JP5539635B2 - Fluorescent dye - Google Patents
Fluorescent dye Download PDFInfo
- Publication number
- JP5539635B2 JP5539635B2 JP2008205238A JP2008205238A JP5539635B2 JP 5539635 B2 JP5539635 B2 JP 5539635B2 JP 2008205238 A JP2008205238 A JP 2008205238A JP 2008205238 A JP2008205238 A JP 2008205238A JP 5539635 B2 JP5539635 B2 JP 5539635B2
- Authority
- JP
- Japan
- Prior art keywords
- group
- groups
- fluorescent dye
- alkyl
- sulfonyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000007850 fluorescent dye Substances 0.000 title claims description 69
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 48
- -1 thiazolium group Chemical group 0.000 claims description 45
- 125000000217 alkyl group Chemical group 0.000 claims description 26
- 150000002148 esters Chemical class 0.000 claims description 20
- 125000003277 amino group Chemical group 0.000 claims description 17
- 125000000623 heterocyclic group Chemical group 0.000 claims description 16
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 claims description 16
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 15
- 125000002029 aromatic hydrocarbon group Chemical group 0.000 claims description 14
- 125000001424 substituent group Chemical group 0.000 claims description 14
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical group C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 12
- 125000003118 aryl group Chemical group 0.000 claims description 12
- 125000002883 imidazolyl group Chemical group 0.000 claims description 12
- 125000001302 tertiary amino group Chemical group 0.000 claims description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 11
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 claims description 11
- 125000001931 aliphatic group Chemical group 0.000 claims description 10
- 125000003545 alkoxy group Chemical group 0.000 claims description 10
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 10
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 10
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 10
- 125000002971 oxazolyl group Chemical group 0.000 claims description 10
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 claims description 10
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical group C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 claims description 9
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical group C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 claims description 9
- 125000005647 linker group Chemical group 0.000 claims description 9
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical group C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 8
- 125000003172 aldehyde group Chemical group 0.000 claims description 8
- 125000005907 alkyl ester group Chemical group 0.000 claims description 8
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical group C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 claims description 8
- VPKDCDLSJZCGKE-UHFFFAOYSA-N carbodiimide group Chemical group N=C=N VPKDCDLSJZCGKE-UHFFFAOYSA-N 0.000 claims description 8
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 8
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical group C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 claims description 7
- RAXXELZNTBOGNW-UHFFFAOYSA-N 1H-imidazole Chemical group C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 7
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 claims description 7
- 150000007980 azole derivatives Chemical class 0.000 claims description 6
- 238000004040 coloring Methods 0.000 claims description 6
- 125000003700 epoxy group Chemical group 0.000 claims description 6
- IQPQWNKOIGAROB-UHFFFAOYSA-N isocyanate group Chemical group [N-]=C=O IQPQWNKOIGAROB-UHFFFAOYSA-N 0.000 claims description 6
- 125000003226 pyrazolyl group Chemical group 0.000 claims description 6
- 125000000168 pyrrolyl group Chemical group 0.000 claims description 6
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical group O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 claims description 5
- BCMCBBGGLRIHSE-UHFFFAOYSA-N 1,3-benzoxazole Chemical group C1=CC=C2OC=NC2=C1 BCMCBBGGLRIHSE-UHFFFAOYSA-N 0.000 claims description 4
- 125000000355 1,3-benzoxazolyl group Chemical group O1C(=NC2=C1C=CC=C2)* 0.000 claims description 4
- HYZJCKYKOHLVJF-UHFFFAOYSA-N 1H-benzimidazole Chemical group C1=CC=C2NC=NC2=C1 HYZJCKYKOHLVJF-UHFFFAOYSA-N 0.000 claims description 4
- ZCQWOFVYLHDMMC-UHFFFAOYSA-N Oxazole Chemical group C1=COC=N1 ZCQWOFVYLHDMMC-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical group C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 4
- 150000001263 acyl chlorides Chemical group 0.000 claims description 4
- 125000003785 benzimidazolyl group Chemical group N1=C(NC2=C1C=CC=C2)* 0.000 claims description 4
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical group O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 claims description 4
- 125000005843 halogen group Chemical group 0.000 claims description 4
- 125000002560 nitrile group Chemical group 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 125000004193 piperazinyl group Chemical group 0.000 claims description 4
- 125000003386 piperidinyl group Chemical group 0.000 claims description 4
- 125000001453 quaternary ammonium group Chemical group 0.000 claims description 4
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 claims description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 3
- 229910052717 sulfur Inorganic materials 0.000 claims description 3
- 125000004434 sulfur atom Chemical group 0.000 claims description 3
- 230000032050 esterification Effects 0.000 claims 1
- 238000005886 esterification reaction Methods 0.000 claims 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 38
- 108090000623 proteins and genes Proteins 0.000 description 28
- 102000004169 proteins and genes Human genes 0.000 description 28
- 239000000975 dye Substances 0.000 description 27
- 230000015572 biosynthetic process Effects 0.000 description 20
- 238000000034 method Methods 0.000 description 20
- 238000003786 synthesis reaction Methods 0.000 description 20
- 238000002372 labelling Methods 0.000 description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 16
- 150000004714 phosphonium salts Chemical class 0.000 description 16
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- NOUIINDKKXZYDN-UHFFFAOYSA-N 2-oxido-4,5-dihydrooxadiazol-2-ium Chemical compound [O-][N+]1=NCCO1 NOUIINDKKXZYDN-UHFFFAOYSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000001514 detection method Methods 0.000 description 11
- 239000002244 precipitate Substances 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 229910021645 metal ion Inorganic materials 0.000 description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 238000010992 reflux Methods 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 8
- 229920002554 vinyl polymer Polymers 0.000 description 8
- NTPLXRHDUXRPNE-UHFFFAOYSA-N 4-methoxyacetophenone Chemical compound COC1=CC=C(C(C)=O)C=C1 NTPLXRHDUXRPNE-UHFFFAOYSA-N 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 125000004432 carbon atom Chemical group C* 0.000 description 7
- 239000011541 reaction mixture Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 6
- 108010035532 Collagen Proteins 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 5
- 125000002091 cationic group Chemical group 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 210000004209 hair Anatomy 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 230000019491 signal transduction Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 125000000129 anionic group Chemical group 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 125000005594 diketone group Chemical group 0.000 description 4
- 125000004185 ester group Chemical group 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 4
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 4
- 229920006395 saturated elastomer Polymers 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- 108091023037 Aptamer Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 102000007982 Phosphoproteins Human genes 0.000 description 3
- 108010089430 Phosphoproteins Proteins 0.000 description 3
- 238000007239 Wittig reaction Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000000460 chlorine Substances 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 229910001414 potassium ion Inorganic materials 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- TXTWXQXDMWILOF-UHFFFAOYSA-N (2-ethoxy-2-oxoethyl)azanium;chloride Chemical compound [Cl-].CCOC(=O)C[NH3+] TXTWXQXDMWILOF-UHFFFAOYSA-N 0.000 description 2
- HZTPKMIMXLTOSK-UHFFFAOYSA-N 2-bromohexanoic acid Chemical compound CCCCC(Br)C(O)=O HZTPKMIMXLTOSK-UHFFFAOYSA-N 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000003923 Protein Kinase C Human genes 0.000 description 2
- 108090000315 Protein Kinase C Proteins 0.000 description 2
- WTKZEGDFNFYCGP-UHFFFAOYSA-N Pyrazole Chemical class C=1C=NNC=1 WTKZEGDFNFYCGP-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 150000001350 alkyl halides Chemical class 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- DIKBFYAXUHHXCS-UHFFFAOYSA-N bromoform Chemical compound BrC(Br)Br DIKBFYAXUHHXCS-UHFFFAOYSA-N 0.000 description 2
- 125000004106 butoxy group Chemical group [*]OC([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 125000004218 chloromethyl group Chemical group [H]C([H])(Cl)* 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 150000003977 halocarboxylic acids Chemical class 0.000 description 2
- 230000002140 halogenating effect Effects 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 125000003253 isopropoxy group Chemical group [H]C([H])([H])C([H])(O*)C([H])([H])[H] 0.000 description 2
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 125000002950 monocyclic group Chemical group 0.000 description 2
- 125000001624 naphthyl group Chemical group 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000004115 pentoxy group Chemical group [*]OC([H])([H])C([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 2
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 2
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 2
- 125000003367 polycyclic group Chemical group 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 125000002572 propoxy group Chemical group [*]OC([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 2
- 230000008054 signal transmission Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000010288 sodium nitrite Nutrition 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical group S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 125000003944 tolyl group Chemical group 0.000 description 2
- 125000005023 xylyl group Chemical group 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- 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 1
- CSDSSGBPEUDDEE-UHFFFAOYSA-N 2-formylpyridine Chemical class O=CC1=CC=CC=N1 CSDSSGBPEUDDEE-UHFFFAOYSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical group [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 description 1
- 235000012765 Cannabis sativa ssp. sativa var. spontanea Nutrition 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 229920003043 Cellulose fiber Polymers 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000010831 Cytoskeletal Proteins Human genes 0.000 description 1
- 108010037414 Cytoskeletal Proteins Proteins 0.000 description 1
- 102100030011 Endoribonuclease Human genes 0.000 description 1
- 101710199605 Endoribonuclease Proteins 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical group [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- XNPOFXIBHOVFFH-UHFFFAOYSA-N N-cyclohexyl-N'-(2-(4-morpholinyl)ethyl)carbodiimide Chemical compound C1CCCCC1N=C=NCCN1CCOCC1 XNPOFXIBHOVFFH-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical group [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101710113029 Serine/threonine-protein kinase Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N anhydrous guanidine Natural products NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- 101150036080 at gene Proteins 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- SIPUZPBQZHNSDW-UHFFFAOYSA-N bis(2-methylpropyl)aluminum Chemical compound CC(C)C[Al]CC(C)C SIPUZPBQZHNSDW-UHFFFAOYSA-N 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229910052796 boron Chemical group 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 229950005228 bromoform Drugs 0.000 description 1
- 235000009120 camo Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 235000005607 chanvre indien Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 229940126543 compound 14 Drugs 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011840 criminal investigation Methods 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 150000001982 diacylglycerols Chemical class 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 230000003724 hair brightness Effects 0.000 description 1
- 125000001188 haloalkyl group Chemical group 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000011487 hemp Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- RWXCVESEMJNNMF-UHFFFAOYSA-N oxadiazolo[5,4-b]pyridine Chemical class C1=CN=C2ON=NC2=C1 RWXCVESEMJNNMF-UHFFFAOYSA-N 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- UHZYTMXLRWXGPK-UHFFFAOYSA-N phosphorus pentachloride Chemical compound ClP(Cl)(Cl)(Cl)Cl UHZYTMXLRWXGPK-UHFFFAOYSA-N 0.000 description 1
- FAIAAWCVCHQXDN-UHFFFAOYSA-N phosphorus trichloride Chemical compound ClP(Cl)Cl FAIAAWCVCHQXDN-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- QJZUKDFHGGYHMC-UHFFFAOYSA-N pyridine-3-carbaldehyde Chemical compound O=CC1=CC=CN=C1 QJZUKDFHGGYHMC-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 239000004627 regenerated cellulose Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000006798 ring closing metathesis reaction Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
- 239000012209 synthetic fiber Substances 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 229940042055 systemic antimycotics triazole derivative Drugs 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- HFRXJVQOXRXOPP-UHFFFAOYSA-N thionyl bromide Chemical compound BrS(Br)=O HFRXJVQOXRXOPP-UHFFFAOYSA-N 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
- Cosmetics (AREA)
Description
本発明は、核酸、タンパク質、ペプチド類、そして糖類等の生体分子の検出に用いる蛍光色素であって、固体状態で発光し、水溶性を有する蛍光色素に関する。 The present invention relates to a fluorescent dye used for detection of biomolecules such as nucleic acids, proteins, peptides, and saccharides, which emits light in a solid state and has water solubility.
