WO2004094447A1 - チオヌクレオシド-s-ニトロシル誘導体 - Google Patents
チオヌクレオシド-s-ニトロシル誘導体 Download PDFInfo
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- WO2004094447A1 WO2004094447A1 PCT/JP2004/003337 JP2004003337W WO2004094447A1 WO 2004094447 A1 WO2004094447 A1 WO 2004094447A1 JP 2004003337 W JP2004003337 W JP 2004003337W WO 2004094447 A1 WO2004094447 A1 WO 2004094447A1
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- Prior art keywords
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- derivative
- nitrosyl
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- substituted
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- 239000005450 thionucleoside Substances 0.000 title claims abstract description 15
- -1 amino, hydroxy Chemical group 0.000 claims abstract description 66
- 150000003839 salts Chemical class 0.000 claims abstract description 22
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 15
- 125000005843 halogen group Chemical group 0.000 claims abstract description 14
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 13
- 125000002252 acyl group Chemical group 0.000 claims abstract description 11
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims abstract description 5
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims abstract description 5
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 claims abstract 2
- 239000002253 acid Substances 0.000 claims description 51
- 125000004432 carbon atom Chemical group C* 0.000 claims description 21
- 230000000295 complement effect Effects 0.000 claims description 19
- 125000003277 amino group Chemical group 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 15
- 238000012546 transfer Methods 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 230000002378 acidificating effect Effects 0.000 claims description 8
- 230000035772 mutation Effects 0.000 claims description 8
- 125000001841 imino group Chemical group [H]N=* 0.000 claims description 7
- 231100000350 mutagenesis Toxicity 0.000 claims description 7
- 238000002703 mutagenesis Methods 0.000 claims description 7
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 7
- 229910052717 sulfur Inorganic materials 0.000 claims description 6
- 125000004434 sulfur atom Chemical group 0.000 claims description 6
- 150000007513 acids Chemical class 0.000 claims description 5
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 4
- 239000007795 chemical reaction product Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 4
- 239000001257 hydrogen Substances 0.000 abstract description 4
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 abstract 1
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical group O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 105
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- 150000001875 compounds Chemical class 0.000 description 45
- 238000006243 chemical reaction Methods 0.000 description 32
- 238000004128 high performance liquid chromatography Methods 0.000 description 27
- 239000000243 solution Substances 0.000 description 24
- 150000007523 nucleic acids Chemical class 0.000 description 23
- 108020004707 nucleic acids Proteins 0.000 description 21
- 102000039446 nucleic acids Human genes 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- LTYUPYUWXRTNFQ-UHFFFAOYSA-N 5,6-diamino-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=C1C=C(N)C(N)=C2 LTYUPYUWXRTNFQ-UHFFFAOYSA-N 0.000 description 13
- 125000003729 nucleotide group Chemical group 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 12
- 239000002773 nucleotide Substances 0.000 description 12
- 238000006276 transfer reaction Methods 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 238000010586 diagram Methods 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 239000007987 MES buffer Substances 0.000 description 8
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 229940104302 cytosine Drugs 0.000 description 6
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 6
- 241000894007 species Species 0.000 description 6
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 6
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 5
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 5
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 5
- 230000007515 enzymatic degradation Effects 0.000 description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- 125000001424 substituent group Chemical group 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 150000003833 nucleoside derivatives Chemical class 0.000 description 4
- 125000003808 silyl group Chemical group [H][Si]([H])([H])[*] 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- ZAYHVCMSTBRABG-UHFFFAOYSA-N 5-Methylcytidine Natural products O=C1N=C(N)C(C)=CN1C1C(O)C(O)C(CO)O1 ZAYHVCMSTBRABG-UHFFFAOYSA-N 0.000 description 3
- ZAYHVCMSTBRABG-JXOAFFINSA-N 5-methylcytidine Chemical compound O=C1N=C(N)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZAYHVCMSTBRABG-JXOAFFINSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- ZIIQCSMRQKCOCT-YFKPBYRVSA-N S-nitroso-N-acetyl-D-penicillamine Chemical compound CC(=O)N[C@@H](C(O)=O)C(C)(C)SN=O ZIIQCSMRQKCOCT-YFKPBYRVSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- CIHUOJZDKZENCP-UHFFFAOYSA-N acetonitrile;triethylazanium;acetate Chemical compound CC#N.CC(O)=O.CCN(CC)CC CIHUOJZDKZENCP-UHFFFAOYSA-N 0.000 description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 239000003471 mutagenic agent Substances 0.000 description 3
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 3
- 230000009635 nitrosylation Effects 0.000 description 3
