JP6275253B2 - 蛍光プローブ、一重項酸素検出剤、又は一重項酸素検出方法 - Google Patents
蛍光プローブ、一重項酸素検出剤、又は一重項酸素検出方法 Download PDFInfo
- Publication number
- JP6275253B2 JP6275253B2 JP2016529417A JP2016529417A JP6275253B2 JP 6275253 B2 JP6275253 B2 JP 6275253B2 JP 2016529417 A JP2016529417 A JP 2016529417A JP 2016529417 A JP2016529417 A JP 2016529417A JP 6275253 B2 JP6275253 B2 JP 6275253B2
- Authority
- JP
- Japan
- Prior art keywords
- substituent
- singlet oxygen
- group
- fluorescent probe
- oxygen detection
- 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.)
- Active
Links
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 title claims description 153
- 239000007850 fluorescent dye Substances 0.000 title claims description 82
- 238000001514 detection method Methods 0.000 title claims description 35
- 239000003795 chemical substances by application Substances 0.000 title claims description 20
- 125000001424 substituent group Chemical group 0.000 claims description 93
- 210000004027 cell Anatomy 0.000 claims description 65
- 150000003839 salts Chemical class 0.000 claims description 44
- 125000000217 alkyl group Chemical group 0.000 claims description 41
- 150000001875 compounds Chemical class 0.000 claims description 32
- 125000003118 aryl group Chemical group 0.000 claims description 30
- 125000003342 alkenyl group Chemical group 0.000 claims description 29
- 125000000304 alkynyl group Chemical group 0.000 claims description 29
- 239000003504 photosensitizing agent Substances 0.000 claims description 28
- 125000005843 halogen group Chemical group 0.000 claims description 26
- KSFOVUSSGSKXFI-GAQDCDSVSA-N CC1=C/2NC(\C=C3/N=C(/C=C4\N\C(=C/C5=N/C(=C\2)/C(C=C)=C5C)C(C=C)=C4C)C(C)=C3CCC(O)=O)=C1CCC(O)=O Chemical group CC1=C/2NC(\C=C3/N=C(/C=C4\N\C(=C/C5=N/C(=C\2)/C(C=C)=C5C)C(C=C)=C4C)C(C)=C3CCC(O)=O)=C1CCC(O)=O KSFOVUSSGSKXFI-GAQDCDSVSA-N 0.000 claims description 24
- 229950003776 protoporphyrin Drugs 0.000 claims description 24
- 210000003470 mitochondria Anatomy 0.000 claims description 22
- 239000002253 acid Substances 0.000 claims description 21
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 14
- 239000012453 solvate Substances 0.000 claims description 11
- 210000001700 mitochondrial membrane Anatomy 0.000 claims description 10
- 125000003545 alkoxy group Chemical group 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 6
- 206010028980 Neoplasm Diseases 0.000 claims description 6
- 201000011510 cancer Diseases 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 230000001678 irradiating effect Effects 0.000 claims description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 6
- 125000004429 atom Chemical group 0.000 claims description 5
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000012800 visualization Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 230000002438 mitochondrial effect Effects 0.000 claims 1
- NVLIIKFLLWSUEI-UHFFFAOYSA-N [7-(dimethylamino)-10-(9,10-dimethylanthracen-2-yl)-5,5-dimethylbenzo[b][1]benzosilin-3-ylidene]-dimethylazanium Chemical compound CN(C)C1=CC=C2C(=C1)[Si](C)(C)C1=CC(C=CC1=C2C1=CC=C2C(C)=C3C=CC=CC3=C(C)C2=C1)=[N+](C)C NVLIIKFLLWSUEI-UHFFFAOYSA-N 0.000 description 47
