CN112624960A - 基于苯并吲哚的ntr-1响应型荧光探针、制备方法及用途 - Google Patents
基于苯并吲哚的ntr-1响应型荧光探针、制备方法及用途 Download PDFInfo
- Publication number
- CN112624960A CN112624960A CN202011479265.6A CN202011479265A CN112624960A CN 112624960 A CN112624960 A CN 112624960A CN 202011479265 A CN202011479265 A CN 202011479265A CN 112624960 A CN112624960 A CN 112624960A
- Authority
- CN
- China
- Prior art keywords
- ntr
- nfp
- probe
- benzindole
- fluorescent 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
- 230000004044 response Effects 0.000 title claims abstract description 31
- 102000020232 neurotensin type 1 receptor Human genes 0.000 title claims abstract description 23
- 108010016501 neurotensin type 1 receptor Proteins 0.000 title claims abstract description 23
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 22
- HIYWOHBEPVGIQN-UHFFFAOYSA-N 1h-benzo[g]indole Chemical compound C1=CC=CC2=C(NC=C3)C3=CC=C21 HIYWOHBEPVGIQN-UHFFFAOYSA-N 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 239000000523 sample Substances 0.000 claims abstract description 70
- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 claims abstract description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 9
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 7
- VOLRSQPSJGXRNJ-UHFFFAOYSA-N 4-nitrobenzyl bromide Chemical compound [O-][N+](=O)C1=CC=C(CBr)C=C1 VOLRSQPSJGXRNJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 27
- 238000001514 detection method Methods 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 210000004881 tumor cell Anatomy 0.000 claims description 5
- 238000003384 imaging method Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 230000001413 cellular effect Effects 0.000 claims description 2
- 206010021143 Hypoxia Diseases 0.000 abstract description 9
- 230000007954 hypoxia Effects 0.000 abstract description 5
- 238000013399 early diagnosis Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 102100033986 Neurotensin receptor type 1 Human genes 0.000 description 64
- 238000006243 chemical reaction Methods 0.000 description 25
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 21
- 239000007787 solid Substances 0.000 description 19
- 238000002189 fluorescence spectrum Methods 0.000 description 18
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 17
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 10
