CN113292527A - Fluorescent probe for identifying thiophenol and preparation method and application thereof - Google Patents
Fluorescent probe for identifying thiophenol and preparation method and application thereof Download PDFInfo
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- CN113292527A CN113292527A CN202110671248.0A CN202110671248A CN113292527A CN 113292527 A CN113292527 A CN 113292527A CN 202110671248 A CN202110671248 A CN 202110671248A CN 113292527 A CN113292527 A CN 113292527A
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- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 title claims abstract description 222
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title abstract description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000000523 sample Substances 0.000 claims abstract description 30
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 28
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000003756 stirring Methods 0.000 claims abstract description 21
- XFVZSRRZZNLWBW-UHFFFAOYSA-N 4-(Diethylamino)salicylaldehyde Chemical compound CCN(CC)C1=CC=C(C=O)C(O)=C1 XFVZSRRZZNLWBW-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000004440 column chromatography Methods 0.000 claims abstract description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 14
- 238000001035 drying Methods 0.000 claims abstract description 13
- YOJMLPKRLNLQFN-UHFFFAOYSA-N 2-methylchromene-2-carbonitrile Chemical compound CC1(OC2=C(C=C1)C=CC=C2)C#N YOJMLPKRLNLQFN-UHFFFAOYSA-N 0.000 claims abstract description 12
- PBOPJYORIDJAFE-UHFFFAOYSA-N 2,4-dinitrobromobenzene Chemical compound [O-][N+](=O)C1=CC=C(Br)C([N+]([O-])=O)=C1 PBOPJYORIDJAFE-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000010438 heat treatment Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000011541 reaction mixture Substances 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 8
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 238000003384 imaging method Methods 0.000 claims description 6
- 238000009987 spinning Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 abstract description 15
- 125000001917 2,4-dinitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C(=C1*)[N+]([O-])=O)[N+]([O-])=O 0.000 abstract description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- NWWWHCCHHJQISH-UHFFFAOYSA-N 2h-chromene-2-carbonitrile Chemical compound C1=CC=C2C=CC(C#N)OC2=C1 NWWWHCCHHJQISH-UHFFFAOYSA-N 0.000 abstract description 2
- 239000000975 dye Substances 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 description 26
- 238000002189 fluorescence spectrum Methods 0.000 description 14
- 238000000862 absorption spectrum Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 7
- 230000005284 excitation Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 description 4
- -1 aliphatic thiols Chemical class 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- UJHSIDUUJPTLDY-UHFFFAOYSA-N (2-nitrophenyl)-phenylmethanone Chemical compound [O-][N+](=O)C1=CC=CC=C1C(=O)C1=CC=CC=C1 UJHSIDUUJPTLDY-UHFFFAOYSA-N 0.000 description 2
- WFQDTOYDVUWQMS-UHFFFAOYSA-N 1-fluoro-4-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C=C1 WFQDTOYDVUWQMS-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 2
- 239000007832 Na2SO4 Substances 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000011698 potassium fluoride Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- WBHQBSYUUJJSRZ-UHFFFAOYSA-M sodium bisulfate Chemical compound [Na+].OS([O-])(=O)=O WBHQBSYUUJJSRZ-UHFFFAOYSA-M 0.000 description 2
- 229910000342 sodium bisulfate Inorganic materials 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- HYHCSLBZRBJJCH-UHFFFAOYSA-M sodium hydrosulfide Chemical compound [Na+].[SH-] HYHCSLBZRBJJCH-UHFFFAOYSA-M 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- WDCYWAQPCXBPJA-UHFFFAOYSA-N 1,3-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC([N+]([O-])=O)=C1 WDCYWAQPCXBPJA-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 231100000111 LD50 Toxicity 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- JNQMTAOUDGKJSS-UHFFFAOYSA-N benzenethiol hydrate Chemical compound [OH-].[SH2+]c1ccccc1 JNQMTAOUDGKJSS-UHFFFAOYSA-N 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 238000010494 dissociation reaction Methods 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 208000027905 limb weakness Diseases 0.000 description 1
- 231100000861 limb weakness Toxicity 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
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- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
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- C—CHEMISTRY; METALLURGY
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- 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"
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- 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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- 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
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Abstract
The invention discloses a fluorescent probe for identifying thiophenol and a preparation method thereof, and the fluorescent probe specifically comprises the following steps: firstly, dissolving 2-methyl benzopyran nitrile and 4- (diethylamino) salicylaldehyde in anhydrous DMF, adding piperidine and acetic acid, heating and stirring under the protection of nitrogen, spin-drying, and separating and purifying by column chromatography to obtain a fluorophore FL-1; fluorophores FL-1 and 2, 4-dinitrobromobenzene were dissolved in anhydrous DMF and K was added2CO3Stirring for 3h under the protection of nitrogen, spin-drying, and separating and purifying by column chromatography to obtain the fluorescent probe for identifying thiophenol. The benzopyran nitrile dye and 4- (diethylamino) salicylaldehyde are used as basic frameworks to construct a fluorophore, 2, 4-dinitrophenyl is used as a recognition group, and the recognition group is connected with the fluorophore through an ether bond. The probe has strong specificity and good specificity for detecting the thiophenolHigh sensitivity and low detection limit.
