CN109293698A - 一种基于苯并噻唑的线粒体pH荧光探针及其制备方法 - Google Patents
一种基于苯并噻唑的线粒体pH荧光探针及其制备方法 Download PDFInfo
- Publication number
- CN109293698A CN109293698A CN201811194600.0A CN201811194600A CN109293698A CN 109293698 A CN109293698 A CN 109293698A CN 201811194600 A CN201811194600 A CN 201811194600A CN 109293698 A CN109293698 A CN 109293698A
- Authority
- CN
- China
- Prior art keywords
- preparation
- probe
- benzothiazole
- htbt2
- fluorescence probe
- 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.)
- Granted
Links
- 239000000523 sample Substances 0.000 title claims abstract description 56
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical compound C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 210000003470 mitochondria Anatomy 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Substances C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 210000004027 cell Anatomy 0.000 claims abstract description 16
- DXYYSGDWQCSKKO-UHFFFAOYSA-N 2-methylbenzothiazole Chemical compound C1=CC=C2SC(C)=NC2=C1 DXYYSGDWQCSKKO-UHFFFAOYSA-N 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims abstract description 8
- 238000012544 monitoring process Methods 0.000 claims abstract description 8
- 230000002438 mitochondrial effect Effects 0.000 claims abstract description 7
- 150000003053 piperidines Chemical class 0.000 claims abstract description 6
- PRYNJOJHKYNLIS-UHFFFAOYSA-N 6-hydroxynaphthalene-2-carbaldehyde Chemical compound C1=C(C=O)C=CC2=CC(O)=CC=C21 PRYNJOJHKYNLIS-UHFFFAOYSA-N 0.000 claims abstract description 5
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052794 bromium Inorganic materials 0.000 claims abstract description 5
- SGRHVVLXEBNBDV-UHFFFAOYSA-N 1,6-dibromohexane Chemical compound BrCCCCCCBr SGRHVVLXEBNBDV-UHFFFAOYSA-N 0.000 claims abstract description 4
- JWAZRIHNYRIHIV-UHFFFAOYSA-N 2-naphthol Chemical compound C1=CC=CC2=CC(O)=CC=C21 JWAZRIHNYRIHIV-UHFFFAOYSA-N 0.000 claims abstract description 4
- 235000019441 ethanol Nutrition 0.000 claims abstract description 4
- 238000010992 reflux Methods 0.000 claims abstract description 4
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims abstract description 4
- 229920002554 vinyl polymer Polymers 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000012265 solid product Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000012043 crude product Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229960001866 silicon dioxide Drugs 0.000 claims description 2
