WO2005085270A1

WO2005085270A1 - Nucleic acid derivative having pyrrolyl group introduced in 5-position of pyridine ring

Info

Publication number: WO2005085270A1
Application number: PCT/JP2005/003469
Authority: WO
Inventors: Mitsuo Sekine; Kohji Seio; Ken-Ichi Miyata; Ryota Mineo
Original assignee: Japan Science And Techonlogy Agency
Priority date: 2004-03-04
Filing date: 2005-03-02
Publication date: 2005-09-15
Also published as: JPWO2005085270A1

Abstract

An artificial nucleic acid which employs a precise copy of the hydrogen bond mode of a protonated cytosine base and has a high triplex-forming ability. It is a nucleic acid derivative represented by the following general formula (I) or a tautomer thereof.

Description

Specification

Nucleic acid derivative with a pyrrolyl group at the 5-position of the pyridine ring

Technical field

The present invention relates to nucleic acid derivatives such as peridine and cytidine having a pyrrolyl group introduced at the 5-position of the pyridine ring.

Background art

[0002] DNA and RNA can have a triple-stranded structure, and for that purpose, the 3N position of the pyrimidine base needs to be protonated. Several artificial nucleic acids capable of forming a triple strand under neutral conditions have been reported so far (Non-Patent Documents 1 and 2). The method accurately imitates the hydrogen-binding mode of 1S protonated cytosine bases and has high!人工 There is no artificial nucleic acid capable of forming triplex yet.

[0003] Non-Patent Document 1: T 丄. Trapane, MS Christopherson, CD Roby, P.O.Ts'o, D.

Wang, J. Am. Chem. Soc, 116, 8412 (1994)

Non-Patent Document 2: V.S.Rana, K.N.Ganesh, Nucleic Acids Res., 28, 1162 (2000) Non-Patent Document 3: A. Avino, M. Frieden, J.C. Morales, B.G. de la Torre, R. G. Garcia,

F. Azorin, J. L. Gelpi, M. Orozco, C. Gonzalez, R. Eritja, Nucleic Acids Res., 30,

2609 (2002)

Disclosure of the invention

Problems to be solved by the invention

[0004] Creation of an artificial nucleic acid that accurately mimics the hydrogen bonding mode of a protonated cytosine base and has a high triple-strand forming ability is urgently required for the development of nucleic acid application techniques such as the antigene method. Thus, the present inventors have conducted intensive studies with the aim of designing a novel modified cytidine derivative and a peridine derivative to create a high triple-stranded nucleic acid, and as a result, completed the present invention.

Means for solving the problem

That is, the present invention provides a nucleic acid derivative represented by the following general formula I: [0006] [chemical 0]

[0007] (In Formula I, Al, A2, and A3 are the same or different and are hydrogen, an alkyl group having 1 to 6 carbon atoms, a halogenated alkyl group having 1 to 3 carbon atoms having 1 halogen, Alkoxy having 1 to 6 carbon atoms, dialkylamino group having two identical or different alkyl groups having 1 to 6 carbon atoms on a nitrogen atom, chlorine, bromine, iodine, fluorine, cyano, nitro, or 1 to 7 carbon atoms A1 and A2, or A2 and A3 together form a cyclic structure, and E has a hydrogen atom, an alkoxycarbonyl group having 15 to 15 carbon atoms, and a substituent. Represents a benzyloxycarbol group or a 9-fluorenylmethoxycarboxy- ゥ group, X represents hydrogen, a hydroxyl group, or an alkyloxy group; Y represents a hydrogen atom, a 4-methoxytrityl group, or Represents a 4 'dimethoxytrityl group, Z may have a substituent on a carbon atom, or may be a phenyl group, a benzoyl group, a hydroxyl group, an amino group, or a nitrogen atom, which may have a substituent on a phenyl group; Represents a dialkylamino group having two identical or different alkyl groups having 1 to 6 carbon atoms on the atom.) Or their isomers.

[0008] The present invention further provides the above-described nucleic acid derivative, wherein Y represents a 4-methoxytrityl group or a 4,4'-dimethoxytrityl group, and the 3'-hydroxyl group of the sugar is phosphoramidite, and the nucleic acid derivative. The present invention relates to a nucleic acid oligomer such as a DNA oligomer or an RNA oligomer containing (a part excluding Y). The invention's effect

[0009] The novel artificial nucleic acid of the present invention induces tautomerism by intramolecular hydrogen bonding between a pyrrolyl group and cytosine 4-position, even if the 3N-position of the cytidine base is not actually protonated. Under the conditions, it is expected that the base recognition can be performed accurately because it has the same hydrogen bonding mode as the protonated cytosine base. In addition, the combined use of a modified ゥ -lysine derivative allows the hydrophobic interaction between pyrrole rings to be achieved under neutral conditions. /, Can acquire the ability to form triple chains.

Brief Description of Drawings

[0010] FIG. 1 shows the structure of a hairpin duplex used in a binding experiment with TFO.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] In the nucleic acid derivative of the present invention, a typical example of Al, A2 and A3 is hydrogen, A1 and A2, or an example of a cyclic structure formed by A2 and A3 is a cycloalkane or benzene ring. Can be done. Preferred examples of E include a hydrogen atom, a benzyloxycarbonyl group and a t-butoxycarbonyl group, preferred examples of X include an alkyloxy group having 1 to 6 carbon atoms such as a methoxy group, and preferred examples of Y include a hydrogen atom Examples of Z and Z include a hydroxyl group (peridine derivative), an amino group (cytosine derivative), and a dialkylamino group such as an N-dimethylaminoethylene group.

[0012] The nucleic acid oligomer containing the nucleic acid derivative of the present invention has a phosphoramidite in the 3 'hydroxyl group of the sugar in the nucleic acid derivative. Those skilled in the art can easily prepare it.

Specifically, the phosphoramidite group has the general formula (式): -P (OR3) N (R4) (R5) (II): (wherein R3 is a phosphate-protecting group, and R4 and R5 Is an alkyl group or an aryl group). Examples of the phosphate-protecting group include 2-cyanoethyl, 412-trophenylethyl, N (trifluoroacetyl) aminobutyl, and 4 [N-methyl-N- (2,2,2, -trifluoro). (Aroacetyl) amino] butyl group, and a 2-cyanoethyl group is preferred. Preferred examples of R4 and R5 are an alkyl group having 14 to 14 carbon atoms, a benzyl group, a phenyl group, or a naphthyl group, and an isopropyl group is particularly preferred. Hereinafter, the present invention will be described in more detail with reference to Examples. The technical scope of the present invention is not limited by these descriptions.