- -1 organic acid salts Chemical class 0.000 description 43
- 229910052710 silicon Inorganic materials 0.000 description 40
- 239000010703 silicon Substances 0.000 description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 39
- 239000000243 solution Substances 0.000 description 36
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 22
- 230000005284 excitation Effects 0.000 description 18
- JNELGWHKGNBSMD-UHFFFAOYSA-N xanthone powder Natural products C1=CC=C2C(=O)C3=CC=CC=C3OC2=C1 JNELGWHKGNBSMD-UHFFFAOYSA-N 0.000 description 16
- 125000004432 carbon atom Chemical group C* 0.000 description 13
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 12
- 239000001301 oxygen Substances 0.000 description 12
- 229910052760 oxygen Inorganic materials 0.000 description 12
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 description 11
- 229960002749 aminolevulinic acid Drugs 0.000 description 11
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 10
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 10
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Natural products C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 9
- 238000011534 incubation Methods 0.000 description 9
- 239000003642 reactive oxygen metabolite Substances 0.000 description 9
- VMPNESUIRKJIRP-UHFFFAOYSA-N 2-bromo-9,10-dimethylanthracene Chemical compound BrC1=CC2=C(C3=CC=CC=C3C(=C2C=C1)C)C VMPNESUIRKJIRP-UHFFFAOYSA-N 0.000 description 8
- 125000005577 anthracene group Chemical group 0.000 description 8
- 229910052801 chlorine Inorganic materials 0.000 description 8
- 125000001309 chloro group Chemical group Cl* 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- AKZFRMNXBLFDNN-UHFFFAOYSA-K meso-tetrakis(n-methyl-4-pyridyl)porphine tetrakis(p-toluenesulfonate) Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1.CC1=CC=C(S([O-])(=O)=O)C=C1.CC1=CC=C(S([O-])(=O)=O)C=C1.CC1=CC=C(S([O-])(=O)=O)C=C1.C1=C[N+](C)=CC=C1C(C=1C=CC(N=1)=C(C=1C=C[N+](C)=CC=1)C1=CC=C(N1)C(C=1C=C[N+](C)=CC=1)=C1C=CC(N1)=C1C=2C=C[N+](C)=CC=2)=C2N=C1C=C2 AKZFRMNXBLFDNN-UHFFFAOYSA-K 0.000 description 8
- 238000002428 photodynamic therapy Methods 0.000 description 8
- 238000010791 quenching Methods 0.000 description 8
- 230000000171 quenching effect Effects 0.000 description 8
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 229910002651 NO3 Inorganic materials 0.000 description 7
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 7
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 7
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 7
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 7
- 238000002189 fluorescence spectrum Methods 0.000 description 7
- 229910052731 fluorine Inorganic materials 0.000 description 7
- 125000001153 fluoro group Chemical group F* 0.000 description 7
- 229910052500 inorganic mineral Inorganic materials 0.000 description 7
- 229910052740 iodine Inorganic materials 0.000 description 7
- 239000011707 mineral Substances 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 7
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 6