- PJKVFARRVXDXAD-UHFFFAOYSA-N 2-naphthaldehyde Chemical compound C1=CC=CC2=CC(C=O)=CC=C21 PJKVFARRVXDXAD-UHFFFAOYSA-N 0.000 claims 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 claims 1
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims 1
- RPGWZZNNEUHDAQ-UHFFFAOYSA-N phenylphosphine Chemical compound PC1=CC=CC=C1 RPGWZZNNEUHDAQ-UHFFFAOYSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 239000011159 matrix material Substances 0.000 abstract description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 abstract 3
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 12
- 229910052698 phosphorus Inorganic materials 0.000 description 7
- RUVJFMSQTCEAAB-UHFFFAOYSA-M 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]benzimidazol-2-ylidene]prop-1-enyl]-3-methyl-1,3-benzoxazol-3-ium;chloride Chemical compound [Cl-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C(N(C1=CC(Cl)=C(Cl)C=C11)CC=2C=CC(CCl)=CC=2)N1CC1=CC=C(CCl)C=C1 RUVJFMSQTCEAAB-UHFFFAOYSA-M 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 230000005284 excitation Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 4
- 238000010226 confocal imaging Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 238000004679 31P NMR spectroscopy Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 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 1
- BBHJTCADCKZYSO-UHFFFAOYSA-N 4-(4-ethylcyclohexyl)benzonitrile Chemical compound C1CC(CC)CCC1C1=CC=C(C#N)C=C1 BBHJTCADCKZYSO-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 229910001914 chlorine tetroxide Inorganic materials 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000002555 ionophore Substances 0.000 description 1
- 230000000236 ionophoric effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Chemical compound [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- 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
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6536—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and sulfur atoms with or without oxygen atoms, as the only ring hetero atoms
- C07F9/6539—Five-membered rings
- C07F9/6541—Five-membered rings condensed with carbocyclic rings or carbocyclic ring systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1011—Condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种基于苯并噻唑的线粒体pH荧光探针及其制备方法和应用。探针具体名称为2‑(2‑(6‑羟基萘‑2‑基)乙烯基)‑3‑(6‑(三苯基膦酰基)己基)苯并噻唑‑3‑溴化物(HTBT2)。其制备方法：先将2‑甲基苯并噻唑与1,6‑二溴己烷在加热条件下反应制备3‑(6‑溴己基)‑2‑甲基苯并噻唑‑3‑溴化物(BMBI)，然后将制备的BMBI与三苯基膦和乙腈混合回流制备2‑甲基‑3‑(6‑(三苯基磷基)己基)苯并噻唑‑3‑溴化物(MTBI)，最后将MTBI和6‑羟基‑2‑萘甲醛溶于乙醇并加入少量哌啶回流后分离提纯得HTBT2。该探针pKa值为8.04±0.02，与线粒体基质(pH～8.0)非常接近。同时，该探针兼具对pH变化的高度灵敏性、良好的选择性和大的Stokes位移等优点。其可用于细胞线粒体内pH变化的监测。