- 238000001914 filtration Methods 0.000 description 10
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 238000001035 drying Methods 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000012295 chemical reaction liquid Substances 0.000 description 6
- 231100000135 cytotoxicity Toxicity 0.000 description 6
- 230000003013 cytotoxicity Effects 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000000799 fluorescence microscopy Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 4
- 102000004459 Nitroreductase Human genes 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 239000012065 filter cake Substances 0.000 description 4
- 230000001146 hypoxic effect Effects 0.000 description 4
- 108020001162 nitroreductase Proteins 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000000967 suction filtration Methods 0.000 description 4
- BYZKRKXJSNSHEE-UHFFFAOYSA-N 1-ethyl-2-benzo[cd]indolone Chemical compound C1=CC(N(CC)C2=O)=C3C2=CC=CC3=C1 BYZKRKXJSNSHEE-UHFFFAOYSA-N 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 239000003208 petroleum Substances 0.000 description 3
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 101001018064 Homo sapiens Lysosomal-trafficking regulator Proteins 0.000 description 2
- 102100033472 Lysosomal-trafficking regulator Human genes 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 235000010703 Modiola caroliniana Nutrition 0.000 description 2
- 244000038561 Modiola caroliniana Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical compound C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 description 2
- CCERQOYLJJULMD-UHFFFAOYSA-M magnesium;carbanide;chloride Chemical compound [CH3-].[Mg+2].[Cl-] CCERQOYLJJULMD-UHFFFAOYSA-M 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- OKKJLVBELUTLKV-VMNATFBRSA-N methanol-d1 Chemical compound [2H]OC OKKJLVBELUTLKV-VMNATFBRSA-N 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000004537 pulping Methods 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000012312 sodium hydride Substances 0.000 description 2
- 229910000104 sodium hydride Inorganic materials 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 238000004435 EPR spectroscopy Methods 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 125000003118 aryl group Chemical class 0.000 description 1