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 239000000975 dye Substances 0.000 description 6
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 239000003086 colorant Substances 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 238000004020 luminiscence type Methods 0.000 description 5
- 239000012466 permeate Substances 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 4
- MZRVEZGGRBJDDB-UHFFFAOYSA-N N-Butyllithium Chemical compound [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 description 4
- 229910019093 NaOCl Inorganic materials 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 4
- 230000001427 coherent effect Effects 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- WGOPGODQLGJZGL-UHFFFAOYSA-N lithium;butane Chemical compound [Li+].CC[CH-]C WGOPGODQLGJZGL-UHFFFAOYSA-N 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 239000003921 oil Substances 0.000 description 4
- 210000003463 organelle Anatomy 0.000 description 4
- 239000002798 polar solvent Substances 0.000 description 4
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 125000006165 cyclic alkyl group Chemical group 0.000 description 3
- 238000000132 electrospray ionisation Methods 0.000 description 3
- 238000000295 emission spectrum Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 229910052744 lithium Inorganic materials 0.000 description 3
- 210000003712 lysosome Anatomy 0.000 description 3
- 230000001868 lysosomic effect Effects 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000006862 quantum yield reaction Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 239000012099 Alexa Fluor family Substances 0.000 description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 2
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 2
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- 231100000131 acute cytotoxicity Toxicity 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- XXROGKLTLUQVRX-UHFFFAOYSA-N allyl alcohol Chemical compound OCC=C XXROGKLTLUQVRX-UHFFFAOYSA-N 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical group C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 150000008282 halocarbons Chemical class 0.000 description 2
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000003068 molecular probe Substances 0.000 description 2
- 125000001624 naphthyl group Chemical group 0.000 description 2
- 239000012454 non-polar solvent Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- YJTKZCDBKVTVBY-UHFFFAOYSA-N 1,3-Diphenylbenzene Chemical group C1=CC=CC=C1C1=CC=CC(C=2C=CC=CC=2)=C1 YJTKZCDBKVTVBY-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- IANQTJSKSUMEQM-UHFFFAOYSA-N 1-benzofuran Chemical group C1=CC=C2OC=CC2=C1 IANQTJSKSUMEQM-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- 125000005917 3-methylpentyl group Chemical group 0.000 description 1
