JP2006248975A - Nucleoside phosphoroamidite compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleoside phosphoroamidite compound which can be used as an intermediate capable of forming an A type RNA double strand having a good thermal stability. <P>SOLUTION: This nucleoside phosphoroamidite compound is, for example, 5'-O-(4,4'-dimethoxytrityl)-5-[3-(N-trifluoroacetyl)-aminopropyn-1-yl]-2'-O-methyluridine-3'-O-(2-cyanoethyl)-N,N-diisopropylphosphoroamidite. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to nucleoside phosphoramidite compounds. More particularly, the present invention relates to a nucleoside phosphoramidite compound that can be used as an intermediate capable of forming a type A RNA duplex having thermal stability.

  Oligonucleotides are known to hybridize to single stranded RNA or single stranded DNA. Hybridization (hybridization) means that a base of an oligonucleotide is hydrogen-bonded to a base of a target RNA or DNA by a sequence-specific base pair. The pair is said to be complementary.

  When determining the degree of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of the oligonucleotide to bind to the complementary nucleic acid can be confirmed by measuring the melting temperature of a particular hybridization complex. . Melting temperature (Tm) is a physical property characteristic of a double helix and refers to the temperature at which 50% helical (hybridized) vs. coiled (unhybridized) is present.

  Although there is RNA called A-type structure, DNA usually exists in B-type structure, and in general, RNA-RNA duplex is more stable than DNA-DNA duplex Or have a higher melting temperature (Tm). However, DNA-RNA hybrid duplexes are less stable than RNA: RNA duplexes and are more or less stable than DNA DNA duplexes depending on their sequence.

  On the other hand, the use of a DNA chip is known as a technique for exhaustively analyzing the expression state, function, base sequence, etc. of intracellular functional RNA such as miRNA that is supposed to be generated from mRNA or siRNA. . However, the current DNA chip is not suitable for the detection of these because the thermal stability of the DNA-RNA duplex formed by siRNA or miRNA which are short strands is particularly low. Furthermore, the target RNA is more likely to form a higher order structure than DNA, so that a probe that is not expected to have a very high thermal stability cannot unravel the higher order structure, resulting in a decrease in detection sensitivity. is there.

  For DNA duplexes, methods for stabilizing nucleic acid duplexes by using modified nucleic acids have been developed. As an effective method for stabilizing nucleic acid duplexes using modified nucleic acids, the introduction of a cationic side chain into the base part of the nucleic acid prevents electrostatic repulsion between negative charges generated on the phosphate group. A technique for reducing and stabilizing nucleic acid duplexes is known (Non-patent Document 1). Various modified nucleic acids having cationic side chains have been incorporated into B-type DNA duplexes, and their behavior has been studied. Modified nucleic acids having cationic side chains that have acquired the ability to stabilize double strands On the other hand, the one that destabilizes the double chain has also been reported (Non-patent Document 2).

  For example, when a compound represented by the following formula (I) (a compound having an aminoheptyl group at the 5-position of the uracil ring of deoxyuridine) is incorporated into a B-type DNA duplex, B-type DNA into which deoxyuridine is incorporated The difference in melting temperature from the duplex (ΔTm value) is almost equivalent to −0.1 ° C. (Non-patent Document 3). Further, a compound represented by the following formula (II) (a compound in which an amino group is introduced at a position close to the uracil ring of the substituent at the 5-position of the uracil ring of the compound represented by formula (I)) is converted into a B-type DNA duplex. When incorporated in, the difference in melting temperature (ΔTm value) from the B-type DNA duplex in which deoxyuridine is incorporated is −3.9 ° C., and the duplex is destabilized (Non-patent Document 2) ). In addition, when a compound represented by the following formula (III) (a compound having an aminopropyl group at the 5-position of the uracil ring of deoxyuridine) is incorporated into a B-type DNA duplex, B-type DNA into which deoxyuridine is incorporated The difference in melting temperature from the duplex (ΔTm value) is −0.6 ° C., and the duplex is slightly destabilized (Non-patent Document 4). In addition, when a compound represented by the following formula (IV) (a compound in which the substituent bonded to the 5-position of the uracil ring has an unsaturated bond as compared with the compound (III)) is incorporated into the B-type DNA duplex, The difference in melting temperature (ΔTm value) from the B-type DNA duplex incorporating deoxyuridine is +2 to 4 ° C., and the duplex formation is greatly stabilized (Non-patent Document 4).

  As described above, in the case of a B-type DNA duplex, modification of side chains and the like for stabilizing the duplex has been clarified to some extent, but the structure is different from that of a B-type DNA duplex. There are currently few reports on the A-type RNA duplex, and the optimal modification that can improve the stabilization of the A-type RNA duplex has not yet been clarified.