Description
技术领域
本发明涉及线粒体pH荧光探针，具体为一种基于苯并噻唑的线粒体pH荧光探针及其制备方法，及其在监测活细胞线粒体中pH变化方面的应用。
背景技术
线粒体是真核细胞中一种非常重要的细胞器，与细胞***和凋亡，信息传递，离子调节等生理过程息息相关，尤其是细胞代谢所需的大部分能量来源于线粒体的氧化还原过程，因而线粒体也被称为细胞的“能量工厂”。正常生理条件下，线粒体基质内pH(pHmito)值约为8.0。细胞的许多生理活动，如细胞信息传递，Na+/K+/Ca2+平衡，以及活性氧的产生离不开线粒体pHmito平衡，更重要的是，研究表明线粒体自噬和凋亡过程伴随着线粒体酸化。此外，pHmito异常与人类的一些疾病和癌症密切相关，如心血管疾病、神经退行性疾病和雷伊综合征等。因此对线粒体内pHmito进行灵敏准确的实时监测具有非常重要的意义。
近年来，文献报道了一些有机小分子荧光探针用于pHmito变化的检测，但是这些探针大多数的pKa与线粒体pH(～8.0)相差较远，因而灵敏度较低。同时，这些探针大多数的发射位于可见区(<600nm)，样品本身在这一波段具有背景吸收。此外，Stokes位移较小，容易受到激发光源的干扰。因此，有必要开发一种pKa与线粒体pHmito接近的远可见-近红外pH荧光探针用于线粒体的pH变化的监测。
发明内容
本发明的目的之一是提供一种基于苯并噻唑的线粒体pH荧光探针及其制备方法；目的之二是提供该探针的用途，即在监测活细胞线粒体中pH变化方面的应用。
本发明提供的一种基于苯并噻唑的线粒体pH荧光探针，其结构式为：
其合成路线为：
本发明提供的一种基于苯并噻唑的线粒体pH荧光探针的制备方法，包括如下步骤：
(1)将2-甲基苯并噻唑和1,6-二溴己烷按摩尔比1:1.5-10混合后，140℃下于封管内反应5h；待反应冷却至室温后在CH2Cl2中用***析出得到灰色固体产物3-(6-溴己基)-2-甲基苯并噻唑-3-溴化物(BMBI)；
(2)将BMBI和三苯基膦按摩尔比2:3-5溶于少量CH3CN中；将混合溶液在N2保护下回流24h；待溶液冷却后，过滤，将固体溶于CH2Cl2并用水萃取，除去水后得灰色固体产物2-甲基-3-(6-(三苯基磷基)己基)苯并噻唑-3-溴化物(MTBI)；
(3)将6-羟基-2-萘甲醛、MTBI和哌啶按摩尔比12-15:10:1溶于少量乙醇中并回流24h；除去溶剂得到2-(2-(6-羟基萘-2-基)乙烯基)-3-(6-(三苯基膦酰基)己基)苯并噻唑-3-溴化物(HTBT2)的粗品；将HTBT2粗品浓缩后，经硅胶柱分离得到纯品。
步骤(1)中所述的2-甲基苯并噻唑和1,6-二溴己烷的摩尔比优选为1:5。
步骤(2)中所述的BMBI和三苯基膦的摩尔比优选为2:3。
步骤(3)中所述的6-羟基-2-萘甲醛、MTBI和哌啶的摩尔比优选为15:10:1。
本发明的探针HTBT2具有优异的线粒体靶向定位能力，可用于线粒体内pH变化的监测。
与现有的线粒体pH荧光探针相比，本发明合成的探针HTBT2具有以下优点：(1)探针HTBT2的pKa为8.04±0.02，pH线性响应范围为7.20-8.70，使其在监测线粒体pH(～8.0)波动时具有较高的灵敏度；(2)探针的最大荧光发射位于612nm附近，位于远可见-近红外区，能够有效减小对细胞的光损伤和降低生物样品自发荧光的干扰；(3)探针HTBT2具有极大的Stokes位移(～176nm)，能有效降低来自激发光的干扰；(4)探针HTBT2对pH响应具有良好的选择性，不受常见的阴、阳离子，氨基酸活性氧(ROS)和活性氮(RNS)等物质的干扰；(5)该探针具有优异的线粒体靶向定位能力，可利用激光共聚焦成像技术实现对线粒体pH的实时监测；(6)该探针合成步骤简单，成本低廉，具有潜在的商品化应用价值。
附图说明
图1.本发明探针HTBT2随pH值变化的紫外吸收光谱图。
图2.本发明探针HTBT2在自然光下识别OH-前后颜色变化，颜色由浅黄色为黄绿色。
图3.本发明探针HTBT2随pH值变化的荧光发射光谱图。
图4.本发明探针HTBT2在紫外灯下识别OH-前后颜色变化，颜色由无色变为橙红色。
图5.本发明探针HTBT2在612nm处的荧光强度随pH值变化的Boltzmann函数关系，pKa为8.04±0.02。
图6.本发明探针HTBT2的荧光强度随pH值变化线性范围为pH 7.20-8.70。
图7.本发明探针HTBT2在常见阴阳离子和生物体内一些常见氨基酸、活性氧、活性氮等物质存在下，对OH-的选择性。
图8.本发明探针HTBT2在人肝癌细胞(SMMC 7721)中与市售线粒体特异选择性染料MitoTracker Green的共定位成像图。
图9.本发明探针HTBT2分别在pH 7.00，pH 7.40，pH 8.00，pH 8.50和pH 9.00时，与SMMC 7721细胞共同孵育40min的激光共聚焦成像图。
图10.本发明探针HTBT2与SMMC7721细胞共同孵育30min后加入NH4Cl处理，分别在0min、5min、15min、20min、25min和30min时的激光共聚焦成像。
