JP5196448B2 - Reactive compounds containing aminooxy groups - Google Patents
Reactive compounds containing aminooxy groups Download PDFInfo
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- JP5196448B2 JP5196448B2 JP2009547151A JP2009547151A JP5196448B2 JP 5196448 B2 JP5196448 B2 JP 5196448B2 JP 2009547151 A JP2009547151 A JP 2009547151A JP 2009547151 A JP2009547151 A JP 2009547151A JP 5196448 B2 JP5196448 B2 JP 5196448B2
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- 150000001875 compounds Chemical class 0.000 title claims description 105
- 125000002344 aminooxy group Chemical group [H]N([H])O[*] 0.000 title claims description 28
- 238000002372 labelling Methods 0.000 claims description 46
- 150000003839 salts Chemical class 0.000 claims description 39
- 239000003153 chemical reaction reagent Substances 0.000 claims description 37
- 125000003172 aldehyde group Chemical group 0.000 claims description 36
- 125000003118 aryl group Chemical group 0.000 claims description 36
- 125000001424 substituent group Chemical group 0.000 claims description 28
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 27
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 claims description 27
- 125000001976 hemiacetal group Chemical group 0.000 claims description 24
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 24
- 125000000468 ketone group Chemical group 0.000 claims description 22
- 125000003277 amino group Chemical group 0.000 claims description 17
- 238000009739 binding Methods 0.000 claims description 17
- 229960002685 biotin Drugs 0.000 claims description 14
- 239000011616 biotin Substances 0.000 claims description 14
- 125000006239 protecting group Chemical group 0.000 claims description 11
- 125000000217 alkyl group Chemical group 0.000 claims description 10
- 125000002947 alkylene group Chemical group 0.000 claims description 10
- 125000002252 acyl group Chemical group 0.000 claims description 8
- 125000000027 (C1-C10) alkoxy group Chemical group 0.000 claims description 6
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 6
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 claims description 6
- 125000004653 anthracenylene group Chemical group 0.000 claims description 6
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 6
- 125000005843 halogen group Chemical group 0.000 claims description 6
- 125000004957 naphthylene group Chemical group 0.000 claims description 6
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 6
- 125000005562 phenanthrylene group Chemical group 0.000 claims description 5
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 claims description 5
- 125000006376 (C3-C10) cycloalkyl group Chemical group 0.000 claims 2
- 238000006467 substitution reaction Methods 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 description 87
- 239000000243 solution Substances 0.000 description 58
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 56
- 125000000962 organic group Chemical group 0.000 description 42
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 39
- -1 DNA Chemical class 0.000 description 33
- 108020004414 DNA Proteins 0.000 description 28
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 27
- 102000039446 nucleic acids Human genes 0.000 description 26
- 108020004707 nucleic acids Proteins 0.000 description 26
- 150000007523 nucleic acids Chemical class 0.000 description 26
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 24
- OOLJDXIORAOCRO-UHFFFAOYSA-N C1C=CC2=C(C1(C(=O)CONC(C3=CC=CC=C3)(C4=CC=CC=C4)C5=CC=CC=C5)N)C=CC=C2N Chemical compound C1C=CC2=C(C1(C(=O)CONC(C3=CC=CC=C3)(C4=CC=CC=C4)C5=CC=CC=C5)N)C=CC=C2N OOLJDXIORAOCRO-UHFFFAOYSA-N 0.000 description 23
- 108091034117 Oligonucleotide Proteins 0.000 description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 23
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 18
- 230000009257 reactivity Effects 0.000 description 18
- 208000035657 Abasia Diseases 0.000 description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- 239000000126 substance Substances 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 238000005481 NMR spectroscopy Methods 0.000 description 15
- 125000005647 linker group Chemical group 0.000 description 15
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 14
- 239000012300 argon atmosphere Substances 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 125000004432 carbon atom Chemical group C* 0.000 description 12
- 125000000524 functional group Chemical group 0.000 description 12
- 239000002244 precipitate Substances 0.000 description 12
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 11
- 239000010410 layer Substances 0.000 description 11
- 238000007254 oxidation reaction Methods 0.000 description 11
- 108091033319 polynucleotide Proteins 0.000 description 11
- 102000040430 polynucleotide Human genes 0.000 description 11
- 239000002157 polynucleotide Substances 0.000 description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 description 10
- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 description 10
- 239000002585 base Substances 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000012156 elution solvent Substances 0.000 description 9
- 125000004672 ethylcarbonyl group Chemical group [H]C([H])([H])C([H])([H])C(*)=O 0.000 description 9
- 239000000499 gel Substances 0.000 description 9
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 9
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- MXHRCPNRJAMMIM-UHFFFAOYSA-N desoxyuridine Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-UHFFFAOYSA-N 0.000 description 8
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- AWKZBUKPPAAXCR-UHFFFAOYSA-N NC(CC=C1)(C(CON)=O)C2=C1C(N)=CC=C2 Chemical compound NC(CC=C1)(C(CON)=O)C2=C1C(N)=CC=C2 AWKZBUKPPAAXCR-UHFFFAOYSA-N 0.000 description 7
- 230000000295 complement effect Effects 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 229920002401 polyacrylamide Polymers 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000006260 foam Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 230000003100 immobilizing effect Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000010898 silica gel chromatography Methods 0.000 description 6
- 125000003396 thiol group Chemical group [H]S* 0.000 description 6
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 150000001299 aldehydes Chemical group 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 5
- 125000001261 isocyanato group Chemical group *N=C=O 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 238000000967 suction filtration Methods 0.000 description 5
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 5
- 125000004890 (C1-C6) alkylamino group Chemical group 0.000 description 4
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 4
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 4
- 229910052794 bromium Inorganic materials 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 4
- 239000000460 chlorine Substances 0.000 description 4
- 229910052801 chlorine Inorganic materials 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 229940125773 compound 10 Drugs 0.000 description 4
- 229940125782 compound 2 Drugs 0.000 description 4
- 229940126214 compound 3 Drugs 0.000 description 4
- 125000000753 cycloalkyl group Chemical group 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 229910052731 fluorine Inorganic materials 0.000 description 4
- 239000011737 fluorine Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 4
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 125000004433 nitrogen atom Chemical group N* 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 125000000213 sulfino group Chemical group [H]OS(*)=O 0.000 description 4
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 4
- 125000005300 thiocarboxy group Chemical group C(=S)(O)* 0.000 description 4
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 4
- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 description 3
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 description 3
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- ONBQEOIKXPHGMB-VBSBHUPXSA-N 1-[2-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 ONBQEOIKXPHGMB-VBSBHUPXSA-N 0.000 description 3
- UNILWMWFPHPYOR-KXEYIPSPSA-M 1-[6-[2-[3-[3-[3-[2-[2-[3-[[2-[2-[[(2r)-1-[[2-[[(2r)-1-[3-[2-[2-[3-[[2-(2-amino-2-oxoethoxy)acetyl]amino]propoxy]ethoxy]ethoxy]propylamino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-1-oxopropan-2-yl Chemical compound O=C1C(SCCC(=O)NCCCOCCOCCOCCCNC(=O)COCC(=O)N[C@@H](CSC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)C(=O)NCC(=O)N[C@H](CO)C(=O)NCCCOCCOCCOCCCNC(=O)COCC(N)=O)CC(=O)N1CCNC(=O)CCCCCN\1C2=CC=C(S([O-])(=O)=O)C=C2CC/1=C/C=C/C=C/C1=[N+](CC)C2=CC=C(S([O-])(=O)=O)C=C2C1 UNILWMWFPHPYOR-KXEYIPSPSA-M 0.000 description 3
- CYNCHYUHEQCZSM-UHFFFAOYSA-N 2-(tritylamino)oxyacetic acid Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(NOCC(=O)O)C1=CC=CC=C1 CYNCHYUHEQCZSM-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
- YQFOTBGRGFJUMI-UHFFFAOYSA-N NC1(C(CON)=O)C(C=CC=C2)=C2C=CC1 Chemical compound NC1(C(CON)=O)C(C=CC=C2)=C2C=CC1 YQFOTBGRGFJUMI-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- WREOTYWODABZMH-DTZQCDIJSA-N [[(2r,3s,4r,5r)-3,4-dihydroxy-5-[2-oxo-4-(2-phenylethoxyamino)pyrimidin-1-yl]oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N(C=C\1)C(=O)NC/1=N\OCCC1=CC=CC=C1 WREOTYWODABZMH-DTZQCDIJSA-N 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 125000004429 atom Chemical group 0.000 description 3
- VPKDCDLSJZCGKE-UHFFFAOYSA-N carbodiimide group Chemical group N=C=N VPKDCDLSJZCGKE-UHFFFAOYSA-N 0.000 description 3
- UXTMROKLAAOEQO-UHFFFAOYSA-N chloroform;ethanol Chemical compound CCO.ClC(Cl)Cl UXTMROKLAAOEQO-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- 125000002228 disulfide group Chemical group 0.000 description 3
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- 125000005842 heteroatom Chemical group 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 3
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- 229920001223 polyethylene glycol Polymers 0.000 description 3
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- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 235000011152 sodium sulphate Nutrition 0.000 description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 125000004434 sulfur atom Chemical group 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- KYHUBFIOWQTECH-LAEOZQHASA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-n'-(2-aminooxyacetyl)pentanehydrazide Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NNC(=O)CON)SC[C@@H]21 KYHUBFIOWQTECH-LAEOZQHASA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- KXDHJXZQYSOELW-UHFFFAOYSA-N Carbamic acid Chemical group NC(O)=O KXDHJXZQYSOELW-UHFFFAOYSA-N 0.000 description 2
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
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- 125000005548 pyrenylene group Chemical group 0.000 description 1
- 125000002098 pyridazinyl group Chemical group 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 125000005551 pyridylene group Chemical group 0.000 description 1
- VTGOHKSTWXHQJK-UHFFFAOYSA-N pyrimidin-2-ol Chemical compound OC1=NC=CC=N1 VTGOHKSTWXHQJK-UHFFFAOYSA-N 0.000 description 1
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- 239000008223 sterile water Substances 0.000 description 1
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- 235000002906 tartaric acid Nutrition 0.000 description 1
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
- C07C279/18—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to carbon atoms of six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C235/16—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
- C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
- C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
- G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する分子に対して高い反応性を有する化合物、該化合物を含む標識試薬、及び該化合物を用いた生体分子固定化用支持体に関する。 The present invention relates to a compound having high reactivity with a molecule having an aldehyde group, a hemiacetal group, a carboxyl group or a keto group, a labeling reagent containing the compound, and a support for immobilizing a biomolecule using the compound. .
アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基に反応する構造として、一級アミノ基、アミノオキシ基などが知られている。一級アミノ基の場合は、不安定なシッフベースを形成するため、その結合を還元して安定化させる必要がある。一方、アミノオキシ基は高い反応性を有し、アミノ基のように還元剤を必要としない。このため、アルデヒド基を有する分子を効率的に検出する試薬として、アミノオキシ基をもった試薬がこれまで広く利用されてきた（非特許文献１、非特許文献２）。この試薬（ＡＲＰ；Ａｌｄｅｈｙｄｅ Ｒｅａｃｔｉｖｅ Ｐｒｏｂｅ）は、アミノオキシ基とビオチンがリンカーを介して結合したシンプルな構造を有する。この試薬を、標的分子に反応させて結合させ、ビオチン−アビジン複合体形成を利用して、蛍光又は発光など分光学的な検出法により標的分子を検出することができる。
この試薬を用いて定量する代表的な分子として、ＲＮＡ及びＤＮＡなどの核酸がある。ＲＮＡには、遺伝子をコードしたｍＲＮＡ、タンパク質合成に関与するｒＲＮＡ、及びｔＲＮＡの他に、種々の機能性ＲＮＡが存在していることが近年明らかになってきた。これらの機能性ＲＮＡは、細胞内でタンパク質の発現などにおいて機能し、癌化にも関与していることが報告されている。そのため、ｍＲＮＡとこれら機能性ＲＮＡの細胞内量を正確に定量することは、遺伝子相互作用の詳細な解明や疾病の診断にもつながる。また、ＤＮＡは種々の環境因子によって損傷を受け、その代表的な損傷に塩基部が失われたアベイシックサイト（アプリニックサイト；ＡＰサイト）がある。アベイシックサイトはヘミアセタール構造とアルデヒド構造の平衡状態で存在し、上記ＡＲＰ試薬を作用させるとアルデヒド構造に反応するため、アベイシックサイトを検出することが可能になっている。種々の化学物質、環境因子のＤＮＡに対する損傷度を測定して見積もることで、食品に含まれる化学物質、紫外線等の遺伝子変異原性評価にも役立つ。
生体分子の中でも特にＤＮＡ及びＲＮＡを正確に定量するには、標的分子に対して高い反応性を有する試薬が必要である。しかしながら、これまでに用いられてきた試薬はその反応効率が不十分で、多量の試薬を作用させる必要があった。そのため、多くの条件下で標的分子を定量する必要がある場合には、コスト高となり時間を要していた。この他、糖鎖などの反応にもアミノオキシ基が用いられているが、反応性が不十分であった。
そのため、生体分子のなかでも特にこれらＲＮＡ、ＤＮＡなどの分子に対し、少量でも迅速に反応する新規な試薬の開発が望まれていた。
Representative molecules to be quantified using this reagent include nucleic acids such as RNA and DNA. In recent years, it has become clear that various functional RNAs exist in addition to mRNA encoding a gene, rRNA involved in protein synthesis, and tRNA. It has been reported that these functional RNAs function in the expression of proteins in cells and are involved in canceration. Therefore, accurate quantification of intracellular amounts of mRNA and these functional RNAs leads to detailed elucidation of gene interactions and diagnosis of diseases. In addition, DNA is damaged by various environmental factors, and there is an abasic site (aponic site; AP site) in which the base portion is lost due to typical damage. The abasic site exists in an equilibrium state of a hemiacetal structure and an aldehyde structure, and reacts with the aldehyde structure when the ARP reagent is acted on, so that the abasic site can be detected. By measuring and estimating the degree of damage of various chemical substances and environmental factors to DNA, it is useful for evaluating chemical mutagenicity such as chemical substances contained in foods and ultraviolet rays.
In order to accurately quantify DNA and RNA among biomolecules in particular, a reagent having high reactivity with a target molecule is required. However, the reagents used so far have inadequate reaction efficiency, and a large amount of reagents must be allowed to act. Therefore, when it is necessary to quantify the target molecule under many conditions, the cost is high and time is required. In addition, aminooxy groups are used for reactions such as sugar chains, but the reactivity is insufficient.
Therefore, it has been desired to develop a novel reagent that reacts quickly with a small amount of biomolecules, particularly RNA and DNA.
本発明の課題は、アルデヒド基などのアミノオキシ基と反応性の官能基を有する生体分子に対して高い反応性を有する化合物を提供することである。
本発明者らは、アミノオキシ基、芳香族基及び親水性基を有する化合物が、アルデヒド基などのアミノオキシ基と反応性の官能基を有する生体分子に対して高い反応性を有することを見出し、本発明を完成するに至った。
すなわち、本発明は以下の発明を包含する。
（１）一般式１：
Ｒ１−ＮＨ−Ｏ−Ｌ１−Ｄ−Ｌ２−Ａ （１）
（式中、Ｒ１は、水素原子、アルキル基又はアミノ基の保護基であり、Ｄは、２価の芳香族基であり、Ｌ１は、直接結合又はリンカー基であり、Ｌ２は、直接結合又はリンカー基であり、Ａは親水性基を含む有機基である）
で表される化合物又はその塩。
（２）Ａが、親水性基として、置換又は無置換のグアニジノ基、置換又は無置換のポリエチレングリコール基、カルボキシル基、アミノオキシ基及びヒドロキシル基からなる群から選択される少なくとも１つの基を含む、（１）記載の化合物又はその塩。
（３）Ａが、親水性基として、置換又は無置換のグアニジノ基を含む、（２）記載の化合物又はその塩。
（４）Ａが、以下の一般式２：
（式中、Ｘ１及びＸ２は、水素原子又は有機基である）
で表される、（１）〜（３）のいずれかに記載の化合物又はその塩。
（５）Ｄが、置換若しくは無置換のフェニレン基、置換若しくは無置換のアントリレン基、置換若しくは無置換のナフチレン基、置換若しくは無置換のフェナントリレン基、置換若しくは無置換のアントラキノリレン、又は置換若しくは無置換のアクリジニレンである、（１）〜（４）のいずれかに記載の化合物又はその塩。
（６）Ｄが、以下の一般式：
（式中、一方の結合部位がＬ１又はＯに結合し、他方の結合部位がＬ２又はＡに結合する）
で表される２価の芳香族基、及びこれらの芳香族基において芳香環が１〜３個の置換基を有する芳香族基から選択される、（５）記載の化合物又はその塩。
（７） Ｌ１が、以下の一般式３又は４：
（式中、Ｒ３は、置換若しくは無置換のＣ１−９アルキレン基又は−（ＣＨ２）ｏ−（ＯＣＨ２ＣＨ２）ｐ−（ＣＨ２）ｑ−であり、ここで、ｏ〜ｑは、それぞれ独立して０〜１５の整数であり、ｏ＋ｐ＋ｑは、１〜１５である）
のいずれかで表される２価の基であり、Ｌ２が、以下の一般式５又は６：
（式中、Ｒ４は、置換若しくは無置換のＣ１−９アルキレン基又は−（ＣＨ２）ｒ−（ＯＣＨ２ＣＨ２）ｓ−（ＣＨ２）ｔ−であり、ここで、ｒ〜ｔは、それぞれ独立して０〜１５の整数であり、ｒ＋ｓ＋ｔは、１〜１５であるである）
のいずれかで表される２価の基である、（１）〜（６）のいずれかに記載の化合物又はその塩。
（８）以下の一般式７：
（式中、Ｒ１は、水素原子又はアミノ基の保護基であり、ｎは、１〜５の整数であり、ｍは、１〜５の整数であり、Ｒａ〜Ｒｆは、それぞれ独立して、水素原子又は置換基であり、Ｘ１及びＸ２は、水素原子又は有機基である）
で表される、（１）記載の化合物又はその塩。
（９）以下の一般式８：
（式中、Ｒ１は、水素原子又はアミノ基の保護基であり、ｎは、１〜５の整数であり、Ｒａ〜Ｒｆは、それぞれ独立して、水素原子又は置換基であり、Ｘ２は、水素原子又は有機基である）
で表される、（１）記載の化合物又はその塩。
（１０）以下の一般式：
（式中、Ｒ１及びＲ５は、それぞれ独立して、水素原子又はアミノ基の保護基であり、ｎ及びｉは、それぞれ独立して、１〜５の整数であり、Ｒａ〜Ｒｆは、それぞれ独立して、水素原子又は置換基である）
で表される、（１）記載の化合物又はその塩。
（１１）Ａが標識基をさらに含む、（１）〜（９）のいずれかに記載の化合物又はその塩。
（１２）Ｘ１及びＸ２の少なくとも一方が有機基であり、該有機基が標識基である、（４）、（８）、又は（９）記載の化合物又はその塩。
（１３）アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する生体分子を標識するための試薬であって、（１１）又は（１２）記載の化合物又はその塩を含む前記試薬。
（１４）（１１）又は（１２）記載の化合物と、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する生体分子とが結合してなる標識化生体分子であって、該化合物のアミノオキシ基と該生体分子のアルデヒド基、ヘミアセタール基、カルボキシル基又はケト基とが反応して共有結合を形成している、前記標識化生体分子。
（１５）アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する生体分子を固定化するための生体分子固定化用支持体であって、担体及び該担体表面に存在する（１）〜（１０）のいずれかに記載の化合物又はその塩の層を含む、前記支持体。
本発明により、アルデヒド基などのアミノオキシ基と反応性の官能基を有する生体分子に対して高い反応性を有する化合物が提供される。An object of the present invention is to provide a compound having high reactivity with a biomolecule having a functional group reactive with an aminooxy group such as an aldehyde group.
The present inventors have found that a compound having an aminooxy group, an aromatic group and a hydrophilic group has high reactivity with a biomolecule having a functional group reactive with an aminooxy group such as an aldehyde group. The present invention has been completed.
That is, the present invention includes the following inventions.
(1) General formula 1:
R 1 -NH-O-L 1 -D-L 2 -A (1)
(Wherein R 1 is a hydrogen atom, an alkyl group or an amino group protecting group, D is a divalent aromatic group, L 1 is a direct bond or a linker group, and L 2 is A direct bond or a linker group, and A is an organic group containing a hydrophilic group)
Or a salt thereof.
(2) A includes at least one group selected from the group consisting of a substituted or unsubstituted guanidino group, a substituted or unsubstituted polyethylene glycol group, a carboxyl group, an aminooxy group, and a hydroxyl group as a hydrophilic group. (1) A compound or a salt thereof.
(3) The compound or salt thereof according to (2), wherein A contains a substituted or unsubstituted guanidino group as a hydrophilic group.
(4) A is the following general formula 2:
(Wherein, X 1 and X 2 are a hydrogen atom or an organic group)
The compound or its salt in any one of (1)-(3) represented by these.
(5) D is a substituted or unsubstituted phenylene group, a substituted or unsubstituted anthrylene group, a substituted or unsubstituted naphthylene group, a substituted or unsubstituted phenanthrylene group, a substituted or unsubstituted anthraquinolylene, or substituted or unsubstituted The compound or salt thereof according to any one of (1) to (4), which is unsubstituted acridinylene.
(6) D is the following general formula:
(Wherein one binding site binds to L 1 or O and the other binding site binds to L 2 or A)
The compound or its salt as described in (5) selected from the bivalent aromatic group represented by these, and the aromatic group in which an aromatic ring has 1-3 substituents in these aromatic groups.
(7) L 1 is the following general formula 3 or 4:
(Wherein R 3 is a substituted or unsubstituted C 1-9 alkylene group or — (CH 2 ) o — (OCH 2 CH 2 ) p — (CH 2 ) q —, where o to q Are each independently an integer of 0 to 15, and o + p + q is 1 to 15)
A divalent group represented by any one of, L 2 is the following general formula 5 or 6:
(Wherein R 4 is a substituted or unsubstituted C 1-9 alkylene group or — (CH 2 ) r — (OCH 2 CH 2 ) s — (CH 2 ) t —, where r to t Are each independently an integer of 0-15, and r + s + t is 1-15.)
The compound or its salt in any one of (1)-(6) which is a bivalent group represented by either.
(8) The following general formula 7:
(In the formula, R 1 is a hydrogen atom or a protecting group for an amino group, n is an integer of 1 to 5, m is an integer of 1 to 5, and R a to R f are each independent. And a hydrogen atom or a substituent, and X 1 and X 2 are a hydrogen atom or an organic group)
The compound or its salt as described in (1) represented by these.
(9) The following general formula 8:
(In the formula, R 1 is a hydrogen atom or a protecting group for an amino group, n is an integer of 1 to 5, and R a to R f are each independently a hydrogen atom or a substituent, X 2 is a hydrogen atom or an organic group)
The compound or its salt as described in (1) represented by these.
(10) The following general formula:
(In the formula, R 1 and R 5 are each independently a hydrogen atom or an amino-protecting group, n and i are each independently an integer of 1 to 5, and R a to R f Each independently represents a hydrogen atom or a substituent)
The compound or its salt as described in (1) represented by these.
(11) The compound or salt thereof according to any one of (1) to (9), wherein A further comprises a labeling group.
(12) The compound or salt thereof according to (4), (8), or (9), wherein at least one of X 1 and X 2 is an organic group, and the organic group is a labeling group.
(13) A reagent for labeling a biomolecule having an aldehyde group, a hemiacetal group, a carboxyl group, or a keto group, the reagent comprising the compound or a salt thereof according to (11) or (12).
(14) A labeled biomolecule obtained by binding a compound according to (11) or (12) and a biomolecule having an aldehyde group, a hemiacetal group, a carboxyl group or a keto group, wherein the aminooxy of the compound The labeled biomolecule, wherein a group and a aldehyde group, a hemiacetal group, a carboxyl group, or a keto group of the biomolecule react to form a covalent bond.
(15) A biomolecule immobilization support for immobilizing a biomolecule having an aldehyde group, a hemiacetal group, a carboxyl group or a keto group, which is present on the carrier and the surface of the carrier (1) to (10) The said support body containing the layer of the compound or its salt in any one of).
According to the present invention, a compound having high reactivity with a biomolecule having a functional group reactive with an aminooxy group such as an aldehyde group is provided.