- 239000002777 nucleoside Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 235000010288 sodium nitrite Nutrition 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- OTDJAMXESTUWLO-UUOKFMHZSA-N 2-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-oxolanyl]-3H-purine-6-thione Chemical group C12=NC(N)=NC(S)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OTDJAMXESTUWLO-UUOKFMHZSA-N 0.000 description 2
- 125000001999 4-Methoxybenzoyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1OC([H])([H])[H])C(*)=O 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
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- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
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- 238000010306 acid treatment Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
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- 125000003118 aryl group Chemical group 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
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- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 2
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- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 125000001153 fluoro group Chemical group F* 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
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- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 125000004170 methylsulfonyl group Chemical group [H]C([H])([H])S(*)(=O)=O 0.000 description 2
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- 125000003232 p-nitrobenzoyl group Chemical group [N+](=O)([O-])C1=CC=C(C(=O)*)C=C1 0.000 description 2
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- 229940113082 thymine Drugs 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
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- STXBQXQLMWLXMY-DJLDLDEBSA-N 3-[2-amino-9-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]sulfanylpropanenitrile Chemical compound C12=NC(N)=NC(SCCC#N)=C2N=CN1[C@H]1C[C@H](O)[C@@H](CO)O1 STXBQXQLMWLXMY-DJLDLDEBSA-N 0.000 description 1
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101710135349 Venom phosphodiesterase Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Chemical class Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000003668 acetyloxy group Chemical group [H]C([H])([H])C(=O)O[*] 0.000 description 1
- 125000004423 acyloxy group Chemical group 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 125000005257 alkyl acyl group Chemical group 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000002490 anilino group Chemical group [H]N(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- XMQFTWRPUQYINF-UHFFFAOYSA-N bensulfuron-methyl Chemical compound COC(=O)C1=CC=CC=C1CS(=O)(=O)NC(=O)NC1=NC(OC)=CC(OC)=N1 XMQFTWRPUQYINF-UHFFFAOYSA-N 0.000 description 1
- DQEPMTIXHXSFOR-UHFFFAOYSA-N benzo[a]pyrene diol epoxide I Chemical compound C1=C2C(C3OC3C(C3O)O)=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 DQEPMTIXHXSFOR-UHFFFAOYSA-N 0.000 description 1
- 125000001231 benzoyloxy group Chemical group C(C1=CC=CC=C1)(=O)O* 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 125000006267 biphenyl group Chemical group 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 229960001701 chloroform Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- DMSZORWOGDLWGN-UHFFFAOYSA-N ctk1a3526 Chemical compound NP(N)(N)=O DMSZORWOGDLWGN-UHFFFAOYSA-N 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- MWKFXSUHUHTGQN-UHFFFAOYSA-N decan-1-ol Chemical group CCCCCCCCCCO MWKFXSUHUHTGQN-UHFFFAOYSA-N 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- HZUOAKQENIFWLY-UHFFFAOYSA-N n-(2-oxo-1h-pyrimidin-6-yl)nitrous amide Chemical compound O=NNC=1C=CNC(=O)N=1 HZUOAKQENIFWLY-UHFFFAOYSA-N 0.000 description 1
- 125000003136 n-heptyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000004888 n-propyl amino group Chemical group [H]N(*)C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- 125000003170 phenylsulfonyl group Chemical group C1(=CC=CC=C1)S(=O)(=O)* 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 125000002130 sulfonic acid ester group Chemical group 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000002813 thiocarbonyl group Chemical group *C(*)=S 0.000 description 1
- ICRHORQIUXBEPA-UHFFFAOYSA-N thionitrous acid Chemical compound SN=O ICRHORQIUXBEPA-UHFFFAOYSA-N 0.000 description 1
- 239000005451 thionucleotide Substances 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000003774 valeryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
- C07D473/36—Sulfur atom
- C07D473/38—Sulfur atom attached in position 6
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
Definitions
- the present invention belongs to the field of technology for manipulating nucleic acids, and in particular, is a novel compound useful for obtaining an oligonucleic acid that recognizes a specific base and transfers a nitrosyl group in a sequence-specific manner. —Related to ditrosyl derivatives. Background art
- nitric oxide plays an important role as an intracellular messenger (eg, LJ Ignarro, Pharmacology & Toxicology, 67 (1), 1, (1990)). It is also believed that S-nitrosothiol acts as a nitric oxide (NO) carrier in vivo (eg, DLH Williams, Acc. Chem. Res., 32, 869 (1999)). Nitric oxide (NO) is thought to act as an oxidizing agent in vivo, react with bases in DNA or RNA, and cause point mutation, in addition to its role in signal transduction. Chemical experiments have shown that mutations are induced nonspecifically (eg, JL Caulfield, JS Wishnok, SR Tannenbaum, J.