- FRDRBZVVFVGENG-UHFFFAOYSA-N 5,10,15,20-tetrakis(1-methyl-2H-pyridin-4-yl)-21,23-dihydroporphyrin Chemical compound CN1CC=C(C=C1)c1c2ccc(n2)c(C2=CCN(C)C=C2)c2ccc([nH]2)c(C2=CCN(C)C=C2)c2ccc(n2)c(C2=CCN(C)C=C2)c2ccc1[nH]2 FRDRBZVVFVGENG-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- MMXQXLWTZDSATD-UHFFFAOYSA-N CN1[CH-][NH+](C)C=C1 Chemical compound CN1[CH-][NH+](C)C=C1 MMXQXLWTZDSATD-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 239000007818 Grignard reagent Substances 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000007259 addition reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 125000005428 anthryl group Chemical group [H]C1=C([H])C([H])=C2C([H])=C3C(*)=C([H])C([H])=C([H])C3=C([H])C2=C1[H] 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 239000003849 aromatic solvent Substances 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000006267 biphenyl group Chemical group 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 150000004292 cyclic ethers Chemical class 0.000 description 1
- 125000000582 cycloheptyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000001511 cyclopentyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- BLHLJVCOVBYQQS-UHFFFAOYSA-N ethyllithium Chemical compound [Li]CC BLHLJVCOVBYQQS-UHFFFAOYSA-N 0.000 description 1
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 125000003983 fluorenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3CC12)* 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 150000004795 grignard reagents Chemical class 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-M iodide Chemical compound [I-] XMBWDFGMSWQBCA-UHFFFAOYSA-M 0.000 description 1
- 125000002510 isobutoxy group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])O* 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000004491 isohexyl group Chemical group C(CCC(C)C)* 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000000555 isopropenyl group Chemical group [H]\C([H])=C(\*)C([H])([H])[H] 0.000 description 1
- 125000003253 isopropoxy group Chemical group [H]C([H])([H])C([H])(O*)C([H])([H])[H] 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- UBJFKNSINUCEAL-UHFFFAOYSA-N lithium;2-methylpropane Chemical compound [Li+].C[C-](C)C UBJFKNSINUCEAL-UHFFFAOYSA-N 0.000 description 1
- 238000010859 live-cell imaging Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 150000002689 maleic acids Chemical class 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- AUHZEENZYGFFBQ-UHFFFAOYSA-N mesitylene Substances CC1=CC(C)=CC(C)=C1 AUHZEENZYGFFBQ-UHFFFAOYSA-N 0.000 description 1
- 125000001827 mesitylenyl group Chemical group [H]C1=C(C(*)=C(C([H])=C1C([H])([H])[H])C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- SKTCDJAMAYNROS-UHFFFAOYSA-N methoxycyclopentane Chemical compound COC1CCCC1 SKTCDJAMAYNROS-UHFFFAOYSA-N 0.000 description 1
- DVSDBMFJEQPWNO-UHFFFAOYSA-N methyllithium Chemical compound C[Li] DVSDBMFJEQPWNO-UHFFFAOYSA-N 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000002950 monocyclic group Chemical group 0.000 description 1
- 150000002780 morpholines Chemical class 0.000 description 1
- 125000006606 n-butoxy group Chemical group 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000003506 n-propoxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 125000001792 phenanthrenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3C=CC12)* 0.000 description 1