Example 1

[0014] 3,5,1-O-Diacetyl-5- [N- (tert-butoxycarbol) pyrroyl-2-yl] deoxyperidine

[0015] [Formula 1]

3 ', 5,1-O-diacetyl-5-ododoxydiridine (219 mg, 0.5 mmol), N- (tert-butoxycarbol) -pyrrole-2-boronic acid (106 mg, 0.5 mmol) ), Radium acetate (5.6 mg, 0.025 mmol), triphenylphosphine-3,3 ', 3, trisulfoacid trisdium salt (20 mg, 0.035 mmol), sodium carbonate (53 mg, 1. Ommol) is dissolved in degassed distilled water-acetonitrile (2: 1, 5 mL). After stirring at 60 ° C for 30 minutes, N (tert-butoxycarbol) pyrrolidine 2-boronic acid (106 mg ゝ 0.5 mmol), palladium acetate (5.6 mg ゝ 0.025 mmol), triphenylphosphine 3 , 3 ', 3 ,,-Trisulfonic acid trisodium salt (20 mg, 0.035 mmol) is added. After stirring at 60 ° C for 30 minutes, dilute with water (10 mL) and distill off the solvent under reduced pressure until the volume becomes half. After washing the form (20 mL), the organic layer is washed with a saline solution and a saturated aqueous solution of sodium hydrogencarbonate. The organic layer is recovered, dried using sodium carbonate, and the solvent is distilled off under reduced pressure. Silica gel column chromatography using C200 silica gel Purify by chromatography (ethyl acetate, hexane) to obtain the title compound (146 mg, 62%).

[0017] 1H NMR (CDC13) δ 1.50 (9Hs) 1.91 (3Hs) 2.01 (3Hs) 2.09— 2.27 (1Hm), 2.45-2.54 (1Hm), 4.23—4.34 (3Hm), 5.18— 5.22 (1H m), 6.14-6.16 (2H m), 6.34 (1H m), 7.31-7.32 (1H br), 7.48 (1H s), 8.37 (1H br)

Example 2

[0018] 5- [N- (tert-butoxycarbol) pyrroyl-2-yl] deoxydziridine (I-Dai-dani-yakuhin 2)

[0020] 3,5 O-Diacetyl-5- [N- (tert-butoxycarbol) pyrroyl-2-yl] deoxyduridine (I-drug 1) (3.94 g, 8.25 mmol) was concentrated in ethanol. After dissolving in aqueous ammonia (1: 1 vZv 80 mL) and stirring at room temperature for 8 hours, the solvent is distilled off under reduced pressure. After removing pyridine (50 mL) and 60N silica gel (10 g), the solvent is distilled off under reduced pressure. The title compound (3.06 g, 94%) is obtained by silica gel column chromatography using 60N silica gel (form-mouth methanol).

^{[0021] J H NMR (DMSO} -d6) δ 1.41 (9Η s), 2.09- 2.14 (2Η m), 3.49- 3.61 (2Η m),

3.75-3.80 (1 m), 4.22-4.29 (1 m), 4.99 (1 t J = 4.9 Hz) 5.23 (1 H d J = 4.3 Hz) 6.13-6.22 (3 H m), 7.91 (1 H s), 11.5 ( 1H br)

Example 3 [0022] 5 (pyrrole 2 yl) deoxyperidine

[0023]

[0024] 5- [N- (tert-butoxycarbyl) pyrroyl-2-yl] deoxyperidine (I-conjugated product 2) (393 mg, lmmol) is azeotropically dehydrated with anhydrous acetonitrile, and then dried with anhydrous methanol (1 OmL). Add sodium methoxide (3.1 M methanol solution, 3.23 mL, 10 mmo 1) and stir at room temperature for 1 hour. After dilution by adding water (10 mL), the mixture was neutralized with an aqueous hydrochloric acid solution. The precipitate formed when the solvent is roughly distilled off is filtered and washed with cold water. Drying under reduced pressure gives the title compound (241 mg, 83%).

^{[0025] J H NMR (DMSO-} d6) δ 2.09- 2.25 (2H, m), 3.56- 3.67 (2Η, m), 3.79- 3.81 (1Η, m), 4.24- 4.31 (1Ηゝm), 5.15 ( 1H, br) ゝ 5.26 (1H, br) 2 6.02-6.03 (1H, br) ゝ 6.22 (1H, dd, J = 6.8Hz, J = 6.6Hz) ゝ 6.38 (1H, br) ゝ 6.75 (1H, br) ) ゝ 8.12 (1H, s) ゝ 10.85 (1H, br) ゝ 11.54 (1H ゝ br)

Example 4

[0026] 5, —0— (4,4, dimethoxytrityl) -5— [N— (tert-butoxycarbol) pyrroyl-2-yl] deoxyperidine (compound 4) [0027] [Formula 4]

[0028] 3 ,, 5, 1-diacetyl-5- [N- (tert-butoxycarbyl) pyrroyl-2-yl] deoxydiziridine (I-Dai-drug 1) (270 mg, 0.55 mmol) in pyridine Dissolve in concentrated aqueous ammonia (1: 2, v Zv, 10 mL) and stir at room temperature for 8 hours. After evaporating the solvent under reduced pressure, azeotropic dehydration is performed using anhydrous pyridine. Anhydrous pyridine (6 mL) and 4,4'-dimethoxytrityl chloride (203 mg, 0.6 mmol) are added, and the mixture is stirred at room temperature for 2 hours. After diluting with chloroform (15 mL), wash the organic layer three times with saturated aqueous sodium bicarbonate. The organic layer is collected, dried over sodium sulfate, and the solvent is distilled off under reduced pressure. Purify by silica gel column chromatography using C200 silica gel (hexane ethyl acetate, 1% pyridine) to obtain the title compound (360 mg, 94%).