図１ａはＤＮＡのアベイシックサイトを表し、図１ｂはＤＮＡのアベイシックサイトへの反応を表し、図１ｃはＲＮＡの３’末端酸化反応を表す。
図２は、３’末端が酸化されたＲＮＡ（Ｆ−Ｌｒ１７Ｘｏｘ）に対するＡＲＰとａｏＮｇの結合反応を示す。ｓｓは１本鎖を表し、括弧内に反応部位で形成される塩基対（ＸＹ）の組み合わせを示す。Ｇｒａｙ ｂａｒ，ＡＲＰ；ｂｌａｃｋ ｂａｒ，ａｏＮｇ。
図３は、アベイシックサイトを有するＤＮＡへのＡＲＰとａｏＮｇの結合反応を示す。アベイシックサイトを有するＤＮＡの一本鎖（ｓｓ）及び二本鎖（ｄｓ）に対し、３７℃、４２℃、４７℃で反応を行ったときの速度定数のグラフを示す。
図４は、２’−デオキシウリジンを含むＤＮＡに対するＡＲＰ、ａｏＮｇ、ａｏＮｇ−ｂｉｏの結合反応を示す。２’−デオキシウリジンを含むＤＮＡ２本鎖をＵＤＧで処理した後に、標識試薬（ＡＲＰ、ａｏＮｇ、ａｏＮｇ−ｂｉｏ）を添加し、各時間の反応産物の精製率を調べた。反応液のポリアクリルアミドゲルによる分析の結果と、各時間の反応率のグラフを示した。
図５は、アベイシックサイトを含む２本鎖ＤＮＡ間の架橋反応の結果を示す。２’−デオキシウリジンを含むＤＮＡをＵＤＧで処理してアベイシックサイトを生成させた後にａｏＮａｏを反応させた。反応液のポリアクリルアミドゲルによる分析の結果と、各時間の反応率のグラフを示した。
本明細書は、本願の優先権の基礎である特願２００７−３３０３１５号の明細書、特許請求の範囲、及び図面に記載された内容を包含する。FIG. 1a shows the basic site of DNA, FIG. 1b shows the reaction to the basic site of DNA, and FIG. 1c shows the 3 ′ end oxidation reaction of RNA.
FIG. 2 shows the binding reaction of ARP and aoNg to RNA (F-Lr17Xox) oxidized at the 3 ′ end. ss represents a single strand, and indicates a combination of base pairs (XY) formed at the reaction site in parentheses. Gray bar, ARP; black bar, aoNg.
FIG. 3 shows the binding reaction of ARP and aoNg to DNA having a basic site. The graph of the rate constant when reacting at 37 degreeC, 42 degreeC, and 47 degreeC with respect to the single strand (ss) and double strand (ds) of DNA which has an abasic site is shown.
FIG. 4 shows the binding reaction of ARP, aoNg, and aoNg-bio to DNA containing 2′-deoxyuridine. After DNA double strands containing 2′-deoxyuridine were treated with UDG, labeling reagents (ARP, aoNg, aoNg-bio) were added, and the purification rate of the reaction product at each time was examined. The result of the analysis of the reaction solution by polyacrylamide gel and the graph of the reaction rate at each time are shown.
FIG. 5 shows the results of a cross-linking reaction between double-stranded DNAs containing a basic site. DNA containing 2′-deoxyuridine was treated with UDG to form a basic site, and then aoNao was reacted. The result of the analysis of the reaction solution by polyacrylamide gel and the graph of the reaction rate at each time are shown.
This specification includes the contents described in the specification, claims, and drawings of Japanese Patent Application No. 2007-330315 which is the basis of the priority of the present application.
一実施形態において本発明は、一般式１：
Ｒ１−ＮＨ−Ｏ−Ｌ１−Ｄ−Ｌ２−Ａ （１）
（式中、Ｒ１は、水素原子、アルキル基（好ましくはＣ１−６アルキル基）又はアミノ基の保護基であり、Ｄは、２価の芳香族基であり、Ｌ１は、直接結合又はリンカー基であり、Ｌ２は、直接結合又はリンカー基であり、Ａは、親水性基を含む有機基である）で表される化合物又はその塩（以下、本発明の化合物と称する場合がある）に関する。
塩としては、例えば、無機酸との塩、有機酸との塩、及び塩基性又は酸性アミノ酸との塩などが挙げられる。無機酸との塩の好適な例としては、塩酸、臭化水素酸、硝酸、硫酸、リン酸などとの塩が挙げられる。有機酸との塩の好適な例としては、酢酸、トリフルオロ酢酸、フマル酸、シュウ酸、酒石酸、マレイン酸、クエン酸、コハク酸、リンゴ酸、メタンスルホン酸、ベンゼンスルホン酸、ｐ−トルエンスルホン酸などとの塩が挙げられる。塩基性アミノ酸との塩の好適な例としては、アルギニン、リジン、オルニチンなどとの塩が挙げられる。酸性アミノ酸との塩の好適な例としては、アスパラギン酸、グルタミン酸などとの塩が挙げられる。
本発明の化合物は、無水物であっても、溶媒和物であってもよい。一般式１の化合物又はその塩の溶媒和物もまた本発明の化合物に包含される。ここで溶媒は溶質（一般式１の化合物又はその塩）の生物活性を妨げるものでなければ特に制限されない。適当な溶媒の具体例としては、水、メタノール、エタノール及び酢酸が含まれる。好ましくは、溶媒は水である。
一般式１のＲ１におけるアミノ基の保護基は、特に制限されないが、例えば、アシル基、カルバメート基、トリアルキルシリル基、フタリル基、カルボキシアルキルカルボニル基、トシル基、トリフルオロアセチル基、トリチル基、及びモノ又はジ置換トリチル基が挙げられ、好ましくはアルキル基である。これらの基は置換されていてもよい。
Ｒ１は、好ましくは炭素数１〜１０、より好ましくは炭素数１〜６の、置換若しくは無置換のアルキル基である。アルキル基は、直鎖でも分岐鎖でもよい。ここで、置換基としては、例えば、フッ素、塩素、臭素及びヨウ素から選ばれるハロゲン原子、ヒドロキシル基、メルカプト基、アミノ基、Ｃ１−６アルキル基、Ｃ１−６アルキルアミノ基、カルバモイル基、チオカルボキシ基、スルホ基、スルフィノ基、イソシアナト基、ニトロ基、シアノ基、Ｃ２−６アルケニル基、Ｃ３−１０シクロアルキル基、Ｃ１−１０アルコキシ基、Ｃ１−１０アシル基、Ｃ１−１０アルコキシカルボニル基並びにカルボキシル基等を挙げることができる。置換基が複数存在する場合、各置換基は同一でも異なっていてもよい。置換基の数は、好ましくは１〜３個である。
本明細書において「Ｃ１−６アルキル基」等の記載における「Ｃ１−６」等の表現は、その基が１〜６個の炭素原子を含むことをさす、該基は直鎖でも分岐鎖でもよい。
一般式１のＤにおける２価の芳香族基は、ベンゼン環を有するものであればよく、縮合環中にベンゼン環を有するものでもよい。２価の芳香族基としては、５〜２５個の炭素原子、好ましくは６〜２０個の炭素原子を含む単環式又は多環式の２価の芳香族基が挙げられる。より具体的には、置換又は無置換のフェニレン基、置換又は無置換のピリジレン基、置換又は無置換のピリダジニル基、置換又は無置換のピリミジニレン基、置換又は無置換のピラジニレン基、置換又は無置換のフリレン基、置換又は無置換のチエニレン基、置換又は無置換のピロリレン基、置換又は無置換のイミダゾリレン基、置換又は無置換のチアゾリレン基、置換又は無置換のオキサゾリレン基、置換又は無置換のナフチレン基、置換又は無置換のアントリレン基、置換又は無置換のピレニレン基、置換又は無置換のインダニレン基、置換又は無置換のテトラヒドロナフチレン基、置換又は無置換のキノリレン基、イソキノリレン基、置換又は無置換のシンノリニレン基、置換又は無置換のキナゾリニレン基、置換又は無置換のキノキサリニレン基、置換又は無置換のナフチリジニレン基、置換又は無置換のフタラジニレン基、置換又は無置換のインドリレン基、置換又は無置換のイソインドリレン基、置換又は無置換のベンゾフリレン基、置換又は無置換のベンゾチエニレン基、置換又は無置換のインダゾリレン基、置換又は無置換のベンゾイミダゾリレン基、置換又は無置換のベンゾチアゾリレン基、置換若しくは無置換のフェナントリレン基、置換若しくは無置換のアントラキノリレン、又は置換若しくは無置換のアクリジニレンが挙げられる。Ｄとしては、置換又は無置換のフェニレン基、置換又は無置換のアントリレン基、置換又は無置換のナフチレン基、及び置換若しくは無置換のフェナントリレン基、置換若しくは無置換のアントラキノリレン、又は置換若しくは無置換のアクリジニレンが好ましく、特に置換又は無置換のアントリレン基及び置換又は無置換のナフチレン基が好ましい。
より具体的には、Ｄは、以下の一般式：
（式中、一方の結合部位がＬ１又はＯに結合し、他方の結合部位がＬ２又はＡに結合する）
で表される２価の芳香族基、及びこれらの芳香族基において芳香環が１〜３個の置換基を有する芳香族基から選択される。
あるいは、Ｄは、以下の一般式：
（式中、一方の結合部位がＬ１又はＯに結合し、他方の結合部位がＬ２又はＡに結合する）
で表される２価の芳香族基、及びこれらの芳香族基において芳香環が１〜３個の置換基を有する芳香族基から選択される。
あるいは、Ｄとしては、以下の一般式：
で表される２価の芳香族基、及びこれらの芳香族基において芳香環が１〜３個の置換基を有する芳香族基もまた好ましい。
一般式１のＤにおける芳香族基の置換基としては、例えば、フッ素、塩素、臭素及びヨウ素から選ばれるハロゲン原子、ヒドロキシル基、メルカプト基、アミノ基、オキソ基、Ｃ１−６アルキル基、Ｃ１−６アルキルアミノ基、カルバモイル基、チオカルボキシ基、スルホ基、スルフィノ基、イソシアナト基、ニトロ基、シアノ基、Ｃ２−６アルケニル基、Ｃ３−１０シクロアルキル基、Ｃ１−１０アルコキシ基、Ｃ１−１０アシル基、Ｃ１−１０アルコキシカルボニル基並びにカルボキシル基等を挙げることができる。置換基が複数存在する場合、各置換基は同一でも異なっていてもよい。置換基の数は、好ましくは１〜１０個、好ましくは１〜３個である。
一般式１のＬ１におけるリンカー基は、アミノオキシ基の酸素原子と芳香族基Ｄを結合する基であって、式１の化合物の反応性を阻害しないものであれば特に制限されない。リンカー基は、通常、２価の有機基、例えば主鎖に１〜１５個、好ましくは１〜１２個の炭素原子並びに／又は複素原子（酸素、窒素又は硫黄原子）を含む２価の有機基である。主鎖に含まれる原子の数は、リンカー基がつなぐ原子間において最短距離を形成する鎖に含まれる原子の数をさす。Ｌ１としては、例えば、−（ＣＨ２）ｏ−（ＯＣＨ２ＣＨ２）ｐ−（ＣＨ２）ｑ−が挙げられる。ここで、ｏ〜ｑは、それぞれ独立して０〜１５の整数であり、ｏ＋ｐ＋ｑは、１〜１５である。
Ｌ１としては、例えば、以下の一般式３又は４：
（式中、Ｒ３は、置換若しくは無置換のＣ１−９アルキレン基又は−（ＣＨ２）ｏ−（ＯＣＨ２ＣＨ２）ｐ−（ＣＨ２）ｑ−であり、ここで、ｏ〜ｑは、それぞれ独立して０〜１５の整数であり、ｏ＋ｐ＋ｑは、１〜１５である）
で表される２価の基も例示できる。一般式３においては、Ｒ３がアミノオキシ基の酸素原子に結合し、−ＮＨ−の窒素原子が芳香族基Ｄに結合する。一般式４においては、Ｒ３がアミノオキシ基の酸素原子に結合し、−（ＣＯ）−の炭素原子が芳香族基Ｄに結合する。
ここでＣ１−９アルキレン基の置換基としては、例えば、フッ素、塩素、臭素及びヨウ素から選ばれるハロゲン原子、ヒドロキシル基、メルカプト基、アミノ基、Ｃ１−６アルキル基、Ｃ１−６アルキルアミノ基、カルバモイル基、チオカルボキシ基、スルホ基、スルフィノ基、イソシアナト基、ニトロ基、シアノ基、Ｃ２−６アルケニル基、Ｃ３−１０シクロアルキル基、Ｃ１−１０アルコキシ基、Ｃ１−１０アシル基、Ｃ１−１０アルコキシカルボニル基並びにカルボキシル基等を挙げることができる。置換基が複数存在する場合、各置換基は同一でも異なっていてもよい。置換基の数は、好ましくは１〜３個である。
一般式１のＬ２におけるリンカー基は、芳香族基Ｄと親水性基を含む有機基Ａとを結合する基であって、式１の化合物の反応性を阻害しないものであれば特に制限されない。リンカー基は、通常、２価の有機基、例えば主鎖に１〜１５個、好ましくは１〜１２個の炭素原子及び／又は複素原子（酸素、窒素又は硫黄原子）を含む２価の有機基である。Ｌ２としては、例えば、−（ＣＨ２）ｒ−（ＯＣＨ２ＣＨ２）ｓ−（ＣＨ２）ｔ−が挙げられる。ここで、ｒ〜ｔは、それぞれ独立して０〜１５の整数であり、ｒ＋ｓ＋ｔは、１〜１５である。
Ｌ２としては、例えば、以下の一般式５又は６：
（式中、Ｒ４は、置換若しくは無置換のＣ１−９アルキレン基又は−（ＣＨ２）ｒ−（ＯＣＨ２ＣＨ２）ｓ−（ＣＨ２）ｔ−であり、ここで、ｒ〜ｔは、それぞれ独立して０〜１５の整数であり、ｒ＋ｓ＋ｔは、１〜１５である）
で表される２価の基も例示できる。一般式５においては、−ＮＨ−の窒素原子が芳香族基Ｄに結合し、Ｒ４が親水性基を有する有機基Ａに結合する。一般式６においては、−（ＣＯ）−の炭素原子が芳香族基Ｄに結合し、Ｒ４が親水性基を有する有機基Ａに結合する。Ｃ１−９アルキレン基の置換基は、一般式３又は４について記載したのと同様である。
一般式１のＡにおける親水性基を含む有機基は、親水性基を少なくとも１つ含む有機基であれば特に制限されず、親水性基を複数、例えば、２〜３個含んでいてもよい。Ａが複数の親水性基を含む場合、それらは同一でも異なっていてもよい。本発明の化合物においてＡが親水性基を含むことにより、化合物の水溶性が向上し、標的分子との相互作用を向上させることができる。その結果、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する分子との反応性を向上させることができる。
有機基Ａは、好ましくは親水性基として、置換又は無置換のグアニジノ基、置換又は無置換のポリエチレングリコール基、カルボキシル基、アミノオキシ基及びヒドロキシル基からなる群から選択される少なくとも１つの親水性基、より好ましくは置換又は無置換のグアニジノ基を含む。
グアニジノ基及びポリエチレングリコール基の置換基としては、例えば、フッ素、塩素、臭素及びヨウ素から選ばれるハロゲン原子、ヒドロキシル基、メルカプト基、アミノ基、Ｃ１−６アルキル基、Ｃ１−６アルキルアミノ基、カルバモイル基、チオカルボキシ基、スルホ基、スルフィノ基、イソシアナト基、ニトロ基、シアノ基、Ｃ２−６アルケニル基、Ｃ３−１０シクロアルキル基、Ｃ１−１０アルコキシ基、Ｃ１−１０アシル基、Ｃ１−１０アルコキシカルボニル基、カルボキシル基並びにグアニジノ基等を挙げることができる。
置換又は無置換のグアニジノ基は、例えば、以下の一般式２：
（式中、Ｘ１及びＸ２は、水素原子又は有機基である）
で表される。ここでＸ１及びＸ２における有機基は、グアニジノ基の親水性を妨げるものでなければ特に制限されず、例えば、上記グアニジノ基の置換基が例示できる。
本発明において、置換又は無置換のグアニジノ基には、以下の一般式２’及び２”：
（式中、Ｘ３〜Ｘ７は、水素原子又は有機基である）
で表される基も包含される。ここでＸ３〜Ｘ７における有機基は、グアニジノ基の親水性を妨げるものでなければ特に制限されず、例えば、上記グアニジノ基の置換基が例示できる。
本発明の化合物におけるアミノオキシ基は、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基、特にアルデヒド基と高い反応性で共有結合を形成できることから、本発明の化合物は、アミノオキシ基は、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する化合物、例えば生体分子に、高い反応性で結合させることができる。親水性基として置換又は無置換のグアニジノ基を含む本発明の化合物は、核酸、特に２本鎖の核酸に対し、高い反応性で結合させることができる。
有機基Ａは、さらに標識基を有していてもよい。本発明の化合物は、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する生体分子と高い反応性をもって結合しうることから、有機基Ａが標識基を有する本発明の化合物をこのような生体分子と反応させることにより、該生体分子を効率的に標識することができる。従って、一実施形態において本発明は、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する生体分子を標識するための試薬であって、標識基を有する本発明の化合物を含む前記標識試薬に関する。本発明はまた、本発明の化合物と、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する生体分子とが結合してなる標識化生体分子であって、該化合物のアミノオキシ基と該生体分子のアルデヒド基、ヘミアセタール基、カルボキシル基又はケト基とが反応して共有結合を形成している、前記標識化生体分子に関する。
アルデヒド基、ヘミアセタール基、カルボキシル基、又はケトン基を有する生体分子としては、ポリペプチド、ポリヌクレオチド及び糖鎖などが挙げられる。ポリペプチドには、ペプチド、オリゴペプチド及びタンパク質が包含される。ポリヌクレオチドには、ＤＮＡ、ＲＮＡ及び非天然型核酸等の核酸が包含され、これらは１本鎖でも２本鎖でもよく、又、ポリヌクレオチドにはオリゴヌクレオチドも包含される。本発明においてポリヌクレオチドの塩基長は、通常２〜１００００塩基、好ましくは２〜１０００塩基である。核酸の任意の位置に、アルデヒド基、ヘミアセタール基、カルボキシル基、又はケトン基を複数導入または生成させることが可能であり、こうして得られたアルデヒド基、ヘミアセタール基、カルボキシル基、又はケトン基を有する非天然型核酸も本発明においてポリヌクレオチドに包含される。また、ポリヌクレオチドには、ＤＮＡ及びＲＮＡの誘導体、例えば、３’末端のヒドロキシル基が酸化されたアルデヒド基を有するＲＮＡ、アベイシックサイトを有するＤＮＡ、及びそのヒドロキシル基が酸化されてアルデヒド基となったものも包含される。特に、核酸におけるこれらの基の導入、生成、及びそれらの基に対する標識に関して以下に記載する。
核酸におけるアルデヒド基の生成方法は幾つかの種類に分けることができる。その代表的な方法の一つに、アベイシックサイト（またはアプリニックサイト（ＡＰサイト））形成を通してのアルデヒド構造の形成がある。核酸のアベイシックサイトは、核酸の塩基部分が除去された糖部分の構造を示し（図１−ａ）、閉環と開環構造の平衡で存在している。アベイシックサイトは、閉環状態ではヘミアセタール構造であるが、開環型ではアルデヒド構造となっており、アミノオキシ基を作用させるとそのアルデヒド構造に反応する（図１−ｂ）。
このアベイシックサイトは、天然の核酸や非天然型核酸を酸性条件で処理することによって生成させることができる。非天然型核酸としては、２−ピリミジドン体（Ｔｅｔｒａｈｅｄｒｏｎ Ｌｅｔｔｅｒｓ，３１，１７５−１７８（１９９０））、２’−デオキシ−キサントシン体（Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，３１，１０４５−１０５１（２００３））、１−デアザ−２’−デオキシグアノシン体（Ｏｒｇ Ｌｅｔｔ，７，７０９−７１２（２００５））などが挙げられる。また、その他にも特別な反応条件でヘミアセタール構造の保護基を脱保護させてアベイシックサイトを形成する２’−デオキシ−Ｄ−リボース誘導体（非特許文献６；非特許文献７）なども知られている。
また、上記のように誘導体を用いてアベイシックサイトを生成させる他に、損傷塩基をオリゴヌクレオチド又はポリヌクレオチドに導入後、その損傷塩基を除去する酵素で処理し、アベイシックサイトを生成させる方法もある。この損傷塩基には、２’−デオキシウリジン、５−ヒドロキシメチル−２’−デオキシシチジンなどが挙げられ、２’−デオキシウリジンに対してはウラシルＮグリコシラーゼを作用させることによってアベイシックサイトを生成させることができる。２’−デオキシウリジンは、化学合成によっても、またそのトリリン酸体（ｄＵＴＰ）を基質に用いてＤＮＡポリメラーゼによる伸張反応を行うことによっても、オリゴヌクレオチドおよびポリヌクレオチド中に複数導入することが可能である。この２’−デオキシウリジンをＤＮＡ中に取り込ませてウラシルＮグリコシラーゼを作用させて生成したアベイシックサイトを標識後にＤＮＡを断片化させ、その断片を化学的に標識するキット（Ｒｉｂｏ−ＳＰＩＡ（登録商標））も市販されている（特表２００５−５３４３０４）。この市販キット中に含まれる標識試薬と本発明の試薬の構造は異なり、本発明の試薬を用いることによって、より迅速、かつ高収率で標的核酸を標識することが可能となる。
アベイシックサイトを経由せずにアルデヒド基を核酸中に生成させる方法もある。例えばあらかじめ非天然型核酸をオリゴヌクレオチドに導入し、その後酸化反応を行うことでアルデヒド基を生成させることが可能である（Ｔｅｔｒａｈｅｄｒｏｎ Ｌｅｔｔｅｒｓ，３７，９０６７−９０７０（１９９６））。また、ＲＮＡの場合には、天然型であってもアルデヒド基を形成可能である。例えばＲＮＡの３’末端の２’位、３’位の水酸基を過ヨウ素酸等によって酸化させ、その３’末端にアルデヒド基を発生させることが可能である（図１−ｃ）。この場合、２’位と３’位にアルデヒド基が生じるため、アミノオキシ基及び標識基を有する化合物を作用させると２’位または３’位においてＲＮＡが標識される。
ケトン基を持つ非天然型核酸も報告されており（Ｏｒｇ Ｌｅｔｔ，３，３９８３−３９８６（２００１））、これらを導入した核酸に対し、本発明のアミノオキシ基を有する化合物を反応させることでケト基に対して標識することも可能である。
標識基は、標識物質を含む基をさす。標識物質としては、生体分子の標識に慣用される標識物質、例えば、放射性同位元素、色素（蛍光色素、発光色素）、ジゴキシゲニン（ＤＩＧ）、ビオチン、メチレンブルー、フェロセンなどを例示できるがこれらに限定されない。
本発明の化合物が標識基を含む態様において、有機基Ａが、上記一般式２のグアニジノ基を含むか、又は一般式２のグアニジノ基である場合は、Ｘ１及びＸ２のどちらかが有機基であり、該有機基が上記標識基であることが好ましい。有機基Ａが、上記一般式２’又は２”のグアニジノ基であるかこれを含む場合も、Ｘ３〜Ｘ５のいずれか、又はＸ６若しくはＸ７が標識基であることが好ましい。
その場合、標識物質は、グアニジノ基の窒素原子に直接結合していてもよいし、リンカーを介して結合していてもよい。リンカーとしては、特に制限されないが、通常、２価の有機基、例えば主鎖に１〜１５個、好ましくは１〜１２個の炭素原子及び／又は複素原子（酸素、窒素又は硫黄原子）を含む２価の有機基である。例えば、−（ＣＨ２）ｖ−（ＯＣＨ２ＣＨ２）ｗ−（ＣＨ２）ｘ−ＮＨ−が挙げられる。ここで、ｖ、ｗ及びｘは、それぞれ独立して０〜１５の整数であり、ｖ＋ｗ＋ｘは、１〜１５である。好ましくは、−（ＣＨ２）２−（ＯＣＨ２ＣＨ２）２−ＮＨ−である。
標識物質としてビオチンを含む標識基（一般式２においては、Ｘ１又はＸ２）としては、以下の式：
で表される基を例示することができる。
有機基Ａが、標識基が結合したグアニジノ基を含む有機基である本発明の化合物は、特に、核酸に標識を付すために好適に用いることができる。
本発明の標識試薬によって標識された生体分子は、様々な検出系によって定量可能である。そのため、本標識試薬は検出系、検出機器の影響を受けず、高い汎用性をもつため、広く利用される可能性を有する。
あるいは、有機基Ａは、生体分子基を含んでいてもよい。生体分子基は、糖鎖、ポリヌクレオチド（オリゴヌクレオチドを含む）、ポリペプチド（例えば、酵素）などの生体分子を含む基をさす。Ａがポリヌクレオチドを含む場合、ＰＣＲのプライマーとして用いることができる。例えば、本発明の化合物を標的分子に結合させた後、鋳型ＤＮＡと蛍光またはビオチンなどで標識された基質（ｄＮＴＰ）共存下でプライマーの伸長反応を行った場合、ポリヌクレオチドに蛍光物質（またはビオチンなど）が取り込まれ、それによって標的分子が標識される。
有機基Ａが、上記一般式２のグアニジノ基を含むか、又は一般式２のグアニジノ基である場合は、Ｘ１及びＸ２のどちらかが有機基であり、該有機基が上記生体分子基であることが好ましい。有機基Ａが、上記一般式２’の基を含むか、又は一般式２’の基である場合は、Ｘ３〜Ｘ５のいずれかが有機基であり、該有機基が上記生体分子基であることが好ましい。上記一般式２”の基を含むか、又は一般式２”の基である場合は、Ｘ６及びＸ７のどちらかが有機基であり、該有機基が上記生体分子基であることが好ましい。その場合、生体分子は、グアニジノ基の窒素原子に直接結合していてもよいし、リンカーを介して結合していてもよい。リンカーは、上記標識基におけるリンカーと同様である。
本発明の化合物におけるアミノオキシ基は、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する分子、例えば生体分子に、高い反応性で結合することから、担体の表面に本発明の化合物の層を形成し、本発明の化合物を介して生体分子を担体に固定化することができる。従って、一実施形態において本発明は、アルデヒド基、ヘミアセタール基、カルボキシル基又はケト基を有する生体分子を固定化するための生体分子固定化用支持体であって、担体及び該担体表面に存在する本発明の化合物の層を含む、前記支持体に関する。
本発明の化合物の層の形成は、単に本発明の化合物を担体上に塗布することにより実施してもよいし、担体上の官能基と共有結合を形成することにより実施してもよい。
担体上の官能基と共有結合を形成する場合、本発明の化合物における有機基Ａは、担体への結合に適した官能基を含むことが好ましい。そのような官能基としては、固定化しようとする担体上に存在する官能基と共有結合を形成しうる基が挙げられ、例えば、活性エステル基、エポキシ基、アルデヒド基、カルボジイミド基、イソチオシアネート基又はイソシアネート基と共有結合しうる基（例えば、アミノ基、アミノオキシ基など）、あるいはマレイミド基又はジスルフィド基と反応しうる基（例えば、メルカプト基など）等が挙げられる。これらの官能基は保護された形態でもよい。保護された形態とは、官能基の水素原子が保護基で置換された形態を意味する。アミノ基、アミノオキシ基などの保護基としては、特に制限されないが、アルキル基、アシル基、カルバメート基、トリアルキルシリル基、フタリル基、カルボキシアルキルカルボニル基、トシル基、トリフルオロアセチル基、トリチル基、及びモノ又はジ置換トリチル基が挙げられる。モノ置換トリチル基としては、例えば、モノアルコキシトリチル基、好ましくは炭素数１〜４、より好ましくは炭素数１のアルコキシ基を有するモノアルコキシトリチル基、具体的には、モノメトキシトリチル基、モノエトキシトリチル基、モノプロポキシトリチル基、モノイソプロポキシトリチル基及びモノブトキシトリチル基が挙げられる。
本発明の化合物を結合させる担体の材料としては、例えば、石英ガラス、ホウ珪酸ガラス及びソーダライムガラスなどのガラス、シリコン、金属、繊維、木材、紙、セラミックス、プラスチック（例えば、ポリエステル樹脂、ポリエチレン樹脂、ポリプロピレン樹脂、ＡＢＳ樹脂、ナイロン、アクリル樹脂、フッ素樹脂、ポリカーボネート樹脂、ポリウレタン樹脂、メチルペンテン樹脂、フェノール樹脂、メラミン樹脂、エポキシ樹脂、塩化ビニル樹脂）が挙げられる。本発明においては、ガラス、シリコン、セラミックス又はプラスチックを使用するのが好ましい。上記担体の表面に本発明の化合物の層を形成する。担体に官能基を導入する場合、導入する官能基としては、例えば、活性エステル基、エポキシ基、アミノ基、クロロ基、ジスルフィド基、アルデヒド基、マレイイミド基、カルボジイミド基、イソチオシアナト基、イソシアナト基等が挙げられる。アミノ基またはアミノオキシ基を有する本発明の化合物を結合する場合は、活性エステル基、エポキシ基、アルデヒド基、カルボジイミド基、イソチオシアネート基、イソシアネート基が導入された担体を用いるのが好ましく、メルカプト基を有する本発明の化合物を結合する場合は、マレイミド基、ジスルフィド基が導入された担体を用いるのが好ましい。
担体の形状は、特に制限されず、基板状、糸状、球状、ビーズ状、多角形状、粉末状、多孔質状などが挙げられ、本発明においては基板状が好ましい。
置換又は無置換のグアニジノ基を含む有機基Ａを有する本発明の化合物は、核酸に特異的に結合し得ることから、当該化合物を担体に結合させることにより、優れた核酸固定化用支持体を製造することができる。得られた核酸固定化用支持体にＤＮＡなどの核酸を固定化することにより、効率的にマイクロアレイを製造するができる。
本発明の化合物により、特に、ＲＮＡ及びＤＮＡなどの核酸への標識反応を効率化することができ、遺伝子検出に要する時間を短縮することができる。さらに本発明の化合物の高い反応性によって、検出感度の向上、及び検出値の定量化が可能となり、遺伝子に関わるより詳細な情報を正確に得ることができる。また、担体上に本発明の化合物の層を形成し、その表面上で標的生体分子との結合反応を行う場合にも、担体上で効率的に標的生体分子の捕捉が行えるようになり、生体分子の同定に応用することができる。
以下、本発明を実施例により説明するが、本発明の範囲は実施例に限定されない。In one embodiment, the present invention provides compounds of general formula 1:
R 1 -NH-OL 1 -D-L 2 -A (1)
(Wherein R 1 Is a hydrogen atom, an alkyl group (preferably C 1-6 An alkyl group) or a protecting group for amino group, D is a divalent aromatic group, L 1 Is a direct bond or a linker group and L 2 Is a direct bond or a linker group, and A is an organic group containing a hydrophilic group) or a salt thereof (hereinafter sometimes referred to as a compound of the present invention).