- NHKJPPKXDNZFBJ-UHFFFAOYSA-N phenyllithium Chemical compound [Li]C1=CC=CC=C1 NHKJPPKXDNZFBJ-UHFFFAOYSA-N 0.000 description 1
- 230000001443 photoexcitation Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002568 propynyl group Chemical group [*]C#CC([H])([H])[H] 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 125000001725 pyrenyl group Chemical group 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000012958 reprocessing Methods 0.000 description 1
- 239000001022 rhodamine dye Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 125000005920 sec-butoxy group Chemical group 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000002640 tocopherol group Chemical group 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/0803—Compounds with Si-C or Si-Si linkages
- C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
- C07F7/0812—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring
- C07F7/0816—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring said ring comprising Si as a ring atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/10—Compounds having one or more C—Si linkages containing nitrogen having a Si-N linkage
Description
ケイ素ローダミン骨格と、一重項酸素捕捉後には一重項酸素捕捉前と比較して該蛍光プローブの蛍光性が上昇する骨格とを有する化合物、又はその塩、水和物若しくは溶媒和物を含有する、蛍光プローブ。
で示される、項1に記載の蛍光プローブ。
で示される、項2に記載の蛍光プローブ。
(1)細胞を一重項酸素が発生する条件下で培養する工程、
(2)前記工程(1)で得た培養液と、請求項1〜3のいずれかに記載の蛍光プローブとを含有し、且つ、前記蛍光プローブの濃度が10〜500nmol/Lである混合培養液を作製、培養する工程、及び
(3)前記工程(2)で得た混合培養液に光照射する工程
を備える、一重項酸素検出方法。
で示される化合物。
で示される、項11に記載の化合物。
本発明の一重項酸素検出用蛍光プローブは、ケイ素ローダミン骨格と、一重項酸素捕捉後には一重項酸素捕捉前と比較して該蛍光プローブの蛍光性が上昇する骨格とを有する化合物、又はその塩、水和物若しくは溶媒和物を含有する。特に、本発明の一重項酸素検出用蛍光プローブは、ケイ素ローダミン骨格と、一重項酸素捕捉後には一重項酸素捕捉前と比較して該蛍光プローブの蛍光性が上昇する骨格とを有する化合物、又はその塩、水和物若しくは溶媒和物のみからなることが好ましい。
で示される化合物、又はその塩、水和物若しくは溶媒和物を含有することが好ましく、一般式(1A):
で示される化合物、又はその塩、水和物若しくは溶媒和物を含有することがより好ましい。特に、一般式(1)で示される化合物、又はその塩、水和物若しくは溶媒和物のみからなることが好ましく、一般式(1A)で示される化合物、又はその塩、水和物若しくは溶媒和物のみからなることがより好ましい。なお、この一般式(1)及び(1A)で示される化合物は、文献未記載の新規化合物である。
一般式(1A1)で示される化合物、又はその塩、水和物若しくは溶媒和物のみからなることがより好ましい。
本発明の蛍光プローブは、特に制限されないが、例えば、ケイ素キサントン化合物又はその誘導体と、ハロゲン原子で置換した上記した特定の骨格を有する化合物とを反応させることにより得られる。
で示されるケイ素キサントン化合物と、一般式(3):
で示されるアントラセン化合物とを、塩基の存在下で反応させることにより製造することができる。
本発明の一重項酸素検出用蛍光プローブは、上記したように、一重項酸素捕獲前には蛍光(発光)せず、一重項酸素捕獲後には特定の光に対して蛍光(発光)を示す。一方、他の活性酸素種存在下においては、このような挙動は示さず、蛍光(発光)を示さない。
本発明において、一重項酸素を検出する方法としては、特に制限されないが、
(1)細胞を一重項酸素が発生する条件下で培養する工程、
(2)前記工程(1)で得た培養液と、本発明の蛍光プローブとを含有し、且つ、前記蛍光プローブの濃度が10〜500nmol/Lである混合培養液を作製、培養する工程、及び
(3)前記工程(2)で得た混合培養液に光照射する工程
を備えることが好ましい。
工程(1)においては、まず、対象となる細胞を、一重項酸素が発生する条件下で培養する。
工程(2)においては、工程(1)で得た培養液に本発明の蛍光プローブを添加することが好ましい。この際、本発明の蛍光プローブの使用量は、得られる混合培養液中の濃度が10〜500nmol/L程度(特に20〜100nmol/L程度)となるように調整することが好ましい。この際、混合培養液のpHを6.0〜8.0程度に調整するため、緩衝剤(ヘペス緩衝剤、トリス緩衝剤、トリシン−水酸化ナトリウム緩衝剤、リン酸系緩衝剤、リン酸緩衝生理食塩水等)を使用してもよい。
工程(3)においては、工程(2)で得た混合培養液に光照射を行う。
本発明の一重項酸素検出用蛍光プローブは、上記のとおり、一重項酸素を選択的に検出するために用いられるものであるが、特に、ミトコンドリアの内膜近傍で発生する一重項酸素を場所選択的に検出することができる。これは、ミトコンドリアの直径が通常200〜400nm程度であり、水中の一重項酸素の拡散可能距離である300nm程度と同程度であることにも起因する。この仕組みを応用し、例えば、ライソゾムマーカーとして広く知られているモルホリン誘導体や特定の細胞内タンパク質を標識化できるような官能基を本発明の蛍光プローブに修飾することによって、細胞内特定小器官(ミトコンドリア等)及びタンパク質の周辺で発生する一重項酸素を光空間分解限界値(数百nm程度)に達する空間選択的に検出することが期待される。