- 230000004791 biological behavior Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical group C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 1
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical group OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000000623 heterocyclic group Chemical class 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/80—[b, c]- or [b, d]-condensed
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- C09K2211/1014—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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
技术领域
本发明涉及荧光探针、制备方法及用途,具体涉及基于苯并吲哚的NTR-1响应型荧光探针、制备方法及用途。
背景技术
硝基还原酶(NTR)属于黄素酶,通常含有黄素单核苷酸单元或黄素腺嘌呤二核苷酸单元。在NADPH或NADH存在时,硝基还原酶可以将硝基取代杂环和芳香环上的硝基还原,生成相应的亚硝酸盐、羟胺或氨基衍生物。已有研究证明,NTR活性与细胞内含氧量相关。在缺氧的细胞或组织中,能观察到NTR的过表达。而缺氧是肿瘤细胞快速增殖的重要病理过程,已经成为实体瘤的主要特征。缺氧微环境在肿瘤发生和转移过程中起着重要作用,可以促进恶性生物学行为,包括肿瘤细胞增殖、侵袭、转移和凋亡。因此,NTR的含量可以反映肿瘤中O2水平。另外,已有报道称NTR在药物筛选和临床诊断等领域都有很好的应用前景。因此,检测肿瘤细胞和组织中硝基还原酶活性在研究其生物功能和临床诊疗中具有重要的意义。
传统的NTR检测方法包括:电子顺磁共振法(EPR)、核磁共振法(NMR)和克拉克电极法等。但是这些方法大都需要复杂精密的仪器,样品处理复杂,并且不能实时监测,分辨率较低,这在很大程度上限制了传统检测方法的应用。而荧光成像技术的出现使NTR的检测找到了新方向。尽管近年来,已经有一系列用于检测NTR的有机小分子荧光探针被开发出来,但是这些探针仍存在稳定性差、灵敏度较低、荧光量子产率较低、受细胞自身条件影响大等缺点,因此开发出结构新颖,性能更好的新型有机小分子荧光探针仍具有重要意义。
发明内容
发明目的:本发明目的是提供可快速、高灵敏度检测NTR的荧光探针。该探针以EMC作为探针骨架,引入芳香族硝基作为NTR特异性识别基团。当以烟酰胺腺嘌呤二核苷酸(NADH)为电子供体时,探针NFP-1能与NTR发生特异性反应,表现出显著的荧光增强特性。该探针具有较好的稳定性,选择性高,细胞毒性较小,响应时间为25 min,检测限低至17ng/mL。细胞成像实验证明,探针NFP-1能对活细胞本发明基于 NTR可以作为衡量细胞缺氧程度的重要指标,有助于肿瘤早期诊断。
本发明另一目的是提供所述探针的制备方法和用途。
技术方案:基于苯并吲哚的NTR-1响应型荧光探针,结构如下:
进一步地,根据权利要求1所述的基于苯并吲哚的NTR-1响应型荧光探针,其特征在于:检测限为17ng/mL。
进一步地,在不同的pH值范围内,探针NTR-1具有不同的荧光强度。
基于苯并吲哚的NTR-1响应型荧光探针的制备方法,先将对羟基苯甲醛与对硝基溴化苄反应合成以醚键连接的稳定中间体NFP-1-M,然后再将1-乙基-2-甲基苯并[cd] 吲哚-1-氯化物与中间体NFP-1-M缩合,生成探针NFP-1,工艺如下:
具体地:
(1)1-乙基-2-甲基苯并[cd]吲哚-1-氯化物(EMC)的合成:
1,8-萘内酰亚胺溶于DMF中,加入氢化钠。将体系升温至室温后,逐滴加入碘乙烷并于室温下搅拌。用乙酸乙酯萃取反应混合物,用饱和食盐水洗涤有机层并用无水硫酸钠干燥,过滤后减压浓缩滤液,所得固体通过硅胶柱层析分离纯化,得到1-乙基苯并[cd] 吲哚-2(1H)-酮。
将1-乙基苯并[cd]吲哚-2(1H)-酮溶于无水THF中,逐滴加入甲基氯化镁。在氮气保护下,混合溶液加热回流,经TLC监测反应完全。将反应液冷却到室温,缓慢滴加少量水猝灭,之后加入少量NaOH溶液将反应液的pH调至碱性,用乙酸乙酯萃取三次,向合并的有机相中加入少量HCl溶液将pH调至酸性,水萃取,向合并的水相中加入大量丙酮并搅拌,有大量固体析出。抽滤,滤饼用丙酮洗涤后烘干,得到EMC。
(2)探针NFP-1的合成:
将对羟基苯甲醛和K2CO3溶于干燥的DMF中,升温搅拌,再加入对硝基溴化苄和碘化钾,继续搅拌反应完全。将体系冷却至室温后加入乙酸乙酯萃取,合并有机相并用无水硫酸钠干燥,过滤后减压浓缩滤液,所得固体溶解在少量CH2Cl2中,加入石油醚搅拌,有固体析出,抽滤后固体干燥,得到中间体NFP-1-M。
将NFP-1-M和化合物EMC溶于乙醇/乙腈的混合溶液中,室温下避光搅拌过夜,至反应完全。将混合液过滤,滤液中缓慢加入适量甲基叔丁基醚后,有固体析出,过滤后收集固体,并加THF打浆除去杂质,再次过滤,用少量THF洗涤滤饼,烘干,得到产物NFP-1(紫红色固体)。