  On the other hand, another method for forming a stable duplex of B-type DNA by using a modified nucleic acid is modification in which the 2 ′ hydroxyl group is 2′-O-alkylated (methylated, methoxyethylated, etc.) By using nucleic acids or modified nucleic acids (BNA, ENA, etc.) in which the sugar moiety conformation is chemically immobilized by crosslinking the 2′-position oxygen atom and the 4′-position carbon atom of the nucleic acid. Is known to adopt a conformation that facilitates incorporation of an A-type RNA duplex and to form a stable duplex (Non-Patent Documents 5, 6 and 7). However, there are currently few reports on the stabilization of A-type RNA duplexes.

J. Biochemistry, 1982, 21, 5458-5462 Chem. Pharm. Bull. 1987, 35, 3558-3567 C.J.Am.Chem.Soc. 1993, 115, 7128-7134 J.Am.Chem.Soc. 1998, 120, 12165-12166 Tetrahedron Lett. 1997, 38, 8735 J. Chem. Commun. 1998, 455 Helv. Chem Acta. 1995, 78, 486

  Accordingly, an object of the present invention is to provide a nucleoside phosphoramidite compound that can be used as an intermediate capable of forming an A-type RNA duplex having thermal stability.

In order to achieve the above object, the present inventors have intensively studied, and as a result, have found that a specific nucleoside phosphoramidite compound can achieve the above object.
The present invention has been made based on the above findings, and provides a nucleoside phosphoramidite compound represented by the following general formula (1).

(In the above formula, R ¹ represents a phosphate protecting group, R ² represents a dialkylamino group in which two identical or different alkyl groups having 1 to 6 carbon atoms are bonded on a nitrogen atom, and R ³ represents an alkoxy group. Or bonded to the 4′-position carbon of ribose to form a ring, and R ⁴ is an alkyl group having 3 or more carbon atoms, an alkenyl group having 3 or more carbon atoms, or a group having 3 or more carbon atoms. Represents an alkynyl group, the alkyl group, alkenyl group or alkynyl group may be bonded to a trifluoroacetylamino group, R ⁵ represents a protecting group for a hydroxyl group, and X represents an oxygen atom or a sulfur atom.)
The present invention also provides an oligonucleic acid containing a modified nucleoside as a constituent component by using the nucleoside phosphoramidite compound.

The nucleoside phosphoramidite compound of the present invention can be used as an intermediate capable of forming an A-type RNA duplex having thermal stability.
The oligonucleic acid of the present invention has thermal stability and can be used as an antisense drug, an antigene drug, a probe for detecting a specific gene, a primer for starting amplification, or the like.

Hereinafter, the nucleoside phosphoramidite compound of the present invention will be described.
The nucleoside phosphoramidite compound of the present invention is represented by the following general formula (1).

In the above general formula (1), R ¹ represents a phosphate protecting group. Any phosphoric acid protecting group can be used without particular limitation as long as it is used in the phosphoramidite method, and examples thereof include a methyl group, a 2-cyanoethyl group, and a 2-trimethylsilylethyl group.

R ² represents a dialkylamino group in which two identical or different alkyl groups having 1 to 6 carbon atoms are bonded on a nitrogen atom. Two alkyl groups may be bonded to each other to form a ring. Examples of such a dialkylamino group include a diethylamino group, a diisopropylamino group, and a dimethylamino group.

R ³ is an alkoxy group, or may be bonded to the 4′-position carbon of ribose to form a ring. The alkoxy group is preferably an alkoxy group having 1 to 6 carbon atoms. Examples of such an alkoxy group include a methoxy group, an ethoxy group, a propoxy group, a 1-butyloxy group, a 1-pentyloxy group, 1- Hexyloxy group and the like, and branched alkoxy groups such as 2-propyloxy group, isobutyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group, cyclopropylmethyl Also included are alkoxy groups in which part or all of the side chain is cyclized, such as oxy groups.

R ⁴ represents an alkyl group having 3 or more carbon atoms, an alkenyl group having 3 or more carbon atoms, or an alkynyl group having 3 or more carbon atoms, and the alkyl group, alkenyl group or alkynyl group binds a trifluoroacetylamino group. It may be.
Here, the alkyl group is preferably an alkyl group having 3 to 6 carbon atoms. Examples of such an alkyl group include alkyl groups such as a propyl group, an n-butyl group, an isobutyl group, a pentyl group, and a hexyl group. A cycloalkyl group such as an isopropyl group, a sec-butyl group, a tert-butyl group, a neopentyl group, a cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group. Examples of the alkenyl group include a propenyl group and a butynyl group. Examples of the alkynyl group include a propynyl group.

R ⁵ represents a hydroxyl-protecting group. The hydroxyl protecting group is not particularly limited as long as it is used in the phosphoramidite method, and examples thereof include a dimethoxytrityl group and a monomethoxytrityl group. X is an oxygen atom or a sulfur atom. When X is an oxygen atom, the nucleoside phosphoramidite compound of the present invention is a uracil derivative, and when it is a sulfur atom, it is a thiouracil derivative.