2時間未満のインキュベーションを必要とする処理には、ヘペス20mMを含有する無血清培地を用い、4時間以上のインキュベーションを必要とする処理には、細胞生存率を維持するため、ヘペス20mMを含有する血清含有培地を用いた。ミトコンドリアをラベルするため、Mito Tracker(登録商標)Green FMを50nMに希釈し、30分間インキュベートした。一方、Alexa Fluor(登録商標)488又はAlexa Fluor(登録商標)647でラベルしたデキストラン10000MW(それぞれA488-及びA647-デキストラン)を50μg/mLに希釈し、24時間インキュベートしてリソソームを染色した。
KillerRedベクターを発現させるため、Lipofectamine(登録商標)2000(invitrogen)の一般的なプロトコルにしたがった。具体的には、2×105のHela細胞を35mmのガラスボトムディッシュに継代培養し、次の日には約50%のコンフルエントとなった。
HeLa細胞及びRAW264.7マクロファージは、それぞれ、理研BRC、及び大阪大学産業科学研究所の西教授から得た。特に表記していない限り、本発明で行った細胞実験は、HeLa細胞を用いて行った。HeLa細胞及びRAW264.7マクロファージを、5%CO2下の加湿インキュベーター中、37℃で、10%ウシ胎児血清(10099-141、Gibco)を補充したダルベッコ変法イーグル培地(D6429、Sigma)中で培養した。
ケイ素キサントン
1H-NMR (400 MHz, CD3OD) δ: 0.65 (s, 6H), 3.05 (s, 3H), 3.18 (s, 3H), 6.74 (dd, 2H, J = 9.6, 2.3 Hz), 7.27 (d, 2H, J = 9.6 Hz), 7.39 (d, 2H, J = 2.3 Hz), 7.44 (d, 1H, J = 8.2 Hz), 7.59-7.61 (m, 2H), 8.24 (s, 1H), 8.39-8.46 (m, 2H), 8.55 (d, 1H, 8.7 Hz). HRMS (ESI) = m/z calculated for C35H37N2Si 513.2721; found 513.2724。
TMPyP4とともに、インキュベートした実施例1のSi−DMAの蛍光スペクトル及び発光スペクトルについて、経時変化を図1に示す。なお、Si−DMAの濃度は50μM、TMPyP4の濃度は5μMであり、光照射は、510〜550nmの波長の光を用いて、0.07W/cm2の強度で、一重項酸素を発生させるために磁気攪拌しながら行った。
実施例1のSi−DMAは、色素の自己酸化を引き起こすことなく、選択的に一重項酸素に応答できるか否かを確認した。
1.1O2:10mMのNaOCl及び10mMのH2O2
2.O2 ●−:10mMのKO2
3.H2O2:10mMのH2O2
4.HOCl:10mMのNaOCl
5.ROO●:10mMの2,2−アゾビス(2−アミンジノプロパン)ジヒドロクロリド
6.●OH:1mMのFeSO4・7H2O及び10MのH2O2
を上記で得たSi−DMAの5μMの溶液に添加することにより作製した。
Rose Bengalと比較例1のSi-Meのサンプルを、1×1×4cm3の石英セルに投入した1:9のDMSO及びメタノール溶液中で作製した(Rose Bengalの濃度は21.5μM、Si−Meの濃度は250μMである)。
実施例1で得たSi-DMAの光照射時の蛍光増加をモニターするために、オリンパスIX81倒立蛍光顕微鏡及び640nmのCWレーザー(コヒーレント)を使用し、活性酸素種を照射しつつプローブをモニターした。ガラス底(ibidi)を有する35mmのμ−ディッシュにHela細胞を投入し、油浸対物(オリンパス、PlanApo 100x/1.40オイル)を介して励起した。発光像を収集し、ダイクロイックビームスプリッタ(セムロック、DI02-R635)及びバンドパスフィルタ(クロマ、HQ690/70)を介して、EMCCDカメラ(ローパーサイエンティフィック、Evolve 512)によって記録した。データ収集中、インキュベーター(37℃、5%CO2)を、Chamlide TC(生細胞計測器)を用いて同じ雰囲気を維持した。擬似蛍光画像は、OriginPro 9.1(OriginLab)及びImage Jを用いて得た動画ファイルを再処理して得た。
処理の前夜に、35mmディッシュに、0.2×106のHela細胞を播種し、150μg/mLの5−アミノレブリン酸、10%のFBS、20mMのヘペス緩衝剤を含有する細胞培養液で4時間インキュベートし、PpIXを含むHela細胞を準備した。さらに、25nMのSi−DMA、20mMのヘペス緩衝剤を含有する細胞培養液で1時間インキュベートした。オリンパスIX81倒立顕微鏡に搭載された高視野蛍光顕微鏡システムを使用し、Coherent社640nmCWレーザーで0.6W/cm2で30秒間光照射した。Roper Scientific社Evolve 512(EMCCDカメラ)で得られた信号は、Image J、originProプログラムを使用し、イメージ再処理した。結果を図10a)に示す。
Claims (16)
- 一重項酸素検出用蛍光プローブであって、
一般式(1):
で示される化合物、又はその塩、水和物若しくは溶媒和物を含有する、蛍光プローブ。 - 前記R1〜R8がいずれもメチル基であり、n及びmがいずれも0である、請求項1又は2に記載の蛍光プローブ。
- 請求項1〜3のいずれかに記載の蛍光プローブを含有する、一重項酸素検出剤。
- 前記蛍光プローブを10〜500nmol/L含有する、請求項4に記載の一重項酸素検出剤。
- ミトコンドリアの内膜近傍に発生する一重項酸素検出剤である、請求項4又は5に記載の一重項酸素検出剤。
- ミトコンドリアのリアルタイム可視化剤である、請求項4又は5に記載の一重項酸素検出剤。
- 細胞内に発生する一重項酸素を検出する方法であって、
(1)細胞を一重項酸素が発生する条件下で培養する工程、
(2)前記工程(1)で得た培養液と、請求項1〜3のいずれかに記載の蛍光プローブとを含有し、且つ、前記蛍光プローブの濃度が10〜500nmol/Lである混合培養液を作製、培養する工程、及び
(3)前記工程(2)で得た混合培養液に光照射する工程
を備える、一重項酸素検出方法。 - 前記工程(1)において、一重項酸素が発生する条件が、光増感剤を含有する条件である、請求項8に記載の一重項酸素検出方法。
- 前記光増感剤が、プロトポルフィリンIXである、請求項9に記載の一重項酸素検出方法。
- 前記細胞が癌細胞である、請求項8〜10のいずれかに記載の一重項酸素検出方法。
- 前記細胞内のミトコンドリアの内膜近傍に発生する一重項酸素を場所選択的に検出する、請求項8〜11のいずれかに記載の一重項酸素検出方法。
- 一般式(1):
で示される化合物。 - 前記R1〜R8がいずれもメチル基であり、n及びmがいずれも0である、請求項13又は14に記載の化合物。
- 請求項1〜3のいずれかに記載の蛍光プローブを含有するか、請求項4〜7のいずれかに記載の一重項酸素検出剤を含有するか、又は請求項13〜15のいずれかに記載の化合物を含有することを特徴とする、細胞試験用試薬。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014124543 | 2014-06-17 | ||