图11.本发明探针HTBT2与SMMC7721细胞共同孵育40min后，分别再加入H2O2和NAC(N-乙酰半胱氨酸)孵育1h后的激光共聚焦成像。
具体实施方式
实施例1
1，化合物3-(6-溴己基)-2-甲基苯并噻唑-3-溴化物(BMBI)、化合物2-甲基-3-(6-(三苯基磷基)己基)苯并噻唑-3-溴化物(MTBI)和探针2-(2-(6-羟基萘-2-基)乙烯基)-3-(6-(三苯基膦酰基)己基)苯并噻唑-3-溴化物(HTBT2)的制备：
(1)将2-甲基苯并噻唑(15mmol，1.90mL)，1,6-二溴己烷(75mmol，12.11mL)的混合溶液在，140℃下于封管内反应5h。待反应冷却至室温后在CH2Cl2中用***析出得到灰色固体产物(5.43g，92％)。1H NMR(400MHz,CDCl3)δ8.32(d,J＝8.1Hz,1H),8.06(d,J＝8.4Hz,1H),7.81(t,J＝7.7Hz,1H),7.69(t,J＝7.7Hz,1H),5.09–4.75(m,2H),3.49(s,3H),3.40(t,J＝6.5Hz,2H),2.25–1.75(m,8H).13C NMR(101MHz,CDCl3)δ175.69,140.93,130.00,129.23,128.67,124.72,116.57,51.00,33.77,32.28,28.67,27.68,25.98,19.30.MS(ESI-MS):m/z Calcd 313.0402[M]+；found 312.0416,314.0387[M]+；
(2)将BMBI(10mmol，3.97g)和三苯基膦(15mmol，3.93g)和150mL CH3CN的混合溶液在N2保护下回流24h。待溶液冷却后，过滤，将固体溶于CH2Cl2并用水萃取，除去水后得灰色固体产物(5.96g，91％)，未经进一步纯化直接用于下一步反应。1H NMR(400MHz,CDCl3)δ8.54(d,J＝8.6Hz,1H),8.19(d,J＝8.0Hz,1H),8.03–7.54(m,17H),5.20–5.01(m,2H),3.78(t,J＝14.4Hz,2H),3.53(s,3H),2.04(d,J＝19.0Hz,2H),1.87(s,4H),1.73(s,2H).13CNMR(101MHz,CDCl3)δ175.31(s),141.10(s),135.05(d,4J(C,P)＝3.0Hz,ArC),133.75(d,J＝10.0Hz,2J(C,P),ArC),130.55(d,3J(C,P)＝12.5Hz,ArC),130.34(s),128.78(s),128.70(s),123.86(s),118.31(d,1J(C,P)＝71.2Hz,ArC),117.81(s),50.83(s),29.11(d,3J(C,P)＝16.8Hz,PCH2CH2CH2),28.35(s),25.13(s),22.46(d,1J(C,P)＝50.4Hz,PCH2),21.93(d,2J(C,P)＝4.4Hz,PCH2 CH2),19.34(s).31P NMR(162MHz,CDCl3)δ24.45.MS(ESI-MS):m/z Calcd247.6069[M]2+；found 247.6070[M]2+
(3)将6-羟基-2-萘甲醛(1.29g,7.5mmol)，MTBI(3.28g,5mmol)和哌啶(0.5mmol)的混合物在30mL乙醇溶液中回流24h。然后将除去溶剂后的初产物并通过柱色谱纯化，含有10％的甲醇的二氯甲烷为洗脱剂得到黄色固体产物(0.93g,23％)。1H NMR(400MHz,DMSO-d6)δ10.02(s,1H),8.39(d,J＝22.8Hz,2H),7.95–7.69(m,3H),7.69–7.49(m,24H),7.24(ddd,J＝84.1,56.2,31.5Hz,1H),3.14(d,J＝14.7Hz,1H),3.06–2.93(m,3H),1.63(dt,J＝11.2,5.6Hz,3H),1.53(dt,J＝10.7,5.4Hz,2H),1.42–0.96(m,1H).31P NMR(162MHz,CDCl3)δ24.45.MS(ESI-MS):m/z Calcd 324.6279[M]2+；found 324.6273[M]2+
实施例2
将实施例1中的探针HTBT2浓度保持在200μM，在不同pH的Tris–盐酸缓冲液(VDMSO:VH2O＝2:1,0.05M)体系中测量其吸收光谱(图1)。随着pH值的从9.30降低至6.40，566nm处的吸收峰逐渐降低，436nm处的吸收峰相应增强，并且在479nm处存在一个等吸收点。溶液的颜色也由原来的黄色变为了黄绿色(图2)。
实施例3
将实施例1中的探针浓度保持在10μM，在不同pH的Tris–盐酸缓冲液(VDMSO:VH2O＝2:1,0.05M)体系中测量其荧光发射光谱，固定激发波长为436nm(图3)。随着pH值由9.30降低到6.40，溶液在612nm处新的荧光发射峰并逐渐增强。紫外灯下溶液的颜色由无色变为橙红色(图4)。通过HTBT2在612nm处的荧光强度值与pH值的Boltzmann函数拟合，计算pKa值为8.04±0.02(图5)，pH响应线性范围为7.20-8.70。线性回归方程为F＝764314.81192-79080.76184×pH,相关系数R2＝0.9991(图6)。
实施例4