近年、ヒトゲノムの全容が明らかにされ、遺伝子治療、遺伝子診断などを目的としたポストゲノム研究が盛んに行われている。例えば、DNA解析は、DNAマイクロアレイ基盤上に固定されたプローブDNAと、蛍光色素等で標識されたサンプルDNAとをハイブリダイズさせて二本鎖を形成させ、サンプルDNAの検出を行っている。これは蛍光色素で標識した核酸をPCR伸長し、基盤上でハイブリダイゼーションを行った後に測定する手法である。最近では、より多くのアミノ基を有するプライマーを用いた手法、DNAにアミノ基を導入する手法がとられている。 In recent years, the entire human genome has been clarified, and post-genomic research aimed at gene therapy, genetic diagnosis, etc. has been actively conducted. For example, in DNA analysis, probe DNA immobilized on a DNA microarray substrate is hybridized with sample DNA labeled with a fluorescent dye or the like to form a double strand, and sample DNA is detected. This is a method in which a nucleic acid labeled with a fluorescent dye is subjected to PCR extension and hybridization is performed on a substrate, followed by measurement. Recently, a technique using a primer having a larger number of amino groups and a technique for introducing amino groups into DNA have been employed.
標識には、蛍光色素が広く使用されており、高い蛍光強度を有すること、乾燥状態(固体状態)でも発光すること、そして水溶性を有することなどが要求されている。蛍光色素としては、例えば、Cy3やCy5が使用されている(例えば、非特許文献1)。
しかしながら、乾燥状態でも発光する蛍光色素は油溶性であり、水溶性が低いという問題がある。そのため、試料溶液に蛍光色素が十分溶解することができず、標識率が上がらない結果、十分な蛍光強度が得られないという問題があった。 However, fluorescent dyes that emit light even in a dry state are oil-soluble and have a problem of low water solubility. As a result, the fluorescent dye cannot be sufficiently dissolved in the sample solution, and as a result, the labeling rate does not increase. As a result, there is a problem that sufficient fluorescence intensity cannot be obtained.
上記の課題を解決するため、本発明者は鋭意努力した結果、アゾール誘導体から成る有機EL色素に二重結合を含むリンカーを介して窒素カチオン含有基又は窒素含有基を導入することにより、蛍光色素の水溶性を向上させ、標識時の蛍光強度を大幅に向上させることが可能なことを見出して本発明を完成させたものである。
すなわち、本発明に係る一の蛍光色素は、以下の一般式(1)で表されるアゾール誘導体から成ることを特徴とする。
To solve the above problems, the present inventors have been made extensive efforts, by introducing nitrogen cation-containing group or a nitrogen-containing group in the organic EL dye comprising azole induced body or found through a linker containing a double bond, The present invention has been completed by finding that the water-solubility of a fluorescent dye can be improved and the fluorescence intensity at the time of labeling can be greatly improved.
That is, one fluorescent dye according to the present invention is characterized by comprising an azole derivative represented by the following general formula (1) .
ここで、R1は、一般式L-Mで示され、Mは、置換基を有しても良いピリジニウム基、2級アミノ基、3級アミノ基、4級アミノ基、ピペリジニウム基、ピペラジニウム基、イミダゾリウム基、チアゾリウム基、オキサゾリウム基、キノリウム基、ベンゾイミダゾリウム基、ベンゾチアゾリウム基又はベンゾオキサゾリウム基である窒素カチオン含有基、あるいは置換基を有しても良いピリジン基、2級アミノ基、3級アミノ基、ピペリジン基、ピペラジン基、イミダゾール基、チアゾール基、オキサゾール基、キノリン基、ベンゾイミダゾール基、ベンゾチアゾール基又はベンゾオキサゾール基である窒素含有基を示し、Lは、-(CH=CR6)n-で表され、ここでnは1から5の整数からなり、R6は水素原子あるいは置換基として、メチル基、エチル基等のアルキル基、アルキル基を有するスルホ基、イミダゾリウム基、ピリジニウム基、ピペリジニウム基、フラン基等の複素環基、2級アミノ基、3級アミノ基そして4級アミノ基等のアミノ基、ヒドロキシ基、アルコキシ基、アルデヒド基、カルボキシル基又は芳香族基を示し、Mと発色部とを連結するリンカー、R2及びR3は、それぞれ独立に、水素原子、ハロゲン原子、置換基としてアルキル基、アルコキシ基、アルキルエステル基、リン酸エステル基、硫酸エステル基、ニトリル基、ヒドロキシル基、シアノ基、スルホニル基、芳香族炭化水素基又は複素環基を有してもよい芳香族炭化水素基又は脂肪族炭化水素基又は複素環基を示し、Xは硫黄原子または酸素原子を示す。
Here, R 1 is represented by the general formula LM, and M is an optionally substituted pyridinium group, secondary amino group, tertiary amino group, quaternary amino group, piperidinium group, piperazinium group, imidazo Nitrogen cation-containing group which is a lithium group, a thiazolium group, an oxazolium group, a quinolium group, a benzoimidazolium group, a benzothiazolium group or a benzoxazolium group, or a pyridine group which may have a substituent, a secondary amino group Represents a nitrogen-containing group which is a group, tertiary amino group, piperidine group, piperazine group, imidazole group, thiazole group, oxazole group, quinoline group, benzimidazole group, benzothiazole group or benzoxazole group, and L is-(CH = CR 6) is represented by n-, wherein n is comprised integer from 1 to 5, as R 6 is a hydrogen atom or a substituent, a methyl group, an ethyl group, Alkyl groups, sulfo groups having alkyl groups, imidazolium groups, pyridinium groups, piperidinium groups, furan groups and other heterocyclic groups, secondary amino groups, tertiary amino groups and amino groups such as quaternary amino groups, hydroxy groups, A linker that represents an alkoxy group, an aldehyde group, a carboxyl group or an aromatic group, and connects M and the coloring part, R 2 and R 3 are each independently a hydrogen atom, a halogen atom, a substituent, an alkyl group, an alkoxy group , An alkyl ester group, a phosphate ester group, a sulfate ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or an aliphatic hydrocarbon group which may have an aromatic hydrocarbon group or a heterocyclic group Represents a hydrogen group or a heterocyclic group, and X represents a sulfur atom or an oxygen atom .
また、上記のR 2 及びR 3 における前記複素環基には、それぞれ独立に、チオフェン基、フラン基、ピロール基、イミダゾール基、オキサゾール基、チジアゾール基、ピラゾール基、ピリジン基及びキノリン基からなる群から選択された1種を用いることができる。
In addition, the said heterocyclic group in the above R 2 and R 3, the group independently, consisting of thiophene, furan group, a pyrrole group, an imidazole group, an oxazole group, Chijiazoru group, pyrazole groups, pyridine groups and
また、上記のR2とR3には、スルホニル基を有するアリール基を用いることができる。 In addition, an aryl group having a sulfonyl group can be used for the above R 2 and R 3 .
また、上記窒素カチオン含有基あるいは上記窒素含有基に、イソチオシアネート基、イソシアネート基、無水マレイン酸基、エポキシ基、ハロゲン化スルホニル基、塩化アシル基、ハロゲン化アルキル基、グリオキザル基、アルデヒド基、トリアジン基、カルボジイミド基、活性エステル化したカルボニル基、スルホニル基、カルボキシル基、4級アンモニウム基およびピリジニウム基から選択される1種の、生体分子と結合する反応性基を結合させることもできる。
In addition, the nitrogen cation-containing group or the nitrogen-containing group includes an isothiocyanate group, an isocyanate group, a maleic anhydride group, an epoxy group, a halogenated sulfonyl group, an acyl chloride group, a halogenated alkyl group, a glyoxal group, an aldehyde group, or a triazine. One kind of reactive group that binds to a biomolecule may be bound , selected from a group, a carbodiimide group, an active esterified carbonyl group, a sulfonyl group, a carboxyl group, a quaternary ammonium group, and a pyridinium group .
本発明の蛍光色素は、共役系を有し、1種以上のヘテロ原子を含むアゾール誘導体から成る有機EL色素であり、二重結合を含むリンカーを介して結合した窒素カチオン含有基又は窒素含有基を有する。その高い水溶性により、生体分子に対する標識率を向上させることができ、高感度の生体分子の検出が可能となる。これにより使用する標識色素の量を大幅に低減できることから、標的分子の検出費用を大幅にコストダウンすることも可能となる。また、有機EL色素は固体状態(固体及び半固体を含む)で高い量子収率を有しているので、マイクロアレイなどの基盤上、もしくはビーズ上の乾燥状態でも高い蛍光強度を与える。また、有機EL色素はCy3やCy5に比べ安価であるので、より低コストで生体分子の検出を行うことができる。また、有機EL色素の置換基を変えることにより励起波長及び発光波長を変化させることができるので、蛍光波長の選択の自由度が増加し、レッド、オレンジ、イエロー、グリーン、ブルーなど多くの蛍光波長を用いることができる。これにより、ストークスシフトの大きい(励起波長と蛍光波長の差が大きい)2種以上の蛍光色素を用いることが可能となり、一つの試料中に含まれる複数の標的核酸を同時に検出することも可能となる。また、Cy3やCy5は冷凍保存する必要があるのに対し、有機EL色素は化学的に安定であり、常温での長期保存に耐えることができるので、取り扱いが容易である。
Fluorescent dye of the present invention has a conjugated system, an azole-induced body or we made organic EL dye containing one or more hetero atom, or a nitrogen cation-containing group bonded through a linker containing a double bond Has a nitrogen-containing group. Due to its high water solubility, the labeling rate for biomolecules can be improved, and highly sensitive biomolecules can be detected. As a result, the amount of the labeling dye to be used can be greatly reduced, so that the cost for detecting the target molecule can be greatly reduced. In addition, since the organic EL dye has a high quantum yield in a solid state (including solid and semi-solid), it gives a high fluorescence intensity even in a dry state on a substrate such as a microarray or on a bead. In addition, since organic EL dyes are cheaper than Cy3 and Cy5, biomolecules can be detected at a lower cost. In addition, since the excitation wavelength and emission wavelength can be changed by changing the substituent of the organic EL dye, the degree of freedom in selecting the fluorescence wavelength increases, and many fluorescence wavelengths such as red, orange, yellow, green, and blue can be obtained. Can be used. This makes it possible to use two or more fluorescent dyes having a large Stokes shift (the difference between the excitation wavelength and the fluorescence wavelength is large) and to simultaneously detect a plurality of target nucleic acids contained in one sample. Become. In addition, while Cy3 and Cy5 need to be stored frozen, organic EL dyes are chemically stable and can withstand long-term storage at room temperature, making them easy to handle.
以下、本発明の実施の形態について詳細に説明する。
本発明の蛍光色素の発色部は、共役系を有し、1種以上のヘテロ原子を含むアゾール誘導体から成る有機EL色素である。
Hereinafter, embodiments of the present invention will be described in detail.
Coloring part of the fluorescent dyes of the present invention has a conjugated system, an azole-induced body or we made organic EL dye containing one or more hetero atom.
アゾール誘導体から成る蛍光色素は、以下の一般式(1)、(2)、(3)で示すことができる。 A fluorescent dye composed of an azole derivative can be represented by the following general formulas (1), (2), and (3).