  Specific examples of the nucleoside phosphoramidite compound of the present invention include compounds represented by the following formula (2) (5′-O- (4,4′-dimethoxytrityl) -5- [3- (N-trifluoroacetyl). ) -Aminopropyn-1-yl] -2'-O-methyluridine 3'-O- (2-cyanoethyl) -N, N-diisopropylphosphoramidite) or a compound represented by (3) (5'- O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyl] -2'-O-methyluridine 3'-O- (2-cyanoethyl) -N, N -Diisopropyl phosphoramidite.

  The nucleoside phosphoramidite compounds of the present invention can be synthesized using methods known to those skilled in the art. For example, it is compoundable according to the method described in the Example of this specification.

Next, the oligonucleic acid of the present invention will be described. The oligonucleic acid of the present invention contains a modified nucleoside as a constituent by using the nucleoside phosphoramidite compound of the present invention. The type, number, combination, position, etc. of the nucleoside phosphoramidite compound of the present invention contained in the oligonucleic acid of the present invention can also be appropriately selected by those skilled in the art according to the intended purpose and use.
The oligonucleic acid of the present invention can be synthesized by any method known to those skilled in the art using the nucleoside phosphoramidite compound of the present invention. Although the oligonucleic acid of the present invention varies depending on its use, it usually comprises about 10 to 30 nucleotide units, and contains at least one nucleoside phosphoramidite compound of the present invention as its structural unit. The oligonucleic acid of the present invention contains the nucleoside phosphoramidite compound of the present invention, has excellent thermal stability, and can be used for various applications described below.

The oligonucleic acid of the present invention has the property of being excellent in thermal stability, and can be used for a hybridization method between a probe for gene analysis and a nucleic acid by utilizing the property of being excellent in thermal stability. Such a hybridization method is used in various measuring means known to those skilled in the art in the fields of PCR method and DNA chip.
The oligonucleic acid of the present invention comprises a DNA primer for PCR (primer for starting amplification of a specific gene), a DNA pharmaceutical material (antisense DNA, decoy DNA, a material for gene repair using oligo DNA), an RNA probe for gene analysis, It can also be used as RNA drug material (antisense RNA, antigene RNA, ribozyme, gene expression control using RNAi), DNA chip or RNA chip material.

Examples of the administration form when the oligonucleic acid of the present invention is administered as a pharmaceutical material include oral administration using tablets, capsules, granules, powders or syrups, or parenteral administration using injections or suppositories. . These preparations are produced by known methods using conventionally known additives such as lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.
When used as a pharmaceutical material, the amount used varies depending on symptoms, age, administration method, etc., and can be appropriately selected.

Hereinafter, the present invention will be described in more detail with reference to examples. Needless to say, the scope of the present invention is not limited to such examples.
Comparative production example 1
5'-O- (4,4'-dimethoxytrityl) -5- [N- (trifluoroacetyl) -methylaminomethyl] -2'-O-methyluridine 3'-O- (2-cyanoethyl) -N Of N, N-diisopropyl phosphoramidite
2′-O-methyluridine (9.80 g, 40.1 mmol) was dissolved in 0.5 M aqueous KOH solution (80 mL), paraformaldehyde (20.0 g, 667 mmol) was added, and the mixture was stirred at a temperature of 65 ° C. for 1 week. After the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography (chloroform: methanol = 96: 4) and further recrystallized with methanol to obtain 5-hydroxymethyl-2′-O-methyluridine. (4.40 g, yield; 40%) was obtained.
mp 183.0-185.0 ° C; ¹ H NMR (270 MHz, DMSO) δ3.33 (3H, s, 2'-OCH ₃ ), 3.52-3.64 (2H, m, 5'-H), 3.78-3.84 (2H, m, 3'-H, 4'- H), 4.11 (3H, bs, CH 2, OH), 4.88 (1H, bs, OH), 5.08 (1H, bs, OH), 5.14-5.16 (1H, m , 2'-H), 5.89 (1H, d, 1'-H, J _{1 ', 2'} = 5.6 Hz), 7.81 (1H, s, 6-H), 11.35 (1H, s, 3-NH) ¹³ C NMR (67.8 MHz, DMSO) δ56.30, 57.46, 60.83, 68.45, 82.42, 85.25, 85.69, 114.39, 136.88, 150.51, 162.64; ESI-mass m / z Calcd for C ₁₁ H ₁₇ N ₂ O ₇ 289.1036; Observed [M + H] 289.1034

5-Hydroxymethyl-2′-O-methyluridine (1.0 g, 3.65 mmol) obtained as described above was dissolved in 4.0 M HCl dioxane solution (10 mL) and stirred at a temperature of 0 ° C. for 2.5 hours. . After the solvent was distilled off under reduced pressure, HCl was azeotroped with anhydrous dioxane. The residue was dissolved in anhydrous dioxane (10 mL), 40% CH ₃ NH ₂ (methanol solution) (20 mL) was added, and the mixture was stirred at room temperature for 10 hr. The solvent was distilled off under reduced pressure, the residue was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (30 mL), trifluoroacetic anhydride (772 μL, 5.47 mmol) was added, and Stir for 3 hours. The reaction was stopped by adding 1 ml of methanol, and then the solvent was distilled off under reduced pressure. The residue was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (30 mL), 4,4'-dimethoxytrityl chloride (1.36 g, 4.02 mmol) was added, and the mixture was added at room temperature under argon atmosphere at room temperature. Stir for hours. After stopping the reaction by adding methanol, the reaction solution was diluted with 200 ml of chloroform and washed twice with water. Furthermore, the organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 50: 50), and 5′-O- (4,4′-dimethoxytrityl) -5- [N- (trifluoroacetyl) -methylaminomethyl. ] -2′-O-methyluridine was obtained (900 mg, yield: 36%).
¹ H NMR (270 MHz, CDCl ₃ ) δ 3.18 (3H, s, CH ₃ -N), 3.46-3.85 (14H, m, 2'-OCH ₃ , OCH ₃ of DMTr, CH ₂ , 4'-H , 5'−H), 4.06−4.11 (1H, m, 3′−H), 4.23−4.26 (1H, m, 2′−H), 5.92 (1H, d, 1′−H, J _{1 ′, 2 '} = 2.1 Hz), 6.79-7.45 (13H, m, ArH of DMTr), 7.87 (1H, s, 6-H); ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ36.80, 46.84, 55.17, 58.77 , 62.65, 68.96, 83.28, 83.48, 86.66, 87.56, 108.41, 113.20, 114.05, 118.28, 126.89, 127.91, 128.24, 130.26, 135.56, 135.68, 141.61, 144.63, 149.77, 156.63, 157.16, 158.57, 163.2s; E m / z Calcd for C ₃₅ H ₃₇ F ₃ N ₃ O ₉ 722.2301; Observed [M + H] 722.2301

5′-O- (4,4′-dimethoxytrityl) -5- [N- (trifluoroacetyl) -methylaminomethyl] -2′-O-methyluridine (300 mg, 0.43 mmol) was azeotropically dehydrated three times with anhydrous acetonitrile and once with anhydrous dichloromethane, and then dissolved in anhydrous dichloromethane (4.3 mL), and 1-H tetrazole (30.1 mg, 0.43 mmol) and diisopropylamine (60 μL, 0.43 mmol) was added, and the mixture was stirred at room temperature for 10 minutes under an argon atmosphere. Further, (2-cyanoethoxy) -bis- (N, N-diisopropylamino) phosphine (273 μL, 0.86 mmol) was added, and the mixture was stirred at room temperature for 3 hours under an argon atmosphere. The reaction was stopped by adding 1 ml of water, diluted with 30 ml of chloroform and washed 3 times with saturated saline. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was diluted with a mixed solvent of diethyl ether: diisopropyl ether = 1: 1, washed 5 times with 0.1 M aqueous sodium hydroxide solution, the organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. . The residue was purified by recycle preparative HPLC (acetonitrile) and 5'-O- (4,4'-dimethoxytrityl) -5- [N- (trifluoroacetyl) -methylaminomethyl] -2'-O- Methyluridine 3′-O- (2-cyanoethyl) -N, N-diisopropyl phosphoramidite (a compound represented by the following formula (4)) was obtained (210 mg, yield: 55%).
¹ H NMR (270 MHz, CDCl ₃ ) δ0.95-0.97 (4H, m), 1.10-1.15 (12H, m), 2.35-2.39 (1H, m), 2.60-2.70 (1H, m), 3.17 ( 3H, s), 3.30-3.50 (7H, m), 3.71-3.83 (7H, m), 3.90-3.98 (1H, m), 4.18-4.25 (1H, m), 5.90-5.99 (1H, m), 6.75-6.90 (4H, m), 7.20-7.46 (13 H, m), 7.83, 7.90 (1H, 2s); ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ20.10, 20.27, 22.85, 24.42, 24.54, 36.59, 43.08, 43.24, 43.42, 46.84, 55.16, 55.20, 58.36, 58.51, 58.78, 62.92, 68.34, 70.01, 82.32, 82.57, 86.69, 88.04, 108.24, 108.34, 112.95, 113.17, 126.98, 127.89, 128.36, 128.42 130.32, 130.38, 135.65, 141.59, 144.57, 149.51, 158.60, 158.79, 162.68; ³¹ P NMR (109.4 MHz, CDCL ₃ ) δ151.13, 151.15; ESI-mass m / z Calcd for C ₄₄ H ₅₄ F ₃ N ₅ O ₁₀ P 900.3560; Observed [M + H] 900.3580

  The synthesis reaction of Comparative Production Example 1 is represented by the following formula.

Production Example 2
3 ′, 5′-bis-acetyl-5- [3- (N-trifluoroacetyl) -aminopropyl] -2′-O-methyluridine (400 mg, 0.80 mmol) was added to pyridine, 28% aqueous ammonia ( It was dissolved in a mixed solvent (8 mL) of v / v = 1: 1) and stirred at a temperature of 50 ° C. for 8 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in ethanol (8 mL), trifluoroacetic acid ethyl ester (191 μL, 1.60 mmol) and triethylamine (455 μL, 2.40 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure, the residue was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (8 mL), and 4,4'-dimethoxytrityl chloride (325 mg, 0.96 mmol) was added. The mixture was stirred at room temperature for 3 hours under an argon atmosphere. After stopping the reaction by adding 1 ml of methanol, the reaction solution was diluted with 50 ml of chloroform and washed 3 times with 5% aqueous sodium hydrogen carbonate solution. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Distilled off. The residue was purified by silica gel chromatography (hexane: ethyl acetate = 50: 50), and 5′-O- (4,4′-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyl. ] -2′-O-methyluridine was obtained (450 mg, yield: 80%).
¹ H NMR (270 MHz, CDCl ₃ ) δ 1.69−1.74 (2H, m, CH ₂ ), 2.14−2.19 (2H, m, CH ₂ ), 2.32−2.40 (2H, m, CH ₂ ), 3.53 ( 3H, s, 2'-O- Me), 3.70-3.85 (8H, m, OCH 3 of DMTr, 5'-H), 3.91-3.99 (2H, m, 3'-H, 4'-H), 4.23−4.31 (1H, m, 2′−H), 5.76 (1H, d, 1′−H, J _{1 ′, 2 ′} = 3.3 Hz), 6.77−6.80 (4H, d, ArH of DMTr, J _{1 ', 2'} = 8.9 Hz), 7.15-7.39 (10H, m, ArH of DMTr, NH), 7.59 (1H, s, 6-H); ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ24.22, 28.18 , 42.70, 55.19, 58.63, 60.91, 68.43, 69.99, 82.89, 84.80, 89.19, 113.09, 114.40, 126.24, 127.82, 128.42, 129.72, 136.68, 138.33, 146.52, 149.80, 157.86, 162.82; ESI-mass m / z Calcd for C ₃₆ H ₃₈ F ₃ N ₃ NaO ₉ 736.2458; Observed [M + Na] 736.2736

5'-O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyl] -2'-O-methyluridine (250 mg) obtained as described above. , 0.35 mmol) was azeotropically dehydrated three times with anhydrous acetonitrile and once with anhydrous dichloromethane, then dissolved in anhydrous dichloromethane (3.5 mL), and 1-H tetrazole (14.7 mg, 0.21 mmol) and diisopropylamine (29.5 μL, 0.21 mmol) was added, and the mixture was stirred at room temperature for 10 minutes under an argon atmosphere. Furthermore, (2-cyanoethoxy) -bis- (N, N-diisopropylamino) phosphine (133 μL, 0.42 mmol) was added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere. The reaction was stopped by adding 1 ml of water, diluted with 20 ml of chloroform and washed 3 times with saturated saline. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was diluted with a mixed solvent of diethyl ether: diisopropyl ether = 1: 1 and washed 5 times with 0.1 M aqueous sodium hydroxide solution. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by recycle preparative HPLC (acetonitrile) and 5'-O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyl] -2'-O. -Methyluridine 3'-O- (2-cyanoethyl) -N, N-diisopropyl phosphoramidite (compound represented by formula (3)) was obtained (140 mg, yield: 44%).
¹ H NMR (270 MHz, CDCl ₃ ) δ0.91-1.10 (14H, m), 1.19-1.28 (2H, m), 1.50-1.88 (2H, m), 2.26-2.31 (1H, m), 2.52− 2.57 (1H, m), 2.81−3.23 (2H, m), 3.43 (3H, s), 3.43−3.57 (2H, m), 3.68, 3.69 (6H, 2s), 3.80−3.91 (1H, m), 4.05−4.19 (1H, m), 4.32−4.48 (1H, m), 5.90−6.06 (1H, m), 7.17−7.35 (10H, m), 7.44, 7.50 (1H, 2s); ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ20.05, 20.16, 20.24, 20.32, 23.05, 24.39, 24.50, 24.59, 28.28, 38.27, 42.95, 43.13, 43.31, 55.17, 57.70, 57.99, 58.18, 58.50, 58.74, 61.64, 62.40, 70.11 , 70.33, 82.39, 82.82, 83.52, 86.70, 86.84, 87.32, 113.12, 113.79, 114.02, 117.47, 117.99, 127.24, 127.86, 128.22, 128.39, 128.94, 130.18, 130.30, 135.05, 135.13, 136.38, 144.05, 156.69, 15 , 158.71; ³¹ P NMR (109.4 MHz, CDCL ₃ ) δ 151.17, 151.23; ESI-mass m / z Calcd for C ₄₅ H ₅₆ F ₃ N ₅ O ₁₀ P 914.3717; Observed [M + H] 914.3711

  The synthesis reaction of Production Example 1 is represented by the following formula.

Production Example 2
5'-O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyn-1-yl] -2'-O-methyluridine 3'-O- ( Synthesis of 2-cyanoethyl) -N, N-diisopropyl phosphoramidite 3 ′, 5′-bis-acetyl-5-iodo-2′-O-methyluridine (2.0 g, 4.3 mmol), a known compound After azeotropic dehydration 3 times with 3 ml of anhydrous acetonitrile and once with 3 ml of anhydrous dimethylformamide, the product was dissolved in anhydrous dimethylformamide (23 mL), and ultrasonic deaeration was performed 5 times while reducing the pressure, followed by substitution with argon. Triethylamine (1.63 mL, 8.6 mmol), tetrakis (triphenylphosphine) palladium (0) (248 mg, 0.215 mmol) and copper iodide (81.9 mg, 0.43 mmol) were added, and the mixture was stirred at room temperature for 10 minutes. N-trifluoroacetyl-aminopropyne (730 μL, 6.4 mmol) is dissolved in degassed anhydrous dimethylformamide (20 mL) and added dropwise to the reaction system using a dropping funnel. Stir for hours. The reaction solution was diluted with 150 ml of ethyl acetate, washed 3 times with 5% aqueous sodium hydrogen carbonate solution and twice with 1% aqueous ammonia. The organic layer was collected and dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 60: 40, hexane: chloroform = 20: 80) 3 ′, 5′-bis-acetyl-5- [3- (N-trifluoroacetyl) -aminopropyn-1-yl] -2′-O-methyluridine was obtained (1.41 g, yield: 67 %).
¹ H NMR (270 MHz, CDCl3) δ 2.13, 2.17 (6H, 2s, Ac), 3.48 (3H, s, 2'-O-Me), 4.04-4.07 (1H, m, 5'-H), 4.29-4.42 (5H, m, 3'- H, 4'-H, 5'-H, CH 2 -NH), 4.89-4.94 (1H, m, 2'-H), 5.90 (1H, d, 1 '-H, J _{1', 2 '} = 2.3 Hz), 7.69 (1H, bs, NH), 7.92 (1H, s, 6-H), 9.77 (1H, s, 3-NH); ¹³ C NMR ( 67.8 MHz, CDCl ₃ ) δ20.56, 20.68, 30.26, 59.00, 61.77, 69.37, 75.23, 79.35, 81.77, 87.87, 88.63, 99.02, 113.55, 117.78, 142.96, 148.98, 156.75, 157.31, 162.02, 1770.23, 170.32; ESI-mass m / z Calcd for C ₁₉ H ₂₁ F ₃ N ₃ O ₉ 492.1230; Observed [M + H] 492.1290

3 ′, 5′-bis-acetyl-5- [3- (N-trifluoroacetyl) -aminopropyn-1-yl] -2′-O-methyluridine (870 mg) obtained as described above. , 1.77 mmol) was dissolved in a mixed solvent (18 mL) of pyridine and 28% aqueous ammonia (v / v = 1: 1) and stirred at a temperature of 50 ° C. for 8 hours. After the solvent was distilled off under reduced pressure, the residue was dissolved in ethanol (18 mL), ethyl trifluoroacetate (844 μL, 7.08 mmol) and triethylamine (1.68 mL, 8.85 mmol) were added, and the mixture was stirred at room temperature for 2 hours. did. The solvent was distilled off under reduced pressure, the residue was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (18 mL), and 4,4'-dimethoxytrityl chloride (718.3 mg, 2.12 mmol) was added. The mixture was stirred at room temperature for 3 hours under an argon atmosphere. After stopping the reaction by adding methanol, the reaction solution was diluted with 50 ml of chloroform and washed 3 times with 5% aqueous sodium hydrogen carbonate solution. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. did. The residue was purified by silica gel chromatography (hexane: ethyl acetate = 50: 50), and 5′-O- (4,4′-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopro Pin-1-yl] -2′-O-methyluridine was obtained (800 mg, yield: 50%).
¹ H NMR (270 MHz, CDCl ₃ ) δ 3.47 (2H, s, CH ₂ ), 3.63 (3H, s, 2′-O—CH ₃ ), 3.77-3.81 (8H, m, OCH ₃ of DMTr, 5'−H), 3.92−3.97 (1H, m, 4′−H), 4.08−4.11 (1H, m, 3′−H), 4.52−4.55 (1H, m, 2′−H), 5.93 ( 1H, s, 1'-H), 6.82-6.86 (4H, m, ArH of DMTr), 7.21-7.54 (10H, m, ArH of DMTr, NH), 8.25 (1H, s, 6-H); ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ30.17, 55.12, 58.74, 61.64, 68.45, 75.02, 83.72, 86.84, 87.51, 98.95, 113.31, 117.63, 121.87, 126.87, 127.81, 128.00, 129.88, 129.94, 135.40, 135.43 , 143.20, 144.52, 149.31, 155.66, 156.22, 156.77, 157.33, 158.51, 158.55, 162.46; ESI-mass m / z Calcd for C ₃₆ H ₃₄ F ₃ N ₃ NaO ₉ 732.2145; Observed [M + Na] 732.2127

5'-O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyn-1-yl] -2'-O- obtained as described above. Methyluridine (400 mg, 0.56 mmol) was azeotropically dehydrated three times with anhydrous acetonitrile and once with anhydrous dichloromethane, then dissolved in anhydrous dichloromethane (5.6 mL), and 1-H tetrazole (23.8 mg, 0.34 mmol) and diisopropyl Amine (47.8 μL, 0.34 mmol) was added, and the mixture was stirred at room temperature for 10 minutes under an argon atmosphere. Further, (2 cyanoethoxy) -bis- (N, N-diisopropylamino) phosphine (212 μL, 0.67 mmol) was added, and the mixture was stirred at room temperature for 3 hours under an argon atmosphere. The reaction was stopped by adding 1 ml of water, diluted with 30 ml of chloroform and washed 3 times with saturated saline. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was diluted with a mixed solvent of diethyl ether: diisopropyl ether = 1: 1 and washed 5 times with 0.1 M aqueous sodium hydroxide solution. The powder precipitated at this time was dissolved in anhydrous tetrahydrofuran (5.6 mL), trifluoroacetic acid ethyl ester (267 μL, 2.24 mmol) and triethylamine (850 mL, 4.48 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the residue was purified by recycle preparative HPLC (acetonitrile), and 5′-O- (4,4′-dimethoxytrityl) -5- [3- (N-trifluoroacetyl)- Aminopropyn-1-yl] -2′-O-methyluridine 3-O- (2-cyanoethyl) -N, N-diisopropyl phosphoramidite (compound represented by formula (2)) was obtained (200 mg). Yield: 40%).
¹ H NMR (270 MHz, CDCl ₃ ) δ0.96-0.98 (4H, m), 1.07-1.11 (10H, m), 2.29-2.31 (1H, m), 2.51-2.55 (1H, m), 3.31- 3.52 (6H, m), 3.52−3.83 (7H, m), 3.98−4.05 (1H, m), 4.13−4.22 (1H, m), 4.40−4.59 (1H, m), 5.57−5.86 (1H, m ), 6.75-6.78 (4H, m), 7.04 (1H, bs), 7.07-7.41 (9H, m), 8.15, 8.19 (1H, 2s,); ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ 7.47 , 20.27, 20.31, 20.37, 24.36, 24.43, 24.47, 24.54, 24.64, 30.12, 43.04, 43.23, 55.13, 58.18, 58.37, 58.40, 58.46, 58.83, 58.87, 75.34, 75.51, 76.53, 82.74, 86.64, 86.80, 86.84 , 86.86, 88.14, 88.60, 98.61, 98.91, 113.22, 113.33, 117.56, 117.59, 117.75, 126.81, 126.85, 127.90, 127.93, 128.10, 130.03, 135.32, 135.39, 135.44, 135.46, 143.40, 144.49, 144.63, 149.3 , 156.10, 156.65, 158.51, 158.55, 162.13, 162.27; ³¹ P NMR (109.4 MHz, CDCL ₃ ) δ 150.58, 150.97; ESI-mass m / z Calcd for C ₄₅ H ₅₁ F ₃ N ₅ NaO ₁₀ P 932.3223; Observed [M + Na] 932.3892
The synthesis reaction of Production Example 2 is represented by the following formula.

Example 1
Using the nucleoside phosphoramidite compounds obtained in Production Examples 1 and 2 and Comparative Production Example 1, 2′-O-methylated 9 of sequence 5′-CGUUU ^* UUGC-3 ′ (SEQ ID NO: 1) An oligonucleotide incorporating a modified nucleic acid at the U ^* site in the monomer was synthesized. Oligonucleotides were synthesized by an ordinary ABI 392 synthesizer (Applied Biosystems) by the usual phosphoramidite method, cut out from the solid phase carrier, and deprotected at the base, and then separated by a Sep-pak C18 cartridge column. Trityl ON simple purification was performed, and further purification was performed with anion HPCL as necessary. Each synthesized oligonucleotide and a separately synthesized complementary strand (sequence 5′-GCAAAACG-3 ′ (SEQ ID NO: 2) or 5′-GCAAGACG-3 ′ (SEQ ID NO: 3)) are mixed and mixed in a solution. At the time of duplex formation, a measurement sample dissolved in 500 μL of phosphate buffer (10 mM sodium phosphate, pH 7.0, 150 mM NaCl, 0.1 mM EDTA) so that the concentration of the duplex becomes 2 μM. For the measurement, Pharma Spec UV-1700 (manufactured by Shimadzu Corporation) was used, and the measurement was performed by first holding the sample at a temperature of 80 ° C. for 30 minutes, and then setting the oligonucleotide in a random coil state. Annealing is performed by changing the temperature to 5 ° C. at 1.0 ° C./min, and then the temperature is increased at a rate of 1.0 ° C./min, and UV absorbance is measured every 1.0 ° C. It was carried out by.
The UV absorbance obtained by the measurement was plotted against the temperature change to obtain a double chain melting curve. After smoothing the melting curve using the Stabilzky-Golay method (25 points), the curve was linearly differentiated to obtain the inflection point, and the inflection point was defined as the double chain melting temperature (Tm value). In addition, in the value obtained from the curve at the time of annealing of the double chain and the value obtained from the curve at the time of melting the double chain, when a deviation of 1.0 ° C. or more is observed, The equilibrium was considered to be incomplete, and the measurement was performed again while changing the conditions as appropriate, such as changing the temperature change rate to 0.5 ° C./min.

  When the compound obtained in Comparative Production Example 1 was incorporated into an A-type RNA duplex, the difference in melting temperature (ΔTm value) was −1 compared to the case where 2′-O-methyluridine was incorporated. The temperature was 2 ° C., and even when the compound obtained in Comparative Production Example 1 was incorporated into the A-type RNA duplex, duplex stabilization was not observed. When the compound obtained in Production Example 1 was incorporated into the A-type RNA duplex, the difference in melting temperature (ΔTm value) was + 3.2 ° C. compared to the case where 2′-O-methyluridine was incorporated. It was found that the A-type RNA duplex was stabilized. This indicates that the amino group and the phosphate group are close to each other without distorting the duplex, and in order to stabilize the A-type RNA duplex, a linker having 3 carbon atoms must be introduced. Is shown to be good. Further, when the compound obtained in Production Example 2 was incorporated into an A-type RNA duplex, the difference in melting temperature (ΔTm value) was +6.9 compared to the case where 2′-O-methyluridine was incorporated. It was observed that the temperature of the A-type RNA duplex was greatly stabilized.

Example 2
The Tm value when the base at the complementary position was G was subtracted from the Tm value when the base at the complementary position in Production Examples 1 and 2 and Comparative Production Example 1 was A, and the value was defined as the base discrimination ability.

The results are shown in FIG. FIG. 1 is a graph showing measurement results of base discrimination ability. In the nucleoside phosphoramidite compound obtained in Comparative Production Example 1, ΔTm _AG is 7.4 ° C., and Production Example 1 and Production Example In the nucleoside phosphoramidite compound obtained in 2, ΔTm _AG was 8.1 ° C. and 10.3 ° C., respectively. It is known that in the modified form that stabilizes the A-type RNA duplex, the base discrimination ability is often lowered. However, when the nucleoside phosphoramidite compound of the present invention is used, the base discrimination ability is not lowered. Rather, it was found that the base discrimination ability was improved.

It is a graph which shows the measurement result of base discrimination ability.

Claims (9)

A nucleoside phosphoramidite compound represented by the following general formula (1).

(In the above formula, R ¹ represents a phosphate protecting group, R ² represents a dialkylamino group in which two identical or different alkyl groups having 1 to 6 carbon atoms are bonded on a nitrogen atom, and R ³ represents an alkoxy group. Or bonded to the 4′-position carbon of ribose to form a ring, and R ⁴ is an alkyl group having 3 or more carbon atoms, an alkenyl group having 3 or more carbon atoms, or a group having 3 or more carbon atoms. Represents an alkynyl group, the alkyl group, alkenyl group or alkynyl group may be bonded to a trifluoroacetylamino group, R ⁵ represents a protecting group for a hydroxyl group, and X represents an oxygen atom or a sulfur atom.) The nucleoside phosphoramidite compound according to claim 1, wherein R ¹ is a methyl group, a 2-cyanoethyl group, or a 2-trimethylsilylethyl group. The nucleoside phosphoramidite compound according to claim 1, wherein R ² is a diisopropylamino group. The nucleoside phosphoramidite compound according to claim 1, wherein R ³ is a methoxy group. The nucleoside phosphoramidite compound according to claim 1, wherein R ⁴ is a propyl group or a propanyl group bonded to a trifluoroacetylamino group. The nucleoside phosphoramidite compound of Claim 1 represented by following formula (2) or (3).

The oligonucleic acid which contains the modified nucleoside as a structural component by using the nucleoside phosphoramidite compound of any one of Claims 1-6. A probe for gene analysis comprising the oligonucleic acid according to claim 7. A primer for starting amplification of a specific gene using the oligonucleic acid according to claim 7.

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