JP2014124543 | 2014-06-17 | ||
PCT/JP2015/067522 WO2015194606A1 (ja) | 2014-06-17 | 2015-06-17 | 蛍光プローブ、一重項酸素検出剤、又は一重項酸素検出方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
JPWO2015194606A1 JPWO2015194606A1 (ja) | 2017-06-29 |
JP6275253B2 true JP6275253B2 (ja) | 2018-02-07 |
Family
ID=54935586
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2016529417A Active JP6275253B2 (ja) | 2014-06-17 | 2015-06-17 | 蛍光プローブ、一重項酸素検出剤、又は一重項酸素検出方法 |
Country Status (3)
Country | Link |
---|---|
US (1) | US10564164B2 (ja) |
JP (1) | JP6275253B2 (ja) |
WO (1) | WO2015194606A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20210009919A (ko) * | 2019-07-18 | 2021-01-27 | 아주대학교산학협력단 | 미토콘드리아를 표적으로 하는 신규한 근적외선 형광 프로브 및 이를 포함하는 종양 진단 및 치료 겸용 조성물 |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6919653B2 (ja) * | 2016-06-22 | 2021-08-18 | 富士フイルム和光純薬株式会社 | ケイ素含有複素環式化合物及び消光剤 |
JOP20190161A1 (ar) * | 2016-12-27 | 2017-06-16 | Profusa Inc | مستشعرات جلوكوز بطيف قريب من الأشعة تحت الحمراء |
EP3793618A4 (en) * | 2018-05-18 | 2022-03-02 | Singular Genomics Systems, Inc. | SILICON CONTAINING DETECTABLE COMPOUNDS |
KR102228538B1 (ko) * | 2018-06-01 | 2021-03-15 | 주식회사 엘지화학 | 염료 화합물 및 포토폴리머 조성물 |
EP4055023A4 (en) * | 2019-11-07 | 2024-01-03 | Singular Genomics Systems Inc | DETECTABLE SILICON-CONTAINING COMPOUNDS AND THEIR USES |
CN116715636B (zh) * | 2023-08-04 | 2023-12-05 | 内蒙古大学 | 一种阴离子-π型荧光探针及其制备方法和应用 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU3053599A (en) | 1998-03-31 | 1999-10-25 | Tetsuo Nagano | Reagent for singlet oxygen determination |
WO2002018362A1 (fr) | 2000-08-31 | 2002-03-07 | Daiichi Pure Chemicals Co., Ltd. | Reactif pour determiner l'oxygene singlet |
JP4206378B2 (ja) | 2002-07-08 | 2009-01-07 | 哲雄 長野 | 蛍光プローブ |
WO2010126077A1 (ja) | 2009-04-30 | 2010-11-04 | 国立大学法人 東京大学 | 近赤外蛍光化合物 |
WO2012029609A1 (ja) | 2010-09-02 | 2012-03-08 | 国立大学法人 群馬大学 | 含ケイ素置換基を導入した化合物、並びにそれを含む一重項酸素発生剤及び癌治療薬 |
WO2012111818A1 (ja) * | 2011-02-18 | 2012-08-23 | 国立大学法人 東京大学 | 蛍光プローブ |
CN104519782A (zh) | 2012-08-23 | 2015-04-15 | 思佰益药业股份有限公司 | 具备准直器的光动力学诊断装置 |
JP6351511B2 (ja) | 2013-01-07 | 2018-07-04 | 国立大学法人 東京大学 | 非対称Siローダミン及びロドールの合成 |
-
2015
- 2015-06-17 WO PCT/JP2015/067522 patent/WO2015194606A1/ja active Application Filing
- 2015-06-17 US US15/320,162 patent/US10564164B2/en not_active Expired - Fee Related
- 2015-06-17 JP JP2016529417A patent/JP6275253B2/ja active Active
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20210009919A (ko) * | 2019-07-18 | 2021-01-27 | 아주대학교산학협력단 | 미토콘드리아를 표적으로 하는 신규한 근적외선 형광 프로브 및 이를 포함하는 종양 진단 및 치료 겸용 조성물 |
KR102283067B1 (ko) | 2019-07-18 | 2021-07-28 | 아주대학교산학협력단 | 미토콘드리아를 표적으로 하는 신규한 근적외선 형광 프로브 및 이를 포함하는 종양 진단 및 치료 겸용 조성물 |
Also Published As
Publication number | Publication date |
---|---|
WO2015194606A1 (ja) | 2015-12-23 |
US20170363636A1 (en) | 2017-12-21 |
JPWO2015194606A1 (ja) | 2017-06-29 |
US10564164B2 (en) | 2020-02-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6275253B2 (ja) | 蛍光プローブ、一重項酸素検出剤、又は一重項酸素検出方法 | |
Niu et al. | Highly photostable two-photon NIR AIEgens with tunable organelle specificity and deep tissue penetration | |