Examples of the salt include a salt with an inorganic acid, a salt with an organic acid, and a salt with a basic or acidic amino acid. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of the salt with an organic acid include acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfone. And salts with acids. Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ornithine and the like. Preferable examples of the salt with acidic amino acid include salts with aspartic acid, glutamic acid and the like.
The compound of the present invention may be an anhydride or a solvate. Solvates of the compound of general formula 1 or salts thereof are also encompassed in the compounds of the present invention. Here, the solvent is not particularly limited as long as it does not interfere with the biological activity of the solute (the compound of general formula 1 or its salt). Specific examples of suitable solvents include water, methanol, ethanol and acetic acid. Preferably the solvent is water.
R in general formula 1 1 The amino-protecting group in is not particularly limited, but examples thereof include acyl groups, carbamate groups, trialkylsilyl groups, phthalyl groups, carboxyalkylcarbonyl groups, tosyl groups, trifluoroacetyl groups, trityl groups, and mono- or di-substituted groups. A trityl group is mentioned, Preferably it is an alkyl group. These groups may be substituted.
R 1 Is preferably a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. The alkyl group may be linear or branched. Here, as the substituent, for example, a halogen atom selected from fluorine, chlorine, bromine and iodine, a hydroxyl group, a mercapto group, an amino group, C 1-6 Alkyl group, C 1-6 Alkylamino group, carbamoyl group, thiocarboxy group, sulfo group, sulfino group, isocyanato group, nitro group, cyano group, C 2-6 Alkenyl group, C 3-10 A cycloalkyl group, C 1-10 Alkoxy group, C 1-10 Acyl group, C 1-10 Examples thereof include an alkoxycarbonyl group and a carboxyl group. When a plurality of substituents are present, each substituent may be the same or different. The number of substituents is preferably 1 to 3.
In this specification, “C 1-6 “C” in the description of “alkyl group” 1-6 The expression "" means that the group contains 1 to 6 carbon atoms, which group may be linear or branched.
The divalent aromatic group in D of General Formula 1 may be any group having a benzene ring, and may have a benzene ring in the condensed ring. Examples of the divalent aromatic group include monocyclic or polycyclic divalent aromatic groups containing 5 to 25 carbon atoms, preferably 6 to 20 carbon atoms. More specifically, a substituted or unsubstituted phenylene group, a substituted or unsubstituted pyridylene group, a substituted or unsubstituted pyridazinyl group, a substituted or unsubstituted pyrimidinylene group, a substituted or unsubstituted pyrazinylene group, a substituted or unsubstituted group Furylene group, substituted or unsubstituted thienylene group, substituted or unsubstituted pyrrolylene group, substituted or unsubstituted imidazolylene group, substituted or unsubstituted thiazolylene group, substituted or unsubstituted oxazolylene group, substituted or unsubstituted naphthylene Group, substituted or unsubstituted anthrylene group, substituted or unsubstituted pyrenylene group, substituted or unsubstituted indanylene group, substituted or unsubstituted tetrahydronaphthylene group, substituted or unsubstituted quinolylene group, isoquinolylene group, substituted or unsubstituted Substituted cinnolinylene group, substituted or unsubstituted quinazolinylene group, substituted or unsubstituted quinoki Linylene group, substituted or unsubstituted naphthyridinylene group, substituted or unsubstituted phthalazinylene group, substituted or unsubstituted indolylene group, substituted or unsubstituted isoindolinylene group, substituted or unsubstituted benzofurylene group, substituted or unsubstituted A benzothienylene group, a substituted or unsubstituted indazolylene group, a substituted or unsubstituted benzimidazolylene group, a substituted or unsubstituted benzothiazolylene group, a substituted or unsubstituted phenanthrylene group, a substituted or unsubstituted anthraquinolylene, or A substituted or unsubstituted acridinylene is mentioned. D is a substituted or unsubstituted phenylene group, a substituted or unsubstituted anthrylene group, a substituted or unsubstituted naphthylene group, a substituted or unsubstituted phenanthrylene group, a substituted or unsubstituted anthraquinolylene, or a substituted or unsubstituted Substituted acridinylene is preferable, and a substituted or unsubstituted anthrylene group and a substituted or unsubstituted naphthylene group are particularly preferable.
More specifically, D represents the following general formula:
(Wherein one binding site is L 1 Or the other binding site is L 2 Or bind to A)
And the aromatic ring in these aromatic groups is selected from aromatic groups having 1 to 3 substituents.
Alternatively, D is the following general formula:
(Wherein one binding site is L 1 Or the other binding site is L 2 Or bind to A)
And the aromatic ring in these aromatic groups is selected from aromatic groups having 1 to 3 substituents.
Alternatively, as D, the following general formula:
A divalent aromatic group represented by the formula (1) and an aromatic group in which the aromatic ring has 1 to 3 substituents are also preferable.
Examples of the substituent of the aromatic group in D of the general formula 1 include a halogen atom selected from fluorine, chlorine, bromine and iodine, a hydroxyl group, a mercapto group, an amino group, an oxo group, C 1-6 Alkyl group, C 1-6 Alkylamino group, carbamoyl group, thiocarboxy group, sulfo group, sulfino group, isocyanato group, nitro group, cyano group, C 2-6 Alkenyl group, C 3-10 A cycloalkyl group, C 1-10 Alkoxy group, C 1-10 Acyl group, C 1-10 Examples thereof include an alkoxycarbonyl group and a carboxyl group. When a plurality of substituents are present, each substituent may be the same or different. The number of substituents is preferably 1 to 10, preferably 1 to 3.
L in general formula 1 1 The linker group in is a group that binds the oxygen atom of the aminooxy group and the aromatic group D, and is not particularly limited as long as it does not inhibit the reactivity of the compound of Formula 1. The linker group is usually a divalent organic group, for example, a divalent organic group containing 1 to 15, preferably 1 to 12 carbon atoms and / or a hetero atom (oxygen, nitrogen or sulfur atom) in the main chain. It is. The number of atoms contained in the main chain refers to the number of atoms contained in the chain that forms the shortest distance between the atoms connected by the linker group. L 1 For example,-(CH 2 ) o -(OCH 2 CH 2 ) p -(CH 2 ) q -. Here, o to q are each independently an integer of 0 to 15, and o + p + q is 1 to 15.
L 1 As, for example, the following general formula 3 or 4:
(Wherein R 3 Is substituted or unsubstituted C 1-9 An alkylene group or-(CH 2 ) o -(OCH 2 CH 2 ) p -(CH 2 ) q Where o to q are each independently an integer of 0 to 15 and o + p + q is 1 to 15)
The bivalent group represented by these can be illustrated. In general formula 3, R 3 Is bonded to the oxygen atom of the aminooxy group, and the nitrogen atom of —NH— is bonded to the aromatic group D. In general formula 4, R 3 Is bonded to the oxygen atom of the aminooxy group, and the carbon atom of — (CO) — is bonded to the aromatic group D.
Where C 1-9 Examples of the substituent of the alkylene group include a halogen atom selected from fluorine, chlorine, bromine and iodine, a hydroxyl group, a mercapto group, an amino group, C 1-6 Alkyl group, C 1-6 Alkylamino group, carbamoyl group, thiocarboxy group, sulfo group, sulfino group, isocyanato group, nitro group, cyano group, C 2-6 Alkenyl group, C 3-10 A cycloalkyl group, C 1-10 Alkoxy group, C 1-10 Acyl group, C 1-10 Examples thereof include an alkoxycarbonyl group and a carboxyl group. When a plurality of substituents are present, each substituent may be the same or different. The number of substituents is preferably 1 to 3.
L in general formula 1 2 The linker group in is a group that binds the aromatic group D and the organic group A containing a hydrophilic group, and is not particularly limited as long as it does not inhibit the reactivity of the compound of Formula 1. The linker group is usually a divalent organic group, for example, a divalent organic group containing 1 to 15, preferably 1 to 12 carbon atoms and / or hetero atoms (oxygen, nitrogen or sulfur atoms) in the main chain. It is. L 2 For example,-(CH 2 ) r -(OCH 2 CH 2 ) s -(CH 2 ) t -. Here, r to t are each independently an integer of 0 to 15, and r + s + t is 1 to 15.
L 2 As, for example, the following general formula 5 or 6:
(Wherein R 4 Is substituted or unsubstituted C 1-9 An alkylene group or-(CH 2 ) r -(OCH 2 CH 2 ) s -(CH 2 ) t Where r to t are each independently an integer of 0 to 15 and r + s + t is 1 to 15)
The bivalent group represented by these can be illustrated. In the general formula 5, a nitrogen atom of —NH— is bonded to the aromatic group D, and R 4 Binds to an organic group A having a hydrophilic group. In the general formula 6, a carbon atom of — (CO) — is bonded to the aromatic group D, and R 4 Binds to an organic group A having a hydrophilic group. C 1-9 The substituents of the alkylene group are the same as those described for the general formula 3 or 4.
The organic group containing a hydrophilic group in A of the general formula 1 is not particularly limited as long as it is an organic group containing at least one hydrophilic group, and may contain a plurality of, for example, 2 to 3, hydrophilic groups. . When A contains a plurality of hydrophilic groups, they may be the same or different. When A contains a hydrophilic group in the compound of the present invention, the water solubility of the compound can be improved and the interaction with the target molecule can be improved. As a result, the reactivity with a molecule having an aldehyde group, a hemiacetal group, a carboxyl group, or a keto group can be improved.
The organic group A is preferably at least one hydrophilic group selected from the group consisting of a substituted or unsubstituted guanidino group, a substituted or unsubstituted polyethylene glycol group, a carboxyl group, an aminooxy group and a hydroxyl group as a hydrophilic group. Groups, more preferably substituted or unsubstituted guanidino groups.
Examples of the substituent of the guanidino group and the polyethylene glycol group include a halogen atom selected from fluorine, chlorine, bromine and iodine, a hydroxyl group, a mercapto group, an amino group, C 1-6 Alkyl group, C 1-6 Alkylamino group, carbamoyl group, thiocarboxy group, sulfo group, sulfino group, isocyanato group, nitro group, cyano group, C 2-6 Alkenyl group, C 3-10 A cycloalkyl group, C 1-10 Alkoxy group, C 1-10 Acyl group, C 1-10 An alkoxycarbonyl group, a carboxyl group, a guanidino group, etc. can be mentioned.
The substituted or unsubstituted guanidino group includes, for example, the following general formula 2:
(Where X 1 And X 2 Is a hydrogen atom or an organic group)
It is represented by Where X 1 And X 2 The organic group in is not particularly limited as long as it does not interfere with the hydrophilicity of the guanidino group, and examples thereof include substituents of the above guanidino group.
In the present invention, the substituted or unsubstituted guanidino group includes the following general formulas 2 ′ and 2 ″:
(Where X 3 ~ X 7 Is a hydrogen atom or an organic group)
The group represented by these is also included. Where X 3 ~ X 7 The organic group in is not particularly limited as long as it does not interfere with the hydrophilicity of the guanidino group, and examples thereof include substituents of the above guanidino group.
The aminooxy group in the compound of the present invention can form a covalent bond with high reactivity with an aldehyde group, a hemiacetal group, a carboxyl group or a keto group, particularly an aldehyde group. It is possible to bind to a compound having a group, a hemiacetal group, a carboxyl group or a keto group, for example, a biomolecule with high reactivity. The compound of the present invention containing a substituted or unsubstituted guanidino group as a hydrophilic group can be bound with high reactivity to a nucleic acid, particularly a double-stranded nucleic acid.
The organic group A may further have a labeling group. Since the compound of the present invention can bind to a biomolecule having an aldehyde group, a hemiacetal group, a carboxyl group, or a keto group with high reactivity, the compound of the present invention in which the organic group A has a labeling group is used as such a living body. By reacting with a molecule, the biomolecule can be labeled efficiently. Therefore, in one embodiment, the present invention relates to a reagent for labeling a biomolecule having an aldehyde group, a hemiacetal group, a carboxyl group or a keto group, the labeling reagent comprising the compound of the present invention having a labeling group. . The present invention also provides a labeled biomolecule formed by binding a compound of the present invention to a biomolecule having an aldehyde group, a hemiacetal group, a carboxyl group, or a keto group, and the aminooxy group of the compound and the biomolecule The present invention relates to the labeled biomolecule in which a aldehyde group, a hemiacetal group, a carboxyl group, or a keto group of the molecule reacts to form a covalent bond.
Examples of biomolecules having an aldehyde group, a hemiacetal group, a carboxyl group, or a ketone group include polypeptides, polynucleotides, and sugar chains. Polypeptides include peptides, oligopeptides and proteins. Polynucleotides include nucleic acids such as DNA, RNA, and non-natural nucleic acids, which may be single-stranded or double-stranded, and polynucleotides include oligonucleotides. In the present invention, the base length of the polynucleotide is usually 2 to 10000 bases, preferably 2 to 1000 bases. A plurality of aldehyde groups, hemiacetal groups, carboxyl groups, or ketone groups can be introduced or generated at any position of the nucleic acid, and the aldehyde group, hemiacetal group, carboxyl group, or ketone group thus obtained can be introduced. The non-natural nucleic acid having is also included in the polynucleotide in the present invention. Polynucleotides also include derivatives of DNA and RNA, such as RNA having an aldehyde group in which the hydroxyl group at the 3 ′ end is oxidized, DNA having an abasic site, and the hydroxyl group being oxidized to an aldehyde group. Are also included. In particular, the introduction, generation and labeling of these groups in nucleic acids is described below.
Methods for generating aldehyde groups in nucleic acids can be divided into several types. One of the typical methods is the formation of an aldehyde structure through the formation of a basic site (or apnic site (AP site)). The abasic site of a nucleic acid shows the structure of a sugar moiety from which the base portion of the nucleic acid has been removed (FIG. 1-a), and exists in an equilibrium between a closed ring and an open ring structure. The abasic site has a hemiacetal structure in the ring-closed state, but has an aldehyde structure in the ring-opening type, and reacts with the aldehyde structure when an aminooxy group is allowed to act (FIG. 1-b).
This abasic site can be generated by treating a natural nucleic acid or a non-natural nucleic acid under acidic conditions. Non-natural nucleic acids include 2-pyrimidone (Tetrahedron Letters, 31, 175-178 (1990)), 2′-deoxy-xanthosine (Nucleic Acids Research, 31, 1045-1051 (2003)), 1-deaza. -2'-deoxyguanosine body (Org Lett, 7, 709-712 (2005)) etc. are mentioned. In addition, 2′-deoxy-D-ribose derivatives (Non-patent Document 6; Non-patent Document 7) that form a basic site by deprotecting a protecting group having a hemiacetal structure under special reaction conditions are also known. It has been.
In addition to generating a basic site using a derivative as described above, there is also a method for generating an abasic site by introducing a damaged base into an oligonucleotide or polynucleotide and then treating with an enzyme that removes the damaged base. is there. Examples of the damaged base include 2′-deoxyuridine, 5-hydroxymethyl-2′-deoxycytidine, and the like, and abasic site is generated by acting uracil N glycosylase on 2′-deoxyuridine. be able to. A plurality of 2′-deoxyuridines can be introduced into oligonucleotides and polynucleotides by chemical synthesis or by performing an extension reaction with DNA polymerase using the triphosphate (dUTP) as a substrate. is there. This 2′-deoxyuridine is incorporated into DNA and a basic site produced by the action of uracil N glycosylase is labeled, then the DNA is fragmented, and the fragment is chemically labeled (Ribo-SPIA (registered trademark)). )) Is also commercially available (Special Table 2005-534304). The structure of the labeling reagent contained in this commercially available kit and the reagent of the present invention is different, and by using the reagent of the present invention, it becomes possible to label the target nucleic acid more rapidly and with a high yield.
There is also a method of generating an aldehyde group in a nucleic acid without going through an abasic site. For example, an aldehyde group can be generated by introducing a non-natural nucleic acid into an oligonucleotide in advance and then performing an oxidation reaction (Tetrahedron Letters, 37, 9067-9070 (1996)). In the case of RNA, an aldehyde group can be formed even in a natural type. For example, it is possible to oxidize the 2′-position and 3′-position hydroxyl group of the 3 ′ end of RNA with periodic acid or the like to generate an aldehyde group at the 3 ′ end (FIG. 1-c). In this case, since an aldehyde group is generated at the 2′-position and the 3′-position, when a compound having an aminooxy group and a labeling group is allowed to act, RNA is labeled at the 2′-position or the 3′-position.
Non-natural nucleic acids having a ketone group have also been reported (Org Lett, 3, 3983-3986 (2001)), and the nucleic acid into which these are introduced is reacted with the compound having an aminooxy group of the present invention. It is also possible to label the group.
The labeling group refers to a group containing a labeling substance. Examples of labeling substances include, but are not limited to, labeling substances commonly used for labeling biomolecules, such as radioisotopes, dyes (fluorescent dyes, luminescent dyes), digoxigenin (DIG), biotin, methylene blue, and ferrocene. .
In an embodiment in which the compound of the present invention contains a labeling group, when the organic group A contains a guanidino group of the above general formula 2 or a guanidino group of the general formula 2, 1 And X 2 Is an organic group, and the organic group is preferably the labeling group. When the organic group A is a guanidino group of the above general formula 2 ′ or 2 ″ or contains this, 3 ~ X 5 Or X 6 Or X 7 Is preferably a labeling group.
In that case, the labeling substance may be directly bonded to the nitrogen atom of the guanidino group or may be bonded via a linker. Although it does not restrict | limit especially as a linker, Usually, a divalent organic group, for example, 1-15 in a main chain, Preferably it contains 1-12 carbon atoms and / or a hetero atom (oxygen, nitrogen, or a sulfur atom). It is a divalent organic group. For example,-(CH 2 ) v -(OCH 2 CH 2 ) w -(CH 2 ) x -NH- is mentioned. Here, v, w, and x are each independently an integer of 0-15, and v + w + x is 1-15. Preferably,-(CH 2 ) 2 -(OCH 2 CH 2 ) 2 -NH-.
A labeling group containing biotin as a labeling substance (in the general formula 2, X 1 Or X 2 ) As the following formula:
The group represented by these can be illustrated.
The compound of the present invention in which the organic group A is an organic group containing a guanidino group to which a labeling group is bonded can be preferably used for labeling a nucleic acid.