ここで、(1)及び(3)ではR1は、そして(2)ではR1とR4の一方は、一般式L-Mで示され、Mは、ピリジニウム基、2級アミノ基、3級アミノ基、4級アミノ基、ピペリジニウム基、ピペラジニウム基、イミダゾリウム基、チアゾリウム基、オキサゾリウム基、キノリウム基、ベンゾイミダゾリウム基、ベンゾチアゾリウム基又はベンゾオキサゾリウム基である窒素カチオン含有基、あるいは置換基を有しても良いピリジン基、2級アミノ基、3級アミノ基、ピペリジン基、ピペラジン基、イミダゾール基、チアゾール基、オキサゾール基、キノリン基、ベンゾイミダゾール基、ベンゾチアゾール基又はベンゾオキサゾール基である窒素含有基を示し、Lは、-(CH=CR6)n-で表され、ここでnは1から5の整数からなり、R6は水素原子あるいは置換基として、メチル基、エチル基等のアルキル基、アルキル基を有するスルホ基、イミダゾリウム基、ピリジニウム基、ピペリジニウム基、フラン基等の複素環基、2級アミノ基、3級アミノ基そして4級アミノ基等のアミノ基、ヒドロキシ基、アルコキシ基、アルデヒド基、カルボキシル基又は芳香族基を示し、Mと発色部とを連結するリンカーを示す。リンカーを用いることが好ましい。リンカーは、発色部と標識対象である生体分子との間の立体障害を緩和させ、結合部と生体分子の標識部位との結合を容易にするので、より高い標識率を与えることが可能だからである。 Here, in (1) and (3), R 1 and in (2) one of R 1 and R 4 is represented by the general formula LM, where M is a pyridinium group, a secondary amino group, a tertiary amino group. Group, quaternary amino group, piperidinium group, piperazinium group, imidazolium group, thiazolium group, oxazolium group, quinolium group, benzoimidazolium group, benzothiazolium group or benzoxazolium group, or a nitrogen cation-containing group A pyridine group, secondary amino group, tertiary amino group, piperidine group, piperazine group, imidazole group, thiazole group, oxazole group, quinoline group, benzimidazole group, benzothiazole group or benzoxazole group which may have a substituent represents nitrogen-containing group is, L is, - (CH = CR 6) is represented by n-, wherein n is comprised integer from 1 to 5, as R 6 is a hydrogen atom or a substituent, Alkyl groups such as til groups and ethyl groups, sulfo groups having alkyl groups, imidazolium groups, pyridinium groups, piperidinium groups, furan groups and other heterocyclic groups, secondary amino groups, tertiary amino groups and quaternary amino groups, etc. The amino group, the hydroxy group, the alkoxy group, the aldehyde group, the carboxyl group, or the aromatic group, and a linker that connects M and the coloring portion. It is preferable to use a linker. The linker relaxes the steric hindrance between the coloring part and the biomolecule to be labeled, and facilitates the binding between the binding part and the labeling part of the biomolecule, so it can give a higher labeling rate. is there.
また、(2)のR1とR4の残部及びR2、R3は、それぞれ独立に、水素原子、ハロゲン原子、置換基としてアルキル基、アルコキシ基、アルキルエステル基、リン酸エステル基、硫酸エステル基、ニトリル基、ヒドロキシル基、シアノ基、スルホニル基、芳香族炭化水素基又は複素環基を有してもよい芳香族炭化水素基又は脂肪族炭化水素基又は複素環基を示す。上記のアルキル基は、例えば炭素数1から6の直鎖状又は分岐状のアルキル基である。また、上記のアルコキシ基は、例えばメトキシ基、エトキシ基、プロポキシ基、イソプロポキシ基、ブトキシ基、ペンチロキシ基又はフェノキシ基である。また、上記のアルキルエステル基は、炭素数1から6の直鎖状又は分岐状のアルキルエステルである。また、上記の芳香族炭化水素基は単環又は多環を含むアリール基、具体的にはフェニル基、トリル基、キシリル基又はナフチル基であり、より好ましくはフェニル基である。また、上記の複素環基は、例えばピロール基、フラン基、チオフェン基、イミダゾール基、オキサゾール基、チアゾール基、ピラゾール基、ピリジン基又はキノリン基であり、より好ましくはフラン基、イミダゾール基又はチオフェン基である。また、上記の脂肪族炭化水素基は、好ましくは炭素数1から6の直鎖状又は分岐状のアルキル基である。好ましくは、R2、R3が、それぞれ独立に、チオフェン基、フラン基、ピロール基、イミダゾール基、オキサゾール基、チジアゾール基、ピラゾール基、ピリジン基及びキノリン基からなる群から選択された1種である。あるいは、好ましくは、R2、R3が、スルホニル基を有するアリール基である。
In addition, the remainder of R 1 and R 4 and R 2 and R 3 in (2) are each independently a hydrogen atom, a halogen atom, a substituent, an alkyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfuric acid group An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have an ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group is shown. The alkyl group is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms. Moreover, said alkoxy group is a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, or a phenoxy group, for example. The alkyl ester group is a linear or branched alkyl ester having 1 to 6 carbon atoms. The aromatic hydrocarbon group is a monocyclic or polycyclic aryl group, specifically a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group. The heterocyclic group is, for example, a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably a furan group, an imidazole group, or a thiophene group. It is. The aliphatic hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. Preferably, R 2 and R 3 are each independently one selected from the group consisting of a thiophene group , a furan group , a pyrrole group , an imidazole group , an oxazole group , a thiazole group , a pyrazole group , a pyridine group, and a quinoline group. is there. Alternatively, preferably, R 2 and R 3 are aryl groups having a sulfonyl group.
また、Xは置換基を有していてもよい炭素原子、窒素原子、硫黄原子、酸素原子、セレン原子又はボロン原子を示す。 X represents a carbon atom, nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom which may have a substituent.
また、R’は芳香環を含んでも良いアルキル基からなる脂肪族炭化水素基あるいは芳香族炭化水素基を示す。ここで、その脂肪族炭化水素基あるいは芳香族炭化水素基には、上記と同様のものを用いることができる。 R 'represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group comprising an alkyl group which may contain an aromatic ring. Here, the aliphatic hydrocarbon group or the aromatic hydrocarbon group may be the same as described above.
また、An-は、Cl-、Br-、I-等のハロゲン化物イオン、CF3SO3 -、BF4 -又はPF6 -を示す。 An − represents a halide ion such as Cl − , Br − or I − , CF 3 SO 3 − , BF 4 − or PF 6 − .
アゾール誘導体として、ジアゾール誘導体の例を示したが、以下の一般式で表されるトリアゾール誘導体を用いることもできる。トリアゾール誘導体を用いても、ジアゾール誘導体の場合と同様の効果を得ることができる。 Although examples of diazole derivatives have been shown as azole derivatives, triazole derivatives represented by the following general formula can also be used. Even if a triazole derivative is used, the same effect as in the case of a diazole derivative can be obtained.
ここで、(9)及び(11)ではR1は、そして(10)ではR1とR7の一方は、一般式L-Mで示され、Mは、ピリジニウム基、2級アミノ基、3級アミノ基、4級アミノ基、ピペリジニウム基、ピペラジニウム基、イミダゾリウム基、チアゾリウム基、オキサゾリウム基、キノリウム基、ベンゾイミダゾリウム基、ベンゾチアゾリウム基又はベンゾオキサゾリウム基である窒素カチオン含有基、あるいは置換基を有しても良いピリジン基、2級アミノ基、3級アミノ基、ピペリジン基、ピペラジン基、イミダゾール基、チアゾール基、オキサゾール基、キノリン基、ベンゾイミダゾール基、ベンゾチアゾール基又はベンゾオキサゾール基である窒素含有基を示し、Lは、-(CH=CR6)n-で表され、ここでnは1から5の整数からなり、R6は水素原子あるいは置換基として、メチル基、エチル基等のアルキル基、アルキル基を有するスルホ基、イミダゾリウム基、ピリジニウム基、ピペリジニウム基、フラン基等の複素環基、2級アミノ基、3級アミノ基そして4級アミノ基等のアミノ基、ヒドロキシ基、アルコキシ基、アルデヒド基、カルボキシル基又は芳香族基を示し、Mと発色部とを連結するリンカーを示す。リンカーを用いることが好ましい。 Here, R 1 in (9) and (11), and one of R 1 and R 7 in (10) is represented by the general formula LM, where M is a pyridinium group, a secondary amino group, a tertiary amino group. Group, quaternary amino group, piperidinium group, piperazinium group, imidazolium group, thiazolium group, oxazolium group, quinolium group, benzoimidazolium group, benzothiazolium group or benzoxazolium group, or a nitrogen cation-containing group A pyridine group, secondary amino group, tertiary amino group, piperidine group, piperazine group, imidazole group, thiazole group, oxazole group, quinoline group, benzimidazole group, benzothiazole group or benzoxazole group which may have a substituent L represents-(CH = CR 6 ) n-, where n is an integer of 1 to 5, and R 6 is a hydrogen atom or a substituent, Alkyl groups such as methyl groups and ethyl groups, sulfo groups having alkyl groups, imidazolium groups, pyridinium groups, piperidinium groups, furan groups and other heterocyclic groups, secondary amino groups, tertiary amino groups and quaternary amino groups, etc. The amino group, the hydroxy group, the alkoxy group, the aldehyde group, the carboxyl group, or the aromatic group, and a linker that connects M and the coloring portion. It is preferable to use a linker.
また、(10)のR1とR7の残部及びR2、R3は、それぞれ独立に、水素原子、ハロゲン原子、置換基としてアルキル基、アルコキシ基、アルキルエステル基、リン酸エステル基、硫酸エステル基、ニトリル基、ヒドロキシル基、シアノ基、スルホニル基、芳香族炭化水素基又は複素環基を有してもよい芳香族炭化水素基又は脂肪族炭化水素基又は複素環基を示す。上記のアルキル基は、例えば炭素数1から6の直鎖状又は分岐状のアルキル基である。また、上記のアルコキシ基は、例えばメトキシ基、エトキシ基、プロポキシ基、イソプロポキシ基、ブトキシ基、ペンチロキシ基又はフェノキシ基である。また、上記のアルキルエステル基は、炭素数1から6の直鎖状又は分岐状のアルキルエステルである。また、上記の芳香族炭化水素基は単環又は多環を含むアリール基、具体的にはフェニル基、トリル基、キシリル基又はナフチル基であり、より好ましくはフェニル基である。また、上記の複素環基は、例えばピロール基、フラン基、チオフェン基、イミダゾール基、オキサゾール基、チアゾール基、ピラゾール基、ピリジン基又はキノリン基であり、より好ましくはフラン基、イミダゾール基又はチオフェン基である。また、上記の脂肪族炭化水素基は、好ましくは炭素数1から6の直鎖状又は分岐状のアルキル基である。好ましくは、R2、R3が、それぞれ独立に、チオフェン基、フラン基、ピロール基、イミダゾール基、オキサゾール基、チジアゾール基、ピラゾール基、ピリジン基及びキノリン基からなる群から選択された1種である。あるいは、好ましくは、R2、R3が、スルホニル基を有するアリール基である。
In addition, the remainder of R 1 and R 7 and R 2 and R 3 in (10) are each independently a hydrogen atom, a halogen atom, a substituent as an alkyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfuric acid group An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have an ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group is shown. The alkyl group is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms. Moreover, said alkoxy group is a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, or a phenoxy group, for example. The alkyl ester group is a linear or branched alkyl ester having 1 to 6 carbon atoms. The aromatic hydrocarbon group is a monocyclic or polycyclic aryl group, specifically a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group. The heterocyclic group is, for example, a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably a furan group, an imidazole group, or a thiophene group. It is. The aliphatic hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. Preferably, R 2 and R 3 are each independently one selected from the group consisting of a thiophene group , a furan group , a pyrrole group , an imidazole group , an oxazole group , a thiazole group , a pyrazole group , a pyridine group, and a quinoline group. is there. Alternatively, preferably, R 2 and R 3 are aryl groups having a sulfonyl group.
