-dimethyl-1,6-hexanediamine 0.1 was added to this solution.
07 ml (0.745 mmol) was added and the mixture was stirred at room temperature overnight. The solvent was distilled off from the reaction solution, and the residue was partitioned once with water and three times with a saturated aqueous solution of potassium dihydrogenphosphate, and washed once with a saturated saline solution. The extracted organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by silica gel column chromatography (solvent: 40% methanol-chloroform containing 0.5% triethylamine). This gave 0.087 g (0.109 mmol, yield 18) of compound 10.
%), As a white foam.

[5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-
2'-deoxyuridine CPG unit (Compound 11)
Synthesis Example] 0.087 mg (0,109 mmol) of Compound 10 and 27.4 mg (0.137 mmol) of 1-
Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N, N-dimethylformamide 3m
1 and dissolve this solution in aminopropyl / CPG (8
5.7 mmol / g, 400 mg, 34,3 μmol)
In addition, the mixture was reacted at room temperature for 2 days. The reaction solution was washed with pyridine, and 3 ml of a 0.1M 4-dimethylaminopyridine pyridine-acetic anhydride (9: 1) mixed solution was added thereto, and the mixture was reacted at room temperature for 12 hours. After washing with ethanol and acetone, it is dried under reduced pressure, which gives compound 11
Was obtained with an activity of 21.2 μmol / g.

[Synthesis Example of Oligodeoxyribonucleotides by DNA Synthesizer]
Applied Biosystems Model 3
81A) and according to the phosphoramidite method. The amidite body was dissolved in anhydrous acetonitrile to a concentration of 0.1 M to 0.12 M, and about 20 equivalents relative to 1 μmol of CPG resin were used for the condensation reaction.

[Preparation and Purification Example of Oligonucleotides (ON-3, ON-4) Having Amino Linker] 1 ml of methanol and 1, 1 ml of methanol were added to the oligonucleotide bound to the solid phase carrier.
400 mg of 6-hexadiamine was added, and 12 at 50 ° C.
After standing for a while, the reaction solution was filtered, and then the filtrate was concentrated under reduced pressure. The residue was collected on a Sephadex G-25 column ((φ
1.7 × 27) cm, solvent: 0.1 M triethylammonium acetate (TEAA) pH 7.0) and after removing excess 1,6-hexadiamine, the absorbance at 254 nm of each fraction was measured, The desired fraction was obtained. As such, a 0.1 M TEAA aqueous solution containing 0-40% acetonitrile (pH:
It was purified by C-18 reverse phase silica gel column chromatography with a concentration gradient of 7.0), and the absorbance at 254 nm of each fraction was measured. The target fractions were collected, the solvent was distilled off under reduced pressure, and then azeotroped with water to remove TEAA. 80% acetic acid was added to the residue, the mixture was allowed to stand at room temperature for 20 minutes, and then the solvent was distilled off. Water was added to the residue and the mixture was washed with ethyl ether, and the aqueous layer was evaporated to remove the residue, and the residue was added to a DEAE-cellulose column (HCO
₃ ^- form) to adsorb and eluted from triethylammonium hydrogen carbonate of 1M. After concentrating the eluate, the residue is
% High-purity oligonucleotides (ON-3, ON-).
4) was obtained.

[Example of introducing palmitoyl group into oligonucleotide having amino linker] Purified oligonucleotide 5 OD ₂₆₀ units was dissolved in 0.05 M bicarbonate buffer (pH 10.3, 70 μl) to prepare palmitic acid N- N, N-dimethylformamide solution of hydroxysuccinimide ester (1 mg / 70 μl) was added,
Stir overnight at room temperature. The reaction solution was concentrated under reduced pressure, 300 μl of water and 10 to 20 μl of 1N hydrochloric acid were added to the residue to adjust the pH of the solution to 3 to 4, and then washed with ethyl acetate 8 to 9 times. Then, 100 μl of saturated aqueous sodium hydrogen carbonate was added to the aqueous layer for neutralization. The aqueous layer was subjected to DE with a concentration gradient of 20% aqueous acetonitrile containing 0-2M ammonium formate.
It was purified by AE-HPLC, whereby a high-purity palmitoyl group-introduced oligonucleotide was obtained (0.294 OD ₂₆₀ units, absorptivity 6%).

[0054]

INDUSTRIAL APPLICABILITY The antisense oligonucleotides of the inventions according to claims 1 and 2 exhibit biological stability and are complementary to target mRNAs in a specific and thermodynamically stable manner. Specificity at issue in antisense oligonucleotides being developed as antiviral agents against AIDS virus, cytomegalovirus, etc. and as anticancer agents against chronic myelogenous leukemia, etc. , It is possible to overcome the requirements for thermal stability and in vivo stability, and it is expected to be used as a more effective drug.

Further, according to the constitution of the invention of claim 3, various functional groups can be introduced into the aminohexylcarbamoyl group, whereby the cell membrane permeability and the effective arrival at the disease target site can be achieved. (Drug delivery)
It is expected to raise the degree.

The nucleosides of the inventions of claims 4 and 5 are used as intermediates in the production of the antisense oligonucleotides of the inventions of claims 1 and 2, and they are used as constituents. The contained antisense oligonucleotide exhibits the above effect.