The biomolecule labeled with the labeling reagent of the present invention can be quantified by various detection systems. For this reason, the present labeling reagent is not affected by the detection system and the detection equipment, and has high versatility, so that it can be widely used.
Alternatively, the organic group A may contain a biomolecular group. The biomolecule group refers to a group containing a biomolecule such as a sugar chain, a polynucleotide (including an oligonucleotide), and a polypeptide (for example, an enzyme). When A contains a polynucleotide, it can be used as a primer for PCR. For example, after the compound of the present invention is bound to a target molecule and a primer extension reaction is carried out in the presence of a template DNA and a substrate labeled with fluorescence or biotin (dNTP), a fluorescent substance (or biotin) is added to the polynucleotide. Etc.) are incorporated thereby labeling the target molecule.
When the organic group A contains a guanidino group of the above general formula 2 or is a guanidino group of the general formula 2, 1 And X 2 Is an organic group, and the organic group is preferably the biomolecular group. When the organic group A contains a group of the above general formula 2 ′ or is a group of the general formula 2 ′, X 3 ~ X 5 Is an organic group, and the organic group is preferably the biomolecular group. If it contains a group of general formula 2 ″ or is a group of general formula 2 ″, X 6 And X 7 Is an organic group, and the organic group is preferably the biomolecular group. In that case, the biomolecule may be directly bonded to the nitrogen atom of the guanidino group or may be bonded via a linker. The linker is the same as the linker in the labeling group.
Since the aminooxy group in the compound of the present invention binds to a molecule having an aldehyde group, a hemiacetal group, a carboxyl group or a keto group, such as a biomolecule, with high reactivity, the layer of the compound of the present invention is formed on the surface of the carrier. And the biomolecule can be immobilized on a carrier via the compound of the present invention. Therefore, in one embodiment, the present invention provides a biomolecule immobilization support for immobilizing a biomolecule having an aldehyde group, a hemiacetal group, a carboxyl group, or a keto group, which is present on the carrier and the surface of the carrier. To the support, comprising a layer of the compound of the invention.
Formation of the layer of the compound of the present invention may be carried out by simply applying the compound of the present invention on a carrier, or by forming a covalent bond with a functional group on the carrier.
When forming a covalent bond with the functional group on a support | carrier, it is preferable that the organic group A in the compound of this invention contains the functional group suitable for the coupling | bonding to a support | carrier. Examples of such a functional group include groups capable of forming a covalent bond with a functional group present on the carrier to be immobilized, such as an active ester group, an epoxy group, an aldehyde group, a carbodiimide group, and an isothiocyanate group. Alternatively, a group that can be covalently bonded to an isocyanate group (for example, an amino group, an aminooxy group, and the like), a group that can react with a maleimide group or a disulfide group (for example, a mercapto group), and the like can be given. These functional groups may be in protected form. The protected form means a form in which a hydrogen atom of a functional group is substituted with a protecting group. The protecting group such as amino group and aminooxy group is not particularly limited, but is alkyl group, acyl group, carbamate group, trialkylsilyl group, phthalyl group, carboxyalkylcarbonyl group, tosyl group, trifluoroacetyl group, trityl group. And mono- or di-substituted trityl groups. Examples of the mono-substituted trityl group include a monoalkoxytrityl group, preferably a monoalkoxytrityl group having 1 to 4 carbon atoms, more preferably an alkoxy group having 1 carbon atom, specifically a monomethoxytrityl group and a monoethoxy group. Examples include a trityl group, a monopropoxytrityl group, a monoisopropoxytrityl group, and a monobutoxytrityl group.
Examples of the material for the carrier to which the compound of the present invention is bound include glass such as quartz glass, borosilicate glass and soda lime glass, silicon, metal, fiber, wood, paper, ceramics, plastic (for example, polyester resin, polyethylene resin). Polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin). In the present invention, glass, silicon, ceramics or plastic is preferably used. A layer of the compound of the present invention is formed on the surface of the carrier. When introducing a functional group into the carrier, examples of the functional group to be introduced include an active ester group, an epoxy group, an amino group, a chloro group, a disulfide group, an aldehyde group, a maleimide group, a carbodiimide group, an isothiocyanato group, an isocyanato group, and the like. Can be mentioned. When linking the compound of the present invention having an amino group or aminooxy group, it is preferable to use a carrier having an active ester group, an epoxy group, an aldehyde group, a carbodiimide group, an isothiocyanate group or an isocyanate group introduced, and a mercapto group In the case of binding the compound of the present invention having a carrier, it is preferable to use a carrier having a maleimide group or a disulfide group introduced therein.
The shape of the carrier is not particularly limited, and examples thereof include a substrate shape, a thread shape, a spherical shape, a bead shape, a polygonal shape, a powder shape, and a porous shape. In the present invention, the substrate shape is preferable.
Since the compound of the present invention having an organic group A containing a substituted or unsubstituted guanidino group can specifically bind to a nucleic acid, an excellent support for immobilizing a nucleic acid can be obtained by binding the compound to a carrier. Can be manufactured. By immobilizing nucleic acids such as DNA on the obtained support for immobilizing nucleic acid, a microarray can be efficiently produced.
With the compound of the present invention, in particular, the labeling reaction to nucleic acids such as RNA and DNA can be made efficient, and the time required for gene detection can be shortened. Furthermore, the high reactivity of the compound of the present invention makes it possible to improve the detection sensitivity and quantify the detected value, so that more detailed information related to the gene can be obtained accurately. In addition, when a layer of the compound of the present invention is formed on a carrier and a binding reaction with a target biomolecule is carried out on the surface, the target biomolecule can be efficiently captured on the carrier, It can be applied to molecular identification.
EXAMPLES Hereinafter, although an Example demonstrates this invention, the scope of the present invention is not limited to an Example.
（実施例１）アミノオキシナフタレン試薬（ａｏＮｇ、化合物６およびａｏＮｇ−ｂｉｏｔｉｎ、化合物１０）の合成
薄層クロマトグラフィーはＫｉｅｓｅｌｇｅｌ ６０Ｆ２５４プレート（Ｍｅｒｃｋ社）上で行った。カラムクロマトグラフィーにはＷａｋｏｇｅｌ Ｃ−２００（和光純薬工業）を用いた。１Ｈ ＮＭＲ及び１３Ｃ ＮＭＲはテトラメチルシランを内部標準とし、ＪＥＯＬ ＪＮＭ−ＥＸ２７０を用いて測定した。
ａｏＮｇ、化合物６の合成
スキーム１
Ｎ １ −（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物２）
アルゴン雰囲気下、１，５−ジアミノナフタレン（化合物１）４４０ｍｇ（２．８０ｍｍｏｌ）及びＮ−トリチルアミノオキシ酢酸１．００ｇ（３．００ｍｍｏｌ）をジメチルホルムアミド（ＤＭＦ）３０ｍｌに溶解し、ＥＤＣ［塩酸１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド］５９０ｍｇ（３．１０ｍｍｏｌ）を加え、室温で２０時間撹拌した。反応液に酢酸エチル２００ｍｌを加え、水７０ｍｌで４回洗浄した。溶液を減圧下濃縮した後、酢酸エチルで３回共沸し、生じた沈殿を酢酸エチル（２０ｍｌ）とヘキサン（１２０ｍｌ）の混合溶液に懸濁させた。沈殿を吸引濾過により濾取して標記化合物（化合物２）１．０３ｇ（収率７２％）を淡茶色粉状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ４７３．３（Ｍ＋）；ＦＡＢ−ＨＲＭＳ 計算値：４７３．２１０３（Ｃ３１Ｈ２７Ｎ３Ｏ２［Ｍ＋］），実測値：４７３．２０９３．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：９．３３（ｂｒ ｓ，１Ｈ，ＮＨ），８．３８（ｂｒ ｓ，１Ｈ，ＮＨ），７．９３（ｄ，１Ｈ，ｎａｐｈ，Ｊ＝８．６Ｈｚ），７．６４（ｄ，１Ｈ，ｎａｐｈ，Ｊ＝７．３Ｈｚ），７．３４−７．２５（ｍ，１６Ｈ，Ｔｒ ａｎｄ ｎａｐｈ），７．２０（ｔ，１Ｈ，ｎａｐｈ，Ｊ＝８．２Ｈｚ），６．９９（ｄ，１Ｈ，ｎａｐｈ，Ｊ＝８．２Ｈｚ），６．７０（ｄ，１Ｈ，ｎａｐｈ，Ｊ＝７．３Ｈｚ），５．７８（ｂｒ ｓ，２Ｈ，ＮＨ２），４．２０（ｓ，２Ｈ，ＣＨ２）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１６８．２６（Ｃ），１４４．９６（Ｃ），１４４．０１（Ｃ），１３２．１３（Ｃ），１２８．７０（ＣＨ），１２７．５７（ＣＨ），１２６．６８（ＣＨ），１２３．０５（Ｃ），１２２．７６（ＣＨ），１２１．０６（ＣＨ），１１９．８８（ＣＨ），１０９．１５（ＣＨ），１０７．５８（ＣＨ），７３．６４（Ｃ），７３．３１（ＣＨ２）。
Ｎ １ −［２−（トリフルオロアセチルアミノ）エチルカルボニル］−Ｎ ５ −（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物３）
アルゴン雰囲気下、Ｎ１−（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物２）１．００ｇ（２．１１ｍｍｏｌ）及びＮ−トリフルオロアセチル−β−アラニン７８０ｍｇ（４．２２ｍｍｏｌ）をジメチルホルムアミド２５ｍｌに溶解し、ＥＤＣ［塩酸１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド］８１０ｍｇ（４．２２ｍｍｏｌ）を加え、室温で１７時間撹拌した。反応液に酢酸エチル２２０ｍｌを加え、水８０ｍｌで４回洗浄した。溶液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：酢酸エチル−ヘキサン）により精製して標記化合物（化合物３）９６１ｍｇ（収率７１％）を白色固体状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ６４０．３（Ｍ＋）；ＦＡＢ−ＨＲＭＳ 計算値：６４０．２２９７（Ｃ３６Ｈ３１Ｆ３Ｎ４Ｏ４［Ｍ＋］），実測値：６４０．２３０８．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１０．０４（ｓ，１Ｈ，ＮＨ），９．６１（ｓ，２Ｈ，ＮＨ ａｎｄ ＮＨ），８．３６（ｓ，１Ｈ，ＮＨ），７．９４（ｄ，１Ｈ，ｎａｐｈ，Ｊ＝８．６Ｈｚ），７．７４−７．６９（ｍ，３Ｈ，ｎａｐｈ），７．５５−７．４８（ｍ，２Ｈ，ｎａｐｈ），７．３５−７．２５（ｍ，１５Ｈ，Ｔｒ），４．２３（ｓ，２Ｈ，ＣＨ２），３．５６（ｂｒ ｔ，２Ｈ，ＣＨ２，Ｊ＝６．８Ｈｚ），２．７８（ｔ，２Ｈ，ＣＨ２，Ｊ＝６．８Ｈｚ）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１６９．２７（Ｃ），１６８．７６（Ｃ），１５６．１２（Ｃ，ｑ，Ｊ＝３５．８Ｈｚ），１４４．０１（Ｃ），１３３．５７（Ｃ），１３２．７３（Ｃ），１２８．７３（ＣＨ），１２８．４６（Ｃ），１２８．４１（Ｃ），１２７．５８（ＣＨ），１２６．７０（ＣＨ），１２５．３６（ＣＨ），１２５．１１（ＣＨ），１２２．０４（ＣＨ），１２０．５２（ＣＨ），１１９．６７（ＣＨ），１１５．８０（Ｃ，ｑ，Ｊ＝２８７．９Ｈｚ），７３．６７（Ｃ），７３．３３（ＣＨ２），３６．０４（ＣＨ２），３４．８０（ＣＨ２）。
Ｎ １ −（２−アミノエチルカルボニル）−Ｎ ５ −（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物４）
Ｎ１−［２−（トリフルオロアセチルアミノ）エチルカルボニル］−Ｎ５−（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物３）９５０ｍｇ（１．４８ｍｍｏｌ）にエタノール４０ｍｌ及び濃アンモニア水２０ｍｌを加えて耐圧ガラス容器に密封し、６０℃で４時間加熱した。溶液を室温に戻した後、減圧下濃縮し、残渣をアセトニトリルで３回共沸して標記化合物（化合物４）を淡黄色泡状物質として得た。本化合物はこれ以上の精製を行わず、次の反応に用いた。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ５４５．４（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：５４５．２５５３（Ｃ３４Ｈ３３Ｎ４Ｏ３［ＭＨ＋］），実測値：５４５．２５７３．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１０．１９（ｓ，１Ｈ，ＮＨ），９．６２（ｓ，１Ｈ，ＮＨ），８．３４（ｓ，１Ｈ，ＮＨ），７．９８（ｄ，１Ｈ，ｎａｐｈ，Ｊ＝８．２Ｈｚ），７．８０（ｂｒ ｓ，２Ｈ，ＮＨ２），７．７５−７．７０（ｍ，３Ｈ，ｎａｐｈ），７．５８−７．４９（ｍ，２Ｈ，ｎａｐｈ），７．３８−７．２５（ｍ，１５Ｈ，Ｔｒ），４．２３（ｓ，２Ｈ，ＣＨ２），３．１６（ｔ，２Ｈ，ＣＨ２，Ｊ＝６．６Ｈｚ），２．８９（ｔ，２Ｈ，ＣＨ２，Ｊ＝６．６Ｈｚ）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１６８．８９（Ｃ），１６８．７８（Ｃ），１４３．９８（Ｃ），１３３．２９（Ｃ），１３２．７７（Ｃ），１２８．７０（ＣＨ），１２８．４９（Ｃ），１２８．２９（Ｃ），１２７．５６（ＣＨ），１２６．７０（ＣＨ），１２５．３２（ＣＨ），１２５．１９（ＣＨ），１２２．１３（ＣＨ），１２１．９４（ＣＨ），１２０．４９（ＣＨ），１１９．８２（ＣＨ），７３．６４（Ｃ），７３．３０（ＣＨ２），３５．１１（ＣＨ２），３２．８２（ＣＨ２）。
Ｎ １ −（２−グアニジノエチルカルボニル）−Ｎ ５ −（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物５）
アルゴン雰囲気下、前反応により合成したＮ１−（２−アミノエチルカルボニル）−Ｎ５−（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物４）全量（１．４８ｍｍｏｌ）をジメチルホルムアミド１５ｍｌに溶解し、塩酸１Ｈ−ピラゾール−１−カルボキシアミジン３２０ｍｇ（２．２０ｍｍｏｌ）及びトリエチルアミン０．６１ｍｌ（４．４０ｍｍｏｌ）を加え、室温で６時間撹拌した。反応液に酢酸エチル１５０ｍｌを加え、水５０ｍｌで４回洗浄した。溶液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：エタノール−クロロホルム）により精製して標記化合物（化合物５）５６７ｍｇ（収率６６％）を淡茶色泡状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ５８７．３（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：５８７．２７７１（Ｃ３５Ｈ３５Ｎ６Ｏ３［ＭＨ＋］），実測値：５８７．２７７２．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１０．１１（ｓ，１Ｈ，ＮＨ），９．６１（ｓ，１Ｈ，ＮＨ），８．３４（ｓ，１Ｈ，ＮＨ），７．９６（ｍ，１Ｈ，ｎａｐｈ），７．７５−７．７０（ｍ，３Ｈ，ｎａｐｈ），７．６７（ｂｒ ｓ，１Ｈ，ＮＨ），７．５７−７．４８（ｍ，２Ｈ，ｎａｐｈ），７．３８−７．２６（ｍ，１５Ｈ，Ｔｒ），７．２５（ｂｒ ｓ，３Ｈ，ＮＨ２ ａｎｄ ＮＨ），４．２３（ｓ，２Ｈ，ＣＨ２），３．４９（ｄｔ，２Ｈ，ＣＨ２，Ｊ＝５．６，６．３Ｈｚ），２．７９（ｔ，２Ｈ，ＣＨ２，Ｊ＝６．３Ｈｚ）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１６９．３９（Ｃ），１６８．６０（Ｃ），１５６．５２（Ｃ），１４３．８３（Ｃ），１３３．３０（Ｃ），１３２．６０（Ｃ），１２８．５５（ＣＨ），１２８．３３（Ｃ），１２８．２２（Ｃ），１２７．４０（ＣＨ），１２６．５３（ＣＨ），１２５．１７（ＣＨ），１２５．０２（ＣＨ），１２１．９３（ＣＨ），１２１．７９（ＣＨ），１２０．３６（ＣＨ），１１９．６２（ＣＨ），７３．４９（Ｃ），７３．１４（ＣＨ２），３６．９７（ＣＨ２），３４．８８（ＣＨ２）。