将实施例1中的探针浓度保持在10μM，分别考察该探针和常见的阴、阳离子以及生命体中一些氨基酸、ROS和RNS等物质的响应情况。如图7所示，该探针对上述物质几乎没有响应，证明本探针对OH-具优异的选择性。图7中物质的顺序和浓度依次为：1,probe；2,F-(1mM)；3,Cl-(10mM)；4,Br-(1mM)；5,I-(1mM)；6,SO4 2-(1mM)；7,S2O3 2-(1mM)；8,SO3 2-(1mM)；9,HS-(1mM)；10,NO3 -(1mM)；11,NO2 -(1mM)；12,Ac-(1mM)；13,HCO3 -(1mM)；14,ClO4 -(1mM)；15,K+(140mM)；16,Cd2+(1mM)；17,Mg2+(1mM)；18,Li+(1mM)；19,Co2+(1mM)；20,Hg2+(1mM)；21,Ba2+(1mM)；22,Ni2+(1mM)；23,H2O2(1mM)；24,O2 -(1mM)；25,HClO(1mM)；26,ONOO-(100μM)；27,L-GSH(1mM)；28,Hcy(1mM)；29,Cys(1mM)
实施例5
为了确认实施例1中的探针HTBT2是否具有线粒体靶向定位能力，我们首先将探针HTBT2与市售线粒体特异选择性染色MitoTracker Green FM进行共定位实验。在pH 7.40的条件下，将贴壁的SMMC 7721细胞与探针HTBT2(终浓度40μM)在37℃，5％CO2的孵育箱中共同孵育40min，然后用磷酸盐缓冲液(pH 7.40)轻轻洗涤3次，移除多余的探针，再加入MitoTracker Green FM(终浓度2μM)继续孵育5min后，在激光共聚焦显微镜下观察二者的共定位情况。其中，HTBT2固定激发波长为458nm，收集绿色发射范围560-660nm；MitoTracker Green FM固定激发波长为488nm，收集绿色发射范围505-540nm。由图8c可知，HTBT2的荧光(红色)分布于细胞质区域，说明探针具有良好的细胞膜通透性。此外，HTBT2的红色荧光与MitoTracker Green FM的绿色荧光(图8a)能够很好地重叠，经软件处理得到黄色荧光(图8d)，表明HTBT2与MitoTracker Green FM具有显著的共定位成像，能够靶向定位于线粒体中。明场成像进一步证实了经HTBT2孵育后细胞的存活性(图8c)，说明HTBT2对细胞具有低毒性。
实施例6
将贴壁的SMMC 7721细胞与实施例1中的探针HTBT2在pH 7.40的条件下，于37℃、5％CO2的孵育箱中共同孵育40min，然后用磷酸盐缓冲液(pH 7.40)轻轻洗涤3次，除去多余的HTBT2，再分别利用pH 7.00、7.40、8.00、8.50和9.00的高K+缓冲液(30mM NaCl、120mmKCl、1mM CaCl2、0.5mM MgSO4、1mM NaH2PO4、5mM葡萄糖、20mM HEPES和20mM NaOAC)和H+/K+离子载体--尼日尼亚菌素继续处理10min，在激光共聚焦显微镜下观察。固定激发波长为458nm，收集红色荧光发射范围560-660nm。pH 9.00时红色通道几乎观察不到荧光(图9o)；随着pH降至7.00时，红色通道逐渐观察到明亮的荧光(图9l,i,f,c)。明场成像进一步证实了经HTBT2孵育后细胞的存活性(图9a,d,g,j,m)。
实施例7
为了证明实施案例1中的探针HTBT2具有对细胞内线粒体pH变化的快速响应能力，我们分别使用NH4Cl(5mM)、H2O2(0.1mM)和NAC(0.5mM)处理被HTBT2染色的SMMC 7721细胞。用NH4Cl处理后的HTBT2细胞荧光逐渐减弱，说明NH4Cl处理后的SMMC7721细胞的线粒体pH逐渐升高(图10)。如图11所示，经H2O2处理后的SMMC7721细胞相较于未处理的细胞pH明显降低，而经NAC处理的细胞pH明显增加。这些结果表明HTBT2能对细胞内线粒体pH的变化做出快速响应。
Claims (6)
1.一种基于苯并噻唑的线粒体pH荧光探针，其特征在于结构式为：
2.如权利要求1所述的pH荧光探针的制备方法，其特征在于包括如下步骤：
(1)将2-甲基苯并噻唑和1,6-二溴己烷按摩尔比1:1.5-10混合后，140℃下于封管内反应5h；待反应冷却至室温后在CH2Cl2中用***析出得到灰色固体产物3-(6-溴己基)-2-甲基苯并噻唑-3-溴化物(BMBI)；
(2)将BMBI和三苯基膦按摩尔比2:3-5溶于少量CH3CN中；将混合溶液在N2保护下回流24h；待溶液冷却后，过滤，将固体溶于CH2Cl2并用水萃取，除去水后得灰色固体产物2-甲基-3-(6-(三苯基磷基)己基)苯并噻唑-3-溴化物(MTBI)；
(3)将6-羟基-2-萘甲醛、MTBI和哌啶按摩尔比12-15:10:1溶于少量乙醇中并回流24h；除去溶剂得到2-(2-(6-羟基萘-2-基)乙烯基)-3-(6-(三苯基膦酰基)己基)苯并噻唑-3-溴化物(HTBT2)的粗品；将HTBT2粗品浓缩后，经硅胶柱分离得到纯品。
3.如权利要求2所述的pH荧光探针的制备方法，其特征在于步骤(1)中所述的2-甲基苯并噻唑和1,6-二溴己烷的摩尔比为1:5。
4.如权利要求2所述的pH荧光探针的制备方法，其特征在于步骤(2)中所述的BMBI和三苯基膦的摩尔比为2:3。
5.如权利要求2所述的pH荧光探针的制备方法，其特征在于步骤(3)中所述的6-羟基-2-萘甲醛、MTBI和哌啶的摩尔比为15:10:1。
6.如权利要求1所述的pH荧光探针用于细胞线粒体内pH变化的监测。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811194600.0A CN109293698B (zh) | 2018-10-15 | 2018-10-15 | 一种基于苯并噻唑的线粒体pH荧光探针及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811194600.0A CN109293698B (zh) | 2018-10-15 | 2018-10-15 | 一种基于苯并噻唑的线粒体pH荧光探针及其制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109293698A true CN109293698A (zh) | 2019-02-01 |