According to the synthesizing methods of the inventions of claims 6 and 7, the nucleosides of the inventions of claims 4 and 5 can be optimally synthesized.

The synthesis unit of each invention according to claims 8 to 11 is used as a synthesis unit when producing the antisense oligonucleotide of each invention according to claims 1 and 2, and the above-mentioned effects are obtained from them. A useful antisense oligonucleotide having

According to the synthesizing methods of the inventions of claims 12 to 15, the oligonucleotide synthesizing units of the inventions of claims 8 to 11 can be optimally synthesized.

[Brief description of drawings]

FIG. 1 is a diagram for explaining a nucleoside synthesis process as a first step and an oligonucleotide synthesis unit synthesis process as a second step when synthesizing an antisense oligonucleotide according to the present invention.

FIG. 2 is a diagram for explaining a process of synthesizing an oligonucleotide synthesizing unit which is also a second step.

Claims (15)

[Claims] 1. A nucleoside having a carboxylic acid ester or an aminohexylcarbamoyl group at the 5-position of deoxyuridine or a derivative thereof and a nucleoside of 5- (N, N-dimethylaminohexyl) carbamoyl-2′-deoxyuridine or a derivative thereof. An antisense oligonucleotide containing as a constituent. 2. An antisense oligonucleotide containing a nucleoside of 5- (N, N-dimethylaminohexyl) carbamoyl-2′-deoxyuridine or a derivative thereof as a constituent component. 3. A functional molecule selected from the group consisting of fatty acids, steroids, lipids and fluorescent chromophores via an amino linker of a uracil base derivative located at the 5′-end or 3′-end of the nucleoside. Introduced claim 1
Alternatively, the antisense oligonucleotide according to claim 2. 4. 5-Trifluoroethoxycarbonyl-
A nucleoside consisting of 2'-deoxyuridine. 5. A nucleoside consisting of 5- (N, N-dimethylaminohexyl) carbamoyl-2′-deoxyuridine. 6. 5-Iodo-2′-deoxyuridine as a starting material 5-trifluoroethoxycarbonyl-
A method for synthesizing a nucleoside for synthesizing 2'-deoxyuridine. 7. 5-Iodo-2'-deoxyuridine as a starting material, 5-trifluoroethoxycarbonyl-
A method for synthesizing a nucleoside for synthesizing 5- (N, N-dimethylaminohexyl) carbamoyl-2'-deoxyuridine via 2'-deoxyuridine. 8. 5-Trifluoroethoxycarbonyl-
An oligonucleotide synthesis unit consisting of 5'-O-dimethoxytrityl-3'-O- (cyanoethoxy) (N, N-diisopropylamino) phosphinyl-2'-deoxyuridine. 9. 5- (N, N-Dimethylaminohexyl) carbamoyl-5′-O-dimethoxytrityl-
An oligonucleotide synthesis unit consisting of 3'-O- (cyanoethoxy) (N, N-diisopropylamino) phosphinyl-2'-deoxyuridine. 10. An oligonucleotide synthesis unit comprising a 5-trifluoroethoxycarbonyl-5′-O-dimethoxytrityl-2′-deoxyuridine CPG unit. 11. 5- (N, N-Dimethylaminohexyl) carbamoyl-5′-O-dimethoxytrityl-
An oligonucleotide synthesis unit comprising a 2'-deoxyuridine CPG unit. 12. 5-Trifluoroethoxycarbonyl-2'-deoxyuridine to 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-
5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-3'-O- via deoxyuridine
(Cyanoethoxy) (N, N-diisopropylamino)
A method for synthesizing an oligonucleotide synthesis unit for synthesizing phosphinyl-2'-deoxyuridine. 13. 5-Trifluoroethoxycarbonyl-2'-deoxyuridine to 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-
Deoxyuridine and 5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxytrityl-2'-deoxyuridine are successively passed through to 5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-. Dimethoxytrityl-3'-O- (cyanoethoxy) (N,
A method of synthesizing an oligonucleotide synthesis unit for synthesizing N-diisopropylamino) phosphinyl-2'-deoxyuridine. 14. 5-Trifluoroethoxycarbonyl-2'-deoxyuridine to 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-
Deoxyuridine and 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-3'-O-succinyl-2'-deoxyuridine are sequentially passed through to 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2 '. -A method for synthesizing an oligonucleotide synthesis unit for synthesizing a deoxyuridine CPG unit. 15. 5-Trifluoroethoxycarbonyl-2'-deoxyuridine to 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-2'-
Deoxyuridine, 5-trifluoroethoxycarbonyl-5'-O-dimethoxytrityl-3'-O-succinyl-2'-deoxyuridine and 5- (N, N-dimethylaminohexyl) carbamoyl-5'-O-dimethoxy. Trityl-3′-O-succinyl-2′-deoxyuridine followed by 5- (N, N-dimethylaminohexyl) carbamoyl-5′-O-dimethoxytrityl-
A method for synthesizing an oligonucleotide synthesis unit for synthesizing a 2'-deoxyuridine CPG unit.