[0029] 1H NMR (CDC13) δ 1.49 (9H, s), 2.21-2.34 (1H, m), 2.42-2.51 (1H, m),

3.27- 3.41 (2Η, m), 3.77 (6Η, s) ゝ 4.05- 4.08 (1H ゝ m), 4.53-4.58 (1H ゝ m), 5,67―5.71 (1H, m), 5.93―5.95 (1H , M), 6.38 (1H, dd, J = 6.0Hz ゝ J = 5.9Hz), 6.73-6.77 (4H, m), 7.18-7.34 (10H, m) ゝ 7.58 (1H, s) ゝ 8.56 (1H, br)

Example 5

[0030] 5, —0— (4,4, dimethoxytrityl) -5— [N— (tert-butoxycarbol) pyrroyl-2-yl] deoxyperidine 3, — (2-cyanoethyl N, N-diisopropyl Phosphoramidite) (compound 5) [0031] [Formula 5]

[0032] 5'-0- (4,4, dimethoxytrityl) -5- [N- (tert-butoxycarbol) pyrroyl-2-yl] deoxydiziridine (I-Dailyid 4) (300 mg, 0. 43 mmol) is azeotropically dehydrated with anhydrous acetonitrile and then dissolved in anhydrous ethylene chloride (5 mL). N, N-diisopropylamine (37 μ 0.26 mmol), 1H-tetrazole (18 mg ゝ 0.26 mmol), (2-cyanoethoxy) -bis (N, N-diisopropylamino) phosphine (165 L, 0.52 mmol) And stir at room temperature for 3 hours. After diluting with chloroform (5 mL), the organic layer is washed with a saturated aqueous sodium hydrogen carbonate solution. The organic layer is recovered, dried using anhydrous sodium sulfate, and the solvent is distilled off under reduced pressure. Dissolve in isopropyl ether-ethyl ether (1: 1, vZv, 10 mL) and wash the organic layer five times with 0.1 M aqueous sodium hydroxide solution. The organic layer is recovered and the solvent is distilled off under reduced pressure. Purification using a recycle HPLC (0.5% in form: black mouth) equipped with a GPC column gives the title compound (282 mg, 73%).

[0033] ^J H NMR (CDC13) δ 1.05-1.15 (12H, m) ゝ 1.47 (9H, s) ゝ 2.21- 2.36 (1H ゝ m),

2.40-2.63 (3H, m) ゝ 3.23-3.38 (2H, m), 3.5 g 3.86 (4H ゝ m), 3.66 (6H, s) ゝ 4.11-4.20 (1H, m), 4.58-4.63 (1H, m ), 5.61-5.64 (1H, m), 5.93-5.96 (1H, m), 6.32-6.40 (1H, m), 6.73-6.78 (4H, m), 7.18-7.34 (10H ゝ m), 7.57,7.62 (1H, 2s) Raw material synthesis example

[0034] 4 N-dimethylaminoethylene 5-ododeoxycytidine

[0035] [Chemical 5-1]

5 Suspension of pseudodoxycytidine (353 mg, 1.0 mmol) in anhydrous methanol (10 mL). Add N, N-dimethylacetamide dimethyl acetal (488 L, 3 mmol) and stir at 50 ° C for 1 hour. After evaporating the solvent under reduced pressure, isopropyl alcohol (10 mL) is added and the resulting precipitate is collected by filtration. Dry under reduced pressure to obtain the title compound (398 mg, 94%).

^{[0037] J H NMR (DMSO} -d6) δ 1.95- 2.05 (1Η, m), 2.12- 2.18 (1Η, m), 2.18 (3Η, s), 3.11 (6Η, s), 3.5 Doo 3.68 (2Η, m), 3.76-3.81 (1Η, m), 4.19-4.26 (1Η, m), 5.08 (1Η, br), 5.19 (1Η, br) ゝ 6.08 (1H, dd, J = 6.6Hz, J = 6.3Hz ) ゝ 8.35 (1H, s)

Example 6

[0038] 4 N-dimethylaminoethylene 5- [N- (tert-butoxycarbol) pyrroyl-2-yl] deoxycytidine (Compound 6) [0039] [Idori 6]

[0040] 4 N-Dimethylaminoethylene 5 odeoxycytidine (1.69 g, 4.0 mmol), N- (tert-butoxycarbol) pyrroyl 2-boronic acid (844 mg, 4. Ommol), palladium acetate (27 mg, 0.12 mmol), triphenylphosphine 3, 3 ', 3, -trisulfonic acid trisodium salt (205 mg, 0.36 mmol), sodium carbonate (848 mg, 8. Ommol ) Is dissolved in degassed distilled water-acetonitrile (2: 1, 40 mL). After stirring at 60 ° C for 30 minutes, N- (tert-butoxycarbol) -pyrroyl-2-boronic acid (211 mg, 1.0 mmol), palladium acetate (27 mg, 0.12 mmol), triphenyl -Luphosphine 3,3 ', 3, -trisulfonic acid trisodium salt (205 mg, 0.36 mmol) is added. After stirring at 60 ° C for 30 minutes, dilute with water (40 mL) and distill off the solvent under reduced pressure until the volume becomes half. After addition of black-mouthed form (50 mL), the organic layer is washed with a saline solution and then with an aqueous solution of saturated sodium bicarbonate. The organic layer is recovered, dried using sodium carbonate, and the solvent is distilled off under reduced pressure. The product was purified by silica gel column chromatography using C200 silica gel (chloroform-form-methanol) to give the title compound (1.13 g, 61

%).

^{[0041] J H NMR (DMSO-} d6) δ 1.36 (9H, s)ゝ1.96- 2.07 (1H. M), 2.15- 2.23 (1Hゝm), 2.79 (3H, br ), 2.98 (3H, br) , 3.51- 3.62 (2H, m), 3.78- 3.81 (1H, m), 4.20- 4.26 (1H, m), 4.95 (1H ゝ t, J = 4.9Hz) ゝ 5.19 (1H, d, J = 4.3Hz) ) ゝ 6.03—6.06 (1H, m), 6.13—6.22 (3H, m), 7.22-7.24 (1Η, m) 7.90 (lH, s)

Example 7

[0042] 5, -O- (4,4, -dimethoxytrityl) -4-N-dimethylaminoethylene-5- [N- (tert-butoxycarbol) pyrroyl-2-yl] deoxycytidine (compound 7 )

[0043] [Formula 7]

[0044] 4 N-Dimethylaminoethylene 5-[[N- (tert-butoxycarbol) pyrroyl-2-yl] deoxycytidine (I-drug 6) (1.13 g, 2.45 mmol) was added to anhydrous acetonitrile. After azeotropic dehydration, dissolve in anhydrous acetonitrile (25 mL). Add triethylamine (517 L, 3.68 mmol) and 4,4, -dimethoxytrityl chloride (996 mg, 2.94 mmol) and stir at room temperature for 10 minutes. After diluting with chloroform (15 mL), wash the organic layer three times with saturated aqueous sodium hydrogen carbonate. The organic layer is collected, dried over sodium sulfate, and the solvent is distilled off under reduced pressure. Purify using silica gel column chromatography using C200 silica gel (hexane methanol with methanol, 0.5% triethylamine) to obtain the title compound (1.19 g, 64%).