Description
Technical Field
The invention belongs to the technical field of fluorescent biosensors, and particularly relates to a fluorescent probe for identifying thiophenol, a preparation method and application thereof.
Background
Thiophenol is an aromatic mercaptan with high reactivity and high toxicity, has irreplaceable critical effect in organic raw materials and is widely used for synthesizing sulfonamides, polymers and other various chemical products, but has no negligible harm to organisms and environment. Studies have shown thiophenol as an environmental contaminant with a semi-lethal dose in fish of 0.01-0.4 mM; and also causes serious health problems when humans are exposed to thiophenols for long periods of time: respiratory disorder, limb weakness, central nerve damage, abnormal kidney and liver function and even death, the half-lethal dose of thiophenol to mice is 6.2 mg/kg. Therefore, it is important to develop a method for rapidly and efficiently detecting thiophenol in environmental and biological samples.
Traditional detection methods (such as gas chromatography, high performance liquid chromatography, electrochemical analysis and the like) do provide extremely high detection accuracy, but they damage the sample to some extent and are time-consuming and labor-consuming. The advent of fluorescent probe technology opens new avenues for the detection of analytes. The fluorescent probe has the advantages of high sensitivity, small damage, rapidness, easy operation and the like, can detect various molecular ions in environmental pollutants, and can specifically identify various biological targets in diseases. In addition, fluorescent probe imaging techniques can also selectively image analytes, assess biocompatibility, and provide the potential for real-time monitoring of a given analyte in vivo and in vitro.
Over the past few decades, many fluorescent probes have been developed based on the various recognition types of thiophenols. There are still some problems: (1) due to the chemical similarity between aliphatic thiols and thiophenols, most probes do not clearly distinguish thiophenols from aliphatic thiols; (2) the relatively short excitation/emission wavelengths of most fluorescent probes cause high background interference in deep tissue images. Therefore, the development of novel near-infrared fluorescent probes that can be used for the specific detection of thiophenol is still imminent.
Disclosure of Invention
The invention aims to provide a fluorescent probe for identifying thiophenol.
Another object of the present invention is to provide a method for preparing the above fluorescent probe for identifying thiophenol.
The third purpose of the invention is to provide the application of the fluorescent probe in the aspect of thiophenol detection in cells and water samples.
The technical scheme adopted by the invention is that the fluorescent probe for identifying thiophenol has a structural formula shown as the following formula (I):
the invention adopts another technical scheme that the preparation method of the fluorescent probe for identifying thiophenol is implemented according to the following steps:
The present invention is also characterized in that,
in step 1, the molar ratio of 2-methylbenzopyranenitrile, 4- (diethylamino) salicylaldehyde, piperidine and acetic acid is 0.5: 0.5: 5: 8.
in step 1, the stirring temperature is 155 ℃ and the stirring time is 30 min.
In step 2, FL-1, 2, 4-dinitrobromobenzene and K2CO3In a molar ratio of 1: 1: 5.
the third scheme adopted by the invention is a fluorescent probe for identifying thiophenol, and the fluorescent probe is applied to the aspects of exogenous thiophenol cell imaging and thiophenol content detection in a water sample.
The invention has the beneficial effects that the benzopyran nitrile dye and 4- (diethylamino) salicylaldehyde are used as basic frameworks to construct the fluorophore, 2, 4-dinitrophenyl is used as a recognition group, and the recognition group and the fluorophore are connected through an ether bond. The probe has the advantages of strong specificity, high sensitivity, low detection limit and the like for detecting the thiophenol. In the application aspect, the probe is used for detecting the thiophenol content of a real environment water sample, and provides a valuable reference for the thiophenol water pollution prevention and control problem in environmental protection. In addition, the probe molecules are successfully subjected to an exogenous thiophenol cell imaging experiment, and a certain foundation is laid for the later research of pathology and toxicology experiments related to thiophenol in living bodies.
