JP2005247771A - Aromatic substituent-introduced modified 4-n-carbamoylcytidine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a modified cytidine that has high double strand-stabilizing effect independent of complementary base and can dissolve the introduced oligonucleotide in water and can function as an ideal universal base. <P>SOLUTION: The modified 4-N-carbamoylcytidine is represented by general formula (I) (wherein Z is an aromatic substituent, Y is H, a halogen, hydroxyl, an amino, an alkyl amino, a dialkylamino, an alkoxy, an alkoxyalkyl, an aryloxy, an arylthio, an alkylthio, cyano, or an acylamino). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a modified cytidine capable of stabilizing a duplex with a complementary strand irrespective of a complementary base by introducing it into oligodeoxyribonucleotide (oligo DNA).

Universal bases provide duplex stability independent of complementary bases. Currently known universal bases do not change depending on the complementary base, but give almost constant stability to the duplex, but the stabilization effect itself is low. Some of them are not suitable for practical use from the viewpoint of water solubility, and an ideal universal base suitable for practical use has not yet been obtained.

  For example, when a conventional modified cytosine base forms a DNA duplex in a DNA oligomer, it forms a Watson-Crick base pair with a guanine base and a mismatch base pair with another base.

Nichols, R .; Andres, P.C .; Zhang, P .; Bergstome, D.E.Nature, 1994, 369, 492-493 Bergstome, D.E .; Zhang, P .; Toma, P.H .; Andrews, P .; Nichols, R .; J. Am. Chem. Soc., 1995,117, 1201

The present invention provides a modified cytidine that solves the above-mentioned problems and has a high double-strand stabilizing effect that does not depend on complementary bases, water solubility of the introduced oligonucleotide, and functions as an ideal universal base. Objective.

That is, the present invention relates to a modified 4-N-carbamoylcytidine represented by the general formula (I):

(In the formula (I), Z represents an aromatic substituent, Y represents a hydrogen atom, halogen, hydroxyl group, amino group, alkylamino group, dialkylamino group, alkoxy group, alkoxyalkyl group, aryloxy group, arylthio group. Represents an alkylthio group, a cyano group, or an acylamino group.

  Furthermore, the present invention relates to a compound in which the 3 ′ hydroxyl group in the modified 4-N-carbamoyl cytidine is phosphoramidite and a nucleic acid oligomer such as a DNA oligomer or an RNA oligomer containing the modified 4-N-carbamoyl cytidine. .

The conventional modified cytosine base formed a Watson-Crick base pair with the guanine base when a DNA duplex was formed in the DNA oligomer, and thus a high Tm value could be obtained. However, the Tm value decreased significantly because it formed mismatched base pairs with other nucleobases. The present invention forms a stable duplex even when synthesizing a double-stranded DNA oligomer in which mismatched base pairs adenine, cytosine, and guanine base are incorporated into the complementary strand of a modified nucleic acid.
This is because 4-N-carbamoylcytidine introduced with an aromatic substituent is incorporated into a DNA oligomer, so that the base at the position complementary to the modified cytosine is driven out of the double-stranded structure, and the aromatic ring and the complementary strand nucleobase are This is probably because a stable duplex was assembled by stacking with adjacent bases.

  Z in the compound of the present invention is any aromatic substituent known to those skilled in the art, and in particular, those selected from the group consisting of phenyl group, naphthyl group, pyrenyl group, quinolyl group, and derivatives thereof are preferable. . Here, the derivative means a compound which is modified with an arbitrary substituent known to those skilled in the art at an arbitrary position of each aromatic group.

As one embodiment of the compound of the present invention, modified 4-N-carbamoylcytidine represented by the following general formula (II) or (III) can be mentioned.

(In the formula (II) or (III), X represents a carbon atom or a nitrogen atom, and A1 to A7 are the same or different and each represents hydrogen, an alkyl group having 1 to 6 carbon atoms, or carbon having 1 to 7 halogen atoms A halogenated alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a dialkylamino group having two identical or different alkyl groups having 1 to 6 carbon atoms on the nitrogen atom, an amino group, a cyano group, or nitro Y represents a hydrogen atom, halogen, hydroxyl group, amino group, alkylamino group, dialkylamino group, alkoxy group, alkoxyalkyl group, aryloxy group, arylthio group, alkylthio group, cyano group, or acylamino group. .)

  Here, a preferred example of A1 to A7 in the general formula (II) or (III) is hydrogen, Y is preferably hydrogen or a hydroxyl group, and the alkyl part of each substituent in Y has 1 to 3 carbon atoms. It is preferable.

  The modified 4-N-carbamoylcytidine of the present invention, a compound in which the 3 ′ hydroxyl group thereof is phosphoramidite-modified, and a nucleic acid oligomer containing the modified 4-N-carbamoylcytidine are described in the art. Based on common technical knowledge in the field, those skilled in the art can easily prepare.

Specifically, the phosphoramidite group has the general formula (IV): -P (OR3) N (R4) (R5)
(Wherein R3 is a protecting group for a phosphate group, and R4 and R5 are an alkyl group or an aryl group). As a protecting group for a phosphate group, a 2-cyanoethyl group, a 4-nitrophenylethyl group, an N- (trifluoroacetyl) aminobutyl group, or 4- [N-methyl-N- (2,2,2, -Trifluoroacetyl) amino] butyl group can be mentioned, and 2-cyanoethyl group is preferred. Preferable examples of R4 and R5 are an alkyl group having 1 to 4 carbon atoms, a benzyl group, a phenyl group, or a naphthyl group, and an isopropyl group is particularly preferable. When the 3 ′ hydroxyl group is converted to phosphoramidite, the 5 ′ hydroxyl group is a 4-methoxytrityl group or 4,4 ′
-It is preferably protected by a protective group such as a dimethoxytrityl group.

  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: Synthesis of 4-N-naphthylcarbamoyldeoxycytidine (dCnphcm)

Deoxycytidine hydrochloride (2.637 mg, 10 mmol) was azeotroped three times with anhydrous pyridine, suspended in anhydrous pyridine (30 ml), trimethylsilyl chloride (3,158 ml, 25 mmol) was added, and the mixture was stirred at room temperature for 5 hours. did. After stirring, dichloromethane (70 ml) and phenyl chloroformate (1.506 ml, 12 mmol) are added and stirred at room temperature for 30 minutes. The reaction is stopped by adding water, washed three times with a saturated aqueous sodium hydrogen carbonate solution (100 ml), and the organic layer is dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, dissolved in pyridine (100 ml), 1-aminonaphthalene (1.117 g, 12 mmol) was added, and the mixture was stirred at 70 ° C. for 1.5 hours. After returning to room temperature, 100 ml of concentrated aqueous ammonia was added and stirred for 3 hours, and then the ammonia was distilled off under reduced pressure. Chloroform 100 ml and water 200 ml were added and stirred to precipitate the target compound, which was obtained by filtration. (2.537g, 64%)

1H-NMR (DMSO, 270 MHz) 2.03-2.12 (1H, m), 2.27-2.36 (1H, m), 3.58-3.63 (2H, m), 3.84 -3.88 (1H, m), 4.22-4.23 (1H, m), 5.06 (1H, t, J = 5.0 MHz), 5.27 (1H, d, J = 4. 3 MHz), 6.18 (1H, t, J = 6.3 MHz), 6.28 (1H, d, J = 7.6 MHz), 7.46-7.70 (4H, m), 7.93- 8.08 (1H, m), 8.22-8.31 (2H, m), 8.39-8.42 (1H, m), 10.5 (1H, s), 12.5 (1H, s)

Example 2: Synthesis of a nucleic acid oligomer containing 4-N-naphthylcarbamoyldeoxycytidine
The DNA chain was elongated using ABI DNA / RNA synthesizer 392 with respect to thymidine (0.1 μmol) bound to the CPG solid phase carrier using thymidine phosphoramidite unit or 4N-naphthyldeoxycytidine phosphoramidite unit. After the synthesis, the solid support was taken out and left overnight in concentrated aqueous ammonia, and then DMTr-on purification using a C18 reverse phase cartridge was performed, followed by lyophilization, and the target product, 5′-TTTCTCXTTCTCT-3 ′ (X = 4) -N-naphthylcarbamoyldeoxycytidine) was obtained. Mass spectrometry (MALDI-TOF) [M + H] Calcd.:3985.7,
Found: 3986.5

Example 3: Base-pairing ability of 4-N-naphthylcarbamoyldeoxycytidine The product of Example 2 and DNA having its complementary base sequence (3'-AAAGAGYAAGAGA-5 'Y = G, C, A, T ) Was mixed with 10 mM phosphate buffer and 1 M NaCl at a concentration of 2 μmol / L, and the temperature dependence of absorbance at a wavelength of 260 nm was measured to determine the UV-melting curve temperature Tm value (° C.). As a result, it was revealed that dCnphcm exhibits a property as a universal base that forms a base pair having the same stability as all bases. These results are shown in Table 1.

The nature of the modified 4-N-naphthylcarbamoylcytidine of the present invention as a universal base is considered to be effective not only as a probe for antisense method and gene analysis, but also for various studies and diagnoses.

  Therefore, the present invention can be widely applied to SNPs analysis, gene diagnosis, gene repair, precision base recognition technology, and the like by exposing specific bases such as SNPs sites present in DNA outside the double strand. .

Claims (15)

Modified 4-N-carbamoyl cytidine represented by general formula (I):

(In the formula (I), Z represents an aromatic substituent, Y represents a hydrogen atom, halogen, hydroxyl group, amino group, alkylamino group, dialkylamino group, alkoxy group, alkoxyalkyl group, aryloxy group, arylthio group. Represents an alkylthio group, a cyano group, or an acylamino group.) The modified 4-N-carbamoyl cytidine according to claim 1, wherein Z is selected from the group consisting of a phenyl group, a naphthyl group, a pyrenyl group, a quinolyl group, and derivatives thereof. The modified 4-N-carbamoyl cytidine according to claim 1 or 2 represented by the general formula (II):

(In the formula (II), X represents a carbon atom or a nitrogen atom, A1 to A7 are the same or different, and are hydrogen, an alkyl group having 1 to 6 carbon atoms, and 1 to 3 carbon atoms having 1 to 7 halogen atoms. Y represents a halogenated alkyl group, an alkoxy group having 1 to 6 carbon atoms, a dialkylamino group having two identical or different alkyl groups having 1 to 6 carbon atoms on the nitrogen atom, an amino group, a cyano group, or a nitro group; Represents a hydrogen atom, halogen, hydroxyl group, amino group, alkylamino group, dialkylamino group, alkoxy group, alkoxyalkyl group, aryloxy group, arylthio group, alkylthio group, cyano group, or acylamino group. The modified 4-N-carbamoyl cytidine according to claim 1 or 2 represented by the general formula (III):

(In the formula (III), X represents a carbon atom or a nitrogen atom, and A1 to A7 are the same or different and each represents hydrogen, an alkyl group having 1 to 6 carbon atoms, or 1 to 3 carbon atoms having 1 to 7 halogen atoms. A halogenated alkyl group, an alkoxy group having 1 to 6 carbon atoms, a dialkylamino group having two identical or different alkyl groups having 1 to 6 carbon atoms on the nitrogen atom, an amino group, a cyano group, or a nitro group, Y represents a hydrogen atom, halogen, hydroxyl group, amino group, alkylamino group, dialkylamino group, alkoxy group, alkoxyalkyl group, aryloxy group, arylthio group, alkylthio group, cyano group, or acylamino group. The modified 4-N-carbamoyl cytidine according to claim 3 or 4, wherein A1 to A7 in the general formula (II) or (III) are hydrogen. The modified 4-N-carbamoyl cytidine according to any one of claims 1 to 5, wherein the alkyl moiety in Y in the general formula (II) or (III) has 1 to 3 carbon atoms. The modified 4-N-carbamoyl cytidine according to any one of claims 1 to 6, wherein Y in the general formula (I) is hydrogen. Modified 4-N-carbamoyl cytidine represented by the formula 4. A compound wherein the 3 'hydroxyl group in the modified 4-N-carbamoylcytidine according to claim 1 is phosphoramidite-ized. The phosphoramidite group has the general formula (IV): -P (OR3) N (R4) (R5)
10. The compound according to claim 9, wherein R3 is a protecting group for a phosphate group, and R4 and R5 are an alkyl group or an aryl group. The protecting group of the phosphoric acid group is a 2-cyanoethyl group, 4-nitrophenylethyl group, N- (trifluoroacetyl) aminobutyl group, or 4- [N-methyl-N- (2,2,2, -tri 11. A compound according to claim 9 or 10 which is a fluoroacetyl) amino] butyl group. The compound according to claim 11, wherein the protecting group of the phosphoric acid group is a 2-cyanoethyl group. The compound as described in any one of Claims 10-12 whose R4 and R5 are a C1-C4 alkyl group, a benzyl group, a phenyl group, or a naphthyl group. 14. The compound according to claim 13, wherein R4 and R5 are isopropyl groups. A nucleic acid oligomer comprising the modified 4-N-carbamoyl cytidine according to claim 1.