^{[0045] J H NMR (CDC13} ) δ 1.38 (9H, s), 2.17- 2.23 (1H, m), 2.24 (3H, s), 2.70- 2.80 (1Ηゝm), 2.88 (3H, br ), 2.94 (3H, br), 3.26—3.41 (2H, m), 3.76 (6H, s), 4.16- 4.20 (1H ゝ m), 4.56- 4.59 (1H ゝ m), 5.64 (1H, br) ゝ 5.95 (1H, t, J = 3.3Hz) ゝ 6.48—6.54 (1H, m),

6.70- 6.76 (4H, m), 7.18- 7.39 (10H, m), 7.77 (1H, s)

Example 8

[0046] 5, —O— (4,4, —dimethoxytrityl) —4—N—dimethylaminoethylene—5— [N— (tert-butoxycarbol) pyrroyl-2-yl] deoxycytidine 3′— (2-cyanoethyl N, N-diisopropyl phosphoramidite) (compound 8)

[0047] [Formula 8]

5, —O— (4,4, —dimethoxytrityl) 4 N—dimethylaminoethylene 5— [N— (tert-butoxycarbol) pyrroyl-2-yl] deoxycytidine (I 7) (286 mg) , 0.48 mmol) are azeotropically dehydrated with anhydrous acetonitrile and then dissolved in anhydrous ethylene chloride (5 mL). N, N-diisopropylamine (41 L, 0.29 mmol), 1H-tetrazole (20 mg, 0.29 mmol), (2-cyanoethoxy) -bis- (N, N-diisopropylamino) phosphine (184 L, 0.29 mmol). (58 mmol) and stirred at room temperature for 3 hours. After dilution with chloroform (5 mL), the organic layer is washed with a saturated aqueous solution of sodium hydrogen carbonate. The organic layer is recovered, dried using anhydrous sodium sulfate, and the solvent is distilled off under reduced pressure. Dissolve in isopropyl ether-ethyl ether (1: 1, vZv, 10 mL), and wash the organic layer with 0.1 M sodium hydroxide. Wash 5 times with an aqueous solution. The organic layer is recovered and the solvent is distilled off under reduced pressure. Purification was carried out using a recycle HPLC (cloth form, 0.5%) equipped with a GPC column to obtain the title compound (282 mg, 73%).

[0049] 1H NMR (CDC13) δ 1.05-1.23 (12H, m) ゝ 1.38 (9H, s) ゝ 2.17-2.80 (4H, m),

3.26- 3.80 (6H, m), 3.78 (6H, s), 4.14-4.22 (1H, m), 4.58- 4.62 (1H, m), 5.63-5.71 (1H, m), 5.90-6.01 (1H, m ), 6.51-6.55 (1H, m), 6.71-6.78 (4H, m), 7.12-7.43 (10H, m) 7.78,7.82 (lH, 2s)

Example 9

[0050] Synthesis of 3′O— (tert-butyldimethylsilyl) -2,1-O-methyl-5- [N— (tert-butoxycarbonyl) pyrroyl-2-yl] -peridine (Compound 9)

[0051] [Formula 9]

[0052] 3'-O- (tert-butyldimethylsilyl) -2'-Omethyl-odouridine (1.18g, 2.37mmol), N-Boc-pyrrole-2-boronic acid (1.Og ゝ3.55 mmol), palladium acetate (27 mg, 0.12 mmol), TPPTS (95 mg, 0.16 mmol) and anhydrous sodium carbonate (502 mg, 4.74 mg) were placed in a two-necked flask, purged with argon, and degassed. A mixed solvent of pure water and acetonitrile 2: 1 (24 ml) was added. 50. After stirring at C for 30 minutes, the mixture was filtered through Hyfloss one-parcel, and the filtrate was distilled off under reduced pressure. Ethyl acetate (30 ml) and water (30 ml) were added to the residue for extraction, and the aqueous layer was extracted twice with ethyl acetate to collect the organic layer. The organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Purification was performed using NH silica gel column chromatography to obtain the title compound (1.1 g, 86%).

^{[0053] J H NMR (CDC1} ) δ 0.08- 0.10 (6H, d, J = 4.29Hz), 0.89 (9H, s), 1.5 (9H, s), 2.65- 2.68 (1H, br), 3.46 (3H, s) ゝ 3.65-3.74 (1H, m), 3.90-3.97 (1H, m),

4.03-4.07 (2H, m), 4.34-4.38 (1H, dd, J = 4.8 Hz 4.9 Hz), 5.60-5.62 (1H, d, J = 4.5 Hz), 6.12-6.16 (2H, m), 7.31- 7.33 (1H, m), 7.62 (1H, s) ゝ 8.23 (1H, s) Example 10

[0054] 3 ', 5, -O-Bis (tert-butyldimethylsilyl) -2, -O-methyl-5- [N- (tert-butoxycarbonyl) pyrroyl-2-yl] -peridine (compound Synthesis of 10)

[0055] [Formula 10]

Compound 9 (4 g, 7.44 mmol) was dissolved in dimethylacetamide (7.5 ml), imidazole (1.7 g, 11.16 mmol), TBDMS—Cl (l. 0 g, 14.88 mmol) , Was added. After stirring at room temperature for 5 hours, water (10 ml) was added to stop the kakogen reaction. Ethyl acetate (30 ml) and water (20 ml) were added for extraction, and the aqueous layer was extracted twice with ethyl acetate to collect the organic layer. The organic layer was washed twice with a saturated sodium hydrogen carbonate solution, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Purification using silica gel column chromatography gave the title compound (4.65 g, 96%).