Drawings
FIG. 1 is a graph of selective ultraviolet absorption spectrum of p-thiophenol in EtOH-PBS system for Compound (I);
FIG. 2 is a selective fluorescence emission spectrum of compound (I) on thiophenol in an EtOH-PBS system;
FIG. 3 is a graph of the selective UV absorption spectrum of para-thiophenol of compound (II) in an EtOH-PBS system;
FIG. 4 is a selective fluorescence emission spectrum of compound (II) on thiophenol in EtOH-PBS system;
FIG. 5 is a graph showing the selective UV absorption spectrum of p-thiophenol in EtOH-PBS system with respect to Compound (III);
FIG. 6 is a selective fluorescence emission spectrum of compound (III) on thiophenol in EtOH-PBS system;
FIG. 7 is a UV absorption spectrum of compound (I) for its anti-interference ability in detecting thiophenol in EtOH-PBS system;
FIG. 8 is a fluorescence emission spectrum of compound (I) in EtOH-PBS system for detecting thiophenol with interference resistance;
FIG. 9 is a graph of the ultraviolet absorption spectrum of compound (I) titrated against thiophenol in an EtOH-PBS system;
FIG. 10 is a fluorescence emission spectrum of compound (I) titrated against thiophenol in an EtOH-PBS system;
FIG. 11 is a graph showing the kinetic response of Compound (I) to thiophenol in an EtOH-PBS system;
FIG. 12 is a graph showing the detection of thiophenol in lake water by Compound (I);
FIG. 13 is a graph showing the detection of thiophenol in tap water by Compound (I);
FIG. 14 is a graph showing the detection of thiophenol in distilled water by Compound (I);
FIG. 15 is a fluorescence emission spectrum of compound (I) on thiophenol detection in three water samples;
FIG. 16 is a cell image of compound (I) on thiophenol in HeLa cells.
Detailed Description
The present invention will be described in detail with reference to the following detailed description and accompanying drawings.
The invention relates to a fluorescent probe for identifying thiophenol, which has a structural formula shown as the following formula (I):
the invention relates to a preparation method of a fluorescent probe for identifying thiophenol, which comprises the following synthetic route:
the method is implemented according to the following steps:
the molar ratio of 2-methylbenzopyranenitrile, 4- (diethylamino) salicylaldehyde, piperidine and acetic acid was 0.5: 0.5: 5: 8;
stirring at 155 deg.C for 30 min;
FL-1, 2, 4-dinitrobromobenzene and K2CO3In a molar ratio of 1: 1: 5.
the principle of detecting thiophenol by the fluorescent probe of the invention is as follows:
due to the strong electron withdrawing effect of 2, 4-dinitrobenzene, the probe molecules themselves are essentially non-fluorescent. When thiophenol is added, it undergoes a nucleophilic reaction with the fluorescent probe, the 2, 4-dinitrophenyl moiety leaves, the fluorophore is released, the PET process is blocked, the fluorescence signal is turned on and the maximum fluorescence emission intensity is achieved at 680 nm. The purposes of detecting the existence of the thiophenol and determining the concentration of the thiophenol are achieved by determining the change of the light intensity.
Example 1
The invention relates to a preparation method of a fluorescent probe for identifying thiophenol, which is implemented according to the following steps:
2-Methylbenzopyranecarbonitrile (100mg,0.28mmol) and 4- (diethylamino) salicylaldehyde (105mg, 0.57mmol) were dissolved in 20mL of anhydrous DMF, and piperidine (0.5mL) and acetic acid (0.5mL) were added. The reaction mixture was heated under nitrogen for 30min with stirring, dried by spinning, and purified by column chromatography (eluent: DCM: PE ═ 1:1) to give a green solid, fluorophore FL-1(110mg, 60%).
Wherein the product is characterized as follows:
1H NMR(400MHz,DMSO-d6,TMS)δ10.19(d,J＝3.7Hz,1H),8.80(s,1H),8.07–7.91(m,2H),7.75(d,J＝9.3Hz,3H),7.11(dd,J＝7.57,3.8Hz,1H),6.87(d,J＝3.6Hz,1H),6.34(d,J＝8.6Hz,2H),3.51–3.41(m,4H),1.23(d,J＝4.4Hz,6H)。
fluorophore FL-1(50mg, 0.13mmol) and 2, 4-dinitrobromobenzene (33.60mg, 0.13mmol) were dissolved in 30mL of anhydrous DMF, and 68mg of K was added2CO3Stirring the reaction mixture for 3h at room temperature under the protection of nitrogen, spin-drying, and purifying by column chromatography (eluent: DCM: PE ═ 1:1) to obtain red solid (52mg, 73%), namely the fluorescent probe (TZ-1); denoted compound (I);
wherein the product is characterized as follows:
1H NMR(400MHz,DMSO-d6,TMS)δ8.95(d,J＝2.8Hz,1H),8.68(dd,J＝8.2,1.08Hz,1H),8.44(dd,J＝9.3,2.84Hz,1H),7.90(d,J＝9.1Hz,1H),7.88–7.84(m,1H),7.67(d,J＝15.8Hz,1H),7.58–7.53(m,1H),7.22(d,J＝15.8Hz,1H),7.15(d,J＝9.3Hz,1H),6.85(s,1H),6.82(dd,J＝9.0,2.40Hz,1H),6.72(d,J＝2.5Hz,1H),3.43(q,J＝7.1Hz,6H),1.11(t,J＝7.0Hz,4H).
MS(ESI)calcd.for C30H23N5O6[M+Na]+:572.1540,found:572.1460；
crystal data:
α＝90°
β＝99.16(3)°
γ＝90°。
comparative example 1
Dissolving 2-methylbenzopyranenitrile and 4- (diethylamino) salicylaldehyde in anhydrous DMF, adding piperidine and acetic acid, heating and stirring the reaction mixture under the protection of nitrogen, performing spin-drying after the reaction is finished, and performing column chromatography separation and purification to obtain a green solid, namely a fluorophore FL-1; the molar ratio of 2-methylbenzopyranenitrile, 4- (diethylamino) salicylaldehyde, piperidine and acetic acid was 0.5: 0.5: 5: 8; stirring at 155 deg.C for 30 min; the fluorophore isDissolving FL-1 and 2-nitrobenzophenone in anhydrous DMF and adding K2CO3FL-1, 2-nitrobenzophenone and K2CO3Is 0.13: 0.2: 0.5; stirring the reaction mixture at room temperature for 3h under the protection of nitrogen, performing spin-drying after the reaction is finished, and performing column chromatography separation and purification to obtain a red solid which is marked as a fluorescent probe (TZ-2); denoted compound (II);
the product was characterized as follows:
1H NMR(400MHz,CDCl3,TMS)δ8.86(dd,J＝8.4,3.2Hz,1H),7.97(dd,J＝8.1,1.6Hz,1H),7.81(d,J＝15.8Hz,1H),7.69-7.64(m,1H),7.59(d,J＝7.5Hz,1H),7.52–7.46(m,2H),7.40-7.36(m,1H),7.22–7.17(m,1H),6.99(dd,J＝8.4,1.1Hz,1H),6.73(s,1H),6.63(d,J＝15.8Hz,1H),6.58(dd,J＝8.9,2.4Hz,1H),6.18(d,J＝2.6Hz,1H),3.36(q,J＝7.1Hz,6H),1.17(t,J＝7.1Hz,4H).
MS(ESI)calcd.for C30H24N4O4[M+Na]+:527.1690,found:527.1698.
comparative example 2
Dissolving 2-methylbenzopyranenitrile and 4- (diethylamino) salicylaldehyde in anhydrous DMF, adding piperidine and acetic acid, heating and stirring the reaction mixture under the protection of nitrogen, performing spin-drying after the reaction is finished, and performing column chromatography separation and purification to obtain a green solid, namely a fluorophore FL-1; the molar ratio of 2-methylbenzopyranenitrile, 4- (diethylamino) salicylaldehyde, piperidine and acetic acid was 0.5: 0.5: 5: 8; stirring at 155 deg.C for 30 min; fluorophores FL-1 and 4-nitrofluorobenzene were dissolved in anhydrous DMF and K was added2CO3FL-1, 4-nitrofluorobenzene and K2CO3Is 0.13: 0.2: 0.5; stirring the reaction mixture at room temperature for 3h under the protection of nitrogen, performing spin-drying after the reaction is finished, and performing column chromatography separation and purification to obtain a red solid which is marked as a fluorescent probe (TZ-3); denoted compound (III);
the product was characterized as follows:
1H NMR(400MHz,CDCl3,TMS)δ8.86(dd,J＝8.6,1.4Hz),8.27–8.25(m),8.24–8.22(m),7.65(ddd,J＝8.8,7.4,1.5Hz),7.62(d,J＝7.5Hz),7.59(s),7.41–7.36(m),7.11–7.09(m),7.08–7.07(m),6.72(s),6.64–6.57(m),6.22(d,J＝2.5Hz),3.39(q,J＝7.1Hz),1.19(t,J＝7.1Hz).
MS(ESI)calcd.for C30H24N4O4[M+Na]+:527.1690,found:527.1482.
selectivity test of the fluorescent probes for example 1, comparative examples 1 and 2 to thiophenol
EtOH-PBS (5:5, v/v) as solvent system was used to prepare 5. mu.M probe, each of which was mixed with 10-fold equivalent of analyte (Na)2S,NaHS,Na2SO4,Na2SO3,NaHSO4,Na2S2O3,Na2NO2,GSH,Cys,Hcy,Na2CO3,NaHCO3,KF,NaCl,NaBr,NaI,CH3COONa, aniline, phenol and thiophenol), and measuring an ultraviolet absorption spectrum and a fluorescence emission spectrum at an excitation wavelength of 570nm of each system, wherein the ultraviolet absorption spectrum and the fluorescence emission spectrum of the compound (I) are shown in fig. 1 and 2, which indicates that only thiophenol is identified; the ultraviolet absorption spectrum and the fluorescence emission spectrum of the compound (II) are shown in figures 3 and 4, which shows that the compound (II) does not identify the thiophenol; the ultraviolet absorption spectrum and the fluorescence emission spectrum of the compound (III) are shown in fig. 5 and 6, which shows that the compound (III) does not identify the thiophenol. In summary, only compound (I) responded significantly to thiophenol upon addition of the different analytes and the maximum fluorescence emission wavelength was 680 nm. The reason is that the number and position of the nitro groups on different recognition units affects the ability to undergo nucleophilic substitution. The compound (I) recognizing two nitro groups has less dissociation energy of ether bond and is more responsive to thiophenol.
Experiment of interference rejection Capacity of fluorescent Probe of example 1
EtOH-PBS (5:5, v/v) is used as a solvent system to prepare a probe with the concentration of 5 mu M, and the probe is firstly mixed with 10 times of equivalent of thiophenol and then respectively mixed with 10 times of equivalent of various interferents (Na)2S,NaHS,Na2SO4,Na2SO3,NaHSO4,Na2S2O3,Na2NO2,GSH,Cys,Hcy,Na2CO3,NaHCO3,KF,NaCl,NaBr,NaI,CH3COONa, aniline, phenol, and thiophenol), and measuring the ultraviolet absorption spectrum and the fluorescence emission spectrum at an excitation wavelength of 570nm of the solution. As a result, as shown in FIGS. 7 and 8, the probe specifically responded to thiophenol and was not affected by other interfering ions.
Titration experiment and detection line assay of fluorescent probe for Phenothiol of example 1
EtOH-PBS (5:5, v/v) is used as a solvent system to prepare a solution with the probe concentration of 5 mu M, and the solution is mixed with 0-16 times of equivalent of thiophenol respectively, and the ultraviolet absorption spectrum and the fluorescence emission spectrum under the excitation wavelength of 570nm are measured. As shown in FIGS. 9 and 10, the fluorescence intensity gradually increased in a gradient manner as the concentration of thiophenol increased in a gradient manner, and the fluorescence intensity was kept constant at a concentration of 40. mu.M. In the range of 0-10. mu.M, the probe TZ-1 has good linear relation to the thiophenol (y is 84.13x +131.89, R20.9981), the detection limit was 0.17 μ M.
Kinetic response of fluorescent probes to thiophenol of example 1
A pure probe solution with the concentration of 5 mu M and a mixed solution of the probe and 10 times of equivalent of thiophenol are prepared by taking EtOH-PBS (5:5, v/v) as a solvent system, and the change of a fluorescence emission spectrum under the excitation wavelength of 570nm along with time is measured, so that the result is shown in figure 11, the fluorescence intensity of the pure probe does not change along with the change of the time, but after 40 mu M of thiophenol is added into the probe solution, the fluorescence is started and gradually enhanced, and the fluorescence intensity is basically stable after 35min, which indicates that the reaction reaches the equilibrium and the fluorescence intensity is enhanced by about 11 times.
Detection of thiophenol in Water sample by fluorescent Probe of example 1
Firstly, filtering collected water samples (lake water, tap water and distilled water) (0.22mm), preparing a solution with the probe concentration of 5 mu M by taking an ethanol-actual water sample (5:5, v/v) as a solvent system, respectively mixing the solution with 0-10 times of equivalent of thiophenol, measuring a fluorescence emission spectrum under the excitation wavelength of 570nm, and recording the fluorescence intensity at 680 nm. As shown in FIG. 12, the fluorescence intensity and the change trend of the three water samples were substantially consistent after adding thiophenol. Shown in FIG. 13And in lake water samples: 32.1407x +136.8720 (R)20.9994); fig. 14 shows, sample of tap water: 31.5199x +144.7906 (R)20.9981); fig. 15 shows, distilled water sample: 31.2250x +143.7232 (R)20.9985); indicating that the concentration of thiophenol is between 0 and 10 mu M, and the fluorescence intensity and the concentration at 680nm have good linear relation. The results of the calculations are shown in tables 2-3, where it can be seen that the recovery of thiophenol is between 90% and 105%. The experiment results show that the probe TZ-1 can be effectively applied to detection of thiophenol in a real environment water sample.
Imaging experiment of fluorescent probe of example 1 for thiophenol in HeLa cells
Inoculating HeLa cells into a culture dish, co-culturing for 4 groups, taking out the cells after culturing for 48h in an incubator, adding 10 mu L of probes into the groups 1, 2, 3 and 4 respectively, continuing to incubate for 15min, pouring out the culture medium, slowly washing for 3 times by using PBS (phosphate buffer solution), adding thiophenols (0 mu M,10 mu M,20 mu M and 30 mu M) with different concentrations respectively, putting the mixture into the cell incubator, continuing to incubate for 30min, taking out, washing for 3 times similarly, and carrying out cell imaging. As shown in fig. 16: cells incubated with the probe alone did not observe any fluorescent signal under the fluorescent field. A red fluorescence signal can be observed by cells incubated by HeLa cells and thiophenol (0 mu M,10 mu M,20 mu M and 30 mu M) with different concentration gradients, and the fluorescence signal is gradually enhanced under a near infrared fluorescence channel, which shows that the fluorescence intensity is positively correlated with the thiophenol concentration.
Claims (6)
1. A fluorescent probe for identifying thiophenol, which is characterized in that the fluorescent probe has a structural formula shown as the following formula (I):
2. the method for preparing a thiophenol-recognizing fluorescent probe according to claim 1, comprising the steps of:
step 1, dissolving 2-methylbenzopyranenitrile and 4- (diethylamino) salicylaldehyde in anhydrous DMF, adding piperidine and acetic acid, heating and stirring a reaction mixture under the protection of nitrogen, performing spin-drying after the reaction is finished, and performing column chromatography separation and purification to obtain a green solid, namely a fluorophore FL-1;
step 2, dissolving fluorophores FL-1 and 2, 4-dinitrobromobenzene in anhydrous DMF, and adding K2CO3And stirring the reaction mixture at room temperature for 3 hours under the protection of nitrogen, spinning and drying after the reaction is finished, and separating and purifying by column chromatography to obtain red solid, namely the fluorescent probe for identifying thiophenol.
3. The method for preparing a thiophenol-recognizing fluorescent probe according to claim 2, wherein in said step 1, the molar ratio of 2-methylbenzopyranenitrile, 4- (diethylamino) salicylaldehyde, piperidine and acetic acid is 0.5: 0.5: 5: 8.
4. the method for preparing a fluorescent probe capable of recognizing thiophenol according to claim 2, wherein in said step 1, the stirring temperature is 155 ℃ and the stirring time is 30 min.
5. The method for preparing the thiophenol-recognizing fluorescent probe as claimed in claim 2, wherein in said step 2, FL-1, 2, 4-dinitrobromobenzene and K are used2CO3In a molar ratio of 1: 1: 5.
6. the thiophenol-recognizing fluorescent probe according to claim 1, wherein said probe is used for imaging exogenous thiophenol cells and detecting thiophenol content in a water sample.
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