また、R'は芳香環を含んでも良いアルキル基からなる脂肪族炭化水素基あるいは芳香族炭化水素基を示す。ここで、その脂肪族炭化水素基あるいは芳香族炭化水素基には、上記と同様のものを用いることができる。 R ′ represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group composed of an alkyl group which may contain an aromatic ring. Here, the aliphatic hydrocarbon group or the aromatic hydrocarbon group may be the same as described above.
また、An-は、Cl-、Br-、I-等のハロゲン化物イオン、CF3SO3 -、BF4 -又はPF6 -を示す。 An − represents a halide ion such as Cl − , Br − or I − , CF 3 SO 3 − , BF 4 − or PF 6 − .
本発明の蛍光色素は、例えば窒素含有基としてピリジン基を含む場合、以下の方法により製造することができる。すなわち、アゾール誘導体又はイミダゾール誘導体のハロアルキル体とトリフェニルホスフィンとを反応させてホスホニウム塩を調製し、このホスホニウム塩とフォルミルピリジンとを用いてウィッティヒ反応により二重結合を介してピリジン基を導入してピリジン体を得る。また、窒素カチオン含有基として、ピリジニウム基を含む場合には、そのピリジン体と、活性エステルのブロモ体とを反応させて、ピリジニウム塩を得ることにより製造することができる。ここで、ハロアルキル体は、アゾール誘導体又はイミダゾール誘導体のヒドロキシ体にハロゲン化剤を反応させる方法を用いることにより得ることができる。ハロゲン化剤には、塩化チオニル、塩化ホスホリル、三塩化リン、五塩化リン、塩化スルフリル、塩素、臭化チオニル、臭素等を用いることができ、好ましくは塩化チオニル又は塩化ホスホリルである。 The fluorescent dye of the present invention can be produced by the following method, for example, when it contains a pyridine group as a nitrogen-containing group. That is, a phosphonium salt is prepared by reacting an azole derivative or imidazole derivative haloalkyl with triphenylphosphine, and a pyridine group is introduced via a double bond by Wittig reaction using this phosphonium salt and formylpyridine. To obtain a pyridine compound. Moreover, when a pyridinium group is included as a nitrogen cation containing group, it can manufacture by making the pyridine body and the bromo body of an active ester react, and obtaining a pyridinium salt. Here, the haloalkyl form can be obtained by using a method in which a halogenating agent is reacted with a hydroxy form of an azole derivative or an imidazole derivative. As the halogenating agent, thionyl chloride, phosphoryl chloride, phosphorus trichloride, phosphorus pentachloride, sulfuryl chloride, chlorine, thionyl bromide, bromine and the like can be used, and thionyl chloride or phosphoryl chloride is preferable.
上記の方法は、窒素カチオン含有基の場合、活性エステルのブロモ体を用い、以下に述べる結合部となる活性エステルを含むピリジニウム塩を得る方法について説明したが、上記において導入したピリジンにヨウ化メチルなどのハロアルカン類、ハロアルケン類あるいはハロカルボン酸類を反応させることによりピリジニウム塩を製造することもできる。この場合、ハロアルカン類、ハロアルケン類の末端にイソチオシアネート基、無水マレイン酸などの、以下に述べる結合部となる官能基を含む場合、生体分子と結合させることができる。また、ハロカルボン酸類を用いた場合、ピリジンに化学結合させることで末端にカルボン酸を有するピリジニウム体となるため、ヒドロキシスクシンイミドなどの活性エステル基を導入することもできる。 In the above-described method, in the case of a nitrogen cation-containing group, a method for obtaining a pyridinium salt containing an active ester to be a bonding portion described below using a bromo form of an active ester has been described, but methyl iodide is introduced into the pyridine introduced above. A pyridinium salt can also be produced by reacting haloalkanes such as haloalkenes, haloalkenes or halocarboxylic acids. In this case, when the terminal of the haloalkanes or haloalkenes contains a functional group that becomes a bonding portion described below, such as an isothiocyanate group or maleic anhydride, it can be bonded to a biomolecule. When halocarboxylic acids are used, a pyridinium compound having a carboxylic acid at the terminal is obtained by chemically bonding to pyridine, so that an active ester group such as hydroxysuccinimide can be introduced.
また、本発明の蛍光色素に生体分子に結合する結合部を設けることもできる。結合部は例えば窒素カチオン含有基又は窒素含有基に設けることができる。結合部は、生体分子と結合する反応性基を有し、その反応性基は共有結合又はイオン結合により生体分子と結合する。 In addition, the fluorescent dye of the present invention can be provided with a binding part that binds to a biomolecule. The bonding portion can be provided, for example, on a nitrogen cation-containing group or a nitrogen-containing group. The binding portion has a reactive group that binds to a biomolecule, and the reactive group binds to the biomolecule through a covalent bond or an ionic bond.
共有結合として、例えばアミド結合、イミド結合、ウレタン結合、エステル結合、又はグアニジン結合を形成する場合、反応性基には、生体分子のアミノ基、イミノ基、チオール基、カルボキシル基又はヒドロキシル基と反応可能な官能基が好ましい。その官能基には、例えば、イソチオシアネート基、イソシアネート基、無水マレイン酸基、エポキシ基、ハロゲン化スルホニル基、塩化アシル基、ハロゲン化アルキル基、グリオキザル基、アルデヒド基、トリアジン基、カルボジイミド基そして活性エステル化したカルボニル基等を用いることができる。好ましくは、イソチオシアネート基、イソシアネート基、エポキシ基、ハロゲン化アルキル基、トリアジン基、カルボジイミド基そして活性エステル化したカルボニル基から選択されたいずれか1種を用いることが好ましい。より好ましくは、イソチオシアネート基、イソシアネート基、エポキシ基、ハロゲン化アルキル基、トリアジン基、カルボジイミド基そして活性エステル化したカルボニル基から選択されたいずれか1種を用いることが好ましい。さらに好ましくはトリアジン基、カルボジイミド基又は活性エステル化したカルボニル基である。これら反応性基と反応する窒素カチオン含有基の官能基としては、例えばカルボキシル基を用いることができる。例えば、活性エステル化したカルボニル基には、N−ヒドロキシ−スクシンイミドエステルやマレイミドエステルを用いることができる。N−ヒドロキシ−スクシンイミドを用い、縮合剤としてDCCを用いることによりN−ヒドロキシ−スクシンイミドエステル体を経由してアミド結合により蛍光色素と生体分子が結合する。また、カルボジイミド基には、N,N'-ジシクロヘキシルカルボジイミド(DCC)や1-シクロヘキシル-3-(2-モルホリノエチル)カルボジイミド等のカルボジイミド試薬を用いることができる。カルボジイミド体を経由してアミド結合により蛍光色素と生体分子とを結合させることができる。 For example, when forming an amide bond, an imide bond, a urethane bond, an ester bond, or a guanidine bond as a covalent bond, the reactive group reacts with an amino group, imino group, thiol group, carboxyl group, or hydroxyl group of a biomolecule. Possible functional groups are preferred. The functional groups include, for example, isothiocyanate groups, isocyanate groups, maleic anhydride groups, epoxy groups, halogenated sulfonyl groups, acyl chloride groups, halogenated alkyl groups, glyoxal groups, aldehyde groups, triazine groups, carbodiimide groups and active groups. An esterified carbonyl group or the like can be used. Preferably, any one selected from an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group, and an active esterified carbonyl group is preferably used. More preferably, any one selected from an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group, and an active esterified carbonyl group is preferably used. More preferred are a triazine group, a carbodiimide group, or an active esterified carbonyl group. As the functional group of the nitrogen cation-containing group that reacts with these reactive groups, for example, a carboxyl group can be used. For example, N-hydroxy-succinimide ester and maleimide ester can be used for the carbonyl group which has been activated esterified. When N-hydroxy-succinimide is used and DCC is used as a condensing agent, the fluorescent dye and the biomolecule are bonded by an amide bond via the N-hydroxy-succinimide ester. As the carbodiimide group, a carbodiimide reagent such as N, N′-dicyclohexylcarbodiimide (DCC) or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide can be used. A fluorescent dye and a biomolecule can be bound by an amide bond via a carbodiimide body.
また、イオン結合を形成する反応性基には、アニオン性基やカチオン性基を用いることができる。アニオン性基としては、例えばスルホニル基やカルボキシル基を用いることができる。これらのアニオン性基は、生体分子のカチオン性基、例えばアミノ基とイオン結合する。また、カチオン性基としては、4級アンモニウム基やピリジニウム基等の窒素カチオン含有基を用いることができる。これらカチオン性基は、生体分子のアニオン性基、例えばカルボキシル基とイオン結合する。なお、本発明においては、発色部に結合した窒素カチオン含有基が反応性基としてのカチオン性基を兼ねることができる。 An anionic group or a cationic group can be used as the reactive group that forms an ionic bond. As the anionic group, for example, a sulfonyl group or a carboxyl group can be used. These anionic groups are ionically bonded to a cationic group of the biomolecule, such as an amino group. Further, as the cationic group, a nitrogen cation-containing group such as a quaternary ammonium group or a pyridinium group can be used. These cationic groups are ionically bonded to an anionic group of the biomolecule, such as a carboxyl group. In the present invention, the nitrogen cation-containing group bonded to the color forming portion can also serve as a cationic group as a reactive group.
本発明の蛍光色素は、標識された固体あるいは半固体状態の生体分子の蛍光を測定する検出方法であれば、あらゆる生体分子の検出方法に適用することができる。従来の蛍光色素に代えて用いることにより、高感度で、化学的に安定で操作性に優れ、さらに低コストの検出方法を提供することができる。本発明の蛍光色素は、生体分子試料に蛍光色素を直接反応させて標識しても良く、あるいは生体分子試料と、本発明の蛍光色素で標識されたプローブとを反応させて標識する方法を用いることもできる。さらに、本発明の蛍光色素で標識した生体分子試料を電気泳動によりサイズ分離する方法を用いることもできる。例えば、核酸を検出対象とするDNAマイクロアレイ法や、プライマーやターミネータを用いるPCR法に用いることができる。 The fluorescent dye of the present invention can be applied to any biomolecule detection method as long as it is a detection method that measures fluorescence of labeled solid or semi-solid biomolecules. By using in place of conventional fluorescent dyes, it is possible to provide a detection method that is highly sensitive, chemically stable, excellent in operability, and low in cost. The fluorescent dye of the present invention may be labeled by directly reacting the biomolecule sample with the fluorescent dye, or a method of labeling by reacting the biomolecule sample with the probe labeled with the fluorescent dye of the present invention is used. You can also. Furthermore, a method of separating the size of a biomolecule sample labeled with the fluorescent dye of the present invention by electrophoresis can also be used. For example, it can be used in a DNA microarray method for detecting nucleic acids or a PCR method using primers and terminators.