Ｎ ５ −（アミノオキシアセチル）−Ｎ １ −（２−グアニジノエチルカルボニル）−１，５−ジアミノナフタレン・２塩酸塩（化合物６）
アルゴン雰囲気下、Ｎ１−（２−グアニジノエチルカルボニル）−Ｎ５−（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物５）５９ｍｇ（０．１０ｍｍｏｌ）を塩化メチレン４．０ｍｌに溶解し、濃塩酸６０μｌを加え、室温で２．５時間撹拌した。反応液中に生じた白色の沈殿物質を塩化メチレンで洗浄した後に、エタノール（２ｍｌ）とジエチルエーテル（３ｍｌ）の混合溶液に懸濁させ、沈殿を吸引濾過により濾取して標記化合物（化合物６）２６ｍｇ（収率６２％）を白色粉状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ３４５．２（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：３４５．１６７５（Ｃ１６Ｈ２１Ｎ６Ｏ３［ＭＨ＋］），実測値：３４５．１６８７．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１１．０１（ｂｒ ｓ，１Ｈ，ＮＨ），１０．４０（ｓ，１Ｈ，ＮＨＣＯ），１０．２４（ｓ，１Ｈ，ＮＨＣＯ），７．９９（ｔ，２Ｈ，ｎａｐｈ，Ｊ＝８．１Ｈｚ），７．７８（ｂｒ ｔ，１Ｈ，ＮＨ，Ｊ＝５．８Ｈｚ），７．７３−７．６９（ｍ，２Ｈ，ｎａｐｈ），７．５６（ｄｄ，１Ｈ，ｎａｐｈ，Ｊ＝４．０，８．３Ｈｚ），７．５３（ｄｄ，１Ｈ，ｎａｐｈ，Ｊ＝４．０，７．９Ｈｚ），７．２８（ｂｒ ｓ，４Ｈ），４．９２（ｓ，２Ｈ，ＣＨ２），３．５０（ｄｔ，２Ｈ，ＣＨ２，Ｊ＝５．８，６．３Ｈｚ），２．８０（ｔ，２Ｈ，ＣＨ２，Ｊ＝６．３Ｈｚ）。
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１６９．５０（Ｃ），１６６．８７（Ｃ），１５６．８１（Ｃ），１３３．４３（Ｃ），１３２．５８（Ｃ），１２８．５３（Ｃ），１２８．４７（Ｃ），１２５．２５（ＣＨ），１２５．１１（ＣＨ），１２２．１３（ＣＨ），１２２．０６（ＣＨ），１２０．７６（ＣＨ），１２０．１９（ＣＨ），７２．１５（ＣＨ２），３７．１７（ＣＨ２），３５．１８（ＣＨ２）。
ａｏＮｇ−ｂｉｏｔｉｎ試薬（以下ａｏＮｇ−ｂｉｏ；化合物１０）の合成
スキーム２
Ｎ １ −〔２−（Ｎ ３ −フルオレニルメチルオキシカルボニルチオウレイド）エチルカルボニル〕−Ｎ ５ −（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物７）
アルゴン雰囲気下、Ｎ１−（２−アミノエチルカルボニル）−Ｎ５−（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物４）（化合物３より前述の方法により合成、１．００ｍｍｏｌ）を塩化メチレン−アセトニトリルの混合溶液（１：２）１５ｍｌに溶解した。別のガラス容器にフルオレニルメチルオキシカルボニルイソチオシアナート３１０ｍｇ（１．１０ｍｍｏｌ）およびジイソプロピルエチルアミン０．１９ｍｌ（１．１０ｍｍｏｌ）を塩化メチレン２０ｍｌに溶解し、ここに上記化合物４の溶液をゆっくりと滴下して加え、さらに室温で１時間撹拌した。反応液を減圧下濃縮した後、生じた沈殿を酢酸エチル（１５ｍｌ）とヘキサン（３０ｍｌ）の混合溶液に懸濁させた。沈殿を吸引濾過により濾取して標記化合物（化合物７）７８０ｍｇ（収率９４％）を淡茶色粉状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ８２５．２（Ｍ＋）；ＦＡＢ−ＨＲＭＳ 計算値：８２５．２９８５（Ｃ５０Ｈ４３Ｎ５Ｏ５Ｓ［Ｍ＋］），実測値：８２５．２９８５．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１１．４２（ｂｒ ｓ，１Ｈ，ＮＨ），１０．１６（ｂｒ ｔ，１Ｈ，ＮＨ，Ｊ＝５．５Ｈｚ），１０．０７（ｂｒ ｓ，１Ｈ，ＮＨ），９．５８（ｂｒ ｓ，１Ｈ，ＮＨ），８．３５（ｂｒ ｓ，１Ｈ，ＮＨ），７．９３（ｍ，１Ｈ，ｎａｐｈ），７．９１−７．８２（ｍ，４Ｈ，Ｆｍｏｃ），７．７３−７．６７（ｍ，３Ｈ，ｎａｐｈ），７．５２−７．３８（ｍ，４Ｈ，ｎａｐｈ ａｎｄ Ｆｍｏｃ），７．３６−７．２４（ｍ，１７Ｈ，Ｆｍｏｃ ａｎｄ Ｔｒ），４．３６−４．３３（ｍ，２Ｈ，Ｆｍｏｃ−ＣＨ２），４．２９−４．２５（ｍ，１Ｈ，Ｆｍｏｃ−ＣＨ），４．２３（ｓ，２Ｈ，ＣＨ２），３．９５（ｄｄ，２Ｈ，ＣＨ２，Ｊ＝５．５，６．１Ｈｚ），２．８９（ｔ，２Ｈ，ＣＨ２，Ｊ＝６．１Ｈｚ）。
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１７９．１４（Ｃ），１６９．９６（Ｃ），１６８．７１（Ｃ），１５３．１０（Ｃ），１４３．９８（Ｃ），１４３．１０（Ｃ），１４０．５０（Ｃ），１３３．４７（Ｃ），１３２．７１（Ｃ），１２８．７０（ＣＨ），１２８．４１（Ｃ），１２８．３１（Ｃ），１２７．６４（ＣＨ），１２７．５６（ＣＨ），１２６．９５（ＣＨ），１２６．６９（ＣＨ），１２５．４２（ＣＨ），１２５．３４（ＣＨ），１２５．１０（ＣＨ），１２１．９３（ＣＨ），１２０．４４（ＣＨ），１１９．９７（ＣＨ），１１９．６３（ＣＨ），７３．６４（Ｃ），７３．３１（ＣＨ２），６７．１４（ＣＨ２），４５．９９（ＣＨ），４０．９３（ＣＨ２），３４．１９（ＣＨ２）。
Ｎ １ −〔２−（Ｎ ３ −｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝−Ｎ ２ −フルオレニルメチルオキシカルボニルグアニジノ）エチルカルボニル〕−Ｎ ５ −（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物８）
アルゴン雰囲気下、Ｎ１−〔２−（Ｎ３−フルオレニルメチルオキシカルボニルチオウレイド）エチルカルボニル〕−Ｎ５−（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物７）４１０ｍｇ（０．５０ｍｍｏｌ）をジメチルホルムアミド１５ｍｌに溶解し、０℃に冷却した。この溶液にジイソプロピルエチルアミン０．２６ｍｌ（１．５０ｍｍｏｌ）を加えた後、（＋）−ビオチニル−３，６−ジオキサオクタンジアミン・２／３酢酸塩３１０ｍｇ（０．７５ｍｍｏｌ）および塩化第二水銀２０５ｍｇ（０．７５ｍｍｏｌ）のジメチルホルムアミド溶液（７ｍｌ）をゆっくりと滴下して加え、さらに０℃で１時間撹拌した。反応液に酢酸エチル８０ｍｌを加えた後、セライト濾過により沈殿物を除去した。ろ液に酢酸エチルを加えて全量を１５０ｍｌとし、水５０ｍｌで４回洗浄した。溶液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：エタノール−クロロホルム）により精製して標記化合物（化合物８）３００ｍｇ（収率５１％）を白色泡状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ １１６６．６（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：１１６６．５１７４（Ｃ６６Ｈ７２Ｎ９Ｏ９Ｓ［ＭＨ＋］），実測値：１１６６．５１６５。
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１０．０５（ｂｒ ｓ，１Ｈ，ＮＨ），９．５８（ｂｒ ｓ，１Ｈ，ＮＨ），８．３５（ｂｒ ｓ，１Ｈ，ＮＨ），７．９５−７．８７（ｍ，３Ｈ，ｎａｐｈ ａｎｄ Ｆｍｏｃ），７．８１（ｂｒ ｔ，１Ｈ，ＮＨ，Ｊ＝５．６Ｈｚ），７．７２−７．６３（ｍ，５Ｈ，ｎａｐｈ ａｎｄ Ｆｍｏｃ），７．５２−７．３７（ｍ，４Ｈ，ｎａｐｈ ａｎｄ Ｆｍｏｃ），７．３４−７．２４（ｍ，１７Ｈ，Ｆｍｏｃ ａｎｄ Ｔｒ），６．４０（ｂｒ ｓ，１Ｈ，ＮＨ），６．３５（ｂｒ ｓ，１Ｈ，ＮＨ），４．３０−４．２３（ｍ，６Ｈ），４．０９（ｍ，１Ｈ），３．５１（ｍ，８Ｈ），３．４０−３．３６（ｍ，４Ｈ），３．１８（ｍ，２Ｈ），３．０５（ｍ，１Ｈ），２．８２−２．７６（ｍ，３Ｈ），２．５６（ｄ，１Ｈ，Ｊ＝１２．２Ｈｚ），２．０５（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），１．６２−１．３７（ｍ，４Ｈ），１．３３−１．２７（ｍ，２Ｈ）。
Ｎ １ −〔２−（Ｎ ３ −｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝−Ｎ ２ −フルオレニルメチルオキシカルボニルグアニジノ）エチルカルボニル〕−Ｎ ５ −（アミノオキシアセチル）−１，５−ジアミノナフタレン（化合物９）
アルゴン雰囲気下、Ｎ１−〔２−（Ｎ３−｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝−Ｎ２−フルオレニルメチルオキシカルボニルグアニジノ）エチルカルボニル〕−Ｎ５−（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物８）２９２ｍｇ（０．２５ｍｍｏｌ）を塩化メチレン１０ｍｌに溶解し、トリフルオロ酢酸２８０μｌ（３．８ｍｍｏｌ）およびトリイソプロピルシラン５１０μｌ（２．５ｍｍｏｌ）を加えて室温で３０分撹拌した。反応液にクロロホルム６０ｍｌを加え、飽和炭酸水素ナトリウム溶液３０ｍｌで２回、水３０ｍｌで１回、飽和食塩水３０ｍｌで１回洗浄し、硫酸ナトリウムにより乾燥した。有機層を減圧下濃縮した後、生じた沈殿をクロロホルム２．５ｍｌとエタノール２．５ｍｌの混合溶媒に溶解し、この溶液を撹拌させたジエチルエーテル２５ｍｌに滴下した。生じた白色の沈殿物質を吸引濾過により濾取して標記化合物（化合物９）１８４ｍｇ（収率８０％）を白色粉状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ９２４．４（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：９２４．４０７８（Ｃ４７Ｈ５８Ｎ９Ｏ９Ｓ［ＭＨ＋］），実測値：９２４．４０５３．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１０．１７（ｂｒ ｓ，１Ｈ，ＮＨ），９．９５（ｂｒ ｓ，１Ｈ，ＮＨ），７．９７−７．８９（ｍ，４Ｈ，ｎａｐｈ ａｎｄ Ｆｍｏｃ），７．８２（ｂｒ ｔ，１Ｈ，ＮＨ，Ｊ＝５．５Ｈｚ），７．７１−７．６６（ｍ，４Ｈ），７．５３（ｔ，２Ｈ，Ｊ＝７．６Ｈｚ），７．４２（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），７．３１（ｍ，２Ｈ），６．４１（ｂｒ ｓ，１Ｈ，ＮＨ），６．３７（ｂｒ ｓ，１Ｈ，ＮＨ），４．４６（ｍ，２Ｈ），４．３７（ｓ，２Ｈ，ＣＨ２），４．３４−４．２６（ｍ，２Ｈ），４．１０（ｍ，１Ｈ），３．５５−３．４７（ｍ，８Ｈ），３．３９−３．３４（ｍ，４Ｈ），３．１７（ｍ，２Ｈ），３．０５（ｍ，１Ｈ），２．８７（ｍ，２Ｈ），２．７９（ｄｄ，１Ｈ，Ｊ＝５．３，１２．２Ｈｚ），２．５６（ｄ，１Ｈ，Ｊ＝１２．２Ｈｚ），２．０４（ｔ，２Ｈ，Ｊ＝７．４Ｈｚ），１．６２−１．３７（ｍ，４Ｈ），１．２７（ｍ，２Ｈ）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１７２．０５（Ｃ），１６９．１６（Ｃ），１６２．５９（Ｃ），１４３．４１（Ｃ），１４０．６４（Ｃ），１３３．３６（Ｃ），１３３．１６（Ｃ），１２８．９２（Ｃ），１２８．５４（Ｃ），１２７．６５（ＣＨ），１２７．０３（ＣＨ），１２５．３１（ＣＨ），１２５．２３（ＣＨ），１２５．０４（ＣＨ），１２２．５０（ＣＨ），１２２．１７（ＣＨ），１２０．５６（ＣＨ），１２０．３８（ＣＨ），１２０．１０（ＣＨ），７３．８８（ＣＨ２），６９．５７（ＣＨ２），６９．２９（ＣＨ２），６９．０１（ＣＨ２），６７．０２（ＣＨ２），６０．９１（ＣＨ），５９．０６（ＣＨ），５５．３１（ＣＨ），４６．２２（ＣＨ），３９．７２（ＣＨ２），３８．２６（ＣＨ２），３４．９７（ＣＨ２），２８．０８（ＣＨ２），２７．９２（ＣＨ２），２５．１４（ＣＨ２）。
Ｎ １ −〔２−（Ｎ ３ −｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝グアニジノ）エチルカルボニル〕−Ｎ ５ −（アミノオキシアセチル）−１，５−ジアミノナフタレン（化合物１０）
アルゴン雰囲気下、Ｎ１−〔２−（Ｎ３−｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝−Ｎ２−フルオレニルメチルオキシカルボニルグアニジノ）エチルカルボニル〕−Ｎ５−（アミノオキシアセチル）−１，５−ジアミノナフタレン（化合物９）５０ｍｇ（０．０５４ｍｍｏｌ）を１，４−ジオキサン３ｍｌとメタノール３ｍｌの混合溶媒に溶解し、ピペリジン２２０μｌ（２．２ｍｍｏｌ）を加えて室温で２０時間撹拌した。反応液を減圧下濃縮した後、残渣を水３０ｍｌに溶解し、クロロホルム２０ｍｌで６回洗浄した。水層を減圧下濃縮後、凍結乾燥して標記化合物（化合物１０）３５ｍｇ（収率９３％）を白色綿状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ７０２．４（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：７０２．３３９７（Ｃ３２Ｈ４８Ｎ９Ｏ７Ｓ［ＭＨ＋］），実測値：７０２．３４０５．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１０．１１（ｂｒ ｓ，１Ｈ，ＮＨ），９．８８（ｂｒ ｓ，１Ｈ，ＮＨ），７．９６（ｄ，１Ｈ，ｎａｐｈ，Ｊ＝８．６Ｈｚ），７．９０（ｄ，１Ｈ，ｎａｐｈ，Ｊ＝８．３Ｈｚ），７．８４（ｔ，１Ｈ，ＮＨ，Ｊ＝５．３Ｈｚ），７．７１−７．６５（ｍ，２Ｈ，ｎａｐｈ），７．５８−７．５０（ｍ，２Ｈ，ｎａｐｈ），６．５７（ｂｒ ｓ，２Ｈ，ＮＨ２Ｏ），６．４１（ｂｒ ｓ，１Ｈ，ＮＨ），６．３７（ｂｒ ｓ，１Ｈ，ＮＨ），４．３０（ｍ，１Ｈ，ｂｉｏ），４．２７（ｓ，２Ｈ，ＣＨ２），４．１１（ｍ，１Ｈ），３．５２（ｍ，８Ｈ），３．３９（ｔ，２Ｈ，Ｊ＝５．８Ｈｚ），３．３１（ｍ，２Ｈ），３．１９（ｍ，２Ｈ），３．０７（ｍ，１Ｈ），２．８４−２．７８（ｍ，３Ｈ），２．５７（ｄ，１Ｈ，Ｊ＝１２．２Ｈｚ），２．０６（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），１．６４−１．４１（ｍ，４Ｈ），１．２９（ｍ，２Ｈ）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１７２．１０（Ｃ），１６９．７３（Ｃ），１６９．６９（Ｃ），１６２．５９（Ｃ），１５５．８６（Ｃ），１３３．３９（Ｃ），１３３．３０（Ｃ），１２９．０３（Ｃ），１２８．４７（Ｃ），１２５．３１（ＣＨ），１２５．２３（ＣＨ），１２２．６３（ＣＨ），１２２．００（ＣＨ），１２０．５３（ＣＨ），１２０．３７（ＣＨ），７４．３０（ＣＨ２），６９．５７（ＣＨ２），６９．３１（ＣＨ２），６９．０１（ＣＨ２），６８．４６（ＣＨ２），６０．９１（ＣＨ），５９．０６（ＣＨ），５５．３１（ＣＨ），４０．９６（ＣＨ２），３９．８３（ＣＨ２），３８．２５（ＣＨ２），３７．２８（ＣＨ２），３４．９８（ＣＨ２），３４．９１（ＣＨ２），２８．０８（ＣＨ２），２７．９３（ＣＨ２），２５．１５（ＣＨ２）。
（実施例２）Ｂｉｓアミノオキシナフタレン試薬（以下ａｏＮａｏ；化合物１２）の合成
スキーム３
Ｎ １ ，Ｎ ５ −ビス（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物１１）
アルゴン雰囲気下、１，５−ジアミノナフタレン（化合物１）１５８ｍｇ（１．００ｍｍｏｌ）をピリジン３０ｍｌに溶解し、Ｎ−トリチルアミノオキシ酢酸３３３ｍｇ（１．００ｍｍｏｌ）およびＥＤＣ［塩酸１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド］２３０ｍｇ（１．２０ｍｍｏｌ）を加え、室温で撹拌した。反応開始３時間後、６時間後にそれぞれＮ−トリチルアミノオキシ酢酸３３３ｍｇ（１．００ｍｍｏｌ）およびＥＤＣ２３０ｍｇ（１．２０ｍｍｏｌ）を追加して、さらに室温で１６時間撹拌した。反応液を減圧下濃縮した後、沈殿を水２０ｍｌに懸濁し、酢酸エチル５０ｍｌで１回、クロロホルム２０ｍｌで３回洗浄した。水層の白色沈殿物質を吸引濾過により濾取し、さらにエタノールおよびエーテルで洗浄して標記化合物（化合物１１）５５０ｍｇ（収率７０％）を白色粉状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ（＋ＮａＩ）７８９．３（ＭＨ＋），８１１．３（［Ｍ＋Ｎａ］＋）；ＦＡＢ−ＨＲＭＳ 計算値：８１１．３２６０（Ｃ５２Ｈ４４Ｎ４Ｏ４Ｎａ［Ｍ＋Ｎａ］＋），実測値：８１１．３２７４。
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：９．６２（ｂｒ ｓ，２Ｈ，ＮＨ），８．３６（ｂｒ ｓ，２Ｈ，ＮＨ），７．７７−７．７１（ｍ，４Ｈ，ｎａｐｈ），７．５２（ｔ，２Ｈ，ｎａｐｈ，Ｊ＝７．９Ｈｚ），７．４１−７．２５（ｍ，３０Ｈ，Ｔｒ），４．２３（ｓ，４Ｈ，ＣＨ２）。
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１６８．８９（Ｃ），１４４．０７（Ｃ），１３２．９０（Ｃ），１２８．７８（ＣＨ），１２７．６３（ＣＨ），１２６．７６（ＣＨ），１２５．４３（ＣＨ），１２３．８０（Ｃ），１２２．１５（ＣＨ），１２０．１１（ＣＨ），７３．６１（Ｃ），７３．２７（ＣＨ２）。
Ｎ １ ，Ｎ ５ −ビス（アミノオキシアセチル）−１，５−ジアミノナフタレン（化合物１２）
アルゴン雰囲気下、Ｎ１，Ｎ５−ビス（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物１１）３９４ｍｇ（０．５０ｍｍｏｌ）をクロロホルム２０ｍｌに懸濁して氷冷し、トリフルオロ酢酸０．９４ｍｌ（１０．０ｍｍｏｌ）およびトリイソプロピルシラン１．０２ｍｌ（５．０ｍｍｏｌ）を加えた。反応液を室温に戻して３．５時間撹拌した。反応液に水４０ｍｌおよびクロロホルム１０ｍｌを加えて沈殿物を溶解した後に分液し、さらに水層をクロロホルム３０ｍｌで２回、酢酸エチル３０ｍｌで１回洗浄した。続いて、水層にアセトニトリル６０ｍｌを加えた後、ＤＯＷＥＷ（１×４−１００（ＯＨ−フォーム）により中和した。ＤＯＷＥＷ（１×４−１００を濾過して除き、さらに８０％のアセトニトリル水溶液でＤＯＷＥＷ（１×４−１００を洗浄した。溶液を合わせて減圧下濃縮した後、残渣を水３０ｍｌに溶解してＣ−１８カラムカラムクロマトグラフィー（溶出溶媒：水−アセトニトリル）により精製することで、標記化合物（化合物１２）１０９ｍｇ（収率７２％）を白色粉状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ３０５．２（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：３０５．１２５０（Ｃ１４Ｈ１７Ｎ４Ｏ４［ＭＨ＋］），実測値：３０５．１２５９．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：９．８８（ｓ，２Ｈ，ＮＨ），７．９３（ｄ，２Ｈ，ｎａｐｈ，Ｊ＝８．６Ｈｚ），７．６６（ｄ，２Ｈ，ｎａｐｈ，Ｊ＝７．２Ｈｚ），７．５４（ｄｄ，２Ｈ，ｎａｐｈ，Ｊ＝７．２，８．６Ｈｚ），６．５７（ｓ，４Ｈ，ＮＨ２），４．２８（ｓ，４Ｈ，ＣＨ２）。
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１６９．７５（Ｃ），１３３．３５（Ｃ），１２９．１１（Ｃ），１２５．４１（ＣＨ），１２２．６６（ＣＨ），１２０．７５（ＣＨ），７４．４２（ＣＨ２）。
（実施例３）オリゴヌクレオチドの合成と精製
オリゴヌクレオチドの合成はＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ ３９４型ＤＮＡ／ＲＮＡシンセサイザー上で行った。０．２又は１μｍｏｌスケールで合成した。ＨＰＬＣにはＧｉｌｓｏｎの装置を用い、分析はＷａｔｅｒｓ９９６フォトダイオードアレイ検出器を用いて行った。
Ａ溶液 ５％ アセトニトリル／０．１Ｍ ＴＥＡＡ（ｐＨ７．０）
Ｂ溶液 ２５％ アセトニトリル／０．１Ｍ ＴＥＡＡ（ｐＨ７．０）
カラム温度：５０℃
カラムは、μ−ボンダスフィアー（Ｃ−１８）カラムΦ３．９ｘ１５０ｍｍ（ウォーターズ製）を使用した。
イオン交換ＨＰＬＣ溶液
Ａ溶液 ２０％ アセトニトリル
Ｂ溶液 ２０％ アセトニトリル、２Ｍ ギ酸アンモニウム
カラム温度：５０℃
カラムは、ＴＳＫ−ＧＥＬ ＤＥＡＥ−２ＳＷ Φ４．６ｘ２５０ｍｍ（東ソー社）を使用した。
ＨＰＬＣ条件
条件１ Ｂ溶液の％ ２０→４０％／２０分
条件２ Ｂ溶液の％ ３５→５５％／２０分
（実施例４）オリゴヌクレオチド（ＲＮＡ）の酸化反応
オリゴヌクレオチド（Ｆ−Ｌｒ１７Ｘ：Ｘ＝Ａ，Ｇ，Ｃ，Ｕ）（１．６ｎｍｏｌ）を、１００ｍＭリン酸バッファー（ｐＨ７）及び８００μＭ過ヨウ素酸ナトリウムに溶解し（反応液総量２００μｌ）、３７℃、９０分で酸化反応を行った。反応後、ＮＡＰ１０カラム（ＧＥヘルスケア）で脱塩し、得られた酸化オリゴヌクレオチド（Ｆ−Ｌｒ１７Ｘｏｘ）をＵＶ定量した。
（実施例５）酸化オリゴヌクレオチド（ＲＮＡ）へのＡＲＰ、ａｏＮｇの標識反応
一本鎖への反応
酸化オリゴヌクレオチド（Ｆ−Ｌｒ１７Ｘｏｘ；Ｘ＝Ａ，Ｇ，Ｃ，Ｕ）（１２ｐｍｏｌ）を１Ｍリン酸バッファー（ｐＨ７）３μｌ及び滅菌水２１μｌに溶解した後、あらかじめ滅菌水に溶解させた２ｍＭ 標識試薬（ＡＲＰ又は実施例１で調製したａｏＮｇ）６μｌを加え、３７℃で反応を開始した。ＡＲＰ（Ａｌｄｅｈｙｄｅ Ｒｅａｃｔｉｖｅ Ｐｒｏｂｅ；ＤＯＪＩＮＤＯ社）は、以下の構造を有する。
一定時間後に２．５μｌをサンプリングしてｌｏａｄｉｎｇ ｓｏｌｕｔｉｏｎ ｍｉｘ ５μｌ（５０ｍＭ ＥＤＴＡ，１０Ｍ 尿素，０．１％ ＢＰＢ；４μｌ、４０ｍＭ グルタルアルデヒド；１μｌを混合して調製）に加えて反応を止めた。その後、変性２０％ポリアクリルアミドゲル（１９：１）で解析した。各バンドのフルオレセインの蛍光強度を測定し、反応効率を算出した。反応の結果を図２に示した。
二本鎖での反応
二本鎖で反応を行う場合は、酸化オリゴヌクレオチド（Ｆ−Ｌｒ１７Ｘｏｘ）（１２ｐｍｏｌ）に相補鎖ＡＳ３２Ｙ（２４ｐｍｏｌ）を加えて、その他の反応条件は一本鎖の反応と同様に行った。
Ｆ−Ｌｒ１７Ｘｏｘの一本鎖（ｓｓ Ｆ−Ｌｒ１７Ｘｏｘ）、及び相補鎖（ＡＳ３２Ｙ）の２本鎖について、ＡＲＰ又はａｏＮｇとの結合率を測定し、見かけの反応速度定数を算出した。ＲＮＡの３’末端の塩基の種類がいずれの場合でも、ａｏＮｇはＡＲＰよりも高い反応性を示した。また、一本鎖よりも二本鎖を形成した場合に、反応効率が上がることが明らかになった。
（実施例６）ＲＮＡの、酸化から標識の連続反応
実施例４、５では、あらかじめＲＮＡを酸化して精製した後に標識反応を行った。次にＲＮＡの酸化から標識までを連続して行うことが可能であるかを確かめる実験を行った。ＲＮＡ（Ｆ−Ｌｒ１７Ｘ；Ｘ＝Ａ，Ｇ，Ｃ，Ｕ）（１２ｐｍｏｌ）を、１５０ｍＭリン酸バッファー（ｐＨ７）及び１５０μＭ過ヨウ素酸ナトリウムに溶解し（反応液総量２０μｌ）、３７℃、６０分で酸化反応を行った。続いて２ｍＭ ＤＴＴ ５μｌを添加して３７℃、３０分加温し、２４０μＭ ａｏＮｇ（又はａｏＮｇ−ｂｉｏ）５μｌを加えて３７℃で標識反応を行った（反応液総量３０μｌ）。実施例５と同様に一定時間後にサンプリングして解析した。反応の結果、酸化に用いた過剰の過ヨウ素酸ナトリウムを還元することで、酸化から標識反応まで連続して行えることを確認した。
（実施例７）アベイシックサイトを含むオリゴヌクレオチドの合成
アベイシックホスホロアミダイト試薬（Ｇｌｅｎ Ｒｅｓ．社）を用いて、アベイシックサイト（Ｚ）を含むオリゴヌクレオチド（Ｆ−２０Ｚ）を合成した。Ｆ−２０Ｚは、５’ Ｆ−ＧＡＡＴＴＧＣＴＴＧＧＡＡＧＡＺＧＴＴＴ ３’を表す。合成したオリゴヌクレオチドは、通常の方法に従い濃アンモニア水で脱保護反応を行った後、４０％酢酸４ｍＬを加えて室温で４時間攪拌しＴＢＤＭＳ基の脱保護を行った。反応後、減圧下酢酸を留去し、さらに水を加えて減圧下で溶媒を留去した。この操作を数回繰り返して酢酸を取り除いた。その後、滅菌水にオリゴヌクレオチドを溶解し、逆相ＨＰＬＣ、イオン交換ＨＰＬＣによって精製した。Ｆ−２０Ｚの相補鎖となるＳｐ３５も同様に合成し精製した（表４、表５）。Ｓｐ３５Ｃは、３’ ＣＧＡＡＡＧＴＡＡＣＣＴＴＡＡＣＧＡＡＣＣＴＴＣＴＣＣＡＡＡＧＡＡＣＧＡ ５’を表す。各ＨＰＬＣの条件は、Ｆ−Ｌｒ１７Ｘ及びＡＳ３２Ｙの精製と同様とした。
条件１ Ｂ溶液の％ ２０→４０％／２０分
条件２ Ｂ溶液の％ ３５→５５％／２０分
条件３ Ｂ溶液の％ ３０→５０％／２０分
（実施例８）アベイシックサイトを含むオリゴヌクレオチドの酸化
実施例７で調製したアベイシックサイトを含むオリゴヌクレオチドＦ−２０Ｚ（２．６ｎｍｏｌ）を、１００ｍＭリン酸バッファー（ｐＨ７）及び１０ｍＭ 過ヨウ素酸ナトリウムに溶解し、３７℃、６０分で酸化反応を行った。反応後、ＮＡＰ５カラム（ＧＥヘルスケア）で脱塩し、濃縮後、変性２０％ポリアクリルアミドゲル（１９：１）によって精製した。ゲル片からはＹＭＣカートリッジ（１００ｍｇ／１ｍｌ）を用いて脱塩した。得られた酸化オリゴヌクレオチド（Ｆ−２０Ｚｏｘ）をＵＶで定量し、反応に供した。
（実施例９）アベイシックサイトへのＡＲＰ、ａｏＮｇの標識反応
一本鎖への反応