Chen et al. | An ESIPT based fluorescence probe for ratiometric monitoring of nitric oxide | |
Zhang et al. | A ratiometric lysosomal pH probe based on the coumarin–rhodamine FRET system | |
JP5090731B2 (ja) | 蛍光プローブ | |
Yu et al. | A TICT based two-photon fluorescent probe for bisulfite anion and its application in living cells | |
Kryman et al. | Synthesis and properties of heavy chalcogen analogues of the Texas reds and related rhodamines | |
Hsieh et al. | Aggregation induced photodynamic therapy enhancement based on linear and nonlinear excited FRET of fluorescent organic nanoparticles | |
Wan et al. | Two spirobifluorene-based fluorescent probes with aggregation-induced emission properties: synthesis and application in the detection of Zn 2+ and cell imaging | |
Zhu et al. | Effect of substituents on Stokes shift of BODIPY and its application in designing bioimaging probes | |
Poon et al. | An amphiphilic ruthenium (II)–polypyridyl appended porphyrin as potential bifunctional two-photon tumor-imaging and photodynamic therapeutic agent | |
JP2017504574A (ja) | ボロンジピロメテン蛍光プローブ、その製造方法及び応用 | |
Zhang et al. | Imidazole functionalized magnesium phthalocyanine photosensitizer: modified photophysics, singlet oxygen generation and photooxidation mechanism | |
US20170015626A1 (en) | Two-photon-absorbing compound | |
Zhang et al. | Indole substituted zinc phthalocyanine: Improved photosensitizing ability and modified photooxidation mechanism | |
Hao et al. | Synthesis of porphyrin-carbohydrate conjugates using" click" chemistry and their preliminary evaluation in human HEp2 cells | |
Guseva et al. | Meso-substituted-BODIPY based fluorescent biomarker: Spectral characteristics, photostability and possibilities for practical application | |
Ma et al. | A novel hydrazino-substituted naphthalimide-based fluorogenic probe for tert-butoxy radicals | |
KR101710899B1 (ko) | 미토콘드리아 및 리소좀 영상화용 이광자 프로브 화합물 및 이를 포함하는 조성물 | |
CN110642882B (zh) | 一种兼具双氧水检测和光动力杀伤癌细胞活性的荧光探针及其制备方法和应用 | |
Wang et al. | Octachloro-fluorescein: Synthesis and photosensitizer performance evaluation | |
Chen et al. | A simple two-photon turn-on fluorescent probe for the selective detection of cysteine based on a dual PeT/ICT mechanism | |
Chen et al. | Two novel two-photon excited fluorescent pH probes based on the A-π-D-π-A system for intracellular pH mapping | |
Wang et al. | Design, synthesis, cell imaging, kinetics and thermodynamics of reaction-based turn-on fluorescent probes for the detection of biothiols | |
Auvray et al. | Silafluorene as a promising core for cell-permeant, highly bright and two-photon excitable fluorescent probes for live-cell imaging |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20161216 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20161216 |
|
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: 20171220 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20180109 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6275253 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
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 |