また、タンパク質を検出対象とする場合、通常、電気泳動後のタンパク質の検出には染色色素が用いられている。泳動後のゲル中に、染色色素、例えばクーマシーブリリアントブルー(CBB)を浸透させてタンパク質を染色し、UVを照射して発光させる方法が用いられる。しかしながら、従来の染色色素を用いる方法は簡便であるが、感度が100ng程度と低く微量のタンパク質の検出には適さない。また、ゲルを介して染色色素を浸透させるため、染色に長時間を要するという問題もある。これに対し、本発明の蛍光色素を用いると高感度であり、微量タンパク質の検出には好適である。さらに、サイズ分離したタンパク質を質量分析して同定することもできる。 In addition, when a protein is a detection target, a staining dye is usually used for detection of the protein after electrophoresis. A method is used in which a dye is dyed, for example, Coomassie Brilliant Blue (CBB), is allowed to permeate into the gel after electrophoresis, and the protein is stained and irradiated with UV to emit light. However, the conventional method using a staining dye is simple, but the sensitivity is as low as about 100 ng, and it is not suitable for detecting a trace amount of protein. Further, since the dye is infiltrated through the gel, there is also a problem that it takes a long time for dyeing. On the other hand, when the fluorescent dye of the present invention is used, it has high sensitivity and is suitable for detecting a trace amount of protein. Further, the size separated protein can be identified by mass spectrometry.
ここで、タンパク質には、アルブミン、グロブリン、グルテリン、ヒストン、プロタミン、そしてコラーゲン等の単純タンパク質、核タンパク質、糖タンパク質、リボタンパク質、リンタンパク質、金属タンパク質等の複合タンパク質のいずれも検出対象とすることができる。例えば、リンタンパク質、糖タンパク質、総タンパク質の染色色素に対応させて3種の蛍光色素を用い、二次元電気泳動で分離したタンパク質試料において、リンタンパク質、糖タンパク質及び総タンパク質を染色することができる。また、TOF-Mass等の質量分析を行うことにより、タンパク質を同定できるので、特殊なタンパク質を生成させる、ガンやウィルスによる感染症などの疾病の診断や治療に応用することが可能である。また、コラーゲンは、動物の結合組織を構成するタンパク質であり、独特の繊維状構造をとる。すなわち、3本のポリペプチド鎖からなり、そのペプチド鎖が寄り集まって三重鎖を形成する。コラーゲンは、一般に極めて免疫原性が低いタンパク質であり、食品、化粧品、医薬品等の分野で広く利用されている。しかし、コラーゲンのペプチド鎖に蛍光色素を導入しても、従来の蛍光色素ではその安定性が十分とは言えず、より安定な蛍光色素が必要とされている。そこで、本発明の蛍光色素を用いてコラーゲンを標識することにより、安定かつ高感度な検出を行うことが可能となる。 Here, the target protein should be any of simple proteins such as albumin, globulin, glutelin, histone, protamine, and collagen, and complex proteins such as nucleoprotein, glycoprotein, riboprotein, phosphoprotein, and metal protein. Can do. For example, phosphoproteins, glycoproteins and total proteins can be stained in protein samples separated by two-dimensional electrophoresis using three types of fluorescent dyes corresponding to the staining dyes of phosphoproteins, glycoproteins, and total proteins. . Moreover, since the protein can be identified by performing mass spectrometry such as TOF-Mass, it can be applied to diagnosis and treatment of diseases such as infections caused by cancer and viruses that produce special proteins. Collagen is a protein constituting the connective tissue of animals and has a unique fibrous structure. That is, it consists of three polypeptide chains, and the peptide chains gather to form a triple chain. Collagen is generally a protein with very low immunogenicity, and is widely used in the fields of food, cosmetics, pharmaceuticals and the like. However, even if a fluorescent dye is introduced into the peptide chain of collagen, the conventional fluorescent dye cannot be said to have sufficient stability, and a more stable fluorescent dye is required. Thus, stable and highly sensitive detection can be performed by labeling collagen with the fluorescent dye of the present invention.
また、タンパク質と特異的に結合する抗体を本発明の蛍光色素で標識することにより、タンパク質を標識することもできる。例えば、IgG抗体をペプシンで処理するとF(ab’)2と呼ばれるフラグメントが得られる。このフラグメントをジチオスレイトール等で還元するとFab’と呼ばれるフラグメントが得られる。Fab’フラグメントは1つもしくは2つのチオール基(-SH)を有している。このチオール基に対してマレイミド基を作用させて特異的な反応を行うことができる。すなわち、本発明の蛍光色素に反応性基としてマレイミド基を導入し、フラグメントのチオール基と反応させることにより抗体を標識することができる。この場合、抗体の生理活性(抗原捕捉能)を失うことがない。 Alternatively, a protein can be labeled by labeling an antibody that specifically binds to the protein with the fluorescent dye of the present invention. For example, when an IgG antibody is treated with pepsin, a fragment called F (ab ′) 2 is obtained. When this fragment is reduced with dithiothreitol or the like, a fragment called Fab ′ is obtained. Fab ′ fragments have one or two thiol groups (—SH). A specific reaction can be carried out by allowing a maleimide group to act on the thiol group. That is, an antibody can be labeled by introducing a maleimide group as a reactive group into the fluorescent dye of the present invention and reacting with the thiol group of the fragment. In this case, the physiological activity (antigen capturing ability) of the antibody is not lost.
なお、本発明の蛍光色素でアプタマーを標識することもできる。アプタマーはオリゴ核酸からなり、塩基配列に依存して種々の特徴ある立体構造をとることができるので、その立体構造を介してタンパク質を含むあらゆる生体分子に結合することができる。この性質を利用し、本発明の蛍光色素で標識したアプタマーを特定のタンパク質に結合させ、被検出物質との結合によるそのタンパク質の構造変化に伴う蛍光変化から間接的に被検出物質を検出することができる。 The aptamer can also be labeled with the fluorescent dye of the present invention. Aptamers are composed of oligonucleic acids and can have various characteristic three-dimensional structures depending on the base sequence, and can bind to any biomolecule including proteins via the three-dimensional structure. Utilizing this property, the aptamer labeled with the fluorescent dye of the present invention is bound to a specific protein, and the detected substance is indirectly detected from the fluorescence change accompanying the structural change of the protein due to the binding with the detected substance. Can do.
また、本発明の蛍光色素を用いて金属イオンの検出を行うこともできる。体内のDNAやタンパク質などの安定性や高次構造の維持、機能発現、そして生体内のすべての化学反応を司る酵素の活性化など、生体内で起こるあらゆる生命現象に金属イオンは関与している。そのため、生体内での金属イオンの動きをリアルタイムで観察できる金属イオンセンサは医療分野を初めとしてその重要性が叫ばれている。従来、生体分子に蛍光色素を導入した金属イオンセンサが知られている。例えば、K+イオン存在化において、K+イオン取り込んで特殊な構造をとる配列を有する核酸を利用する金属イオンセンサが提案されている(J. AM. CHEM. SOC. 2002, 124, 14286-14287)。エネルギートランスファーを起こす蛍光色素を核酸の両端に導入する。通常は色素間距離があるためエネルギートランスファーは起きない。しかし、K+イオン存在下では核酸が特殊な形をとる結果、蛍光色素がエネルギートランスファーを起こす距離に近接することで、蛍光を観察することができる。また、ペプチドに蛍光色素を導入した亜鉛イオンセンサも提案されている(J. Am. Chem. Soc. 1996, 118, 3053-3054)。これらの従来の蛍光色素に代えて本発明の蛍光色素を用いることにより、従来に比べ高感度で取り扱いが容易な金属イオンセンサを提供することが可能となる。なお、生体内に存在する金属イオンであれば、すべての金属イオンを検出することが可能である。 In addition, metal ions can be detected using the fluorescent dye of the present invention. Metal ions are involved in all life phenomena that occur in the body, such as the stability of DNA and proteins in the body, the maintenance of higher-order structures, functional expression, and the activation of enzymes that control all chemical reactions in the body. . Therefore, the importance of a metal ion sensor capable of observing the movement of metal ions in a living body in real time is sought after in the medical field. Conventionally, a metal ion sensor in which a fluorescent dye is introduced into a biomolecule is known. For example, the K + ions present of a metal ion sensor that utilizes a nucleic acid having a sequence that takes the K + ions takes in a special structure has been proposed (J. AM. CHEM. SOC. 2002, 124, 14286-14287 ). A fluorescent dye that causes energy transfer is introduced at both ends of the nucleic acid. Normally, energy transfer does not occur because there is a distance between dyes. However, in the presence of K + ions, as a result of the nucleic acid taking a special form, the fluorescence can be observed by approaching the distance at which the fluorescent dye causes energy transfer. A zinc ion sensor in which a fluorescent dye is introduced into a peptide has also been proposed (J. Am. Chem. Soc. 1996, 118, 3053-3054). By using the fluorescent dye of the present invention in place of these conventional fluorescent dyes, it is possible to provide a metal ion sensor that is more sensitive and easy to handle than in the past. In addition, if it is a metal ion which exists in the living body, it is possible to detect all the metal ions.
また、本発明の蛍光色素を用いて、細胞内のシグナル観察を行うこともできる。内部シグナルや環境情報に対する細胞の応答には、イオンから酵素へと多大な分子が関与している。シグナル伝達過程では特殊なプロテインキナーゼが活性化し、特殊な細胞タンパク質のリン酸化を導くことで様々な細胞応答の初期応答を担っていることが知られている。ヌクレオチドの結合と加水分解はこれらの活性に重大な役割を果たしており、ヌクレオチド誘導体を用いることで、シグナル伝達挙動を素早く観察することが出来る。例えば、プロテインキナーゼC(PKC)は細胞膜におけるシグナル伝達において重要な役割を果たしている。このCa2+依存セリン/スレオニンプロテインキナーゼはジアシルグリセロールやフォスファティジルセリンの様な膜構成脂質上で活性化され、イオンチャネルや細胞骨格タンパク質に存在するセリンやスレオニンをリン酸化することで膜表面電化を変えシグナル伝達を行っている。これらを生細胞において動的に観察することで細胞のシグナル伝達の観察を行うことができる。 Moreover, intracellular signal observation can also be performed using the fluorescent dye of the present invention. A large number of molecules from ions to enzymes are involved in cellular responses to internal signals and environmental information. It is known that a special protein kinase is activated in the signal transduction process, and leads to phosphorylation of a special cellular protein, thereby carrying out an initial response of various cellular responses. Nucleotide binding and hydrolysis play a critical role in these activities, and signal transduction behavior can be quickly observed by using nucleotide derivatives. For example, protein kinase C (PKC) plays an important role in signal transduction at the cell membrane. This Ca 2+ -dependent serine / threonine protein kinase is activated on membrane-constituting lipids such as diacylglycerol and phosphatidylserine, and phosphorylates serine and threonine present in ion channels and cytoskeletal proteins. It changes the electrification and performs signal transmission. By observing these dynamically in living cells, cell signal transduction can be observed.
ここで、ヌクレオチド誘導体は酵素の基質や阻害剤として供給され、孤立性タンパク質の構造と力学の探査、膜結合タンパク酵素の再構成、ミトコンドリアのようなオルガネラ、除膜筋線維のような組織のヌクレオチド結合タンパク質部分に、結合してその調節を行っている。また、最近ではG-タンパク質の阻害剤や活性体のようなシグナル伝達に影響を与える化合物の存在も解ってきている。このヌクレオチド誘導体に本発明の有機EL色素からなる標識色素を導入することで、これらの細胞内シグナル伝達の動的観察を高感度で、かつ取り扱い容易に行うことが可能となる。 Here, nucleotide derivatives are supplied as enzyme substrates and inhibitors, exploring the structure and dynamics of isolated proteins, reconstitution of membrane-bound protein enzymes, organelles like mitochondria, nucleotides in tissues like membrane-removed muscle fibers It binds to the binding protein and regulates it. Recently, the existence of compounds that affect signal transduction, such as inhibitors and activators of G-proteins, has also been elucidated. By introducing a labeling dye comprising the organic EL dye of the present invention into this nucleotide derivative, dynamic observation of these intracellular signal transmissions can be performed with high sensitivity and easy handling.