| CN109293698B CN109293698B (zh) | 2021-02-02 |
Family
ID=65162676
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811194600.0A Active CN109293698B (zh) | 2018-10-15 | 2018-10-15 | 一种基于苯并噻唑的线粒体pH荧光探针及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109293698B (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234040A (zh) * | 2021-05-28 | 2021-08-10 | 中国科学院新疆理化技术研究所 | 一种用于检测pH荧光探针分子及其制备方法 |
| CN114133413A (zh) * | 2021-11-04 | 2022-03-04 | 广东工业大学 | 一种苯并噻唑-三苯胺化合物及其制备方法与应用 |
| WO2023045909A1 (zh) * | 2021-09-22 | 2023-03-30 | 杭州天玑济世生物科技有限公司 | 一类具有萘胺结构的小分子化合物及其应用 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282334A (zh) * | 1997-11-25 | 2001-01-31 | 奥塔戈大学 | 线粒体靶向的抗氧剂 |
| CN104531139A (zh) * | 2015-01-06 | 2015-04-22 | 山西大学 | 一种咔唑类的pH荧光探针及其制备方法和应用 |
| CN106565596A (zh) * | 2016-10-28 | 2017-04-19 | 山西大学 | 一种萘基衍生物作为线粒体靶向型pH荧光探针的应用 |
| CN106632138A (zh) * | 2016-09-23 | 2017-05-10 | 济南大学 | 一种识别肼的小分子荧光探针及其应用 |
| CN108485653A (zh) * | 2018-05-10 | 2018-09-04 | 河南科技大学 | 同时检测过氧化氢和过氧化亚硝基阴离子的近红外荧光探针及其合成方法和应用 |
-
2018
- 2018-10-15 CN CN201811194600.0A patent/CN109293698B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282334A (zh) * | 1997-11-25 | 2001-01-31 | 奥塔戈大学 | 线粒体靶向的抗氧剂 |
| CN104531139A (zh) * | 2015-01-06 | 2015-04-22 | 山西大学 | 一种咔唑类的pH荧光探针及其制备方法和应用 |
| CN106632138A (zh) * | 2016-09-23 | 2017-05-10 | 济南大学 | 一种识别肼的小分子荧光探针及其应用 |
| CN106565596A (zh) * | 2016-10-28 | 2017-04-19 | 山西大学 | 一种萘基衍生物作为线粒体靶向型pH荧光探针的应用 |
| CN108485653A (zh) * | 2018-05-10 | 2018-09-04 | 河南科技大学 | 同时检测过氧化氢和过氧化亚硝基阴离子的近红外荧光探针及其合成方法和应用 |
Non-Patent Citations (3)
| Title |
|---|
| BO LIN等: "A naphthalene-based fluorescent probe with a large Stokes shift for mitochondrial pH imaging", 《ANALYST》 * |
| HAO WANG等: "A dual-site fluorescent probe for separate detection of hydrogen sulfide and bisulfide", 《DYES AND PIGMENTS》 * |
| XIUQIONG CHEN等: "A ratiometric fluorescent probe for palladium detection based on an allyl carbonate group functionalized hemicyanine dye", 《TETRAHEDRON LETTERS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234040A (zh) * | 2021-05-28 | 2021-08-10 | 中国科学院新疆理化技术研究所 | 一种用于检测pH荧光探针分子及其制备方法 |
| CN113234040B (zh) * | 2021-05-28 | 2022-03-25 | 中国科学院新疆理化技术研究所 | 一种用于检测pH荧光探针分子及其制备方法 |
| WO2023045909A1 (zh) * | 2021-09-22 | 2023-03-30 | 杭州天玑济世生物科技有限公司 | 一类具有萘胺结构的小分子化合物及其应用 |