酸化されたＦ−２０Ｚｏｘ（１２ｐｍｏｌ）を１Ｍ リン酸緩衝液（ｐＨ７）３μｌ及び滅菌水１５μｌに溶解し、９０℃で１分加熱し氷冷した。１０秒後に室温で５分放置して３７℃で５分間プレインキュベーションした。続いて、あらかじめ滅菌水に溶解しておいた標識試薬（５ｍＭ ＡＲＰ又はａｏＮｇ）１２μｌを加えて３７℃、４２℃又は４７℃で標識反応を開始した。一定時間後に２．５μｌをサンプリングしてｌｏａｄｉｎｇ ｓｏｌｕｔｉｏｎ ｍｉｘ ５μｌ（５０ｍＭ ＥＤＴＡ，１０Ｍ 尿素，０．１％ ＢＰＢ；４μｌ、２００ｍＭ グルタルアルデヒド；１μｌを混合して調製）に加えて反応を止めた。その後、変性２０％ポリアクリルアミドゲル（１９：１）で解析した。各バンドのフルオレセインの蛍光強度を測定し、反応効率を算出した。反応の結果を図３に示す。
二本鎖での反応
二本鎖で反応を行う場合は、酸化オリゴヌクレオチド（Ｆ−２０Ｚｏｘ）（１２ｐｍｏｌ）に相補鎖Ｓｐ３５Ｃ（１４．４ｐｍｏｌ）を加え、その他の反応条件は一本鎖の反応と同様に行った。反応の結果を図３に示す。
Ｆ−２０Ｚ中のアベイシックサイトＺに対して試薬が反応した。ＤＮＡに対しても、ａｏＮｇの方が高い反応性を示した。特に二本鎖に対してはＡＲＰの反応効率は一本鎖と同じであったが、ａｏＮｇでは反応効率が大きく向上した。
（実施例１０）２’−デオキシウリジン（ｄＵ）を含むオリゴヌクレオチドの合成と精製
５’末端がフルオレセインで標識され、ｄＵを鎖内にもったオリゴヌクレオチド（Ｆ−２０ｄＵ）およびその相補鎖（Ｓｐ２０ｄＵ）は、２’−デオキシウリジンホスホロアミダイト試薬（Ｇｌｅｎ Ｒｅｓ．社）を用いてＤＮＡ自動合成機によって合成した。Ｆ−２０ｄＵは、５’ Ｆ−ＧＡＡＴＴＧＣＴｄＵＧＧＡＡＧＡＧＧＴＴＴ ３’を、Ｓｐ２０ｄＵは、３’ ＣＴＴＡＡＣＧＡｄＵＣＣＴＴＣＴＣＣＡＡＡ ５’を表す。合成オリゴヌクレオチドは通常の方法に従って脱保護した後、逆相ＨＰＬＣで分取、精製した（表６）。ＨＰＬＣの条件などを以下に示す。
Ａ溶液 ５％ アセトニトリル／０．１Ｍ ＴＥＡＡ（ｐＨ７．０）；
Ｂ溶液 ２５％ アセトニトリル／０．１Ｍ ＴＥＡＡ（ｐＨ７．０）
カラム温度：室温
カラムは、Ｉｎｅｒｔｓｉｌ ＯＤＳ−３（Ｃ−１８）カラムΦ３．０ｘ３００ｍｍ（ＧＬ Ｓｃｉｅｎｃｅ社）を使用した。
ＨＰＬＣ条件
条件４ Ｂ溶液の％ ４０→６０％／２０分
（実施例１１）２’−デオキシウリジンを含むＤＮＡへのＡＲＰ、ａｏＮｇ、ａｏＮｇ−ｂｉｏの標識反応
本発明の化合物が２’−デオキシウリジン（ｄＵ）とウラシルＤＮＡグリコシラーゼ（ＵＤＧ）から生成したアベイシックサイトに反応するかどうかを評価する実験を行った。
ｄＵを含むＤＮＡ、Ｆ−２０ｄＵ（１２ｐｍｏｌ）と、その相補鎖（Ｓｐ３５Ｃ；２４ｐｍｏｌ）を、ＵＤＧバッファー（ｘ１０，３μｌ）を含む溶液（総量２９．５μｌ）に溶解させた。Ｓｐ３５Ｃは、３’ ＣＧＡＡＡＧＴＡＡＣＣＴＴＡＡＣＧＡＡＣＣＴＴＣＴＣＣＡＡＡＧＡＡＣＧＡ ５’を表す。反応液を９０℃で１分加熱し氷冷した後、３７℃で５分間プレインキュベーションし、そこにＵＤＧ（１ｕｎｉｔ，０．５μｌ）を加え、総量３０μｌで３７℃、６０分間反応を行った。その後、９０℃で１分加熱して酵素を失活させ、氷冷で１０分、室温で５分放置した。この反応液にあらかじめ溶解させた１０ｍＭに調整した標識試薬（ＡＲＰ、ａｏＮｇ、ａｏＮｇ−ｂｉｏ）６μｌを加えて３７℃で標識反応を開始した。一定時間後に２．５μｌをサンプリングしてｌｏａｄｉｎｇ ｓｏｌｕｔｉｏｎ ｍｉｘ ５μｌ（５０ｍＭ ＥＤＴＡ，１０Ｍ 尿素，０．１％ ＢＰＢ；４μｌ、２００ｍＭ グルタルアルデヒド；１μｌを混合して調製）に加えて反応を止めた。その後、変性２０％ポリアクリルアミドゲル（１９：１）で解析した（図４）。各バンドのフルオレセインの蛍光強度を測定し、反応効率を算出した（図４）。
ａｏＮｇ、ＡＲＰ、ａｏＮｇ−ｂｉｏの反応速度定数は、それぞれ０．５１ｍｉｎ−１、０．０３ｍｉｎ−１、０．１７ｍｉｎ−１であったことから、ａｏＮｇおよびａｏＮｇ−ｂｉｏの方が高い反応効率を示すことが明らかとなった。
（実施例１２）ａｏＮａｏのＤＮＡへの反応
ｄＵを含むＤＮＡ、Ｆ−２０ｄＵ（１２ｐｍｏｌ）と、その相補鎖（Ｓｐ２０ｄＵ；１３．２ｐｍｏｌ）を、ＵＤＧバッファー（ｘ１０，３μｌ）を含む溶液（総量２９．５μｌ）に溶解させた。Ｓｐ２０ｄＵは、３’ ＣＴＴＡＡＣＧＡｄＵＣＣＴＴＣＴＣＣＡＡＡ ５’を表す。反応液を９０℃で１分加熱し氷冷した後、３７℃で５分間プレインキュベーションし、そこにＵＤＧ（１ｕｎｉｔ，０．５μｌ）を加え、総量３０μｌで３７℃、６０分間反応を行った。その後、９０℃で１分加熱して酵素を失活させ、氷冷で１０分、室温で５分放置した。この反応液にあらかじめ溶解させた２ｍＭ ａｏＮａｏ ６μｌを加えて３７℃で標識反応を開始した。同様の反応をＦ−２０ｄＵ、１本鎖のみでも行った。一定時間後に３μｌをサンプリングしてｌｏａｄｉｎｇ ｓｏｌｕｔｉｏｎ ｍｉｘ ５μｌ（５０ｍＭ ＥＤＴＡ，１０Ｍ 尿素，０．１％ ＢＰＢ；４μｌ、２００ｍＭ グルタルアルデヒド；１μｌを混合して調製）に加えて反応を止めた。その後、変性２０％ポリアクリルアミドゲル（１９：１）で解析した（図５）。各バンドのフルオレセインの蛍光強度を測定し、反応効率を算出した（図５）。ａｏＮａｏは２本鎖間を架橋するように反応できることを確認した。
（実施例１３）ａｏＮｄｇ−ｂｉｏｔｉｎ試薬（以下ａｏＮｄｇ−ｂｉｏ；化合物１６）の合成
スキーム４
１−（Ｎ ３ −フルオレニルメチルオキシカルボニルチオウレイド）−Ｎ ５ −（トリチルアミノオキシアセチル）−５−アミノナフタレン（化合物１３）
アルゴン雰囲気下、フルオレニルメチルオキシカルボニルイソチオシアナート８５０ｍｇ（３．０１ｍｍｏｌ）およびジイソプロピルエチルアミン０．５２ｍｌ（３．０１ｍｍｏｌ）を塩化メチレン３０ｍｌに溶解し、０℃に冷却した。ここにＮ１−（トリチルアミノオキシアセチル）−１，５−ジアミノナフタレン（化合物２）１．１９ｇ（２．５１ｍｍｏｌ）の塩化メチレン溶液（３０ｍｌ）をゆっくりと滴下して加え、さらに室温に戻して１．５時間撹拌した。反応液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：酢酸エチル−ヘキサン）により精製して標記化合物（化合物１３）１．０４ｇ（収率５５％）を白色固体状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ７５５．３（ＭＨ＋），７７７．２（［Ｍ＋Ｎａ］＋）；ＦＡＢ−ＨＲＭＳ 計算値：７７７．２５１１（Ｃ４７Ｈ３８Ｎ４Ｏ４ＳＮａ［Ｍ＋Ｎａ］＋），実測値：７７７．２４８６．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１１．８１（ｓ，１Ｈ，ＮＨ），１１．６０（ｓ，１Ｈ，ＮＨ），９．６６（ｓ，１Ｈ，ＮＨ），
８．３７（ｓ，１Ｈ，ＮＨ），７．９４−７．８５（ｍ，６Ｈ），７．７８−７．６８（ｍ，３Ｈ），７．６１−７．５３（ｍ，１Ｈ），７．４９−７．４１（ｍ，２Ｈ），７．３８−７．２７（ｍ，１７Ｈ），４．４９（ｄ，２Ｈ，Ｆｍｏｃ−ＣＨ２，Ｊ＝７．３Ｈｚ），４．３７（ｔ，１Ｈ，Ｆｍｏｃ−ＣＨ，Ｊ＝７．３Ｈｚ），４．２３（ｓ，２Ｈ，ＣＨ２）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１８０．５９（Ｃ），１６８．９６（Ｃ），１５３．５６（Ｃ），１４４．０８（Ｃ），１４３．２３（Ｃ），１４０．６５（Ｃ），１３４．６８（Ｃ），１３３．１４（Ｃ），１２９．７９（Ｃ），１２８．７９（ＣＨ），１２８．６１（Ｃ），１２７．７７（ＣＨ），１２７．６４（ＣＨ），１２７．０７（ＣＨ），１２６．７７（ＣＨ），１２６．１０（ＣＨ），１２５．５８（ＣＨ），１２５．４３（ＣＨ），１２５．３１（ＣＨ），１２２．３４（ＣＨ），１２１．９８（ＣＨ），１２１．２８（ＣＨ），１２０．０８（ＣＨ），１１９．９３（ＣＨ），７３．６２（Ｃ），７３．３０（ＣＨ２），６７．４２（ＣＨ２），４５．９９（ＣＨ）。
１−（Ｎ ３ −｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝−Ｎ ２ −フルオレニルメチルオキシカルボニルグアニジノ）−Ｎ ５ −（トリチルアミノオキシアセチル）−５−アミノナフタレン（化合物１４）
アルゴン雰囲気下、１−（Ｎ３−フルオレニルメチルオキシカルボニルチオウレイド）−Ｎ５−（トリチルアミノオキシアセチル）−５−アミノナフタレン（化合物１３）９１０ｍｇ（１．２０ｍｍｏｌ）をジメチルホルムアミド２０ｍｌに溶解して０℃に冷却した。この溶液に（＋）−ビオチニル−３，６−ジオキサオクタンジアミン５４０ｍｇ（１．４４ｍｍｏｌ）、塩化第二水銀３６０ｍｇ（１．３２ｍｍｏｌ）およびジイソプロピルエチルアミン０．５０ｍｌ（２．８８ｍｍｏｌ）のジメチルホルムアミド溶液（１５ｍｌ）をゆっくりと滴下して加え、さらに０℃で１．５時間撹拌した。反応液に酢酸エチル１５０ｍｌを加えた後、セライト濾過により沈殿物を除去した。ろ液に酢酸エチルを加えて全量を３５０ｍｌとし、水１２０ｍｌで４回、飽和食塩水１２０ｍｌで１回洗浄し、硫酸ナトリウムにより乾燥した。溶液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：エタノール−クロロホルム）により精製して標記化合物（化合物１４）９６３ｍｇ（収率７３％）を白色泡状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ １０９５．５（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：１０９５．４８０３（Ｃ６３Ｈ６７Ｎ８Ｏ８Ｓ［ＭＨ＋］），実測値：１０９５．４７９６．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：９．６１（ｂｒ ｓ，１Ｈ，ＮＨ），８．３３（ｂｒ ｓ，１Ｈ，ＮＨ），７．８９−７．７５（ｍ，６Ｈ），７．６１−７．４７（ｍ，３Ｈ），７．４２−７．２５（ｍ，２０Ｈ），６．３９（ｂｒ ｓ，１Ｈ，ＮＨ），６．３３（ｂｒ ｓ，１Ｈ，ＮＨ），４．３０−４．２４（ｍ，６Ｈ），４．１０（ｍ，１Ｈ），３．４９（ｍ，６Ｈ），３．３８−３．３３（ｍ，４Ｈ），３．１６（ｍ，２Ｈ），３．０６（ｍ，１Ｈ），２．７９（ｄｄ，１Ｈ，Ｊ＝５．３，１２．２Ｈｚ），２．５６（ｄ，１Ｈ，Ｊ＝１２．２Ｈｚ），２．０４（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），１．６３−１．３７（ｍ，４Ｈ），１．３２−１．２３（ｍ，２Ｈ）。
１−（Ｎ ３ −｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝−Ｎ ２ −フルオレニルメチルオキシカルボニルグアニジノ）−Ｎ ５ −（アミノオキシアセチル）−５−アミノナフタレン（化合物１５）
アルゴン雰囲気下、１−（Ｎ３−｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝−Ｎ２−フルオレニルメチルオキシカルボニルグアニジノ）−Ｎ５−（トリチルアミノオキシアセチル）−５−アミノナフタレン（化合物１４）１７０ｍｇ（０．１６ｍｍｏｌ）を塩化メチレン１５ｍｌに溶解して０℃に冷却し、トリフルオロ酢酸１８０μｌ（２．４ｍｍｏｌ）およびトリイソプロピルシラン３３０μｌ（１．６ｍｍｏｌ）を加えた。反応液を室温に戻して３０分撹拌した後、反応液にクロロホルム６５ｍｌを加え、飽和炭酸水素ナトリウム溶液３５ｍｌで１回、水３５ｍｌで１回、飽和食塩水３５ｍｌで１回洗浄し、硫酸ナトリウムにより乾燥した。溶液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：メタノール−クロロホルム）により精製して標記化合物（化合物１５）８６ｍｇ（収率６５％）を白色泡状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ８５３．４（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：８５３．３７０７（Ｃ４４Ｈ５３Ｎ８Ｏ８Ｓ［ＭＨ＋］），実測値：８５３．３７１３．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：９．９０（ｂｒ ｓ，１Ｈ，ＮＨ），８．０２（ｍ，１Ｈ），７．８９−７．７１（ｍ，５Ｈ），７．７８（ｂｒ ｓ，１Ｈ，ＮＨ），７．６２−７．３１（ｍ，８Ｈ），６．５６（ｂｒ ｓ，２Ｈ，ＮＨ２），６．３９（ｂｒ ｓ，１Ｈ，ＮＨ），６．３３（ｂｒ ｓ，１Ｈ，ＮＨ），４．２９−４．２６（ｍ，６Ｈ），４．１０（ｍ，１Ｈ），３．４８（ｍ，６Ｈ），３．３６（ｍ，４Ｈ），３．１６（ｍ，２Ｈ），３．０６（ｍ，１Ｈ），２．８０（ｄｄ，１Ｈ，Ｊ＝５．０，１２．５Ｈｚ），２．５６（ｄ，１Ｈ，Ｊ＝１２．５Ｈｚ），２．０４（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），１．６６−１．３８（ｍ，４Ｈ），１．２８（ｍ，２Ｈ）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１７２．０７（Ｃ），１６９．６８（Ｃ），１６２．６４（Ｃ），１４４．１５（Ｃ），１４０．６３（Ｃ），１２７．４５（ＣＨ），１２６．９８（ＣＨ），１２５．０４（ＣＨ），１２０．０４（ＣＨ），７４．３７（ＣＨ２），６９．４７（ＣＨ２），６９．１４（ＣＨ２），６８．８１（ＣＨ２），６６．０４（ＣＨ２），６０．９９（ＣＨ），５９．１５（ＣＨ），５５．３７（ＣＨ），４６．６７（ＣＨ），３９．７９（ＣＨ２），３８．３７（ＣＨ２），３５．０４（ＣＨ２），２８．１４（ＣＨ２），２７．９９（ＣＨ２），２５．２１（ＣＨ２）。
１−（Ｎ ３ −｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝グアニジノ）−Ｎ ５ −（アミノオキシアセチル）−５−アミノナフタレン（化合物１６、酢酸塩）
アルゴン雰囲気下、１−（Ｎ３−｛２−〔２−（２−ビオチニルアミノエトキシ）エトキシ〕エチル｝−Ｎ２−フルオレニルメチルオキシカルボニルグアニジノ）−Ｎ５−（アミノオキシアセチル）−５−アミノナフタレン（化合物１５）８５ｍｇ（０．１０ｍｍｏｌ）を１，４−ジオキサン４ｍｌとメタノール４ｍｌの混合溶媒に溶解し、ピペリジン０．４０ｍｌ（４．０ｍｍｏｌ）を加えて室温で１６時間撹拌した。反応液を減圧下濃縮した後、残渣を水３５ｍｌに溶解し、酢酸エチル１５ｍｌで３回洗浄した。水層を減圧下濃縮後、Ｃ１８カートリッジカラムクロマトグラフィー（溶出溶媒：アセトニトリル−０．１Ｍ ＴＥＡＡ溶液）により精製して粗精製物（５２ｍｇ）を得た。この粗精製物の一部を２％酢酸水溶液５ｍｌに溶解して、再度Ｃ１８カートリッジカラムクロマトグラフィー（溶出溶媒：アセトニトリル−水）により精製して標記化合物（化合物１６、酢酸塩）９．２ｍｇを白色綿状物質として得た。
ＦＡＢ−ＬＲＭＳ ｍ／ｚ ６３１．３（ＭＨ＋）；ＦＡＢ−ＨＲＭＳ 計算値：６３１．３０２６（Ｃ２９Ｈ４３Ｎ８Ｏ６Ｓ［ＭＨ＋］），実測値：６３１．３００５．
１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ：９．９４（ｓ，１Ｈ，ＮＨ），９．６４（ｂｒ ｓ，１Ｈ，ＮＨ），８．０７（ｍ，１Ｈ，ｎａｐ），７．８２（ｍ，１Ｈ），７．８１（ｄ，１Ｈ，Ｊ＝８．２Ｈｚ，ｎａｐ），７．７３（ｄ，１Ｈ，Ｊ＝６．９Ｈｚ，ｎａｐ），７．６２（ｍ，２Ｈ，ｎａｐ），７．５５（ｍ，２Ｈ），７．５１（ｍ，１Ｈ，ｎａｐ），６．５５（ｓ，２Ｈ，ＮＨ２Ｏ），６．３９（ｓ，１Ｈ，ＮＨ），６．３５（ｓ，１Ｈ，ＮＨ），４．３１（ｍ，１Ｈ），４．２９（ｓ，２Ｈ，ＣＨ２），４．１２（ｂｒ ｄｄ，１Ｈ，Ｊ＝４．３，７．６Ｈｚ），３．５８（ｍ，６Ｈ），３．４２（ｍ，４Ｈ），３．２１（ｍ，２Ｈ），３．０９（ｍ，１Ｈ），２．８１（ｄｄ，１Ｈ，Ｊ＝５．０，１２．５Ｈｚ），２．５７（ｄ，１Ｈ，Ｊ＝１２．５Ｈｚ），２．０７（ｔ，２Ｈ，Ｊ＝７．３Ｈｚ），１．６４−１．４２（ｍ，４Ｈ），１．３０（ｍ，２Ｈ）．
１３Ｃ ＮＭＲ（６７．８ＭＨｚ，ＤＭＳＯ−ｄ６）δ：１７２．１０（Ｃ），１６９．６８（Ｃ），１６２．５９（Ｃ），１５５．７５（Ｃ），１３３．７２（Ｃ），１３０．４４（Ｃ），１２９．３９（Ｃ），１２６．３２（ＣＨ），１２５．８１（ＣＨ），１２３．００（ＣＨ），１１９．８７（ＣＨ），７４．２７（ＣＨ２），６９．６６（ＣＨ２），６９．３８（ＣＨ２），６９．０６（ＣＨ２），６８．５２（ＣＨ２），６０．９２（ＣＨ），５９．０８（Ｃ），５５．３１（ＣＨ），４１．２２（ＣＨ２），３９．７３（ＣＨ２），３８．２７（ＣＨ２），３５．００（ＣＨ２），２８．０８（ＣＨ２），２７．９４（ＣＨ２），２５．１６（ＣＨ２）。
（実施例１４）
実施例１１と同様のオリゴヌクレオチドを用い、同じ条件でａｏＮｄｇ−ｂｉｏの反応効率を調べた。反応の結果、ａｏＮｄｇ−ｂｉｏの反応速度定数は、０．２３ｍｉｎ−１であったことから、ａｏＮｇ、ａｏＮｇ−ｂｉｏと同様に、ＡＲＰよりもａｏＮｄｇ−ｂｉｏの方が高い反応効率を示すことが明らかとなった。
本明細書中で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書中にとり入れるものとする。
［配列表］
Example 1 Synthesis of aminooxynaphthalene reagent (aoNg, Compound 6 and aoNg-biotin, Compound 10)
Thin layer chromatography was performed on Kieselgel 60F254 plates (Merck). For column chromatography, Wakogel C-200 (Wako Pure Chemical Industries) was used.1H NMR and13C NMR was measured using JEOL JNM-EX270 with tetramethylsilane as an internal standard.
aoNg, Synthesis of Compound 6
Scheme 1
N 1 -(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 2)
Under an argon atmosphere, 440 mg (2.80 mmol) of 1,5-diaminonaphthalene (compound 1) and 1.00 g (3.00 mmol) of N-tritylaminooxyacetic acid were dissolved in 30 ml of dimethylformamide (DMF), and EDC [hydrochloric acid 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide] (590 mg, 3.10 mmol) was added, and the mixture was stirred at room temperature for 20 hours. 200 ml of ethyl acetate was added to the reaction solution, and the mixture was washed 4 times with 70 ml of water. The solution was concentrated under reduced pressure, then azeotroped three times with ethyl acetate, and the resulting precipitate was suspended in a mixed solution of ethyl acetate (20 ml) and hexane (120 ml). The precipitate was collected by suction filtration to obtain 1.03 g (yield 72%) of the title compound (Compound 2) as a light brown powder.
FAB-LRMS m / z 473.3 (M+FAB-HRMS calculated value: 473.2103 (C31H27N3O2[M+]), Measured value: 473.2093.
11 H NMR (270 MHz, DMSO-d6) Δ: 9.33 (br s, 1H, NH), 8.38 (br s, 1H, NH), 7.93 (d, 1H, naph, J = 8.6 Hz), 7.64 (d, 1H, naph, J = 7.3 Hz), 7.34-7.25 (m, 16H, Tr and naph), 7.20 (t, 1H, naph, J = 8.2 Hz), 6.99 (d , 1H, naph, J = 8.2 Hz), 6.70 (d, 1H, naph, J = 7.3 Hz), 5.78 (br s, 2H, NH2), 4.20 (s, 2H, CH2).
13C NMR (67.8 MHz, DMSO-d6) Δ: 168.26 (C), 144.96 (C), 144.01 (C), 132.13 (C), 128.70 (CH), 127.57 (CH), 126.68 (CH ), 123.05 (C), 122.76 (CH), 121.06 (CH), 119.88 (CH), 109.15 (CH), 107.58 (CH), 73.64 (C) 73.31 (CH2).
N 1 -[2- (trifluoroacetylamino) ethylcarbonyl] -N 5 -(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 3)
N in argon atmosphere11.00 g (2.11 mmol) of-(tritylaminooxyacetyl) -1,5-diaminonaphthalene (compound 2) and 780 mg (4.22 mmol) of N-trifluoroacetyl-β-alanine are dissolved in 25 ml of dimethylformamide, 810 mg (4.22 mmol) of EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride] was added, and the mixture was stirred at room temperature for 17 hours. To the reaction solution, 220 ml of ethyl acetate was added and washed 4 times with 80 ml of water. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (elution solvent: ethyl acetate-hexane) to obtain 961 mg (yield 71%) of the title compound (Compound 3) as a white solid substance.
FAB-LRMS m / z 640.3 (M+FAB-HRMS calculated value: 640.2297 (C36H31F3N4O4[M+]), Measured value: 640.2308.
11 H NMR (270 MHz, DMSO-d6) Δ: 10.04 (s, 1H, NH), 9.61 (s, 2H, NH and NH), 8.36 (s, 1H, NH), 7.94 (d, 1H, naph, J = 8.6 Hz), 7.74-7.69 (m, 3H, naph), 7.55-7.48 (m, 2H, naph), 7.35-7.25 (m, 15H, Tr), 4.23 (s, 2H, CH2), 3.56 (br t, 2H, CH2, J = 6.8 Hz), 2.78 (t, 2H, CH2, J = 6.8 Hz).
13C NMR (67.8 MHz, DMSO-d6) Δ: 169.27 (C), 168.76 (C), 156.12 (C, q, J = 35.8 Hz), 144.01 (C), 133.57 (C), 132.73 ( C), 128.73 (CH), 128.46 (C), 128.41 (C), 127.58 (CH), 126.70 (CH), 125.36 (CH), 125.11 (CH ), 122.04 (CH), 120.52 (CH), 119.67 (CH), 115.80 (C, q, J = 287.9 Hz), 73.67 (C), 73.33 (CH2), 36.04 (CH2), 34.80 (CH2).
N 1 -(2-Aminoethylcarbonyl) -N 5 -(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (compound 4)
N1-[2- (trifluoroacetylamino) ethylcarbonyl] -N5To 950 mg (1.48 mmol) of-(tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 3), 40 ml of ethanol and 20 ml of concentrated aqueous ammonia were added, sealed in a pressure-resistant glass container, and heated at 60 ° C. for 4 hours. The solution was returned to room temperature and concentrated under reduced pressure, and the residue was azeotroped three times with acetonitrile to obtain the title compound (Compound 4) as a pale yellow foam. This compound was used in the next reaction without further purification.
FAB-LRMS m / z 545.4 (MH+); FAB-HRMS calculated value: 545.2553 (C34H33N4O3[MH+]), Measured value: 545.2573.
11 H NMR (270 MHz, DMSO-d6) Δ: 10.19 (s, 1H, NH), 9.62 (s, 1H, NH), 8.34 (s, 1H, NH), 7.98 (d, 1H, naph, J = 8. 2Hz), 7.80 (br s, 2H, NH2), 7.75-7.70 (m, 3H, naph), 7.58-7.49 (m, 2H, naph), 7.38-7.25 (m, 15H, Tr), 4.23 (S, 2H, CH2), 3.16 (t, 2H, CH2, J = 6.6 Hz), 2.89 (t, 2H, CH2, J = 6.6 Hz).
13C NMR (67.8 MHz, DMSO-d6) Δ: 168.89 (C), 168.78 (C), 143.98 (C), 133.29 (C), 132.77 (C), 128.70 (CH), 128.49 (C ), 128.29 (C), 127.56 (CH), 126.70 (CH), 125.32 (CH), 125.19 (CH), 122.13 (CH), 121.94 (CH) , 120.49 (CH), 119.82 (CH), 73.64 (C), 73.30 (CH2), 35.11 (CH2), 32.82 (CH2).
N 1 -(2-Guanidinoethylcarbonyl) -N 5 -(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 5)
N synthesized by pre-reaction under argon atmosphere1-(2-Aminoethylcarbonyl) -N5-(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (compound 4) (Total amount: 1.48 mmol) was dissolved in 15 ml of dimethylformamide, and 320 mg (2.20 mmol) of 1H-pyrazole-1-carboxyamidine hydrochloride and triethylamine 0 were dissolved. .61 ml (4.40 mmol) was added and stirred at room temperature for 6 hours. 150 ml of ethyl acetate was added to the reaction solution, and the mixture was washed 4 times with 50 ml of water. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (elution solvent: ethanol-chloroform) to obtain 567 mg (yield 66%) of the title compound (Compound 5) as a light brown foam.
FAB-LRMS m / z 587.3 (MH+); FAB-HRMS calculated value: 5872771 (C35H35N6O3[MH+]), Measured value: 5872772.
11 H NMR (270 MHz, DMSO-d6) Δ: 10.11 (s, 1H, NH), 9.61 (s, 1H, NH), 8.34 (s, 1H, NH), 7.96 (m, 1H, naph), 7.75 -7.70 (m, 3H, naph), 7.67 (brs, 1H, NH), 7.57-7.48 (m, 2H, naph), 7.38-7.26 (m, 15H) , Tr), 7.25 (br s, 3H, NH2 and NH), 4.23 (s, 2H, CH2), 3.49 (dt, 2H, CH2, J = 5.6, 6.3 Hz), 2.79 (t, 2H, CH2, J = 6.3 Hz).
13C NMR (67.8 MHz, DMSO-d6) Δ: 169.39 (C), 168.60 (C), 156.52 (C), 143.83 (C), 133.30 (C), 132.60 (C), 128.55 (CH ), 128.33 (C), 128.22 (C), 127.40 (CH), 126.53 (CH), 125.17 (CH), 125.02 (CH), 121.93 (CH) 121.79 (CH), 120.36 (CH), 119.62 (CH), 73.49 (C), 73.14 (CH2), 36.97 (CH2), 34.88 (CH2).
N 5 -(Aminooxyacetyl) -N 1 -(2-Guanidinoethylcarbonyl) -1,5-diaminonaphthalene dihydrochloride (Compound 6)
N in argon atmosphere1-(2-Guanidinoethylcarbonyl) -N5-(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 5) 59 mg (0.10 mmol) was dissolved in 4.0 ml of methylene chloride, 60 μl of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 2.5 hours. The white precipitate formed in the reaction solution was washed with methylene chloride, suspended in a mixed solution of ethanol (2 ml) and diethyl ether (3 ml), and the precipitate was collected by suction filtration to give the title compound (Compound 6). ) 26 mg (62% yield) was obtained as a white powder.
FAB-LRMS m / z 345.2 (MH+); FAB-HRMS calculated value: 345.1675 (C16H21N6O3[MH+]), Measured value: 345.1687.
11 H NMR (270 MHz, DMSO-d6) Δ: 11.01 (br s, 1H, NH), 10.40 (s, 1H, NHCO), 10.24 (s, 1H, NHCO), 7.99 (t, 2H, naph, J = 8) .1 Hz), 7.78 (br t, 1 H, NH, J = 5.8 Hz), 7.73-7.69 (m, 2 H, naph), 7.56 (dd, 1 H, naph, J = 4) 0.03, 8.3 Hz), 7.53 (dd, 1H, naph, J = 4.0, 7.9 Hz), 7.28 (br s, 4H), 4.92 (s, 2H, CH2), 3.50 (dt, 2H, CH2, J = 5.8, 6.3 Hz), 2.80 (t, 2H, CH2, J = 6.3 Hz).
13C NMR (67.8 MHz, DMSO-d6) Δ: 169.50 (C), 166.87 (C), 156.81 (C), 133.43 (C), 132.58 (C), 128.53 (C), 128.47 (C ), 125.25 (CH), 125.11 (CH), 122.13 (CH), 122.06 (CH), 120.76 (CH), 120.19 (CH), 72.15 (CH2), 37.17 (CH2), 35.18 (CH2).
Synthesis of aoNg-biotin reagent (hereinafter aoNg-bio; Compound 10)
Scheme 2
N 1 -[2- (N 3 -Fluorenylmethyloxycarbonylthioureido) ethylcarbonyl] -N 5 -(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 7)
N in argon atmosphere1-(2-Aminoethylcarbonyl) -N5-(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (compound 4) (synthesized by the method described above from compound 3, 1.00 mmol) was dissolved in 15 ml of a mixed solution of methylene chloride-acetonitrile (1: 2). In another glass container, 310 mg (1.10 mmol) of fluorenylmethyloxycarbonyl isothiocyanate and 0.19 ml (1.10 mmol) of diisopropylethylamine are dissolved in 20 ml of methylene chloride, and the solution of the above compound 4 is slowly added dropwise thereto. And then stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the resulting precipitate was suspended in a mixed solution of ethyl acetate (15 ml) and hexane (30 ml). The precipitate was collected by suction filtration to obtain 780 mg (yield 94%) of the title compound (Compound 7) as a light brown powdery substance.
FAB-LRMS m / z 825.2 (M+FAB-HRMS calculated value: 825.2985 (C50H43N5O5S [M+]), Measured value: 825.2985.
11 H NMR (270 MHz, DMSO-d6) Δ: 11.42 (br s, 1H, NH), 10.16 (br t, 1H, NH, J = 5.5 Hz), 10.07 (br s, 1H, NH), 9.58 (br s, 1H, NH), 8.35 (br s, 1H, NH), 7.93 (m, 1H, naph), 7.91-7.82 (m, 4H, Fmoc), 7.73-7 .67 (m, 3H, naph), 7.52-7.38 (m, 4H, naph and Fmoc), 7.36-7.24 (m, 17H, Fmoc and Tr), 4.36-4. 33 (m, 2H, Fmoc-CH2), 4.29-4.25 (m, 1H, Fmoc-CH), 4.23 (s, 2H, CH2), 3.95 (dd, 2H, CH2, J = 5.5, 6.1 Hz), 2.89 (t, 2H, CH2, J = 6.1 Hz).
13C NMR (67.8 MHz, DMSO-d6) Δ: 179.14 (C), 169.96 (C), 168.71 (C), 153.10 (C), 143.98 (C), 143.10 (C), 140.50 (C ), 133.47 (C), 132.71 (C), 128.70 (CH), 128.41 (C), 128.31 (C), 127.64 (CH), 127.56 (CH) , 126.95 (CH), 126.69 (CH), 125.42 (CH), 125.34 (CH), 125.10 (CH), 121.93 (CH), 120.44 (CH), 119.97 (CH), 119.63 (CH), 73.64 (C), 73.31 (CH2), 67.14 (CH2), 45.99 (CH), 40.93 (CH2), 34.19 (CH2).
N 1 -[2- (N 3 -{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} -N 2 -Fluorenylmethyloxycarbonylguanidino) ethylcarbonyl] -N 5 -(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 8)
N in argon atmosphere1-[2- (N3-Fluorenylmethyloxycarbonylthioureido) ethylcarbonyl] -N5410 mg (0.50 mmol) of-(tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 7) was dissolved in 15 ml of dimethylformamide and cooled to 0 ° C. After adding 0.26 ml (1.50 mmol) of diisopropylethylamine to this solution, 310 mg (0.75 mmol) of (+)-biotinyl-3,6-dioxaoctanediamine.2 / 3 acetate and 205 mg of mercuric chloride (0.75 mmol) of a dimethylformamide solution (7 ml) was slowly added dropwise, and the mixture was further stirred at 0 ° C. for 1 hour. After adding 80 ml of ethyl acetate to the reaction solution, the precipitate was removed by celite filtration. Ethyl acetate was added to the filtrate to make a total volume of 150 ml, and it was washed 4 times with 50 ml of water. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (elution solvent: ethanol-chloroform) to obtain 300 mg (yield 51%) of the title compound (Compound 8) as a white foam.
FAB-LRMS m / z 1166.6 (MH+FAB-HRMS calculated value: 1166.5174 (C66H72N9O9S [MH+]), Measured value: 1166.5165.
11 H NMR (270 MHz, DMSO-d6) Δ: 10.05 (br s, 1H, NH), 9.58 (br s, 1H, NH), 8.35 (br s, 1H, NH), 7.95-7.87 (m, 3H) , Naph and Fmoc), 7.81 (brt, 1H, NH, J = 5.6 Hz), 7.72-7.63 (m, 5H, naph and Fmoc), 7.52-7.37 (m , 4H, naph and Fmoc), 7.34-7.24 (m, 17H, Fmoc and Tr), 6.40 (brs, 1H, NH), 6.35 (brs, 1H, NH), 4 .30-4.23 (m, 6H), 4.09 (m, 1H), 3.51 (m, 8H), 3.40-3.36 (m, 4H), 3.18 (m, 2H) ), 3.05 (m, 1H), 2.82-2.76 (m, 3H), 2.56 (d, 1H, J = 1) .2Hz), 2.05 (t, 2H, J = 7.3Hz), 1.62-1.37 (m, 4H), 1.33-1.27 (m, 2H).
N 1 -[2- (N 3 -{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} -N 2 -Fluorenylmethyloxycarbonylguanidino) ethylcarbonyl] -N 5 -(Aminooxyacetyl) -1,5-diaminonaphthalene (Compound 9)
N in argon atmosphere1-[2- (N3-{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} -N2-Fluorenylmethyloxycarbonylguanidino) ethylcarbonyl] -N5292 mg (0.25 mmol) of-(tritylaminooxyacetyl) -1,5-diaminonaphthalene (compound 8) was dissolved in 10 ml of methylene chloride, 280 μl (3.8 mmol) of trifluoroacetic acid and 510 μl (2.5 mmol) of triisopropylsilane. ) And stirred at room temperature for 30 minutes. To the reaction solution, 60 ml of chloroform was added, washed twice with 30 ml of saturated sodium hydrogen carbonate solution, once with 30 ml of water and once with 30 ml of saturated brine, and dried over sodium sulfate. After the organic layer was concentrated under reduced pressure, the resulting precipitate was dissolved in a mixed solvent of 2.5 ml of chloroform and 2.5 ml of ethanol, and this solution was added dropwise to 25 ml of stirred diethyl ether. The resulting white precipitate was collected by suction filtration to give 184 mg (yield 80%) of the title compound (Compound 9) as a white powder.
FAB-LRMS m / z 924.4 (MH+); FAB-HRMS calculated value: 924.4078 (C47H58N9O9S [MH+]), Measured value: 924.4053.
11 H NMR (270 MHz, DMSO-d6) Δ: 10.17 (br s, 1H, NH), 9.95 (br s, 1H, NH), 7.97-7.89 (m, 4H, naph and Fmoc), 7.82 (br t , 1H, NH, J = 5.5 Hz), 7.71-7.66 (m, 4H), 7.53 (t, 2H, J = 7.6 Hz), 7.42 (t, 2H, J = 7.3 Hz), 7.31 (m, 2H), 6.41 (brs, 1H, NH), 6.37 (brs, 1H, NH), 4.46 (m, 2H), 4.37 (S, 2H, CH2), 4.34-4.26 (m, 2H), 4.10 (m, 1H), 3.55-3.47 (m, 8H), 3.39-3.34 (m, 4H), 3.17 (m, 2H), 3.05 (m, 1H), 2.87 (m, 2H), 2.79 (dd, 1H, J = 5.3, 12.2 Hz), 2.56 ( d, 1H, J = 12.2 Hz), 2.04 (t, 2H, J = 7.4 Hz), 1.62-1.37 (m, 4H), 1.27 (m, 2H).
13C NMR (67.8 MHz, DMSO-d6) Δ: 172.05 (C), 169.16 (C), 162.59 (C), 143.41 (C), 140.64 (C), 133.36 (C), 133.16 (C ), 128.92 (C), 128.54 (C), 127.65 (CH), 127.03 (CH), 125.31 (CH), 125.23 (CH), 125.04 (CH) , 122.50 (CH), 122.17 (CH), 120.56 (CH), 120.38 (CH), 120.10 (CH), 73.88 (CH2), 69.57 (CH2), 69.29 (CH2), 69.01 (CH2), 67.02 (CH2), 60.91 (CH), 59.06 (CH), 55.31 (CH), 46.22 (CH), 39.72 (CH2), 38.26 (CH2), 34.97 (CH2), 28.08 (CH2), 27.92 (CH2), 25.14 (CH2).
N 1 -[2- (N 3 -{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} guanidino) ethylcarbonyl] -N 5 -(Aminooxyacetyl) -1,5-diaminonaphthalene (Compound 10)
N in argon atmosphere1-[2- (N3-{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} -N2-Fluorenylmethyloxycarbonylguanidino) ethylcarbonyl] -N5-(Aminooxyacetyl) -1,5-diaminonaphthalene (Compound 9) 50 mg (0.054 mmol) was dissolved in a mixed solvent of 1,4-dioxane 3 ml and methanol 3 ml, and piperidine 220 μl (2.2 mmol) was added. Stir at room temperature for 20 hours. After the reaction solution was concentrated under reduced pressure, the residue was dissolved in 30 ml of water and washed 6 times with 20 ml of chloroform. The aqueous layer was concentrated under reduced pressure and lyophilized to obtain 35 mg (yield 93%) of the title compound (Compound 10) as a white cotton-like substance.
FAB-LRMS m / z 702.4 (MH+FAB-HRMS calculated value: 702.3397 (C32H48N9O7S [MH+]) Measured value: 702.3405.
11 H NMR (270 MHz, DMSO-d6) Δ: 10.11 (br s, 1 H, NH), 9.88 (br s, 1 H, NH), 7.96 (d, 1 H, naph, J = 8.6 Hz), 7.90 (d, 1H, naph, J = 8.3 Hz), 7.84 (t, 1H, NH, J = 5.3 Hz), 7.71-7.65 (m, 2H, naph), 7.58-7.50 (M, 2H, naph), 6.57 (br s, 2H, NH2O), 6.41 (brs, 1H, NH), 6.37 (brs, 1H, NH), 4.30 (m, 1H, bio), 4.27 (s, 2H, CH2), 4.11 (m, 1H), 3.52 (m, 8H), 3.39 (t, 2H, J = 5.8 Hz), 3.31 (m, 2H), 3.19 (m, 2H), 3.07 (m, 1H), 2.84-2.78 (m, 3H), 2.57 (d, 1H, J = 12.2 Hz), 2.06 (t, 2H, J = 7.3 Hz), 1.64-1.41 (m, 4H), 1.29 (m, 2H).
13C NMR (67.8 MHz, DMSO-d6) Δ: 172.10 (C), 169.73 (C), 169.69 (C), 162.59 (C), 155.86 (C), 133.39 (C), 133.30 (C ), 129.03 (C), 128.47 (C), 125.31 (CH), 125.23 (CH), 122.63 (CH), 122.00 (CH), 120.53 (CH) , 120.37 (CH), 74.30 (CH2), 69.57 (CH2), 69.31 (CH2), 69.01 (CH2), 68.46 (CH2), 60.91 (CH), 59.06 (CH), 55.31 (CH), 40.96 (CH2), 39.83 (CH2), 38.25 (CH2), 37.28 (CH2), 34.98 (CH2), 34.91 (CH2), 28.08 (CH2), 27.93 (CH2), 25.15 (CH2).
Example 2 Synthesis of Bisaminooxynaphthalene reagent (hereinafter aoNao; Compound 12)
Scheme 3
N 1 , N 5 -Bis (tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 11)
Under an argon atmosphere, 158 mg (1.00 mmol) of 1,5-diaminonaphthalene (Compound 1) was dissolved in 30 ml of pyridine, and 333 mg (1.00 mmol) of N-tritylaminooxyacetic acid and EDC [1-ethyl-3-hydrochloride ( 3-Dimethylaminopropyl) carbodiimide] 230 mg (1.20 mmol) was added and stirred at room temperature. After 3 hours and 6 hours from the start of the reaction, 333 mg (1.00 mmol) of N-tritylaminooxyacetic acid and 230 mg (1.20 mmol) of EDC were added, respectively, and further stirred at room temperature for 16 hours. After the reaction solution was concentrated under reduced pressure, the precipitate was suspended in 20 ml of water and washed once with 50 ml of ethyl acetate and three times with 20 ml of chloroform. The white precipitated substance in the aqueous layer was collected by suction filtration, and further washed with ethanol and ether to obtain 550 mg (yield 70%) of the title compound (Compound 11) as a white powdery substance.
FAB-LRMS m / z (+ NaI) 789.3 (MH+), 811.3 ([M+Na]+FAB-HRMS calculated value: 811.3260 (C52H44N4O4Na [M+Na]+), Measured value: 811.3274.
11 H NMR (270 MHz, DMSO-d6) Δ: 9.62 (br s, 2H, NH), 8.36 (br s, 2H, NH), 7.77-7.71 (m, 4H, naph), 7.52 (t, 2H, naph, J = 7.9 Hz), 7.41-7.25 (m, 30H, Tr), 4.23 (s, 4H, CH2).
13C NMR (67.8 MHz, DMSO-d6): 168.89 (C), 144.07 (C), 132.90 (C), 128.78 (CH), 127.63 (CH), 126.76 (CH), 125.43 (CH ), 123.80 (C), 122.15 (CH), 120.11 (CH), 73.61 (C), 73.27 (CH2).
N 1 , N 5 -Bis (aminooxyacetyl) -1,5-diaminonaphthalene (Compound 12)
N in argon atmosphere1, N5394 mg (0.50 mmol) of bis (tritylaminooxyacetyl) -1,5-diaminonaphthalene (compound 11) was suspended in 20 ml of chloroform and cooled on ice, 0.94 ml (10.0 mmol) of trifluoroacetic acid and triisopropyl 1.02 ml (5.0 mmol) of silane was added. The reaction solution was returned to room temperature and stirred for 3.5 hours. 40 ml of water and 10 ml of chloroform were added to the reaction solution to dissolve the precipitate, followed by liquid separation. The aqueous layer was washed twice with 30 ml of chloroform and once with 30 ml of ethyl acetate. Subsequently, 60 ml of acetonitrile was added to the aqueous layer, and then DOWE (1 × 4-100 (OH−Foam). DOWE (1 × 4-100 was removed by filtration, and DOWEW (1 × 4-100 was washed with 80% acetonitrile aqueous solution. After combining the solutions and concentrating under reduced pressure, the residue was dissolved in 30 ml of water and dissolved in C Purification by -18 column column chromatography (elution solvent: water-acetonitrile) gave 109 mg (yield 72%) of the title compound (Compound 12) as a white powder.
FAB-LRMS m / z 305.2 (MH+); FAB-HRMS calculated value: 305.1250 (C14H17N4O4[MH+]), Measured value: 305.1259.
11 H NMR (270 MHz, DMSO-d6) Δ: 9.88 (s, 2H, NH), 7.93 (d, 2H, naph, J = 8.6 Hz), 7.66 (d, 2H, naph, J = 7.2 Hz), 7. 54 (dd, 2H, naph, J = 7.2, 8.6 Hz), 6.57 (s, 4H, NH2), 4.28 (s, 4H, CH2).
13C NMR (67.8 MHz, DMSO-d6) Δ: 169.75 (C), 133.35 (C), 129.11 (C), 125.41 (CH), 122.66 (CH), 120.75 (CH), 74.42 (CH2).
Example 3 Oligonucleotide Synthesis and Purification
Oligonucleotide synthesis was performed on an Applied Biosystems type 394 DNA / RNA synthesizer. Synthesized on a 0.2 or 1 μmol scale. The HPLC was performed using a Gilson apparatus and the analysis was performed using a Waters 996 photodiode array detector.
Solution A 5% acetonitrile / 0.1M TEAA (pH 7.0)
Solution B 25% acetonitrile / 0.1M TEAA (pH 7.0)
Column temperature: 50 ° C
As the column, a μ-Bondsphere (C-18) column Φ3.9 × 150 mm (manufactured by Waters) was used.
Ion exchange HPLC solution
Solution A 20% acetonitrile
B solution 20% acetonitrile, 2M ammonium formate
Column temperature: 50 ° C
The column used was TSK-GEL DEAE-2SW Φ4.6 × 250 mm (Tosoh Corporation).
HPLC conditions
Condition 1% of solution B 20 → 40% / 20 minutes
Condition 2% of solution B 35 → 55% / 20 minutes
(Example 4) Oxidation reaction of oligonucleotide (RNA)
Oligonucleotide (F-Lr17X: X = A, G, C, U) (1.6 nmol) was dissolved in 100 mM phosphate buffer (pH 7) and 800 μM sodium periodate (total reaction volume 200 μl), 37 ° C., The oxidation reaction was performed in 90 minutes. After the reaction, desalting was performed using a NAP10 column (GE Healthcare), and the resulting oxidized oligonucleotide (F-Lr17Xox) was subjected to UV quantification.
(Example 5) Labeling reaction of ARP and aoNg to oxidized oligonucleotide (RNA)
Reaction to single strand
An oxidized oligonucleotide (F-Lr17Xox; X = A, G, C, U) (12 pmol) was dissolved in 3 μl of 1M phosphate buffer (pH 7) and 21 μl of sterilized water, and then 2 mM labeling reagent ( 6 μl of ARP or aoNg prepared in Example 1 was added, and the reaction was started at 37 ° C. ARP (Aldehyde Reactive Probe; DOJINDO) has the following structure.
After a certain time, 2.5 μl was sampled and added to 5 μl of loading solution mix (50 mM EDTA, 10 M urea, 0.1% BPB; prepared by mixing 4 μl, 40 mM glutaraldehyde; 1 μl) to stop the reaction. Then, it analyzed with the denaturation 20% polyacrylamide gel (19: 1). The fluorescence intensity of fluorescein in each band was measured and the reaction efficiency was calculated. The results of the reaction are shown in FIG.
Double-stranded reaction
When the reaction was performed in double strand, complementary strand AS32Y (24 pmol) was added to oxidized oligonucleotide (F-Lr17Xox) (12 pmol), and other reaction conditions were the same as in the single strand reaction.
The binding rate of ARP or aoNg was measured for the single strand of F-Lr17Xox (ss F-Lr17Xox) and the double strand of the complementary strand (AS32Y), and the apparent reaction rate constant was calculated. AoNg showed higher reactivity than ARP regardless of the type of base at the 3 'end of RNA. It was also found that the reaction efficiency is increased when double strands are formed rather than single strands.
(Example 6) Continuous reaction from oxidation to labeling of RNA
In Examples 4 and 5, the labeling reaction was performed after the RNA was previously oxidized and purified. Next, an experiment was conducted to confirm whether it was possible to continuously perform the process from RNA oxidation to labeling. RNA (F-Lr17X; X = A, G, C, U) (12 pmol) was dissolved in 150 mM phosphate buffer (pH 7) and 150 μM sodium periodate (total reaction volume 20 μl) at 37 ° C. for 60 minutes. An oxidation reaction was performed. Subsequently, 5 μl of 2 mM DTT was added and the mixture was heated at 37 ° C. for 30 minutes, and 5 μl of 240 μM aoNg (or aoNg-bio) was added, and a labeling reaction was performed at 37 ° C. (total reaction volume 30 μl). In the same manner as in Example 5, sampling was performed after a certain period of time for analysis. As a result of the reaction, it was confirmed that the excess sodium periodate used in the oxidation could be reduced to continuously perform the oxidation to the labeling reaction.