また、本発明の蛍光色素を、組織又は細胞試料中の標的核酸や標的タンパク質の発現レベルの検討に用いる組織又は細胞の染色色素としても用いることができる。すなわち、本発明の染色色素を真核細胞の染色に用いると、乾燥状態でも蛍光を発することから標識後の保存などの点で従来の色素よりも優れた性能を示す。また、真核細胞のみならず、細胞骨格用色素としても十分に用いることが可能である。この他、ミトコンドリア、ゴルジ体、小胞体、ソリゾーム、脂質二重膜などの標識に用いることが可能である。これら、標識された細胞等は、湿潤及び乾燥のあらゆる条件下で観測が可能であるため、汎用性が大きい。観測に際しては、蛍光顕微鏡などを用いることができる。 The fluorescent dye of the present invention can also be used as a staining dye for tissues or cells used for studying the expression level of a target nucleic acid or target protein in a tissue or cell sample. That is, when the staining dye of the present invention is used for staining of eukaryotic cells, it emits fluorescence even in a dry state, and thus exhibits superior performance compared to conventional dyes in terms of storage after labeling. Further, it can be sufficiently used not only as a eukaryotic cell but also as a dye for cytoskeleton. In addition, it can be used for labeling of mitochondria, Golgi apparatus, endoplasmic reticulum, solisome, lipid bilayer membrane and the like. Since these labeled cells can be observed under all wet and dry conditions, they are highly versatile. For observation, a fluorescence microscope or the like can be used.
また、臨床段階で人体より採取された組織は、ミクロトームなどの機器を用いて薄膜にスライスした後、染色されている。ここでは、Cy色素及びAlexa色素が用いられている。しかしながら、既存の色素は安定性が非常に悪く、再診断の際には、再びサンプルを作製する必要がある。また、作製されたサンプルは標本として保存することが不可能である。しかし、上記の従来の色素に比べ本発明の蛍光色素は、非常に安定な色素であるので、染色した組織を標本として保存することが可能である。 In addition, tissues collected from the human body in the clinical stage are stained after being sliced into a thin film using a device such as a microtome. Here, Cy dye and Alexa dye are used. However, the existing dye is very unstable, and it is necessary to prepare a sample again at the time of rediagnosis. In addition, the prepared sample cannot be stored as a specimen. However, since the fluorescent dye of the present invention is a very stable dye as compared with the conventional dyes described above, the stained tissue can be stored as a specimen.
また、ガンや感染症等の診断には、抗体の特異的認識能を利用したイムノアッセイが用いられている。イムノアッセイは、標識抗体を用いて目的の抗原を検出する方法であり、標識物質に酵素を用いる酵素イムノアッセイ(ELISA法)や標識物質に蛍光色素を用いる蛍光イムノアッセイ(FIA法)等が用いられている。ELISA法は、最終的な検出は標識物質である酵素の反応によって生じるさまざまなシグナル(発色、発光、化学発光等)を検出及び定量することにより行う。一方、FIA法は、標識物質である蛍光色素に励起光を照射し、それによる蛍光を検出及び定量することにより行う。FIA法は蛍光色素を用いるため鮮明なコントラストを有し定量性に優れ、またELISA法に比べ、より短時間での検出が可能でかつ操作も簡便であるという特徴を有している。しかしながら、従来の蛍光色素は標識率が低いという問題がある。例えば、抗体に対して200倍モル程度の蛍光色素を用いているが、この条件下においても標識率は50−60%程度であった。そのため、蛍光色素を大量に使用する必要があるため検出費用が高コストになったり、未反応の蛍光色素を除去するための処理工程が必要となり検出に長時間を要するという問題があった。これに対し、本発明の蛍光色素を用いることにより、標識率を向上させることができるので、より高感度の検出を行うことが可能となる。 For the diagnosis of cancer, infectious diseases, etc., an immunoassay utilizing the specific recognition ability of an antibody is used. The immunoassay is a method for detecting a target antigen using a labeled antibody, and an enzyme immunoassay (ELISA method) using an enzyme as a labeling substance or a fluorescent immunoassay (FIA method) using a fluorescent dye as a labeling substance is used. . In the ELISA method, the final detection is performed by detecting and quantifying various signals (color development, luminescence, chemiluminescence, etc.) generated by the reaction of the enzyme as a labeling substance. On the other hand, the FIA method is performed by irradiating a fluorescent dye, which is a labeling substance, with excitation light, and detecting and quantifying the resulting fluorescence. Since the FIA method uses a fluorescent dye, it has a clear contrast and excellent quantification, and has features that it can be detected in a shorter time and is easier to operate than the ELISA method. However, the conventional fluorescent dye has a problem that the labeling rate is low. For example, about 200-fold mol of the fluorescent dye is used with respect to the antibody, and the labeling rate is about 50-60% even under this condition. For this reason, it is necessary to use a large amount of fluorescent dye, so that the detection cost becomes high, and there is a problem that a processing step for removing the unreacted fluorescent dye is required and it takes a long time for detection. On the other hand, since the labeling rate can be improved by using the fluorescent dye of the present invention, detection with higher sensitivity can be performed.
また、本発明の蛍光色素を化粧用組成物に用いることもできる。蛍光色素を含む化粧用組成物は、夜間や室内における演出用の化粧としてだけでなく、蛍光色素の明色化効果を利用して、ファンデーションや毛髪の染色剤等に用いられている。ここで、明色化効果とは、蛍光色素が紫外光を吸収して可視光を放出して、皮膚や毛髪に明るさや鮮やかさを与える効果をいう。日本の室内照明には、昼光色や白色の蛍光灯が使われているが、これらの蛍光灯からの光は、青や緑が主であり赤が少ない。そのため、女性の化粧肌は青白くくすんで見えるという問題がある。これに対し、本発明の蛍光色素を用いることにより、例えば、橙色の光を放出する蛍光色素を用い、鮮やかな赤味の色を発色させてくすみの解消を図ることが可能である。また、毛髪の染色に用いると、蛍光色素は可視領域の放出光線により毛髪の色を変えるだけでなく、毛髪の輝きを増加させることも可能である。 Moreover, the fluorescent dye of the present invention can also be used in a cosmetic composition. Cosmetic compositions containing fluorescent dyes are used in foundations, hair dyeing agents, and the like, not only as makeup for production at night and in the room, but also by using the lightening effect of fluorescent dyes. Here, the lightening effect means an effect in which the fluorescent dye absorbs ultraviolet light and emits visible light to give the skin and hair brightness and vividness. Japanese indoor lighting uses daylight and white fluorescent lamps, but the light from these fluorescent lamps is mainly blue and green, with little red. Therefore, there is a problem that a woman's makeup skin looks pale and dull. On the other hand, by using the fluorescent dye of the present invention, for example, a fluorescent dye that emits orange light can be used to develop a bright reddish color to eliminate dullness. In addition, when used for hair dyeing, the fluorescent dye not only changes the color of the hair by the emitted light in the visible region, but can also increase the shine of the hair.
また、本発明の蛍光色素をマーキング剤に用いることもできる。本発明の蛍光色素を含むマーキング剤は、通常の可視光下では不可視であるが、紫外線等の励起光を照射することにより蛍光色素を発光させて視認することができる。この性質を利用し、犯罪防止や犯罪捜査を目的として、物品や人体等の識別や物質の検出等に使用することができる。マーキング剤の対象物には、偽造や盗難等の犯罪の防止や犯罪捜査の対象となる物品や人体が含まれる。例えば、紙幣、小切手、株券、各種証明書等の重要文書や、自動車、オートバイ、自転車、美術品、家具、ブランド品、衣服等の物品、人体の皮膚、頭髪、爪等の身体表面部分、潜在指紋等の遺留物質等を挙げることができる。さらに、対象物を構成する材料に関しては、上質紙、OCR紙、ノーカーボン紙、アート紙等の紙や、塩化ビニル、ポリエステル、ポリエチレンテレフタレート、ポリプロピレン等のプラスチックや、金属や、ガラスや、セラミックスや、羊毛、木綿、絹、麻等の天然繊維や、再生セルロース繊維、ポリビニルアルコール繊維、ポリアミド繊維、ポリエステル繊維等の合成繊維や、人体皮膚や体液中のタンパク質等を挙げることができる。 Moreover, the fluorescent dye of the present invention can also be used as a marking agent. The marking agent containing the fluorescent dye of the present invention is invisible under normal visible light, but can be visually recognized by emitting the fluorescent dye by irradiating excitation light such as ultraviolet rays. Utilizing this property, it can be used for identification of articles and human bodies, detection of substances, etc. for the purpose of crime prevention and crime investigation. Objects of the marking agent include articles and human bodies that are subject to crime prevention and criminal investigation such as counterfeiting and theft. For example, important documents such as banknotes, checks, stock certificates, various certificates, articles such as automobiles, motorcycles, bicycles, arts, furniture, branded goods, clothes, body surface parts such as human skin, hair, nails, latent Examples include retained substances such as fingerprints. Furthermore, regarding the materials that make up the object, paper such as high-quality paper, OCR paper, carbonless paper, art paper, plastics such as vinyl chloride, polyester, polyethylene terephthalate, and polypropylene, metals, glass, ceramics, And natural fibers such as wool, cotton, silk and hemp, synthetic fibers such as regenerated cellulose fibers, polyvinyl alcohol fibers, polyamide fibers and polyester fibers, and proteins in human skin and body fluids.
以下、実施例を用いて本発明をさらに詳細に説明するが、本発明の範囲は以下の実施例により限定されるものではない。
合成例1.
有機EL色素としてオキサジアゾロピリジン誘導体を用いた。
以下に、4,7-ジフェニル-1,2,5-オキサジアゾロピリジンエチルエステルの合成スキームを示す。
EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, the scope of the present invention is not limited by the following example.
Synthesis Example 1
An oxadiazolopyridine derivative was used as the organic EL dye.
The synthesis scheme of 4,7-diphenyl-1,2,5-oxadiazolopyridine ethyl ester is shown below.
(1)ジケトン誘導体(2)の合成
500mL三口フラスコに4-メトキシアセトフェノン(1)30.0 g (0.25 mol)、亜硝酸ナトリウム0.15 gを酢酸100 mLに溶解した。水浴中、HNO3 100 mLを酢酸100 mLに溶解したものを1時間かけて滴下した。その後、室温で2日間撹拌した。反応混合物を500mLの水にゆっくりと入れ、沈殿を生成させた。沈殿物は濾過し、クロロホルムに溶解した。クロロホルム相を飽和重曹水で洗浄し、10% NaCl 水溶液で2回洗浄した。MgSO4で脱水した後、減圧下、クロロホルムを留去し、オキサジアゾール-N-オキサイド(2)を30.5 g (収率82%)で得た。
(1) Synthesis of diketone derivative (2)
In a 500 mL three-necked flask, 30.0 g (0.25 mol) of 4-methoxyacetophenone (1) and 0.15 g of sodium nitrite were dissolved in 100 mL of acetic acid. In a water bath, 100 mL of HNO 3 dissolved in 100 mL of acetic acid was added dropwise over 1 hour. Thereafter, the mixture was stirred at room temperature for 2 days. The reaction mixture was slowly poured into 500 mL of water to produce a precipitate. The precipitate was filtered and dissolved in chloroform. The chloroform phase was washed with a saturated aqueous sodium bicarbonate solution and twice with a 10% NaCl aqueous solution. After dehydration with MgSO 4 , chloroform was distilled off under reduced pressure to obtain 30.5 g (yield 82%) of oxadiazole-N-oxide (2).
(2)ジケトン誘導体(3)の合成
500mL三口フラスコにオキサジアゾール-N-オキサイド(2)14.7 g (0.05 mol)をアセトニトリル400 mLに溶解した。それにZn 6.0 g、AcOH 7 mL、Ac2O 20mLを添加した。水浴中で反応温度が35℃を超えないように冷却した。6時間撹拌して反応終点とした。反応混合物を濾過し、不溶分を除去した。アセトニトリルを減圧下留去して残渣を得た。残渣をクロロホルムで再結晶し、オキサジアゾールジベンゾイル体(3)を9.6 g (収率69%)で得た。
(2) Synthesis of diketone derivative (3)
14.7 g (0.05 mol) of oxadiazole-N-oxide (2) was dissolved in 400 mL of acetonitrile in a 500 mL three-necked flask. Zn 6.0 g, AcOH 7 mL, and Ac 2 O 20 mL were added thereto. The reaction was cooled in a water bath so that the reaction temperature did not exceed 35 ° C. The reaction was terminated by stirring for 6 hours. The reaction mixture was filtered to remove insolubles. Acetonitrile was distilled off under reduced pressure to obtain a residue. The residue was recrystallized from chloroform to obtain 9.6 g (69% yield) of oxadiazole dibenzoyl compound (3).
(3)オキサジアゾロピリジンエチルエステル(4)の合成
500mL三口フラスコでオキサジアゾールジベンゾイル体(3)10.0 g (0.035 mol)をブタノール300 mLに溶解した。そこへグリシンエチルエステル塩酸塩 32.0 g (0.23 mol)を添加した。24時間加熱還流を行った。ブタノールを減圧下留去し、残渣を得た。残渣を200mLのクロロホルムに溶解し、10% HCl、飽和NaHCO3、10%NaClで洗浄した。MgSO4で乾燥し、溶媒を留去した。得られた残渣をクロロホルムで再結晶し、オキサジアゾロピリジンエチルエステル(4)を7.6 g (収率 65%)で得た。
(3) Synthesis of oxadiazolopyridine ethyl ester (4)
In a 500 mL three-necked flask, 10.0 g (0.035 mol) of the oxadiazole dibenzoyl compound (3) was dissolved in 300 mL of butanol. Thereto was added 32.0 g (0.23 mol) of glycine ethyl ester hydrochloride. The mixture was heated under reflux for 24 hours. Butanol was distilled off under reduced pressure to obtain a residue. The residue was dissolved in 200 mL chloroform and washed with 10% HCl, saturated NaHCO 3 , 10% NaCl. It was dried over MgSO 4 and the solvent was distilled off. The obtained residue was recrystallized from chloroform to obtain 7.6 g (yield 65%) of oxadiazolopyridine ethyl ester (4).
次いで、ジフェニルオキサジアゾロピリジンエチルエステル体(4)をNaBH4存在下、還元反応を行い、ジアミノアルコール体(5)を得、これと塩化チオニルを反応させチアジアゾロピリジンクロロメチル体(6)を得、これにトリフェニルホスフィンを反応させてホスホニウム塩(7)を得、さらにWittig反応によりビニル体(8)を得、そして活性エステルを含むピリジニウム塩(9)(Lが-CH=CH-の場合)を合成した。以下に反応例を示す。 Next, the diphenyloxadiazolopyridine ethyl ester (4) is reduced in the presence of NaBH 4 to obtain a diaminoalcohol (5), which is reacted with thionyl chloride to obtain a thiadiazolopyridine chloromethyl (6) This is reacted with triphenylphosphine to obtain a phosphonium salt (7), and further a vinyl form (8) is obtained by Wittig reaction, and a pyridinium salt (9) containing an active ester (L is —CH═CH— In the case of A reaction example is shown below.
(1)ジアミノアルコール体(5)の合成
エステル体(4)(1.73 g, 5 mmol)とNaBH4(1.30 g, 35 mmol)のエタノール溶液(100 mL)を12時間加熱環流。反応液を水に注入、一夜放置後に沈澱をろ過してジアミノアルコール体 (5)(1.17 g, 80%)を得た。
(1) Synthesis of diaminoalcohol (5) An ethanol solution (100 mL) of ester (4) (1.73 g, 5 mmol) and NaBH 4 (1.30 g, 35 mmol) was heated to reflux for 12 hours. The reaction solution was poured into water and allowed to stand overnight, and then the precipitate was filtered to obtain a diamino alcohol form (5) (1.17 g, 80%).
(2)クロロメチル化閉環体(6)の合成
室温下、アルコール体 (5)(1.17 g)のクロロホルム溶液(60 mL)にSOCl2(6mL)、ピリジンNaBH4(3 mL)をこの順で滴下、その後3時間30分加熱環流。反応液を水に注入、NaHCO3で中和、クロロホルムで抽出。抽出液をMgSO4で乾燥、減圧留去して得た残さをカラム(Kanto C-60; Hexane/CHCl3 = 3/1 (v/v))処理してクロロメチル化閉環体 (6)(1.11 g, 82%)を得た。
(2) Synthesis of chloromethylated closed ring (6) At room temperature, a solution of alcohol (5) (1.17 g) in chloroform (60 mL) was charged with SOCl 2 (6 mL) and pyridine NaBH 4 (3 mL) in this order. Dropping, followed by heating for 3
(3)ホスホニウム塩(7)の合成
クロロメチル体2(112.6 mg, 0.33 mmol)とPh3P(96 mg, 0.37 mmol)のトルエン溶液(5 mL)を3日間加熱環流。沈澱をろ過、エーテルで洗浄してホスホニウム塩 (7)(108 mg, 55%)を得た。
(3) Synthesis of phosphonium salt (7) A toluene solution (5 mL) of chloromethyl compound 2 (112.6 mg, 0.33 mmol) and Ph 3 P (96 mg, 0.37 mmol) was heated to reflux for 3 days. The precipitate was filtered and washed with ether to obtain phosphonium salt (7) (108 mg, 55%).
(4)ビニル体(8)の合成
氷冷下、m-フォルミルピリジン(16 microL, 0.18 mmol)と水酸化カリウム(85% purity, 15 mg)のエタノール溶液(1 mL)にホスホニウム塩(7)(140.5 mg, 0.23 mmol)を加え、その温度で1時間30分撹拌。沈澱をろ過、エタノール、水で洗浄、乾燥してビニル体(8)(44 mg, 62%)を得た。
(4) Synthesis of vinyl compound (8) Under ice-cooling, phosphonium salt (7 mL) was added to an ethanol solution (1 mL) of m-formylpyridine (16 microL, 0.18 mmol) and potassium hydroxide (85% purity, 15 mg). ) (140.5 mg, 0.23 mmol) was added and stirred at that temperature for 1
(5)活性エステルを含むピリジニウム塩(9)の合成
ビニル体(8)(40 mg, 0.10 mmol)とブロムヘキサン酸活性エステル(32 mg, 0.11 mol)のトルエン溶液(2 mL)を5日間加熱環流。沈澱をろ過して活性エステル含有ピリジニウム塩(9)を得た。
(5) Synthesis of pyridinium salt (9) containing active ester A toluene solution (2 mL) of vinyl (8) (40 mg, 0.10 mmol) and bromohexanoic acid active ester (32 mg, 0.11 mol) is heated for 5 days. Reflux. The precipitate was filtered to obtain an active ester-containing pyridinium salt (9).
合成例2.
以下に、4,7-ジ(メトキシフェニル)-1,2,5-オキサジアゾロピリジンエチルエステルの合成スキームを示す。
Synthesis Example 2
The synthesis scheme of 4,7-di (methoxyphenyl) -1,2,5-oxadiazolopyridine ethyl ester is shown below.
(1)ジケトン誘導体(11)の合成
500mL三口フラスコに4-メトキシアセトフェノン(10)37.5 g (0.25 mol)、亜硝酸ナトリウム0.15 gを酢酸100 mLに溶解した。水浴中、HNO3 100 mLを酢酸100 mLに溶解したものを2時間かけて滴下した。その後、室温で2日間撹拌した。反応混合物を500mLの水にゆっくりと入れ、沈殿を生成させた。沈殿物は濾過し、クロロホルムに溶解した。クロロホルム相を飽和重曹水で洗浄し、10% NaCl 水溶液で2回洗浄した。MgSO4で脱水した後、減圧下、クロロホルムを留去し、オキサジアゾール-N-オキサイド(11)を34.5 g (収率78%)で得た。
(1) Synthesis of diketone derivative (11)
In a 500 mL three-necked flask, 37.5 g (0.25 mol) of 4-methoxyacetophenone (10) and 0.15 g of sodium nitrite were dissolved in 100 mL of acetic acid. In a water bath, 100 mL of HNO 3 dissolved in 100 mL of acetic acid was added dropwise over 2 hours. Thereafter, the mixture was stirred at room temperature for 2 days. The reaction mixture was slowly poured into 500 mL of water to produce a precipitate. The precipitate was filtered and dissolved in chloroform. The chloroform phase was washed with a saturated aqueous sodium bicarbonate solution and twice with a 10% NaCl aqueous solution. After dehydration with MgSO 4 , chloroform was distilled off under reduced pressure to obtain 34.5 g (yield 78%) of oxadiazole-N-oxide (11).
(2)ジケトン誘導体(12)の合成
500mL三口フラスコにオキサジアゾール-N-オキサイド(11)17.7 g (0.05 mol)をアセトニトリル400 mLに溶解した。それにZn 12.0 g、AcOH 7 mL、Ac2O 20mLを添加した。水浴中で反応温度が30℃を超えないように冷却した。12時間撹拌して反応終点とした。反応混合物を濾過し、不溶分を除去した。アセトニトリルを減圧下留去して残渣を得た。残渣をクロロホルムで再結晶し、オキサジアゾール-N-オキサイド(12)を10.2 g (収率60%)で得た。
(2) Synthesis of diketone derivative (12)
17.7 g (0.05 mol) of oxadiazole-N-oxide (11) was dissolved in 400 mL of acetonitrile in a 500 mL three-necked flask. Zn 12.0 g, AcOH 7 mL, and Ac 2 O 20 mL were added thereto. The reaction was cooled in a water bath so that the reaction temperature did not exceed 30 ° C. The reaction was terminated by stirring for 12 hours. The reaction mixture was filtered to remove insolubles. Acetonitrile was distilled off under reduced pressure to obtain a residue. The residue was recrystallized from chloroform to obtain 10.2 g (yield 60%) of oxadiazole-N-oxide (12).
(3)オキサジアゾロピリジンエチルエステル(13)の合成
500mL三口フラスコでオキサジアゾール-N-オキサイド(12)15.6 g (0.046 mol)をブタノール300 mLに溶解した。そこへグリシンエチルエステル塩酸塩 32.0 g (0.23 mol)を添加した。24時間加熱還流を行った。ブタノールを減圧下留去し、残渣を得た。残渣を200mLのクロロホルムに溶解し、10% HCl、飽和NaHCO3、10%NaClで洗浄した。MgSO4で乾燥し、溶媒を留去した。得られた残渣をクロロホルムで再結晶し、オキサジアゾロピリジンエチルエステル(13)を13.0 g (収率 70%)で得た。
(3) Synthesis of oxadiazolopyridine ethyl ester (13)
In a 500 mL three-necked flask, 15.6 g (0.046 mol) of oxadiazole-N-oxide (12) was dissolved in 300 mL of butanol. Thereto was added 32.0 g (0.23 mol) of glycine ethyl ester hydrochloride. The mixture was heated under reflux for 24 hours. Butanol was distilled off under reduced pressure to obtain a residue. The residue was dissolved in 200 mL chloroform and washed with 10% HCl, saturated NaHCO 3 , 10% NaCl. It was dried over MgSO 4 and the solvent was distilled off. The obtained residue was recrystallized from chloroform to obtain 13.0 g (yield 70%) of oxadiazolopyridine ethyl ester (13).