所述的基于苯并吲哚的NTR-1响应型荧光探针在制备NTR-1检测试剂中的用途。
进一步地,所述NTR-1检测试剂用于活细胞中NTR检测。
进一步地,所述活细胞为肿瘤细胞。
所述的基于苯并吲哚的NTR-1响应型荧光探针在细胞成像中的用途。
所述的基于苯并吲哚的NTR-1响应型荧光探针在制备肿瘤诊断试剂中的用途。
本发明研究结果显示在NTR浓度为0~2μg/mL范围内,探针NFP-1的I490nm与NTR 浓度呈良好的线性关系(R2=0.9915)。NFP-1的检测限为17ng/mL,灵敏度较高,能对低浓度的NTR进行精确定量的检测。探针NFP-1的稳定性较好,加入NTR后探针的 I490nm随响应时间的增加而增强,能在25min内与NTR完全反应。此外,探针NFP-1 适用于生理pH条件下NTR的检测。并且,其他分析物不会导致NFP-1的荧光发生明显的改变,只有与NTR响应时探针NFP-1才会表现出显著的荧光增强响应。探针NFP-1 对NTR具有较高的选择性。
细胞毒性实验结果表明探针NFP-1细胞毒性较小。细胞荧光成像实验结果显示,常氧条件下培养的细胞中几乎没有发现荧光信号,而缺氧条件下培养的细胞中,在绿色荧光通道观察到较强的荧光信号。荧光成像结果证明,探针NFP-1能够成像活细胞中的内源性NTR,在肿瘤诊断方面有较好的应用潜力。
有益效果:
1.本发明的荧光增强型小分子探针NFP-1。当存在NADH作为电子供体时,该探针能够特异性识别NTR,表现出显著的荧光增强。
2.探针NFP-1的稳定性较好,检测限为17ng/mL,能在25min内实现对NTR的快速、高灵敏度的检测。
3.细胞实验证明,探针NFP-1具有较小的细胞毒性,并且能成功检测活细胞中缺氧诱导产生的NTR。
附图说明
图1探针NFP-1(10μM)与NTR(5μg/mL)和/或NADH(500μM)反应后的a紫外-可见光吸收光谱和b荧光光谱,c存在NADH(500μM)时,探针NFP-1(10μM)与不同浓度NTR响应后的荧光光谱,dNFP-1的荧光强度I490nm随NTR浓度(0~5μg/mL)的变化,插图:I490nm与NTR(0~2μg/mL) 的线性相关,λex=420nm;
图2探针NFP-1(10μM)与/不与NTR(5μg/mL)响应不同时间(0~70 min)后490nm处的荧光强度(I490nm),λex=420nm;
图3不同pH下,探针NFP-1(10μM)与/不与NTR(5μg/mL)响应后 490nm处的荧光强度,λex=420nm;
图4为探针NFP-1与不同分析物反应后的荧光光谱,图4a为探针NFP-1 与不同分析物反应后的荧光光谱,图4b为图4a中490nm处NFP-1与不同分析物反应后的荧光强度,(1)blank;(2)K+(1mM);(3)Na+(1mM);(4)Mg2+(1mM); (5)·OtBu(500μM);(6)TBHP(500μM);(7)ClO-(500μM);(8)O2 ·-(500μM);(9)H2O2 (500μM);(10)Pro(1mM);(11)Gly(1mM);(12)Lys(1mM);(13)Leu(1mM);(14)Ser(1 mM);(15)GSH(1mM);(16)Cys(1mM);(17)NTR(5μg/mL).λex=420nm;
图5探针NFP-1(10μM)对HeLa细胞的MTT分析结果;
图6不同氧气条件下培养的HeLa细胞与NFP-1(10μM)共培养后的共聚焦图像, a、b、c:常氧(20%O2);d、e、f:低氧(1%O2),Green Channel:492nm~577nm,λex=420nm.Scalebar:10μm。
具体实施方式
实施例1探针NFP-1的合成:
1. 1-乙基-2-甲基苯并[cd]吲哚-1-氯化物(EMC)的合成
1,8-萘内酰亚胺(8.03g,47.4mmol)溶于100mL DMF中,在0℃下缓慢加入氢化钠(3.528g,150mmol)。将体系升温至室温后,逐滴加入碘乙烷(7.403g,50mmol) 并于室温下搅拌1h。用乙酸乙酯(150mL×2)萃取反应混合物,用饱和食盐水洗涤有机层并用无水硫酸钠干燥,过滤后减压浓缩滤液,所得固体通过硅胶柱层析(石油醚∶乙酸乙酯=10∶1)分离纯化,得到1-乙基苯并[cd]吲哚-2(1H)-酮3(7.762g黄色固体,收率83%),直接用于下一步反应。
将3(1.972g,10mmol)溶于40mL无水THF(40mL)中,逐滴加入甲基氯化镁 (15mL,45mmol)。在氮气保护下,混合溶液60℃加热回流,经TLC监测反应2h 反应完全。将反应液冷却到室温,缓慢滴加少量水猝灭,之后加入少量NaOH溶液(1M) 将反应液的pH调至碱性,用乙酸乙酯萃取三次,向合并的有机相中加入少量HCl溶液 (1M)将pH调至酸性,用少量水萃取三次,向合并的水相中加入大量丙酮并搅拌,有大量固体析出。抽滤,滤饼用丙酮洗涤后置于40℃烘箱中烘干,得到1.753g EMC (绿色固体),收率76%。m.p.159℃~160℃;1H NMR(400MHz,MeOD)δ8.93(d,J= 7.3Hz,1H),8.79(d,J=8.1Hz,1H),8.47(d,J=6.3Hz,1H),8.46(d,J=7.1Hz,1H),8.23 -8.16(m,1H),8.07-8.02(m,1H),4.83-4.79(m,2H),3.36-3.34(m,3H),1.72(t,J=7.4 Hz,3H).13C NMR(100MHz,D2O):δ170.68,138.33,137.25,134.31,130.84,130.48, 129.40,128.08,127.73,121.41,120.40,42.29,14.51,12.24.MS[ES-API]:calcd for C14H14N+,196.1,found:196.1[M]+。