[0057] ^J H NMR (CDC1) δ 0.13—0.12 (6H, d, J = 2.3 Hz) ゝ 0.05—0.07 (6H, d, J = 7.1 Hz) ゝ

Three

0.71 (9H, s) ゝ 0.88 (9H, s) ゝ 1.45 (9H, s) ゝ 3.48 (3H, s) ゝ 3.64—3.71 (2H, m), 3.90—4.00 (2H, m), 4.19—4.23 ( 1H, m), 5.99 (1H, m), 6.08-6.12 (2H, m), 7.32 (1H, s), 7.70 (1H, s), 9.20 (1H, s)

Example 11

[0058] Synthesis of 3 ', 5, -O-Bis (tert-butyldimethylsilyl) -2, -O-methyl-5- (pyrroyl-2-yl) -peridine (I-Danied Compound 11) [0059] [Formula 11]

[0060] A mixture of 10 (2.66 g, 4. O9 mmol) and sodium methoxide (1. lg, 20. 45 mmol) was placed in a two-necked flask, and purged with argon, followed by mixing THF and methanol 2: 1. Solvent (40 ml) was added. After stirring at room temperature for 1 hour, the reaction was stopped by adding a saturated salt solution of ammonia to the solution, and the solvent was distilled off under reduced pressure. Ethyl acetate (30 ml) and water (30 ml) were added to the residue for extraction, and the mixture was extracted twice from the aqueous layer with ethyl acetate to collect the organic layer. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Purification was performed using silica gel column chromatography to obtain the title compound (1.8 g, 96%).

^{[0061] J H NMR (CDC1} ) δ θ.09- 0.11 (12H, m), 0.90 (18H, s), 3.28 (3H, s), 3.74- 3.81 (2H

Three

, M), 3.91-3.81 (2H, m), 4.01-4.05 (1H, m), 4.23-4.27 (1H, dd, J = 4.6 Hz 5.0 Hz), 6.02-6.04 (1H, d, J = 5.0 Hz ), 6.17-6.20 (1H, d, J = 5.0 Hz), 6.29-6.23 (1H, m), 6.80-6.82 (1H, m), 7.80 (1H, s) ゝ 8.91 (1H, s) ゝ 10.25 ( 1H, s)

Example 12

[0062] Synthesis of 2,1-O-methyl-5- [N- (tert-butoxycarbol) pyrroyl-2-yl] -peridine [0063]

[0064] Compound 10 (54 mg, 0.1 mmol), TEA'3HF (63.6 / zl, 0.4 mmol), TEA (138 / zl, lmmol), and THF (1 ml) were placed in a two-necked flask. The atmosphere was replaced with argon, and the mixture was stirred at room temperature for 19 hours. The solvent was distilled off under reduced pressure. Hexane (15 ml) and water (15 ml) were added to the residue, and the mixture was extracted. The aqueous layer was further extracted with chloroform, and the organic layer was collected. The extract was dried over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure to obtain the title compound (42 mg, 90%).

[0065] 1H NMR (CDC1) δ 1.45 (9H, s), 2.71 (1H, s), 3.36—3.44 (1H, m), 3.50 (3H, s),

Three

3.71-3.93 (2H, m), 3.95-3.97 (2H, m), 4.26-4.30 (1H, m), 5.72-5.73 (1H, d, J = 3.3 Hz), 6.07-6.08 (1H, d, J = 2.4 Hz)

Example 13

[0066] Synthesis of 3 ', 5,1-O- (Bis-tert-butyldimethylsilyl) -2,1-O-methyl-5- (pyrroyl-2-yl) -cytidine (Compound 13)

[0067] [Formula 13]

[0068] The compound II (130 mg, 0.24 mmol) was dissolved in methylene chloride (2 ml), and 0.1 M carbonic acid was dissolved. An aqueous sodium solution (3 ml), TBAB (39 mg 0.12 mmol), TPS-Cl (107 mg 0.35 mmol) were added, and the mixture was stirred for 4 hours. Water (10 ml) and black-mouthed form (10 ml) were extracted from Kagami ぇ, and the aqueous layer was extracted twice with black-mouthed form to collect the organic layer. The organic layer was washed with a saturated aqueous solution of sodium hydrogen carbonate, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. After azeotropic dehydration three times with anhydrous acetonitrile, a 7N ammonia methanol solution (15 ml) was added, and the mixture was stirred for 3 hours. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography, and further purified by HPLC to obtain the title compound (70 mg 53%).

[0069] ^J H NMR (DMSO) δ-0.09-0.05 (6H d J = 10.5 Hz) 0.07-0.08 (6 H d J = 2.0 Hz)

0.75 (9H s), 0.87 (9H s), 3.30 (3H s), 3.64—3.69 (1H m), 3.78—3.85 (3H m) 4.19—4.22 (1H t J = 4.6 Hz) 5.93—5.95 (1H d J = 4.9 Hz), 6.08 (2H br) 6.16 (1H br) 7.54 (2H br) 10.95 (1H s)

Example 14

[0070] Synthesis of 2 O-methyl-5- (pyrroyl-2-yl) -cytidine (Compound 14)

[0071] [Formula 14]

[0072] A mixture of 13 (55 mg, 0.1 mmol) of TEA'3HF (63.6, 10.4 mmol) and THF (1 ml) was placed in a two-necked flask, the atmosphere was replaced with argon, and the mixture was stirred at room temperature for 24 hours. The solvent was distilled off under reduced pressure. Hexane (15 ml) and water (15 ml) were extracted into the residue, and the aqueous layer was washed twice with hexane. The aqueous layer was distilled off under reduced pressure, purified by silica gel column chromatography, and further purified by LC908 to obtain the title compound (29 mg 90%).

[0073] 1H NMR (DMSO) δ 3.49 (3Hs), 3.52-3.76 (2Hm), 3.79-3.38 (2Hm), 4.10—4.14 (1H, m), 5.01—5.06 (2H, m), 5.91—5.93 (1H, d, J = 4.3 Hz) ゝ

6.07-6.14 (2H, m), 6.25 (1H, br), 6.80-6.82 (1H, m), 7.53 (1H, br) ゝ 7.86 (1H, s) ゝ 10.91 (1H, s)

Example 15

[0074] O- (tert-butyldimethylsilyl) -5,1-O- (4,4, dimethoxytrityl) -2,1-O-methyl-5- (N-tert-butyloxycarbolylol Le) peridine (compound 15)

[0075] [Formula 15]

Compound 9 (250 mg, 0.46 mmol) was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (4.6 ml), and added with DMTrCl (190 mg, 0.56 mmol). Stirred at room temperature for hours. Water (1 ml) was added to stop the reaction, and the solvent was distilled off under reduced pressure. Ethyl acetate (15 ml) and a saturated aqueous solution of sodium hydrogen carbonate (20 ml) were added to the residue for extraction, and the aqueous layer was extracted with ethyl acetate and the organic layer was collected. Sodium sulfate was added and dried, and the solvent was distilled off under reduced pressure. Purification was performed using silica gel column chromatography in which pyridine (1%) was added to the elution solvent to give the title compound (386 mg, quant).

[0077] 1H NMR (CDC1) δ 0.04-0.05 (6Η, d, J = 22.8 Hz), 0.81 (9H, s), 1.49 (9H, s),

Three

3.21-3.27 (IH, m), 3.39-3.43 (IH, d, J = 9.3 Hz), 3.75 (6H, s), 3.79-3.83 (IH, dd, J = 4.5 Hz, 4.4 Hz), 4.09 (IH , s), 4.30-4.34 (IH, dd, J = 5.2 Hz, 5.2 Hz), 5.64 (IH, s), 5.77-5.79 (IH, 3.3 Hz, 3.3 Hz), 6.02-6.04 (IH, d, J = 3.8 Hz), 6.70-6.74 (4H, m), 7.16-7.32 (9H, m), 7.66 (IH, s), 8.18 (IH, br)

Example 16

[0078] 5, —O— (4,4, —dimethoxytrityl) -2, —O—methyl-5— (N—tert-butyloxyl Ballpyro-2-yl) peridine (compound 16)

[0079]

[0080] Compound 15 (386 mg, 0.46 mmol) was azeotropically dehydrated three times with anhydrous acetonitrile, then dissolved in anhydrous THF (5 ml), and TEA-3HF (296 mg, 1.84 mmol) and TEA (325 mg) were dissolved. , 3.22 mmol) and stirred at room temperature for 12 hours. Water (1 ml) was added to stop the reaction, and the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate (15 ml) and a saturated aqueous solution of sodium hydrogen carbonate (20 ml), and extracted from the aqueous layer with ethyl acetate, and the organic layer was collected. Sodium sulfate was added and dried, and the solvent was distilled off under reduced pressure. Purification was performed using silica gel column chromatography in which pyridine (1%) was added to the elution solvent to obtain the title compound (300 mg, 90%).

[0081] 1H NMR (CDC1) δ 1.54 (9Η, s), 3.03 (1H, br), 3.61-3.74 (6H, s), 3.96 (1H, s),

Three

4.09 (1H, s), 4.42 (1H, s), 5.64 (1H, s), 5.82--5.85 (1H, dd, J = 3.1 Hz, 3.1 Hz), 6.05-6.07 (1H, d, J = 3.1 Hz ), 6.72-6.75 (4H, d, J = 5.7 Hz), 7.17-7.34 (9H, m), 7.62 (1H, s), 9.61 (1H, br);

Example 17

[0082] 5, -O- (4,4, -dimethoxytrityl) -2, -O-methyl-5- (N-tert-butyloxycarbonylpyrroyl-2-yl) -lysine 3,-(2-cyanoethyl Ν, Ν-diisopropylphospholoamidite) (compound 17)

[Formula 17]

[0083] Compound 16 (300 mg, 0.41 mmol) was azeotropically dehydrated three times with anhydrous acetonitrile, then dissolved in anhydrous methylene chloride (4 ml), and Ν, Ν-diisopropylamine (25 mg, 0.25 mmol), (2-Cyanethoxy) (Ν, Ν-diisopropylamino) phosphine (149 mg, 0.49 mmol) and 1H-tetrazole (17 mg, 0.25 mmol) were added and stirred at room temperature for 5 hours. Water (1 ml) was added to stop the reaction, and the solvent was distilled off under reduced pressure. To the residue was added chloroform (15 ml) and a saturated aqueous solution of sodium hydrogen carbonate (20 ml), and the mixture was extracted. The solvent was distilled off under reduced pressure. The residue was extracted with 0.2N sodium hydroxide (15 ml), a mixed solvent of diisopropyl ether and getyl ether 2: 1 (15 ml), and further washed with 0.2N sodium hydroxide (15 ml) ten times. did. Sodium sulfate was added and dried, and the solvent was distilled off under reduced pressure. Purification using LC908 gave the title compound (261 mg, 70%).

[0084] 1H NMR (CDC1) δ 1.03-1.49 (12H, m), 1.48-1.49 (9H, d, J = 2.8 Hz), 2.35—2.28

Three

(1H, t, 6.5 Hz), 2.63-2.68 (1H, t, J = 6.3 Hz), 3.31-3.74 (8H, m), 3.75-3.76 (6H, m), 3.85-4.27 (3H, m), 4.47-4.49 (1H, m), 5.54-5.62 (1H, m), 5.80-5.84 (1H, m), 6.05-6.14 (1H, m), 6.70-6.75 (4H, m), 7.14-7.30 (9H , m), 7.54-7.62 (1H, s), 8.66 (1H, br)

Example 18

[0085] 4N-Dimethylaminoethylene 2,1-O-methyl-5- (N-tert-butyloxycarboxy-rubyrol 2yl) cytidine (Compound 18) [0086] [Formula 18]

[0087] 4 N-dimethylaminoethylene 2,1-O-methyl-5-odocytidine (700 mg, 1.54

mmol), N-Boc-Pyrrole-2-boronic acid (1.3 g, 6.19 mmol), radium acetate (104 mg, 0.46 mmol), TPPTS (261 mg, 0.46 mmol), anhydrous sodium carbonate (489 mg, 4.62 mmol) was placed in a two-necked flask, purged with argon, and a mixed solvent (15 ml) of degassed pure water and acetonitrile 2: 1 was added. After stirring at 60 ° C. for 30 minutes, the mixture was filtered through Noflo Super Cell, and the filtrate was distilled off under reduced pressure. Subsequently, methanol was added to the residue, followed by filtration, and the solvent was distilled off under reduced pressure. Purification was performed using silica gel column chromatography to obtain a crude product (536 mg, 64%). The proton ratio with the by-product at this time was 1: 0.11.