次いで、ジ(メトキシ)フェニルオキサジアゾロピリジンエチルエステル体(13)をNaBH4存在下、還元反応を行い、ヒドロキシメチル体(14)を得、これと塩化チオニルを反応させオキサジアゾロピリジンクロロメチル体(15)を得、これにトリフェニルホスフィンを反応させてホスホニウム塩(16)を得、そしてWittig反応により活性エステルを含むピリジニウム塩(18)(Lが-CH=CH-の場合)を合成した。以下に反応例を示す。 Next, the di (methoxy) phenyloxadiazolopyridine ethyl ester (13) is subjected to a reduction reaction in the presence of NaBH 4 to obtain a hydroxymethyl compound (14), which is reacted with thionyl chloride to react with oxadiazolopyridine chloromethyl. To obtain a phosphonium salt (16) by reacting with triphenylphosphine, and a pyridinium salt (18) containing an active ester (when L is —CH═CH—) by a Wittig reaction. did. A reaction example is shown below.
(1)ヒドロキシメチル体(14)の合成
氷冷下、エステル体 13(202 mg, 0.50 mmol)のTHF溶液(3 mL)に DIBALのトルエン溶液(Aldrich, conc 1.5 mol/L, 6 μL)を滴下、その後氷冷下で30分、続いて室温で30分撹拌。反応液を水に注入、3% HCl aq を加えて酸性(沈澱が消失)とし、クロロホルムで抽出。抽出液をMgSO4で乾燥、減圧留去して得た残さをシリカゲル(Kanto C-60)に分散させて一夜80度で加熱。このシリカゲルをAcOEtで洗浄、洗浄液を減圧留去して得た残さをカラム(Kanto C-60; Hexane/AcOEt = 2/1 (v/v))処理してヒドロキシメチル体 (14)(69 mg, 41%)を得た。
(1) Synthesis of hydroxymethyl compound (14) Under ice-cooling, DIBAL in toluene (Aldrich, conc 1.5 mol / L, 6 μL) was added to THF solution (3 mL) of ester compound 13 (202 mg, 0.50 mmol). Dropping, followed by stirring for 30 minutes under ice cooling, followed by 30 minutes at room temperature. The reaction mixture was poured into water, acidified with 3% HCl aq (precipitation disappeared), and extracted with chloroform. The extract was dried over MgSO 4 and evaporated under reduced pressure. The residue was dispersed in silica gel (Kanto C-60) and heated at 80 ° C. overnight. The silica gel was washed with AcOEt, and the residue obtained by distilling off the washing solution under reduced pressure was treated with a column (Kanto C-60; Hexane / AcOEt = 2/1 (v / v)) to obtain a hydroxymethyl compound (14) (69 mg , 41%).
(2)クロロメチル体(15)の合成
ヒドロキシメチル体 14(95 mg)とSOCl2(3 mL)のクロロホルム溶液(3 mL)を2時間加熱環流。反応液を水に注入、NaHCO3で中和、クロロホルムで抽出。抽出液をMgSO4で乾燥、減圧留去して得た残さをカラム(Kanto C-60; Hexane/CHCl3 = 5/1 (v/v))処理してクロロメチル体 (15)(quant.)を得た。
(2) Synthesis of chloromethyl compound (15) A chloroform solution (3 mL) of hydroxymethyl compound 14 (95 mg) and SOCl 2 (3 mL) was heated to reflux for 2 hours. The reaction mixture was poured into water, neutralized with NaHCO 3 and extracted with chloroform. The extract was dried over MgSO 4 and evaporated under reduced pressure. The residue was treated with a column (Kanto C-60; Hexane / CHCl3 = 5/1 (v / v)) and treated with chloromethyl compound (15) (quant.) Got.
(3)ホスホニウム塩(16)の合成
クロロメチル体 15(127 mg, 0.44 mmol)とPh3P(126 mg, 0.48 mmol)のトルエン溶液(4 mL)を24時間加熱環流。沈澱をろ過してホスホニウム塩(16)(168.6 mg, 54%)を得た。
(3) Synthesis of phosphonium salt (16) A toluene solution (4 mL) of chloromethyl 15 (127 mg, 0.44 mmol) and Ph 3 P (126 mg, 0.48 mmol) was heated to reflux for 24 hours. The precipitate was filtered to obtain a phosphonium salt (16) (168.6 mg, 54%).
(4)ビニル体(17)の合成
氷冷下、ホスホニウム塩(16)(168.6 mg, 0.26 mmol)と水酸化カリウム(85% purity, 30 mg)のエタノール溶液(3 mL)にm-フォルミルピリジン(27 μL, 0.29 mmol)を加え、その温度で1時間、続いて室温で1時間撹拌。沈澱をろ過、カラム(Kanto C-60; CHCl3/AcOEt = 10/1 (v/v))処理してビニル体(17)(86 mg, 76%)を得た。
(4) Synthesis of vinyl compound (17) Under ice cooling, m-formyl was added to an ethanol solution (3 mL) of phosphonium salt (16) (168.6 mg, 0.26 mmol) and potassium hydroxide (85% purity, 30 mg). Add pyridine (27 μL, 0.29 mmol) and stir for 1 hour at that temperature followed by 1 hour at room temperature. The precipitate was filtered and treated with a column (Kanto C-60; CHCl3 / AcOEt = 10/1 (v / v)) to obtain a vinyl compound (17) (86 mg, 76%).
(5)活性エステルを含むピリジニウム塩(18)の合成
ビニル体(17)(86 mg, 0.20 mmol)とブロムヘキサン酸活性エステル(63 mg, 0.22 mol)のトルエン溶液(2 mL)を3日間加熱環流。沈澱をろ過して活性エステルを含むピリジニウム塩 (18) を得た。
(5) Synthesis of pyridinium salt containing active ester (18) A toluene solution (2 mL) of vinyl (17) (86 mg, 0.20 mmol) and bromohexanoic acid active ester (63 mg, 0.22 mol) was heated for 3 days. Reflux. The precipitate was filtered to obtain a pyridinium salt (18) containing an active ester.
(評価)
ピリジン基を含むビニル体(8)と、活性エステルを含むピリジニウム塩(9)の水溶液中における蛍光スペクトルを測定した。比較として、窒素カチオン含有基も窒素含有基を含まない、エチルエステル(4)の活性エステル体を測定した。結果を図1に示す。ビニル体(8)とピリジニウム塩(9)は、比較の活性エステル体に比べ、約10倍の蛍光強度を有していた。これにより、本発明の蛍光色素を用いることにより、生体分子の高感度の検出が可能であることを確認できた。
(Evaluation)
Fluorescence spectra were measured in an aqueous solution of a vinyl compound (8) containing a pyridine group and a pyridinium salt (9) containing an active ester. As a comparison, an active ester form of ethyl ester (4) in which neither a nitrogen cation-containing group nor a nitrogen-containing group was measured. The results are shown in FIG. The vinyl body (8) and the pyridinium salt (9) had a fluorescence intensity about 10 times that of the comparative active ester body. Thus, it was confirmed that the biomolecule can be detected with high sensitivity by using the fluorescent dye of the present invention.
Claims (5)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008205238A JP5539635B2 (en) | 2008-08-08 | 2008-08-08 | Fluorescent dye |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008205238A JP5539635B2 (en) | 2008-08-08 | 2008-08-08 | Fluorescent dye |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2010037511A JP2010037511A (en) | 2010-02-18 |
JP5539635B2 true JP5539635B2 (en) | 2014-07-02 |
Family
ID=42010385
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2008205238A Expired - Fee Related JP5539635B2 (en) | 2008-08-08 | 2008-08-08 | Fluorescent dye |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP5539635B2 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012111142A1 (en) * | 2011-02-18 | 2012-08-23 | Isobe Shinichiro | Fluorescent dye |
US9056874B2 (en) | 2012-05-04 | 2015-06-16 | Novartis Ag | Complement pathway modulators and uses thereof |
US9475806B2 (en) | 2013-03-14 | 2016-10-25 | Novartis Ag | Complement factor B inhibitors and uses there of |
EP3089963A1 (en) | 2013-10-30 | 2016-11-09 | Novartis AG | 2-benzyl-benzimidazole complement factor b inhibitors and uses thereof |
WO2016008671A1 (en) | 2014-07-15 | 2016-01-21 | Valitacell Limited | A method of measuring antibody concentration in a sample |
US20170313885A1 (en) * | 2014-10-31 | 2017-11-02 | Shinichiro | Alkoxysilyl group-containing organic el dye and a method for producing the same |
JP2016196608A (en) * | 2015-04-06 | 2016-11-24 | 学校法人 中村産業学園 | Fluorescent dye |
CN114105900B (en) * | 2021-11-25 | 2023-07-11 | 浙江工业大学 | Preparation method of 3, 4-diphenyl ketone-1, 2, 5-oxadiazole |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5503836B2 (en) * | 2006-12-26 | 2014-05-28 | 株式会社アイエスティー | Fluorescent dye and method for producing the same |
-
2008
- 2008-08-08 JP JP2008205238A patent/JP5539635B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
JP2010037511A (en) | 2010-02-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5539635B2 (en) | Fluorescent dye | |
JP5881624B2 (en) | Fluorescent dye | |
JP5638734B2 (en) | Labeling dye for biomolecule, labeling kit, and method for detecting biomolecule | |
JP5503836B2 (en) | Fluorescent dye and method for producing the same | |
KR20240031282A (en) | Compound based cyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same | |
JP4801445B2 (en) | Biomolecule detection method and labeling dye and labeling kit used therefor | |
Ma et al. | Insight into fluorescence imaging and bioorthogonal reactions in biological analysis | |
KR102645305B1 (en) | Compound based cyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same | |
US8476443B2 (en) | Fluorescent dyes | |
JP6709734B2 (en) | Alkoxysilyl group-containing organic EL dye and method for producing the same | |
JP2016196608A (en) | Fluorescent dye | |
JP2009014369A (en) | Fluorescence reagent for labeling biomolecule | |
US11597842B2 (en) | Labeling dye and kit including same | |
JP7045752B2 (en) | Fluorescent dye | |
JP2006234772A (en) | Method of detecting protein and fluorescent dye used for same | |
JP7014549B2 (en) | Fluorescent dye | |
JP7479660B2 (en) | Fluorescent dyes | |
US20200239491A1 (en) | Merocyanine-based compound and biomolecular labeling dye, kit and contrast agent composition comprising same | |
KR102593166B1 (en) | Labeling dye and labeling kit comprising the same | |
KR20190043075A (en) | Compound based merocyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same | |
Hong et al. | Applications of Aggregation‐Induced Emission Materials in Biotechnology | |
WO2008059364A2 (en) | Determining the interaction between nucleic acids and nucleic acid binding molecules |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20110804 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20130618 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130814 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20131119 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140114 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20140401 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 5539635 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20140501 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
LAPS | Cancellation because of no payment of annual fees |