2.中间体NFP-1-M的合成:
将对羟基苯甲醛(0.244g,2mmol)和K2CO3(0.277g,2mmol)溶于15ml干燥的DMF中,升温至50℃搅拌10min,再加入对硝基溴化苄(0.518g,2.4mmol)和碘化钾(0.332g,2mmol),继续搅拌4h反应完全。将体系冷却至室温后加入20ml乙酸乙酯萃取2次,合并有机相并用无水硫酸钠干燥,过滤后减压浓缩滤液,所得固体溶解在少量CH2Cl2中,加入石油醚搅拌,有固体析出,抽滤后固体在40℃烘箱中烘干,得到0.375g中间体粗品NFP-1-M(淡黄色固体),直接用于下一步反应。
3.NFP-1的合成:
将粗品NFP-1-M(0.154g,0.6mmol)和化合物EMC(0.116g,0.5mmol)溶于 10mL乙醇/乙腈(9∶1)的混合溶液中,室温下避光搅拌过夜,至反应完全。将混合液过滤,滤液中缓慢加入适量甲基叔丁基醚后,有固体析出,过滤后收集固体,并加1mL THF打浆除去杂质,再次过滤,用少量THF洗涤滤饼,在40℃烘箱中烘干,得到0.198 g产物NFP-1(紫红色固体),收率52%。1H NMR(400MHz,DMSO):δ9.37(s,1H),8.85 (d,J=14.1Hz,1H),8.72(s,1H),8.39(s,2H),8.31(d,J=6.4Hz,4H),8.16(s,1H),7.95(d, J=13.2Hz,2H),7.79(d,J=7.6Hz,2H),7.31(d,J=6.4Hz,2H),5.48(s,2H),4.88(s,2H), 1.52(s,3H).13C NMR(101 MHz,MeOD):δ163.08,162.27,153.82,147.70,143.99,138.87, 136.71,134.53,132.61,130.76,129.88,129.59,128.66,127.83,123.70,123.31,118.34, 115.73,111.71,68.71,41.27,14.29.MS[ES-API]:calcd for C28H23N2O3 +,435.2,found: 435.2[M]+。
实施例2探针NFP-1的光谱性质:
探针NFP-1的所有紫外-可见光吸收光谱和荧光光谱都在溶液DMSO-PBS(1∶9 v/v,10mM,,pH=7.4)中记录。在λex=420nm下收集溶液在430nm~700nm的荧光发射,激发和发射狭缝宽度为5nm/5nm。NFP-1在DMSO中制得1mM母液再稀释使用。NTR溶于超纯水中制成100μg/mL母液备用。如未经特殊说明,所有反应体系中都加入了500μM NADH。
在NFP-1(10μM)中加入NADH(500μM),再与不同浓度的NTR(0-10μg/mL) 37℃孵育30min,记录紫外-可见光吸收光谱和荧光光谱。在选择性研究中,将NFP-1 (10μM)与NTR(5μg/mL)、活性氧(500μM)、部分金属离子(1mM),氨基酸 (1mM)在37℃下孵育30min,然后记录它们的荧光光谱。在动力学响应实验中,记录NFP-1(10μM)与/不与NTR(5μg/mL)孵育的不同时间点(0,1,2,3,4,5,10,15,20,25, 30,40,50,60,70min)的荧光光谱。在pH响应研究中,探针NFP-1(10μM)和NTR(5 μg/mL)在不同pH(1~14)下37℃孵育30min,记录其荧光光谱。
探针NFP-1与NTR和NADH反应前后的紫外吸收光谱和荧光光谱如图1所示。图 1a为紫外-可见光吸收光谱,图1b为荧光光谱,图1c为存在NADH(500μM) 时,探针NFP-1(10μM)与不同浓度NTR响应后的荧光光谱,图1d为NFP-1 的荧光强度I490nm随NTR浓度(0~5μg/mL)的变化,λex=420nm。图1a表明,当探针中只加入NADH(500μM)或NTR(5μg/mL)时,反应体系的吸收峰没有明显的变化,都在500nm左右;而当NADH和NTR同时存在时,在440nm出现一个新的最大吸收峰。图1b表明420nm激发下,探针本身荧光非常弱,几乎没有,仅加入NADH 或NTR也没有显著改变反应体系的荧光强度,而NTR和NADH同时存在时,才能在 490nm附近观察到显著的荧光增强。以上分析表明,当NADH存在时,探针NFP-1上的硝基才能被NTR还原,反应体系表现出荧光增强响应。因此,在后续的荧光响应实验中,反应体系中都加入了500μM NADH作为电子供体。
为了评价探针NFP-1对NTR的浓度响应,记录了NFP-1与不同浓度NTR(0-10 μg/mL)反应后的荧光光谱。如图1c所示,随着NTR浓度的增加,探针NFP-1在490nm 处的荧光强度(I490nm)逐渐增强并且在5μg/mL达到饱和。如图1d所示,在NTR浓度为0~2μg/mL范围内,探针的I490nm与NTR浓度呈良好的线性关系(R2=0.9915)。以上结果表明,探针NFP-1能够对一定浓度的NTR进行有效的定量检测。
对探针NFP-1(10μM)(空白组)在420nm激发下的荧光强度进行3次测定,获得空白组在490nm处荧光强度的标准偏差。根据荧光响应中NFP-1(10μM)与NTR 浓度(0~2μg/mL)的线性回归方程计算检测限。计算公式如下:
DL=3σ/k
其中σ是空白测量的标准偏差,k是NFP-1(10μM)与NTR(0-2μg/mL)反应后 490nm处的荧光强度与不同NTR浓度之间的斜率。根据公式计算出探针NFP-1的检测限为17ng/mL,说明探针NFP-1灵敏度较高,能对低浓度的NTR进行精确定量的检测。
实施例3探针NFP-1的动力学响应:
为了研究探针NFP-1对NTR的动力学响应,记录了探针NFP-1(10μM) 与NTR(5μg/mL)响应不同时间内(0~70min)的荧光强度I490nm的变化。如图2所示。结果显示,探针NFP-1的稳定性较好,加入NTR后探针的I490nm随响应时间的增加而增强,并且在25min达到平台期。以上分析表明,探针NFP-1能在25min内与NTR完全反应。为了确保反应完全进行,在所有响应实验中选择的反应时间为30min。
实施例4探针NFP-1的pH响应:
pH是影响探针荧光响应性能的一个重要因素,因此实验中通过记录不同 pH下NFP-1与NTR(5μg/mL)反应后的荧光光谱来评估pH对探针荧光强度 I490nm的影响。结果如图3所示。结果显示,不同pH下探针NFP-1荧光强度都没有发生显著的改变,只表现出很弱的发射强度,表明探针本身很稳定,背景干扰很小。加入NTR后,探针NFP-1在pH 6~10都表现出了极强的荧光发射,并在pH=7附近达到最大值。结果表明探针NFP-1适用于生理pH条件下NTR的检测。
实施例5探针NFP-1选择性研究:
性能优良的荧光探针能特异识别目标待测物质,并且不受其他活性物质干扰。因此为了评价探针NFP-1的选择性,比较了NFP-1与NTR以及其他分析物反应后的的荧光光谱,记录了探针NFP-1加入不同分析物(K+、Na+、 Mg2+、·OtBu、TBHP、ClO-、O2 ·-、H2O2、Pro、Gly、Lys、Leu、Ser、GSH、 Cys)后的荧光光谱变化。结果如图4所示,图4a为探针NFP-1与不同分析物反应后的荧光光谱,图4b为图4a中490nm处NFP-1与不同分析物反应后的荧光强度。结果表明,与对照组相比,其他分析物不会导致NFP-1的荧光发生明显的改变,只有与NTR响应时探针NFP-1才会表现出显著的荧光增强响应。因此,探针NFP-1对NTR具有较高的选择性。
实施例6细胞毒性实验:
采用MTT法评估了探针NFP-1对HeLa细胞的细胞毒性。如图5所示,探针NFP-1 浓度在25μM时细胞的存活率依旧在90%以上,表明探针NFP-1细胞毒性较小。
实施例7细胞荧光成像实验:
在成像实验前,将实验组HeLa细胞接种在培养皿中置于缺氧条件(1%O2)下培养12h,以促进细胞中的NTR过表达。对照组细胞在常氧条件(20%O2)下培养12h。然后两组细胞分别与探针NFP-1(10μM)孵育30min,PBS缓冲溶液漂洗三次后使用激光共聚焦显微镜拍摄荧光图像。结果如图6所示。实验结果表明,常氧条件下培养的细胞中几乎没有发现荧光信号(图6a~c),而缺氧条件下培养的细胞中,在绿色荧光通道观察到较强的荧光信号(图6d~f)。荧光成像结果证明,探针NFP-1能够成像活细胞中的内源性NTR,在肿瘤诊断方面有较好的应用潜力。
Claims (9)
2.根据权利要求1所述的基于苯并吲哚的NTR-1响应型荧光探针,其特征在于:检测限为17ng/mL。
3.根据权利要求1所述的基于苯并吲哚的NTR-1响应型荧光探针,其特征在于:在不同的pH值范围内,探针NTR-1具有不同的荧光强度。
5.权利要求1所述的基于苯并吲哚的NTR-1响应型荧光探针在制备NTR-1检测试剂中的用途。
6.根据权利要求5所述的用途,其特征在于:所述NTR-1检测试剂用于活细胞中NTR检测。
7.根据权利要求6所述的用途,其特征在于:所述活细胞为肿瘤细胞。
8.权利要求1所述的基于苯并吲哚的NTR-1响应型荧光探针在细胞成像中的用途。
9.权利要求1所述的基于苯并吲哚的NTR-1响应型荧光探针在制备肿瘤诊断试剂中的用途。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011479265.6A CN112624960B (zh) | 2020-12-15 | 2020-12-15 | 基于苯并吲哚的ntr-1响应型荧光探针、制备方法及用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011479265.6A CN112624960B (zh) | 2020-12-15 | 2020-12-15 | 基于苯并吲哚的ntr-1响应型荧光探针、制备方法及用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112624960A true CN112624960A (zh) | 2021-04-09 |
CN112624960B CN112624960B (zh) | 2022-10-14 |
Family
ID=75313170
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011479265.6A Active CN112624960B (zh) | 2020-12-15 | 2020-12-15 | 基于苯并吲哚的ntr-1响应型荧光探针、制备方法及用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112624960B (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113735839A (zh) * | 2021-09-07 | 2021-12-03 | 南通大学 | 二氢呫吨/苯并[cd]吲哚杂合物荧光探针及其制备方法与应用 |
CN116239515A (zh) * | 2022-12-19 | 2023-06-09 | 苏州大学 | 一种荧光探针及其制备方法与应用 |
-
2020
- 2020-12-15 CN CN202011479265.6A patent/CN112624960B/zh active Active
Non-Patent Citations (1)
Title |
---|
杨敏: "X-新型有机小分子荧光探针的构建与应用研究", 《东南大学硕士学位论文》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113735839A (zh) * | 2021-09-07 | 2021-12-03 | 南通大学 | 二氢呫吨/苯并[cd]吲哚杂合物荧光探针及其制备方法与应用 |
CN116239515A (zh) * | 2022-12-19 | 2023-06-09 | 苏州大学 | 一种荧光探针及其制备方法与应用 |
Also Published As
Publication number | Publication date |
---|---|
CN112624960B (zh) | 2022-10-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110540837B (zh) | 一种过氧化氢近红外荧光探针的制备和应用 | |
CN112624960B (zh) | 基于苯并吲哚的ntr-1响应型荧光探针、制备方法及用途 | |
CN109867611B (zh) | 一种用于红酒和活体内硫化氢检测的水溶性双光子硫化氢荧光探针及其制备方法和应用 | |
CN109438319B (zh) | 一种检测亮氨酸氨肽酶的化合物及其制备方法和应用 | |
CN109336815B (zh) | 一种检测细胞内质网内次氯酸的双光子荧光探针 | |
CN110526908B (zh) | 基于2-苯乙烯基吲哚盐类衍生物长波发射可区分检测Cys/Hcy荧光探针及其应用 | |
Liu et al. | Regulating glutathione-responsiveness of naphthalimide-based fluorescent probes by an oxidation strategy | |
CN113354627B (zh) | 一种用于检测粘度的近红外荧光化合物及其制备与应用 | |
CN112778288B (zh) | 一种近红外固态发光的荧光探针及其制备方法与应用 | |
WO2012023487A1 (ja) | 金属錯体、蛍光プローブ | |
CN109369569B (zh) | 一类检测丙酮醛的荧光探针及其制备方法和应用 | |
Mei et al. | A novel fluorescence probe for the selective detection of cysteine in aqueous solutions and imaging in living cells and mice | |
CN114634497B (zh) | 一种半胱氨酸/高半胱氨酸响应的aie荧光探针及其制备方法与应用 | |
CN112457360B (zh) | 一种肝靶向的过氧亚硝酸根荧光探针及制备方法和应用 | |
CN113045599B (zh) | 一种高对比度区分癌细胞/组织的方法及荧光探针的制备 | |
Wang et al. | A benzoindole-based fluorescent probe for nitroreductase imaging in living cells under hypoxia conditions | |
CN114456116A (zh) | 一种以萘酰亚胺为骨架的小分子抗癌剂及其制备方法和应用 | |
CN108840818B (zh) | 一种用于检测硫化氢的比色型咔唑类荧光探针的合成与应用 | |
CN110964022A (zh) | 一种检测过氧亚硝酸根离子的荧光探针及其制备方法和应用 | |
CN113024445A (zh) | 基于吲哚菁类荧光探针、制备方法及用途 | |
CN109776379A (zh) | 一种可用于响应活细胞内和慢性伤口发展过程中pH变化的近红外荧光探针及其制备方法 | |
CN115583920B (zh) | 一种四嗪类化合物及其制备方法和应用 | |
CN115894427B (zh) | 近红外频率上转换荧光探针及其制备方法和在检测生物硫醇中的应用 | |
CN114539214B (zh) | 原位检测醌氧化还原酶荧光探针的设计、合成及活性研究 | |
CN114524794A (zh) | 三苯胺-苯并吡喃鎓盐衍生物nir-bt-p及其合成方法和应用 |
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 |