^{[0088] J H NMR (CDCl} ) δ 1.43 (9Η, s), 2.28 (3Η, s), 2.88 (3H, br), 3.01, (3H, br), 3.54

Three

(3H, s), 3.74-3.78 (IH, m), 3.93--3.97 (IH, m), 4.1 (IH, br), 4.38--4.46 (2H, m), 5.37-5.55 (IH, d, J = 3.6 Hz), 6.04-6.06 (IH, m), 6.12-6.14 (IH, t, J = 3.3 Hz), 7.26-7.28 (IH, m), 7.55 (IH, s)

Example 19

[0089] 4, O— (4,4, —O-dimethoxytrityl) 4 N-dimethylaminoethylene 2, O-methyl-5- (N-tert-butyloxycarbolyl-2-yl) cytidine [0090]

[0091] Compound 18 (536 mg, 1.09 mmol) as a crude product was azeotropically dehydrated three times with acetonitrile, then dissolved in anhydrous methylene chloride (11 ml), and DMTr-Cl (369 mg, 1.09 mmol) was added. The mixture was stirred at room temperature for 4 hours. Water (1 ml) was added to stop the reaction, and the solvent was distilled off under reduced pressure. Chloroform (20 ml) and an aqueous solution of sodium hydrogen carbonate (20 ml) were extracted from the residue, and the aqueous layer was extracted with aqueous chloroform, and the organic layer was collected. Sodium sulfate was added and dried, and the solvent was distilled off under reduced pressure. Purification was performed using silica gel column chromatography in which pyridine (1%) was added to the elution solvent to give the title compound as a crude product.

^{[0092] J H NMR (CDCl} ) δ 1.41 (9Η, s), 2.23 (3Η, s), 2.84 (3H, br), 2.95 (3H, br), 3.61

Three

(3H, s), 3.68 (6H, s), 3.83-3.85 (1H, d, J = 5.6 Hz), 4.01-4.07 (1H, m), 4.03 (1H, br), 5.60 (1H, s), 5.81-5.83 (1H, dd, J = 2.3 Hz, 3.3 Hz), 6.08 (1H, s), 6.69-6.72 (4H, d, J = 8.5 Hz), 7.10-7.43 (9H, m), 7.71 (1H , s)

Example 20

[0093] 4, O— (4,4, —O-dimethoxytrityl) 4 N-dimethylaminoethylene 2, O-methyl-5- (N-tert-butyloxycarbolyl-2-yl) cytidine 3, — ( 2 Cyanethyl Ν, Ν-diisopropylphosphoramidite (compound 20) [0094] [Formula 20]

[0095] Compound 19 (200 mg, 0.26 mmol) as a crude product was azeotropically dehydrated three times with anhydrous acetonitrile, then dissolved in anhydrous methylene chloride (3 ml), and Ν, Ν-diisopropylamine (22 μl , 0.16 mmol), (2-cyanoethoxy) (N, N-diisopropylamino) phosphine (100 g, 0.31 mmol) and 1H-tetrazole (11 mg, 0.31 mmol) were stirred and stirred at room temperature for 5 hours. Water (1 ml) was added to stop the reaction, and the solvent was distilled off under reduced pressure. To the residue was added chloroform (15 ml) and a saturated aqueous solution of sodium hydrogen carbonate (20 ml), and the mixture was extracted. The solvent was distilled off under reduced pressure. The residue was extracted with 0.2N sodium hydroxide (15 ml), a mixed solvent of diisopropyl ether and getyl ether 2: 1 (15 ml), and further washed ten times with 0.2N sodium hydroxide (15 ml). Sodium sulfate was added and dried, and the solvent was distilled off under reduced pressure. Purification using LC908 gave a two-step yield of 11%. (120 mg, 11%)

[0096] ^J H NMR (CDCl) δ 0.97-1.15 (12H, m), 1.39-1.41 (9H, d, J = 5.3 Hz), 2.35-2.28

Three

(1H, t, 6.5 Hz), 2.63-2.68 (1H, t, J = 6.3 Hz), 3.31-3.74 (8H, m), 3.75-3.76 (6H, m), 3.85-4.27 (3H, m), 4.47-4.49 (1H, m), 5.51-5.55 (1H, d, J = 10.7 Hz), 5.75-5.82 (1H, d, J = 19.1 Hz), 6.11-6.22 (1H, m,), 6.70-6.73 (4H, m), 7.15-7.33 (9H, m), 7.66-7.78 (1H, d, J = 32 Hz)

Example 21

[0097] Oligo DNA or 5-pyrrolyl 2, -deoxyperidine, 5-pyrrolyl 2, -deoxycytidine, 5-pyrrolinolate 2, -O-methylperidine, 5-pyrrolyl 2,1-O-methylcytidine Is the synthesis of oligo 2,1 O-methyl RNA

[0098] The synthesis was performed using Compounds 5, 8, 17, and 20 and a commercially available deoxynucleoside phosphoramidite, or a 2,1 O-methylnucleoside phosphoramidite unit and a commercially available CPG solid support. This was performed using the coupled nucleosides and the ABI392 DNA automated synthesizer using standard protocols. After completion of the synthesis, the terminal dimethoxytrityl group was deprotected, and the solid support was immersed in 0.8 M sodium methoxide (methanol solution) for 3 hours to perform deprotection and cleavage. The synthesized oligonucleotide was purified by anion exchange HPLC, and its structure was confirmed by MALDI-T OF mass spectrometry. Table 1 shows examples of the synthesis of oligo DNAs containing 5-pyrrolyl-2-deoxyperidine and 5-pyrrolyl-2,1-deoxycytidine. Table 2 shows examples of the synthesis of oligo-2, -O-methyl RNAs containing 5-pyrrolyl 2, -O-methylperidine and 5-pyrrolyl-1,2,0-methylcytidine. In the table, each symbol of Cpy and Upy represents the corresponding 5-pyrrolyl-lysine derivative and 5-pyrrolylcytidine derivative.

[0099] [Table 1]

isolated [M + H] ⁺

sequence (14 mer) yield found * calcd

TTTTTTCpyTTTCTTT 14% 4229.5 4230.7

TTTTTTCTUpyTCTTT 9% 4215.7 4216.7

TTTTTUpycupyTTCTTT 17% 4267.8 4267.7

TTTUpyTTCTUpyTCTUpyT 10% 4316.0 4318.7

TTTTTCUpyu yupycTTT 25% 4320.0 4318.7

* MALDI- TOF- MASS

[0100] [Table 2]

Example 22

[0101] Oligo DNA or DNA containing 5-pyrrolyl 2, -deoxyperidine, 5-pyrrolyl 2, -deoxycytidine, 5-pyrrolinolae 2, -O-methylperidine, 5-pyrrolyl 2,1-O-methylcytidine Is the performance evaluation of oligo 2,1 O-methyl RNA as triple-stranded nucleic acid (TFO)

[0102] A binding experiment between the hairpin duplex and TFO shown in Fig. 1 was performed, and the stability was evaluated by a UV heat melting curve. Table 3 shows a solution of hairpin duplex (2 μΜ) and the appropriate TF 0 (2 ^ 表) shown in Table 3 dissolved in lOmM sodium codylate buffer (containing 500 mM sodium chloride and lOmM magnesium chloride). Adjusted to pH. The temperature of the solution was raised from 5 degrees Celsius to 80 degrees Celsius, the absorbance at 260 nm was measured, and a UV heat melting curve was drawn. The inflection point of the obtained UV heat melting curve was defined as the melting temperature, and the stability of the triple chain was evaluated. All of the TFOs tested this time were shown to form triplex in the pH range of 6.0 to 7.4.

[0103] [Table 3]

pH

TFO sequence (14 mer) 6.0 6.4 7.0 7.4

TTTTTTCTUpyTCTTT 41.1 35.7 28.8 21.3

TTTTTUpycupyTTCTTT 39.9 33.5 26.6 19.3

TTTUpyTTCTUpyTCTUpyT 39.5 34.6 25.8 17,0

TTTTTCUpyupyupycTTT 35.8 30.5 26.1 14.7

TTTTTTCPVTTTCTTT 36.2 31.3 23,7 17.2

Industrial applicability

The compound of the present invention can accurately mimic the hydrogen bonding mode of a protonated cytosine base and contribute to the creation of an artificial nucleic acid having a high triplex-forming ability. Therefore, it can be used for a wide range of nucleic acid application technologies such as the antigene method, the preparation of fluorescent probes, and gene diagnosis.

Claims

Claims A nucleic acid derivative represented by the following general formula I:

[Chemical 1]

OH X

(In Formula I, Al, A2, and A3 are the same or different and are hydrogen, an alkyl group having 1 to 6 carbon atoms, a halogenated alkyl group having 1 to 7 carbon atoms having 1 to 7 halogens, 6 alkoxy, a dialkylamino group having two identical or different alkyl groups having 1 to 6 carbon atoms on a nitrogen atom, chlorine, bromine, iodine, fluorine, cyano, nitro, or an acyl group having 1 to 7 carbon atoms Or A1 and A2, or A2 and A3 together form a cyclic structure, and E has a hydrogen atom, an alkoxycarbonyl group having 115 carbon atoms, a substituent, or a benzyl group. X represents a hydrogen atom, a hydroxyl group or an alkyloxy group, and Y represents a hydrogen atom, a 4-methoxytrityl group, or a 4,4′-group. Represents a dimethoxytrityl group, and Z is A benzoyl group, a hydroxyl group, an amino group, or a nitrogen atom, which may have a substituent on a carbon atom, may have a substituent on a carbon atom, or may have a substituent on a phenyl group; Represents a dialkylamino group having two identical or different alkyl groups having 1 to 6 carbon atoms.) Or their isomers.

2. The nucleic acid derivative according to claim 1, wherein Al, A2 and A3 are hydrogen.

[3] The nucleic acid derivative according to claim 1, wherein the cyclic structure formed by Al and A2 or A2 and A3 is a cycloalkane or a benzene ring.

[4] The nucleic acid derivative according to any one of [13] to [13], wherein E is a benzyloxycarbol group or a t-butoxycarbol group.

[5] The nucleic acid derivative according to any one of claims 14 to 14, wherein X is alkyloxy having 1 to 6 carbon atoms.

[6] The nucleic acid derivative according to any one of claims 115, wherein Y is a hydrogen atom.

[7] The nucleic acid derivative according to any one of claims 16 to 16, wherein Z is a hydroxyl group, an amino group, or an N-dimethylaminoethylene group.

[8] The method according to any one of claims 117, wherein Y represents a 4-methoxytrityl group or a 4,4′-dimethoxytrityl group, and the 3 ′ hydroxyl group of the sugar is phosphoramidite. Nucleic acid derivatives of

[9] The phosphoramidite group has the general formula (II): — P (OR3) N (R4) (R5) (II)

9. The nucleic acid derivative according to claim 8, wherein R3 is a protecting group for a phosphate group, and R4 and R5 are an alkyl group or an aryl group.

[10] The protecting group for the phosphate group is 2-cyanoethyl group, 412-trophenylethyl group, N (trifluoromethyl) acetylbutyl group, or 4- [N-methyl-N— (2,2,2 10. The nucleic acid derivative according to claim 8 or 9, which is a trifluoroacetyl) amino] butyl group.

[11] The nucleic acid derivative according to claim 10, wherein the phosphate-protecting group is a 2-cyanoethyl group.

[12] The nucleic acid derivative according to any one of claims 6 to 11, wherein R4 and R5 are an alkyl group having 14 to 14 carbon atoms, a benzyl group, a phenyl group, or a naphthyl group.

[13] The nucleic acid derivative according to claim 12, wherein R4 and R5 are isopropyl groups.

[14] 5, -0- (4,4, dimethoxytrityl) -5- [N- (tert-butoxycarbol) pyrroyl-2-yl] deoxyperidine 3, — (2-cyanoethyl N, N-diisopropyl Phosphoramidite).

[15] 5, —O— (4,4, —dimethoxytrityl) 4-N-dimethylaminoethylene-5— [N— (tert-butoxycarbol) pyrroyl-2-yl] deoxycytidine 3 ′ — ( 2-cyanoethyl N, N-diisopropyl phosphoramidite).

[16] 5, —O— (4,4, —dimethoxytrityl) -2, —O—methyl-5— (N—tert-butyloxyl (V-pyrroyl-2-yl) -lysine 3,-(2-cyanoethyl Ν, Ν-diisopropylphosphoramidite).

[17] 4, -0- (4,4, -Ο-dimethoxytrityl) 4 Ν-dimethylaminoethylene 2, Οmethyl-5- (Ν-tert-butyloxycarboxy-rubyrol 2-yl) cytidine 3, — (2 cyanoethyl Ν, Ν-diisopropyl phosphoramidite).

[18] A nucleic acid oligomer comprising the nucleic acid derivative according to any one of [1] to [7].