(Example 7) Synthesis of oligonucleotide containing abasic site
Using an abasic phosphoramidite reagent (Glen Res.), An oligonucleotide (F-20Z) containing an abasic site (Z) was synthesized. F-20Z represents 5 'F-GAATTGCTTTGGAAGAZGTTT 3'. The synthesized oligonucleotide was deprotected with concentrated aqueous ammonia according to a conventional method, and then 4 mL of 40% acetic acid was added and stirred at room temperature for 4 hours to deprotect the TBDMS group. After the reaction, acetic acid was distilled off under reduced pressure, water was further added, and the solvent was distilled off under reduced pressure. This operation was repeated several times to remove acetic acid. Thereafter, the oligonucleotide was dissolved in sterile water and purified by reverse phase HPLC and ion exchange HPLC. Sp35, which is the complementary strand of F-20Z, was synthesized and purified in the same manner (Tables 4 and 5). Sp35C represents 3 'CGAAAGTAACCCTTAACGAACCTCTCTCAAAAGAACGA 5'. The conditions for each HPLC were the same as those for purification of F-Lr17X and AS32Y.
Condition 1% of solution B 20 → 40% / 20 minutes
Condition 2% of solution B 35 → 55% / 20 minutes
Condition 3% of solution B 30 → 50% / 20 minutes
(Example 8) Oxidation of oligonucleotide containing abasic site
Oligonucleotide F-20Z (2.6 nmol) containing an abasic site prepared in Example 7 was dissolved in 100 mM phosphate buffer (pH 7) and 10 mM sodium periodate, and an oxidation reaction was performed at 37 ° C. for 60 minutes. It was. After the reaction, the solution was desalted with a NAP5 column (GE Healthcare), concentrated, and purified by a denaturing 20% polyacrylamide gel (19: 1). The gel pieces were desalted using a YMC cartridge (100 mg / 1 ml). The obtained oxidized oligonucleotide (F-20Zox) was quantified by UV and subjected to the reaction.
(Example 9) Labeling reaction of ARP and aoNg on a basic site
Reaction to single strand
Oxidized F-20Zox (12 pmol) was dissolved in 3 μl of 1M phosphate buffer (pH 7) and 15 μl of sterilized water, heated at 90 ° C. for 1 minute, and ice-cooled. After 10 seconds, the mixture was left at room temperature for 5 minutes and preincubated at 37 ° C. for 5 minutes. Subsequently, 12 μl of a labeling reagent (5 mM ARP or aoNg) previously dissolved in sterilized water was added, and the labeling reaction was started at 37 ° C., 42 ° C. or 47 ° C. After a certain period of time, 2.5 μl was sampled and added to 5 μl of loading solution mix (50 mM EDTA, 10 M urea, 0.1% BPB; prepared by mixing 4 μl, 200 mM glutaraldehyde; 1 μl) to stop the reaction. Then, it analyzed with the denaturation 20% polyacrylamide gel (19: 1). The fluorescence intensity of fluorescein in each band was measured and the reaction efficiency was calculated. The results of the reaction are shown in FIG.
Double-stranded reaction
When the reaction was performed in double strand, complementary strand Sp35C (14.4 pmol) was added to oxidized oligonucleotide (F-20Zox) (12 pmol), and other reaction conditions were the same as in the single strand reaction. The results of the reaction are shown in FIG.
The reagent reacted with a basic site Z in F-20Z. Also for DNA, aoNg showed higher reactivity. Especially for double strands, the reaction efficiency of ARP was the same as that of single strands, but with aoNg, the reaction efficiency was greatly improved.
(Example 10) Synthesis and purification of oligonucleotide containing 2'-deoxyuridine (dU)
Oligonucleotide (F-20dU) labeled with fluorescein at the 5 'end and dU in the chain and its complementary strand (Sp20dU) were prepared using 2'-deoxyuridine phosphoramidite reagent (Glen Res.). The DNA was synthesized by an automatic DNA synthesizer. F-20dU represents 5 'F-GAATTGCTdUGGAAGAGGTTT 3', and Sp20dU represents 3 'CTTAACGAdUCCTTCTCCAAA 5'. The synthetic oligonucleotide was deprotected according to a conventional method, and then separated and purified by reverse phase HPLC (Table 6). The conditions of HPLC etc. are shown below.
Solution A 5% acetonitrile / 0.1M TEAA (pH 7.0);
Solution B 25% acetonitrile / 0.1M TEAA (pH 7.0)
Column temperature: room temperature
As the column, Inertsil ODS-3 (C-18) column Φ3.0 × 300 mm (GL Science) was used.
HPLC conditions
Condition 4% of solution B 40 → 60% / 20 minutes
(Example 11) Labeling reaction of ARP, aoNg, and aoNg-bio to DNA containing 2'-deoxyuridine
An experiment was conducted to evaluate whether the compounds of the present invention react with abasic sites generated from 2'-deoxyuridine (dU) and uracil DNA glycosylase (UDG).
DNA containing dU, F-20dU (12 pmol) and its complementary strand (Sp35C; 24 pmol) were dissolved in a solution (total volume 29.5 μl) containing UDG buffer (x10, 3 μl). Sp35C represents 3 'CGAAAGTAACCCTTAACGAACCTCTCTCAAAAGAACGA 5'. The reaction solution was heated at 90 ° C. for 1 minute and ice-cooled, preincubated at 37 ° C. for 5 minutes, UDG (1 unit, 0.5 μl) was added thereto, and the reaction was performed at 37 ° C. for 60 minutes in a total amount of 30 μl. Thereafter, the enzyme was inactivated by heating at 90 ° C. for 1 minute, and allowed to stand for 10 minutes on ice and 5 minutes at room temperature. The labeling reaction was started at 37 ° C. by adding 6 μl of labeling reagent (ARP, aoNg, aoNg-bio) adjusted to 10 mM dissolved in advance in this reaction solution. After a certain period of time, 2.5 μl was sampled and added to 5 μl of loading solution mix (50 mM EDTA, 10 M urea, 0.1% BPB; prepared by mixing 4 μl, 200 mM glutaraldehyde; 1 μl) to stop the reaction. Then, it analyzed by the denaturation 20% polyacrylamide gel (19: 1) (FIG. 4). The fluorescence intensity of fluorescein in each band was measured and the reaction efficiency was calculated (FIG. 4).
The reaction rate constants of aoNg, ARP, and aoNg-bio are each 0.51 min.-1, 0.03min-10.17min-1Therefore, it was revealed that aoNg and aoNg-bio show higher reaction efficiency.
(Example 12) Reaction of aoNao to DNA
DNA containing dU, F-20dU (12 pmol) and its complementary strand (Sp20dU; 13.2 pmol) were dissolved in a solution (total volume 29.5 μl) containing UDG buffer (x10, 3 μl). Sp20dU represents 3 'CTTAACGAdUCCTTCTCCAAA 5'. The reaction solution was heated at 90 ° C. for 1 minute and ice-cooled, preincubated at 37 ° C. for 5 minutes, UDG (1 unit, 0.5 μl) was added thereto, and the reaction was performed at 37 ° C. for 60 minutes in a total amount of 30 μl. Thereafter, the enzyme was inactivated by heating at 90 ° C. for 1 minute, and allowed to stand for 10 minutes on ice and 5 minutes at room temperature. The labeling reaction was started at 37 ° C. by adding 6 μl of 2 mM aoNao dissolved in advance in this reaction solution. A similar reaction was performed with F-20dU, single strand only. After a certain time, 3 μl was sampled and added to 5 μl of loading solution mix (50 mM EDTA, 10 M urea, 0.1% BPB; prepared by mixing 4 μl, 200 mM glutaraldehyde; 1 μl) to stop the reaction. Then, it analyzed by the denaturation 20% polyacrylamide gel (19: 1) (FIG. 5). The fluorescence intensity of fluorescein in each band was measured and the reaction efficiency was calculated (FIG. 5). It was confirmed that aoNao can react so as to crosslink between the two strands.
Example 13 Synthesis of aoNdg-biotin reagent (hereinafter aoNdg-bio; Compound 16)
Scheme 4
1- (N 3 -Fluorenylmethyloxycarbonylthioureido) -N 5 -(Tritylaminooxyacetyl) -5-aminonaphthalene (Compound 13)
Under an argon atmosphere, 850 mg (3.01 mmol) of fluorenylmethyloxycarbonyl isothiocyanate and 0.52 ml (3.01 mmol) of diisopropylethylamine were dissolved in 30 ml of methylene chloride and cooled to 0 ° C. N here1-(Tritylaminooxyacetyl) -1,5-diaminonaphthalene (Compound 2) 1.19 g (2.51 mmol) in methylene chloride (30 ml) was slowly added dropwise, and the mixture was further returned to room temperature for 1.5 hours. Stir. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (elution solvent: ethyl acetate-hexane) to obtain 1.04 g (yield 55%) of the title compound (Compound 13) as a white solid substance.
FAB-LRMS m / z 755.3 (MH+), 777.2 ([M + Na]+FAB-HRMS calculated value: 777.2511 (C47H38N4O4SNa [M + Na]+), Measured value: 777.2486.
11 H NMR (270 MHz, DMSO-d6) Δ: 11.81 (s, 1H, NH), 11.60 (s, 1H, NH), 9.66 (s, 1H, NH),
8.37 (s, 1H, NH), 7.94-7.85 (m, 6H), 7.78-7.68 (m, 3H), 7.61-7.53 (m, 1H), 7.49-7.41 (m, 2H), 7.38-7.27 (m, 17H), 4.49 (d, 2H, Fmoc-CH2, J = 7.3 Hz), 4.37 (t, 1H, Fmoc-CH, J = 7.3 Hz), 4.23 (s, 2H, CH2).
13C NMR (67.8 MHz, DMSO-d6) Δ: 180.59 (C), 168.96 (C), 153.56 (C), 144.08 (C), 143.23 (C), 140.65 (C), 134.68 (C ), 133.14 (C), 129.79 (C), 128.79 (CH), 128.61 (C), 127.77 (CH), 127.64 (CH), 127.07 (CH) , 126.77 (CH), 126.10 (CH), 125.58 (CH), 125.43 (CH), 125.31 (CH), 122.34 (CH), 121.98 (CH), 121.28 (CH), 120.08 (CH), 119.93 (CH), 73.62 (C), 73.30 (CH2), 67.42 (CH2), 45.99 (CH).
1- (N 3 -{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} -N 2 -Fluorenylmethyloxycarbonylguanidino) -N 5 -(Tritylaminooxyacetyl) -5-aminonaphthalene (Compound 14)
1- (N under argon atmosphere3-Fluorenylmethyloxycarbonylthioureido) -N5910 mg (1.20 mmol) of-(tritylaminooxyacetyl) -5-aminonaphthalene (Compound 13) was dissolved in 20 ml of dimethylformamide and cooled to 0 ° C. To this solution, a dimethylformamide solution of 540 mg (1.44 mmol) of (+)-biotinyl-3,6-dioxaoctanediamine, 360 mg (1.32 mmol) of mercuric chloride and 0.50 ml (2.88 mmol) of diisopropylethylamine ( 15 ml) was slowly added dropwise, and the mixture was further stirred at 0 ° C. for 1.5 hours. After adding 150 ml of ethyl acetate to the reaction solution, the precipitate was removed by Celite filtration. Ethyl acetate was added to the filtrate to a total volume of 350 ml, washed 4 times with 120 ml of water and once with 120 ml of saturated brine, and dried over sodium sulfate. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (elution solvent: ethanol-chloroform) to obtain 963 mg (yield 73%) of the title compound (Compound 14) as a white foam.
FAB-LRMS m / z 1095.5 (MH+); FAB-HRMS calculated value: 1095.4803 (C63H67N8O8S [MH+]), Measured value: 10955.496.
11 H NMR (270 MHz, DMSO-d6) Δ: 9.61 (brs, 1H, NH), 8.33 (brs, 1H, NH), 7.89-7.75 (m, 6H), 7.61-7.47 (m, 3H), 7.42-7.25 (m, 20H), 6.39 (brs, 1H, NH), 6.33 (brs, 1H, NH), 4.30-4.24 (m, 6H), 4.10 (m, 1H), 3.49 (m, 6H), 3.38-3.33 (m, 4H), 3.16 (m, 2H), 3.06 (m, 1H) ), 2.79 (dd, 1H, J = 5.3, 12.2 Hz), 2.56 (d, 1H, J = 12.2 Hz), 2.04 (t, 2H, J = 7.3 Hz) 1.63-1.37 (m, 4H), 1.32-1.23 (m, 2H).
1- (N 3 -{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} -N 2 -Fluorenylmethyloxycarbonylguanidino) -N 5 -(Aminooxyacetyl) -5-aminonaphthalene (Compound 15)
1- (N under argon atmosphere3-{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} -N2-Fluorenylmethyloxycarbonylguanidino) -N5170 mg (0.16 mmol) of-(tritylaminooxyacetyl) -5-aminonaphthalene (compound 14) was dissolved in 15 ml of methylene chloride, cooled to 0 ° C., 180 μl (2.4 mmol) of trifluoroacetic acid and 330 μl of triisopropylsilane (1.6 mmol) was added. The reaction solution was returned to room temperature and stirred for 30 minutes, and then added with 65 ml of chloroform, washed once with 35 ml of saturated sodium bicarbonate solution, once with 35 ml of water, and once with 35 ml of saturated brine, and then washed with sodium sulfate. Dried. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (elution solvent: methanol-chloroform) to obtain 86 mg (yield 65%) of the title compound (Compound 15) as a white foam.
FAB-LRMS m / z 853.4 (MH+FAB-HRMS calculated value: 853.3707 (C44H53N8O8S [MH+]), Measured value: 853.3713.
11 H NMR (270 MHz, DMSO-d6) Δ: 9.90 (brs, 1H, NH), 8.02 (m, 1H), 7.89-7.71 (m, 5H), 7.78 (brs, 1H, NH), 7 .62-7.31 (m, 8H), 6.56 (br s, 2H, NH2), 6.39 (brs, 1H, NH), 6.33 (brs, 1H, NH), 4.29-4.26 (m, 6H), 4.10 (m, 1H), 3. 48 (m, 6H), 3.36 (m, 4H), 3.16 (m, 2H), 3.06 (m, 1H), 2.80 (dd, 1H, J = 5.0, 12. 5Hz), 2.56 (d, 1H, J = 12.5 Hz), 2.04 (t, 2H, J = 7.3 Hz), 1.6-1.38 (m, 4H), 1.28 ( m, 2H).
13C NMR (67.8 MHz, DMSO-d6) Δ: 172.07 (C), 169.68 (C), 162.64 (C), 144.15 (C), 140.63 (C), 127.45 (CH), 126.98 (CH ), 125.04 (CH), 120.04 (CH), 74.37 (CH2), 69.47 (CH2), 69.14 (CH2), 68.81 (CH2), 66.04 (CH2), 60.99 (CH), 59.15 (CH), 55.37 (CH), 46.67 (CH), 39.79 (CH2), 38.37 (CH2), 35.04 (CH2), 28.14 (CH2), 27.99 (CH2), 25.21 (CH2).
1- (N 3 -{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} guanidino) -N 5 -(Aminooxyacetyl) -5-aminonaphthalene (Compound 16, acetate)
1- (N under argon atmosphere3-{2- [2- (2-biotinylaminoethoxy) ethoxy] ethyl} -N2-Fluorenylmethyloxycarbonylguanidino) -N5-(Aminooxyacetyl) -5-aminonaphthalene (Compound 15) 85 mg (0.10 mmol) was dissolved in a mixed solvent of 1,4-dioxane 4 ml and methanol 4 ml, and piperidine 0.40 ml (4.0 mmol) was added. Stir at room temperature for 16 hours. After the reaction solution was concentrated under reduced pressure, the residue was dissolved in 35 ml of water and washed 3 times with 15 ml of ethyl acetate. The aqueous layer was concentrated under reduced pressure and then purified by C18 cartridge column chromatography (elution solvent: acetonitrile-0.1 M TEAA solution) to obtain a crude product (52 mg). A portion of this crude product was dissolved in 5 ml of 2% acetic acid aqueous solution and purified again by C18 cartridge column chromatography (elution solvent: acetonitrile-water) to give 9.2 mg of the title compound (Compound 16, acetate) as white. Obtained as a flocculent material.
FAB-LRMS m / z 631.3 (MH+FAB-HRMS calculated value: 631.3026 (C29H43N8O6S [MH+]), Measured value: 631.3005.
11 H NMR (270 MHz, DMSO-d6) Δ: 9.94 (s, 1H, NH), 9.64 (brs, 1H, NH), 8.07 (m, 1H, nap), 7.82 (m, 1H), 7.81 ( d, 1H, J = 8.2 Hz, nap), 7.73 (d, 1H, J = 6.9 Hz, nap), 7.62 (m, 2H, nap), 7.55 (m, 2H), 7.51 (m, 1H, nap), 6.55 (s, 2H, NH2O), 6.39 (s, 1H, NH), 6.35 (s, 1H, NH), 4.31 (m, 1H), 4.29 (s, 2H, CH2), 4.12 (br dd, 1H, J = 4.3, 7.6 Hz), 3.58 (m, 6H), 3.42 (m, 4H), 3.21 (m, 2H), 3 .09 (m, 1H), 2.81 (dd, 1H, J = 5.0, 12.5 Hz), 2.57 (d, 1H, J = 12.5 Hz), 2.07 (t, 2H, J = 7.3 Hz), 1.64-1.42 (m, 4H), 1.30 (m, 2H).
13C NMR (67.8 MHz, DMSO-d6) Δ: 172.10 (C), 169.68 (C), 162.59 (C), 155.75 (C), 133.72 (C), 130.44 (C), 129.39 (C ), 126.32 (CH), 125.81 (CH), 123.00 (CH), 119.87 (CH), 74.27 (CH2), 69.66 (CH2), 69.38 (CH2), 69.06 (CH2), 68.52 (CH2), 60.92 (CH), 59.08 (C), 55.31 (CH), 41.22 (CH2), 39.73 (CH2), 38.27 (CH2), 35.00 (CH2), 28.08 (CH2), 27.94 (CH2), 25.16 (CH2).
(Example 14)
Using the same oligonucleotide as in Example 11, the reaction efficiency of aoNdg-bio was examined under the same conditions. As a result of the reaction, the reaction rate constant of aoNdg-bio was 0.23 min.-1From the above, it was revealed that aoNdg-bio showed higher reaction efficiency than ARP, like aoNg and aoNg-bio.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
[Sequence Listing]
Claims (11)
Ｒ１−ＮＨ−Ｏ−Ｌ１−Ｄ−Ｌ２−Ａ （１）
（式中、
Ｒ１は、水素原子、アルキル基又はアミノ基の保護基であり、
Ｄは、置換若しくは無置換のフェニレン基、置換若しくは無置換のアントリレン基、置換若しくは無置換のナフチレン基、置換若しくは無置換のフェナントリレン基、置換若しくは無置換のアントラキノリレン基、及び置換若しくは無置換のアクリジニレン基から選択される芳香族基であり、
芳香族基の置換基は、ハロゲン原子、Ｃ １−６アルキル基、ニトロ基、シアノ基、Ｃ２−６アルケニル基、Ｃ３−１０シクロアルキル基、Ｃ１−１０アルコキシ基及びＣ１−１０アシル基からなる群から選択され、
Ｌ１は、直接結合又は以下の一般式３又は４：
のいずれかで表される２価の基であり、Ｌ２は、直接結合又は以下の一般式５又は６：
のいずれかで表される２価の基であり、
Ａは、置換又は無置換のグアニジノ基、アミノオキシ基又は保護されたアミノオキシ基であり、
グアニジノ基の置換基は、−（ＣＨ ２ ） ｖ −（ＯＣＨ ２ ＣＨ ２ ） ｗ −（ＣＨ ２ ） ｘ −ＮＨ−ビオチンであり、ｖ、ｗ及びｘは、それぞれ独立して０〜１５の整数であり、ｖ＋ｗ＋ｘは、１〜１５である）
で表される化合物又はその塩。General formula 1:
R 1 -NH-O-L 1 -D-L 2 -A (1)
(Where
R 1 is a protecting group for a hydrogen atom, an alkyl group or an amino group,
D is a substituted or unsubstituted phenylene group, a substituted or unsubstituted anthrylene group, a substituted or unsubstituted naphthylene group, a substituted or unsubstituted phenanthrylene group, a substituted or unsubstituted anthraquinolylene group, and a substituted or unsubstituted group An aromatic group selected from acridinylene groups of
Substituents of aromatic groups, halogen atoms, C 1-6 alkyl group, nitro group, cyano group, C 2-6 alkenyl group, C 3-10 cycloalkyl, C 1-10 alkoxy group and C 1- is selected from 10 acyl group or Ranaru group,
L 1 is a direct bond or the following general formula 3 or 4:
A divalent group represented by any one of, L 2 is 5 or a direct bond or the following general formula 6:
A divalent group represented by any of the following :
A is a substituted or unsubstituted guanidino group, an aminooxy group or a protected aminooxy group,
The substituent of the guanidino group is — (CH 2 ) v — (OCH 2 CH 2 ) w — (CH 2 ) x —NH-biotin, and v, w and x are each independently an integer of 0 to 15 And v + w + x is 1 to 15 )
Or a salt thereof.
で表される、請求項１又は２記載の化合物又はその塩。A is the following general formula 2:
The compound or its salt of Claim 1 or 2 represented by these.
で表される２価の芳香族基、及びこれらの芳香族基において芳香環が１〜３個の置換基を有する芳香族基から選択される、請求項１〜３のいずれか１項記載の化合物又はその塩。D is the following general formula:
The bivalent aromatic group represented by these, and the aromatic ring in these aromatic groups are selected from the aromatic group which has 1-3 substituents of any one of Claims 1-3 Compound or salt thereof.
で表される、請求項１記載の化合物又はその塩。The following general formula 7:
The compound of Claim 1 represented by these, or its salt.
で表される、請求項１記載の化合物又はその塩。The following general formula 8:
The compound of Claim 1 represented by these, or its salt.
で表される、請求項１記載の化合物又はその塩。The following general formula:
The compound of Claim 1 represented by these, or its salt.
Ｒ １ −ＮＨ−Ｏ−Ｌ １ −Ｄ−Ｌ ２ −Ａ （１）
（式中、
Ｒ １ は、水素原子、アルキル基又はアミノ基の保護基であり、
Ｄは、置換若しくは無置換のフェニレン基、置換若しくは無置換のアントリレン基、置換若しくは無置換のナフチレン基、置換若しくは無置換のフェナントリレン基、置換若しくは無置換のアントラキノリレン基、及び置換若しくは無置換のアクリジニレン基から選択される芳香族基であり、
芳香族基の置換基は、ハロゲン原子、Ｃ １−６ アルキル基、ニトロ基、シアノ基、Ｃ ２−６ アルケニル基、Ｃ ３−１０ シクロアルキル基、Ｃ １−１０ アルコキシ基及びＣ １−１０ アシル基からなる群から選択され、
Ｌ １ は、直接結合または以下の一般式３又は４：
のいずれかで表される２価の基であり、Ｌ ２ は、直接結合または以下の一般式５又は６：
のいずれかで表される２価の基であり：
Ａは、標識化されたグアニジノ基である）
で表される化合物又はその塩を含む前記試薬。A reagent for labeling a biomolecule having an aldehyde group, a hemiacetal group, a carboxyl group or a keto group, which has the general formula (1):
R 1 -NH-O-L 1 -D-L 2 -A (1)
(Where
R 1 is a protecting group for a hydrogen atom, an alkyl group or an amino group,
D is a substituted or unsubstituted phenylene group, substituted or unsubstituted anthrylene group, substituted or unsubstituted naphthylene group, substituted or unsubstituted phenanthrylene group, substituted or unsubstituted anthraquinolylene group, and substituted or unsubstituted An aromatic group selected from acridinylene groups of
The substituent of the aromatic group includes a halogen atom, a C 1-6 alkyl group, a nitro group, a cyano group, a C 2-6 alkenyl group, a C 3-10 cycloalkyl group, a C 1-10 alkoxy group, and a C 1-10. Selected from the group consisting of acyl groups;
L 1 is a direct bond or the following general formula 3 or 4:
Wherein L 2 is a direct bond or the following general formula 5 or 6:
Is a divalent group represented by:
A is a labeled guanidino group)
The said reagent containing the compound or its salt represented by these .
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| US20210032282A1 (en) * | 2014-12-12 | 2021-02-04 | National Institute Of Advanced Industrial Science And Technology | Nucleic acid complex, method for forming nucleic acid hybridization, pharmaceutical composition, nucleic acid probe, and complementary-strand nucleic acid complex |
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