- An object of the present invention is to provide a thionucleoside nitrosyl derivative, an oligonucleic acid that specifically transfers a nitrosyl group to a target nucleic acid, and a sequence-specific mutagenesis method.
- the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, nitrosylation is performed using an oligonucleotide incorporating a structure in which a basic structure of a natural nucleobase is converted to a thiocarbonyl group.
- an oligonucleotide containing a novel nitrosyl compound was successfully synthesized. Furthermore, they have found that this oligonucleotide transfers a nitrosyl group in a sequence-specific and base-specific manner, and have completed the present invention.
- the present invention is as follows.
- R i represents a report, 2-doxylipose or a derivative thereof
- R 2 represents a hydrogen atom, an amino group, a hydroxyl group, a halogen atom, an R 3 -oxy group or R 3 Represents an amino group (R 3 represents an alkyl group having 1 to 15 carbon atoms which may be substituted or an acyl group having 1 to 15 carbon atoms which may be substituted).
- Ri represents report, 2-deoxylipose or a derivative thereof
- R 2 represents a hydrogen atom, an amino group, a hydroxyl group, a halogen atom, an R 3 -oxy group or an R 3 -amino group
- R 3 is a substituted Represents an alkyl group having 1 to 15 carbon atoms which may be substituted or an acyl group having 1 to 15 carbon atoms which may be substituted.
- R 1 represents a report, 2-doxylipose or a derivative thereof, and R 2 ′ represents an oxygen atom, a sulfur atom, or an imino group.
- Ri represents a report, 2-deoxylipose or a derivative thereof
- R 2 represents a hydrogen atom, an amino group, a hydroxyl group, a halogen atom, an R 3 -oxy group or an R 3 -amino group (where R 3 is Represents an alkyl group having 1 to 15 carbon atoms which may be substituted or an acyl group having 1 to 15 carbon atoms which may be substituted.).
- a method for producing a thionucleoside S-nitrosyl derivative comprising reacting a thionucleoside represented by the formula (1) with an S-nitrosyl compound.
- Ri represents a report, 2-deoxylipose or a derivative thereof
- R 2 represents a hydrogen atom, an amino group, a hydroxyl group, a halogen atom, an R 3 -oxy group or an R 3 -amino group (where R 3 is Represents an alkyl group having 1 to 15 carbon atoms which may be substituted or an acyl group having 1 to 15 carbon atoms which may be substituted.).
- a method for producing a thionucleoside S-nitrosyl derivative comprising reacting a thionucleoside represented by the formula (1) with an S-nitrosyl compound.
- Ri represents a report, 2-doxylipose or a derivative thereof, and R 2 ′ represents an oxygen atom, a sulfur atom, or an imino group.
- a method for producing a thionucleoside S-nitrosyl derivative comprising reacting a thionucleoside represented by the formula (1) with an S-nitrosyl compound.
- Oligonucleic acids of the present invention include, for example, those having a length of at least 12 bases.
- a method for mutagenizing a base sequence comprising reacting the oligonucleic acid according to (7) with a complementary strand thereof and treating the resulting reaction product under acidic conditions.
- the corresponding base is transferred by transferring a nitrosyl group contained in the oligonucleic acid to a corresponding base on the complementary strand side.
- a mutagenic agent or base for a base sequence comprising at least one selected from the group consisting of the derivative according to any one of (1) to (3) and the oligonucleic acid according to (7). Sequence mutagenesis kit.
- FIG. 1 is a diagram showing the transfer of a nitrosyl group by the derivative of the present invention.
- FIG. 2 is a diagram showing a hydrolysis reaction of an N-ditroso compound to a carbonyl group.
- FIG. 3 is a diagram showing a synthesis step of the nucleoside derivative of the present invention.
- FIG. 4 is a view showing the result of HPLC of the nucleoside derivative of the present invention.
- FIG. 5 is a diagram showing a step of synthesizing a ditrosylated oligonucleic acid of the present invention.
- FIG. 6 is a diagram showing the results of HPLC for the production of nitrosylated oligonucleic acids.
- FIG. 7 is a graph showing an increase curve of the fluorescence intensity of the nitrosylated oligonucleic acid DAF-2.
- FIG. 8 is a diagram showing the sequence of an oligonucleotide and its complementary strand and their reactions.
- FIG. 9 is a diagram showing the results of HPLC in which a NO-transfer reaction was performed.
- FIG. 10 is a graph showing a time-dependent change in the NO-transfer reaction performed using the oligonucleotide ⁇ .
- FIG. 11 is an HPLC diagram in which the reaction product of Oligonucleotide 7 and Oligonucleotide 9 with DAF-2 was analyzed to confirm the presence of NO.
- FIG. 12 is a diagram showing that cytidine was mutated to deoxyperidine and cytidine diazoate by HPLC analysis of the enzymatically degraded product of 9 generated by NO-transfer to oligonucleotide 8.
- FIG. 13 shows that 5-methyl cytidine was mutated to thymidine and 5-methyl cytidine diazolate by HPLC analysis of the enzymatic degradation product of 9 generated by NO-transfer on oligonucleotide 8.
- the derivative of the present invention is characterized in that it has a structure in which a ditosil group is bonded to a sulfur atom of a thionucleoside. Further, the derivative of the present invention is characterized in that it recognizes a specific base and binds to it, thereby transferring its own nitrosyl group to the other base.
- R i represents a report, 2-deoxylipose or a derivative thereof.
- R 2 represents a hydrogen atom, an amino group, a hydroxyl group, a halogen atom, an R 3 -oxy group, or an R 3 -amino group.
- R 2 ′ represents an oxygen atom, a sulfur atom or an imino group.
- R 3 represents an alkyl group having 1 to 15 carbon atoms which may be substituted or an acyl group having 1 to 15 carbon atoms which may be substituted.
- the alkyl group or the acyl group preferably has 1 to 10 carbon atoms, and more preferably 1 to 5 carbon atoms.
- the alkyl group may be linear, branched or cyclic, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, n- Examples include a hexyl group, a cyclohexyl group, an n-heptyl group, an n-octyl group, and an n-dodecyl group.
- the acryl group having 1 to 15 carbon atoms refers to a linear or branched alkyl group which may be substituted, a cycloalkyl carbonyl group which may be substituted, a benzoyl group which may be substituted, and the like. Represents
- alkylacyl groups include formyl, acetyl, propionyl, n-butyryl, isoptyryl, 2-methylbutyryl, 3-methylbutyryl, pivaloyl, valeryl, 2-methylvaleryl, dimethylpropyl, Examples include heptyl, octyl, and decanol groups.
- cycloalkylcarbonyl group examples include a cyclopropanecarbonyl group, a cyclohexanecarbonyl group, and a cyclopentanecarbonyl group.
- the substituent on the phenyl group may be unsubstituted, or any one of the 2-, 3-, and 4-positions on the phenyl group may be substituted.
- a substituent may be present at a plurality of positions, and in that case, the substituents may be the same or different.
- Substituents on the phenyl group include, for example, an alkyl group (methyl group, ethyl group, isopropyl group, etc.); an alkyloxy group (methoxy group, ethoxy group, n-propyl group).
- Substituted or unsubstituted amino group (nitro group, amino group, methylamino group, ethylamino group, n-propylamino group, i-propylamino group, dimethylamino group, ethylamino group, etc.); halogen group (fluoro group) Acryl group (formyl group, acetyl group, propionyl group, benzoyl group, etc.); acyloxy group (formyloxy group, acetyloxy group, propionyloxy group, benzoyloxy group, etc.); amide group (A formamide group, an acetoamide group, a benzamide group, etc.); and an aromatic ring (a phenyl group, etc.).
- benzoyl group which may be substituted include a benzoyl group, a 2-methoxybenzoyl group, a 3-methoxybenzoyl group, a 4-methoxybenzoyl group, a 2-methylbenzoyl group, a 3-methylbenzoyl group, and a benzoyl group.
- Ribose or deoxylipose has the following formula (VII):
- R 4 represents a hydroxyl group (in the case of report) or a hydrogen atom (in the case of deoxylipose).
- R 5 and R 6 are independent of each other and are the same or different and represent, for example, a hydrogen atom, a halogen group, or a substituted or unsubstituted hydroxyl group, in any case of ribose and deoxylipose and their derivatives.
- a halogen group means a fluorine atom, a chlorine atom, a bromine atom or Represents an iodine atom.
- a hydroxyl group substituted at R 4 and / or R 5 include a hydroxyl group substituted with a substituent that can be a general hydroxyl-protecting group (for example, carboxylate ester, sulfonate ester, ether, urethane, silyl group) Etc.).
- Hydroxyl protecting groups include alkyl groups (methyl, ethyl, n-propyl, isopropyl, n-butyl, i-butyl, t-butyl, pentyl, benzyl, 2-methoxybenzyl) Group, 3-methoxybenzyl group, 4-methoxybenzyl group, 2-methylbenzyl group, 3-methylbenzyl group, 4-methylbenzyl group, methoxyethyl group, ethoxyxethyl group, benzyloxymethyl group, benzyloxymethyl group, acetoxoxymethyl Group, acetoxityl group, benzoyloxymethyl group, benzoyloxyshethyl group, etc.); aryl group (phenyl group, 2-methoxyphenyl group, 3-methoxyphenyl group, 4-methoxyphenyl group, 4-phenyl group) Diphenyl group, 2-pyridinyl group, 3-pyridinyl group, 4-pyr
- Preferred examples of the compound represented by the formula (I) of the present invention include those in which R 1 is a dexoxy ribose and R 2 is an amino group. More preferably, R 1 is a 3 ′, 5′-bis-t-butyldimethylsilyl of report or dexoxylipose. And R 2 is an amino group.
- Preferred examples of the compound represented by the formula (II) of the present invention include those in which R 1 is dexoxy and R 2 is an amino group. More preferably, R 1 is a 3 ′, 5′-bis-t-butyldimethylsilyl derivative of report or dexoxylipose, and R 2 is an amino group.
- Preferred examples of the compound represented by the formula (III) of the present invention include those in which R 1 is a dexoxy report and R 2 ′ is an oxygen atom or an imino group. More preferably, R 1 is a 3 ′, 5′-bis-t-butyldimethylsilyl derivative of lipose or deoxylipose, and R 2 ′ is an oxygen atom or an imino group.
- the compound of the present invention represented by the general formula (1), ( ⁇ ) or (III) can be synthesized by devising a known reaction.
- the compound shown in (IV) When the compound shown in (IV) is poorly water-soluble, the compound is dissolved in an organic solvent (for example, acetonitrile or methanol), and triethylamine is added thereto in an amount equivalent to 10 times the amount of the raw material to react.
- organic solvent for example, acetonitrile or methanol
- triethylamine is added thereto in an amount equivalent to 10 times the amount of the raw material to react.
- the nitrosylating reagent include S-nitroso-N-acetylpenicillamine, nitric oxide and the like.
- the compound shown in (IV) When the compound shown in (IV) is poorly water-soluble, the compound is dissolved in an organic solvent (for example, acetonitrile or methanol), and triethylamine is added thereto in an amount equivalent to 10 times the amount of the raw material to react.
- organic solvent for example, acetonitrile or methanol
- triethylamine examples include S-nitroso-N-acetylpenicillamine, nitric oxide and the like.
- Nitrosylation reagents include, for example, S-nitroso-N-acetylpenicillamine, nitric oxide and the like.
- the compound of the present invention represented by the general formula (1), ( ⁇ ) or (III) is an acid addition salt or a base.
- Such salts may form addition salts, and such salts are also included in the scope of the present invention.
- physiologically acceptable salts are preferable.
- the base addition salt include salts of amines such as triethylamine, dimethylamine, ammonia and getylamine, and salts of metals such as sodium, potassium, calcium and magnesium.
- acid adducts include salts of mineral acids such as hydrochloric acid, sulfuric acid, or perchloric acid; or oxalic acid, fumaric acid, maleic acid, acetic acid, propionic acid, methanesulfonic acid, P-toluenesulfonic acid, etc. And salts with organic acids and the like.
- the compound of the present invention represented by the general formula (1), (II) or (III) is a nucleoside, when phosphoric acid is ester-bonded to the nucleoside, it becomes a nucleotide, which is a component of the nucleic acid.
- the nucleotide thus obtained can be incorporated into an appropriate oligonucleic acid and used as an oligonucleic acid that transfers a nitrosyl group in a sequence-specific manner.
- “Transferring a nitrosyl group” means that a nitrosyl group (a ditrosyl group present in the compound of the present invention) in the oligonucleic acid of the present invention is replaced with a base in a nucleic acid on the other side that reacts with the oligonucleic acid. It means transfer to the opposite base (also called target base) that forms a double strand (complex) (Fig. 1).
- the sequence of the oligonucleic acid of the present invention is not particularly limited, and it can be designed according to the nucleic acid sequence to be type III or can be designed to be a random base sequence.
- the oligonucleic acid can be designed so as to be complementary to the target base sequence of the gene (in this case, the oligonucleic acid side of the present invention). From the viewpoint, the nucleotide sequence of the gene becomes the complementary strand of the oligonucleic acid).
- a nitrosyl group is introduced into any of the bases.
- a nucleic acid having a sequence can be synthesized (in this case, from the viewpoint of the oligonucleic acid of the present invention, the synthesized nucleic acid becomes a complementary strand of the oligonucleic acid).
- the length of the oligonucleic acid sequence of the present invention is not particularly limited as long as the nitrosyl group in the compound of the present invention can be transferred to its target base. In the present invention, it preferably has at least 12 bases, and more preferably 15 to 22 bases.
- the position at which the compound of the present invention is incorporated into the oligonucleic acid may be at any position. In particular, in the case of expressing sequence specificity, the compound of the present invention is preferably placed at the third position from both ends of oligo nucleic acid It is good to incorporate it so that it is located inside. For example, in FIG. 5, the oligonucleic acid 7 of the present invention has 16 bases, of which the compound of the present invention is located at the fifth position.
- the compound of the present invention contained in the oligonucleic acid of the present invention does not need to be at one site, but may be at multiple sites. That is, in the oligonucleic acid of the present invention, the number of target base groups for introducing a nitrosyl group is not particularly limited. When the derivative is present at a plurality of locations, the derivative may be continuous at two or more locations or may be discontinuous.
- Oligonucleic acids can be produced by known methods, commercially available reagents for nucleic acid synthesis, and nucleic acid synthesizers.
- a synthesis example using the formula (IV) [where Ri is 2-doxylipose] is described below.
- a 5 ' ⁇ 0- ⁇ -dimethoxytrityl-3 ⁇ - ⁇ -(] 3-cyanoethyl-diisopropylphosphorotemidite) compound of formula (IV) (about 80 mmol) is dissolved in anhydrous acetonitrile, Attach to a synthesizer (Applied Biosystems 394 DNA / RNA Synthesizer).
- Purity assay and structure confirmation are performed, for example, by MALDI-TOF MS (Perseptive Biosystems, Voyager Elite, 3-hydroxy 2-picolinic Molecular weight root by acid-diammonium hydrogen citrate matrix)! ] Can be done on a regular basis.
- R 2 bonded to the heterocyclic ring in the general formula (1), (II) or (III) is a hydrogen atom, an amino group, a hydroxyl group, a halogen atom, R 3 _ Okishi group, R 3- Amino groups or other hetero atoms, (R 3 is a is as before SL) it is also possible to introduce.
- the compound of the present invention represented by the general formula (1), (II) or (III) is capable of recognizing and binding to a specific base of a gene according to the type of the base.
- the oligonucleic acid produced from (1), (II) or (III) and containing the compound as a constituent can be hybridized with a single-stranded nucleic acid containing such a base to form a double-stranded chain.
- Hybridization is performed under stringent conditions.
- the stringent conditions include, for example, a salt (sodium) concentration of 50 to 900 mM, a temperature force S of 10 to 50 ° C., preferably a salt (sodium) concentration of 50 to 150 mM, and a temperature of 50 to 150 mM. Conditions at 25 degrees C.
- the base subjected to the nitrosyl group transfer reaction is not particularly limited, and may be either a purine base (adenine, guanine) or a pyrimidine base (cytosine, thymine).
- a purine base represented by the formula (I) for example, guanine
- Compound (I) recognizes the corresponding cytosine base and is useful for specifically performing a nitrosyl transfer reaction on a nucleic acid containing this base.
- “corresponding” means complementary to each base.
- the base corresponding to guanine is cytosine.
- cytidine can be converted to deoxyperidine in a sequence-specific manner (FIG. 2). That is, cytosine in the oligonucleic acid is mutated to peracil. The conversion reaction at this time is performed under acidic conditions.
- the acidic condition is adjusted to pH 2 to 6, preferably 4 to 5, by adding hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid or the like to a reaction solution such as an aqueous solution or an organic buffer solution. Further, the pH can be adjusted using various organic acids.
- the reaction temperature is from 10 to 30 ° (: preferably from 15 to 25 ° C, and the reaction time is from 10 to 40 hours, preferably from 20 to 30 hours. It can be used as a so-called drug delivery compound, which transports NO active species to a specific place by a sequence-specific nitrosyl group transfer reaction.
- the compound or oligonucleic acid of the present invention can be used as a mutagenic agent or a mutagenesis kit for a base sequence.
- the mutagenic agent or kit of the present invention contains at least one of the compound or oligonucleic acid of the present invention, and may contain all of them.
- “Base sequence” means the base sequence of nucleic acids such as DNA, cDNA, RNA, and mRNA, and can be of any type, whether it is of biological origin or artificially synthesized. Nucleic acids are bacteria, yeast, animal cells, plant cells, insect cells, or It can be collected from a mammal or the like by a known method, and anything having a nucleic acid such as a genome or a plasmid can be used.
- the oligonucleic acid is arranged so that the compound of the present invention is located at the same position as the position of the base to be mutagenized in the base sequence of the type I nucleic acid, that is, so as to hybridize to the type II base sequence.
- the compound of the present invention is located at the same position as the position of the base to be mutagenized in the base sequence of the type I nucleic acid, that is, so as to hybridize to the type II base sequence.
- the kit of the present invention can contain, in addition to the compound of the present invention, a hybridization solution, a washing buffer, a fluorescent reagent, and the like.
- HPLC eluate was separated by a splitter, and about l / 40 (about 5 HL) was introduced into the mass spectrometer.
- Mass spectrometry was performed in positive mode ESI-TOF. MS condition (Applied Biosystem Mariner System 5299) was completed with Spray Tip Potential, 4006, Nozzle potential 300, Nozzle temperature 140.
- Oligonucleotide 6 (150 ninol) and SNAP (3 imol) were dissolved in carbonate buffer (pH 10.0) 3001 and left at room temperature for 12 hours. After confirming the progress of the reaction by HPLC, a newly generated peak was isolated under the same HPLC conditions. As a result, oligonucleotide 7 (SEQ ID NO: 2) shown in Scheme 7 of FIG. 5 was obtained. The yield was 31% by UV absorbance at 260 nm. In addition, as a result of molecular weight measurement by MALDI-TOF MASS, a measured value of molecular weight 4796.69 corresponding to the theoretical value of 4797.76 was observed. [Example 3] Generation test of [NO +] by decomposition of nucleotide 7
- MES buffer solution of oligonucleotide 7 (Fig. 5) (0.05 M MES, 0.1 M NaCl, adjusted to pH 5 and pH 7, 1.5 mL, oligonucleotide 7: 0.5 M), and add DAF-2 ( diaminofluoroscein, final concentration 4 M) at 25 ° C Stirred.
- the fluorescence spectrum (excitation wavelength 488 ⁇ , fluorescence wavelength 500-600 nm) of this reaction solution was measured over time. At the same time, 11 was sampled over time from this solution and injected into HPLC to observe the change from oligonucleotide 7 to oligonucleotide 6.
- reaction was carried out using 10 M of each of ODN (7) and ODN (8,10-12), or ImM daltathione at pH 7, 25 ° C. in 50 mM MES buffer containing 100 mM NaCl.
- ODN (6) SEQ ID NO: 1
- ODN (7) SEQ ID NO: 2
- ODN (8) SEQ ID NO: 3
- ODN (9) SEQ ID NO: 4
- MES buffer solution (0.05 M MES, 0.1 M NaCl, pH 7) containing about 20 iM of each of oligonucleotide 7 and oligonucleotide 8 was reacted at 25 ° C. for 8 hours. Thereafter, the solution was adjusted to pH 5 and left at the same temperature for another 30 hours. Oligonucleotide 9 was isolated from the reaction mixture by HPLC and lyophilized.
- HPLC conditions are as follows.
- the isolated oligonucleotide 9 was hydrolyzed with an enzyme, and the resulting nucleoside was analyzed by HPLC. Enzymatic degradation and HPLC, conditions are as follows.
- BAP bacteria alkaline phosphatase
- VPDE venom phosphodiesterase
- Buffer Ol M Tris, Ol M NaCl, 14 mM MgCl 2 , 37 ° C pH 7.
- A 0.1M TEAA buffer
- B CH 3 CN, 10% to 30% / 20min, 40% / 30min, linear gradient
- a MES buffer solution (0.05 M MES, 0.1 M NaCl, pH 7) containing about 20 M of each of oligonucleotide 7 and oligonucleotide 8 was reacted at 25 ° C. for 8 hours. Then adjust the solution to pH 5 and add CaCl 2 ZnCl 2 or MgCl 2 (5 mM) and left at the same temperature for an additional day. Oligonucleotide 9 was isolated from the reaction mixture by HPLC and lyophilized. This was hydrolyzed with BAP and BPDE, and then subjected to HPLC.
- the enzyme reaction conditions and HPLC conditions are as follows.
- BAP bacteria alkaline phosphatase
- VPDE venom phospnodiesterase
- Buffer 0.1 M Tris, 0.1 M NaCl, 14 mM MgCl 2 , 37 ° C pH 7.
- the present invention By applying the NO-delivery method, it becomes possible to selectively perform site-directed mutagenesis at the translation or polymerization step. Industrial applicability
- a thionucleoside-S-nitrosyl derivative is provided.
- INDUSTRIAL APPLICABILITY The derivative of the present invention can transfer its silosyl group in a sequence-specific and base-specific manner and can substitute a base of a complementary strand.
- SEQ ID NO: 1 Synthetic nucleotide
- n represents a nucleotide derivative of a, g , c or t (location: 5)
- n represents a thionucleotide nitrosyl derivative of a, g, c or t (location: 5) '
- SEQ ID NO: 3 Synthetic nucleotide
- SEQ ID NO: 3 n represents a, g, c or t (location: 12).
- SEQ ID NO: 4 Synthetic nucleotide
- SEQ ID NO: 4 n represents a, g, c or t nitrosyl derivative (location: 12).
- SEQ ID NO: 5 synthetic nucleotide
- SEQ ID NO: 6 synthetic nucleotide
- SEQ ID NO: 7 synthetic nucleotide
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US10/554,158 US7495095B2 (en) | 2003-04-22 | 2004-03-12 | Thionucleoside-S-nitrosyl derivative |
JP2005505689A JP4665758B2 (ja) | 2003-04-22 | 2004-03-12 | チオヌクレオシド−s−ニトロシル誘導体 |
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US9714265B2 (en) | 2015-01-28 | 2017-07-25 | Massachusetts Institute Of Technology | Mutagenic nucleoside analogs and uses thereof |
EP3452055A4 (en) | 2016-05-06 | 2019-11-06 | Tod M. Woolf | IMPROVED METHODS OF GENOME EDITING WITH AND WITHOUT PROGRAMMABLE NUCLEASES |
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Non-Patent Citations (1)
Title |
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AMADO S. ET AL: "Kinetics and mechanism of the formation and reactions of S-nitroso derivatives of some heterocyclic thiones", JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS 2, no. 4, 2001, pages 441 - 447, XP002979742 * |
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WO2020042729A1 (zh) * | 2018-08-30 | 2020-03-05 | 浙江大学 | 6-双硫取代-2'-脱氧鸟苷类化合物及其制备方法和应用 |
US11247994B2 (en) | 2018-08-30 | 2022-02-15 | Zhejiang University | 6-dithio-substituted-2′-deoxyguanosine compound, preparation method thereof and use thereof |
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US7495095B2 (en) | 2009-02-24 |
JP4665758B2 (ja) | 2011-04-06 |
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