| CN114133413A (zh) * | 2021-11-04 | 2022-03-04 | 广东工业大学 | 一种苯并噻唑-三苯胺化合物及其制备方法与应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109293698B (zh) | 2021-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Fluorescence imaging of SO2 derivatives in Daphnia magna with a mitochondria-targeted two-photon ratiometric fluorescent probe | |
| Zhu et al. | A highly specific and ultrasensitive two-photon fluorescent probe for imaging native hypochlorous acid in living cells | |
| CN105693591B (zh) | 一种比率型pH荧光探针及其制备方法与应用 | |
| CN109293698A (zh) | 一种基于苯并噻唑的线粒体pH荧光探针及其制备方法 | |
| Qu et al. | Construction of a novel far-red fluorescence light-up probe for visualizing intracellular peroxynitrite | |
| CN107056769A (zh) | 一种l‑半胱氨酸荧光探针及其制备方法 | |
| Nie et al. | Dual-site fluorescent probe to monitor intracellular nitroxyl and GSH-GSSG oscillations | |
| CN110003060A (zh) | 一种丙二腈衍生物类近红外硫化氢荧光探针及其制备方法与应用 | |
| CN106977450B (zh) | 一种萘基双光子荧光探针及其制备方法和应用 | |
| CN110698454B (zh) | 一种异佛尔酮类硫化氢荧光探针及其制备方法与应用 | |
| Chen et al. | A highly sensitive fluorescent probe based on the Michael addition mechanism with a large Stokes shift for cellular thiols imaging | |
| CN113501818B (zh) | 一种荧光探针分子及其制备方法与应用 | |
| Fang et al. | Coumarinocoumarin-based fluorescent probe for the sensitive and selective detection of hydrazine in living cells and zebra fish | |
| CN102344449B (zh) | 并杂环萘酰亚胺及其制备方法与应用 | |
| CN106632212A (zh) | 一种检测细胞内半胱氨酸的荧光探针 | |
| Fu et al. | The unique substitution-cyclization reaction cascade inspired highly selective H2Sn probe development | |
| CN106518855B (zh) | 一种以半川菁及黄酮醇为荧光团的二氧化硫衍生物比例荧光探针及其应用 | |
| Cui et al. | A rhodamine B-based turn on fluorescent probe for selective recognition of mercury (II) ions | |
| Wu et al. | Novel near-infrared frequency up-conversion luminescence probe for monitoring biothiols in vitro and in vivo | |
| Zeng et al. | A fluorescent sensor for selective detection of hypochlorite and its application in Arabidopsis thaliana | |
| Han et al. | A ratiometric fluorescent probe for monitoring the changes in the level of hydrogen polysulfides in mitochondria during stimulus-induced redox imbalance | |
| He et al. | A water-soluble two-photon fluorescent probe for rapid and reversible monitoring of redox state | |
| CN108640902A (zh) | 一种识别纯水体系内二氧化硫的荧光探针及其应用 | |
| CN106977585B (zh) | 一种线粒体定位用于光动力疗法的双光子荧光探针库及其应用 | |
| CN108641710A (zh) | 一种检测蛋白质硫巯基化的荧光探针及其制备方法和应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |