CN110386898B - Quinoline ring derivative fluorescent probe and preparation method and application thereof - Google Patents
Quinoline ring derivative fluorescent probe and preparation method and application thereof Download PDFInfo
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- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 title claims abstract description 10
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 6
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 150000001875 compounds Chemical class 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 30
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 18
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- MLUCVPSAIODCQM-NSCUHMNNSA-N crotonaldehyde Chemical compound C\C=C\C=O MLUCVPSAIODCQM-NSCUHMNNSA-N 0.000 claims description 7
- MLUCVPSAIODCQM-UHFFFAOYSA-N crotonaldehyde Natural products CC=CC=O MLUCVPSAIODCQM-UHFFFAOYSA-N 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- JPJALAQPGMAKDF-UHFFFAOYSA-N selenium dioxide Chemical compound O=[Se]=O JPJALAQPGMAKDF-UHFFFAOYSA-N 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
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- 239000007787 solid Substances 0.000 claims description 5
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical class [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 3
- 239000012043 crude product Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- ZIUSEGSNTOUIPT-UHFFFAOYSA-N ethyl 2-cyanoacetate Chemical compound CCOC(=O)CC#N ZIUSEGSNTOUIPT-UHFFFAOYSA-N 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000003472 neutralizing effect Effects 0.000 claims description 3
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- 238000010992 reflux Methods 0.000 claims description 3
- 238000004809 thin layer chromatography Methods 0.000 claims description 3
- MLIREBYILWEBDM-UHFFFAOYSA-M 2-cyanoacetate Chemical compound [O-]C(=O)CC#N MLIREBYILWEBDM-UHFFFAOYSA-M 0.000 claims description 2
- 238000003760 magnetic stirring Methods 0.000 claims description 2
- RNRTUXHMHDKLEH-UHFFFAOYSA-N 6-(dimethylamino)quinoline-2-carbaldehyde Chemical compound N1=C(C=O)C=CC2=CC(N(C)C)=CC=C21 RNRTUXHMHDKLEH-UHFFFAOYSA-N 0.000 claims 1
- JGRWYZQONRFIFH-UHFFFAOYSA-N n,n,2-trimethylquinolin-6-amine Chemical compound N1=C(C)C=CC2=CC(N(C)C)=CC=C21 JGRWYZQONRFIFH-UHFFFAOYSA-N 0.000 claims 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 48
- 239000003068 molecular probe Substances 0.000 abstract description 48
- 238000001514 detection method Methods 0.000 abstract description 12
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 abstract description 9
- 210000000170 cell membrane Anatomy 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
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- 230000007246 mechanism Effects 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 238000011534 incubation Methods 0.000 description 9
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- 238000002474 experimental method Methods 0.000 description 6
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- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 6
- 229910002091 carbon monoxide Inorganic materials 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
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- 230000005284 excitation Effects 0.000 description 4
- 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 4
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- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
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- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
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- WNPNNLQNNJQYFA-UHFFFAOYSA-N [2-(3,4-dihydroxyphenyl)-2-hydroxyethyl]azanium;2,3,4-trihydroxy-4-oxobutanoate Chemical compound OC(=O)C(O)C(O)C(O)=O.NCC(O)C1=CC=C(O)C(O)=C1 WNPNNLQNNJQYFA-UHFFFAOYSA-N 0.000 description 2
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- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethyl-succinimide Natural products CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- SMWDFEZZVXVKRB-UHFFFAOYSA-N anhydrous quinoline Natural products N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 1
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- 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
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D215/38—Nitrogen atoms
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- 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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- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
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- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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Abstract
The invention relates to a quinoline ring derivative fluorescent probe and a preparation method and application thereof, and the compound name is 2-cyano-3- (6- (dimethylamino) quinoline-2-yl) acrylate. The compound provided by the invention has the advantages of small molecular mass and simple structure, can be used as a molecular sensor, sensitively and selectively detects sulfite in living cells, and starts a fluorescence reaction. The detection mechanism is not influenced by sulfide and sulfhydryl compounds, has the characteristic of high selectivity, and more remarkably, the fluorescent molecular probe is almost non-toxic to cells and has very good cell membrane permeation effect, and can penetrate through a living cell membrane within 2min to start fluorescence reaction. The method provides a new method for detecting the endogenous sulfite of the living cells, and has very important practical significance.
Description
The invention relates to a quinoline ring derivative fluorescent molecular probe, a preparation method thereof and application thereof in detection of sulfite.
Background
SO 2 Since the last century gases were classified as toxic pollutants, recent studies have shown that this molecule may be another important gas signaling molecule, highly exogenous SO, involved in various pathological and physiological processes following nitric oxide, carbon monoxide and hydrogen sulfide 2 The level can cause cardiovascular diseases, nervous system diseases and cancers. The U.S. food and drug administration states that products containing levels of sulfite that reach or exceed 10 micrograms/ml need to be specifically labeled. On the other hand, hydrogen sulfide and biological sulfur are also passed through in cytoplasm and mitochondriaAlcohol oxidation and the like to produce SO 2 SO also occurs in aqueous media 3 2- And HSO 3 - . Normal endogenous SO 2 Levels of endogenous SO that are abnormal and have the effect of regulating blood pressure 2 The level is also closely related to diseases such as nervous system diseases and cancer. Therefore, it is important to develop a rapid, sensitive, selective sulfite detection probe or method.
An effective, sensitive and low-toxicity fluorescent molecular probe is obtained by preparing quinoline ring derivatives, and a series of experiments with the same classification show that the fluorescent molecule has good performance and high potential practical application value.
Disclosure of Invention
The invention aims to provide a novel quinoline ring derivative fluorescent molecular probe, a preparation method thereof and practical application thereof.
The technical scheme of the invention is as follows:
a quinoline cyclic amine derivative fluorescent molecular probe is characterized by having the following structure:
A method for preparing the quinoline ring derivative comprises the following steps:
And 3, dissolving the product obtained in the step 2 and ethyl cyanoacetate in an ethanol solution, stirring at room temperature for 1h, washing the obtained ethanol mixture with cold ethanol for 3 times, and recrystallizing the obtained solid in a mixed solution of ethanol and acetone at a volume ratio of 9: 1 of ethanol to acetone to obtain the target compound.
The invention has the advantages that: the compound has very sensitive effect on detecting sulfite, and has the advantages of quick detection process, stable performance, low toxicity and the like. Experiments show that the fluorescent molecule can effectively and quickly detect sulfur dioxide molecules of internal and external sources, and most importantly, the fluorescent molecule has very short cell membrane permeation time which only needs 2 min; the invention synthesizes probe molecules with fluorescence characteristics by using crotonaldehyde as a lead compound, can effectively detect the content of sulfur dioxide molecules in cells, and has wide application prospect.
Detailed Description
The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples at all.
The first embodiment is as follows: preparation of 2-cyano-3- (6- (dimethylamino) quinolin-2-yl) acrylate
Adding 36.7mmol, 5g of 4-N, N-dimethylaniline into 6mol, 66mL of hydrochloric acid solution to fully dissolve the 4-N, N-dimethylaniline, adding 73.5mmol, 6mL of crotonaldehyde, magnetically stirring to uniformly mix, reacting at normal temperature for 1h, detecting the degree of reaction progress by TLC (thin layer chromatography), wherein the mass ratio of the substances is 4-N, N-dimethylaniline to crotonaldehyde is 1: 2, adding 35mL of toluene into the reaction solution, further refluxing at 115 ℃ for reacting overnight, cooling to room temperature, removing a toluene layer, neutralizing a water layer with a saturated sodium hydroxide solution, extracting the obtained solution with dichloromethane, washing twice with a saturated sodium chloride solution, drying, filtering with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying the crude product by silica gel column chromatography to obtain a brown yellow solid product; adding selenium dioxide into a solution of dioxane and water with a volume ratio of 10: 1 (dioxane to water) of 140mL and 40mL of water, heating at 80 ℃ for 30min, adding 18.8mmol and 3.5g of the obtained product, magnetically stirring to uniformly mix, reacting at 80 ℃ for 4h, cooling to room temperature, filtering through diatomite, washing filter residues with a small amount of dichloromethane, concentrating the filtrate under reduced pressure, and separating by silica gel column chromatography, wherein an eluent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6: 1 to obtain a product; 3.0mmol, 0.6g of the obtained product and 3.3mmol, 0.37g of ethyl cyanoacetate were dissolved in an ethanol solution, the mixture was stirred at room temperature for 1 hour, the obtained ethanol mixture was washed with cold ethanol for 3 times, and the obtained solid was recrystallized from a mixture of ethanol and acetone in a volume ratio of ethanol to acetone of 9: 1, to obtain 0.73g of the objective compound as red powder with a yield of 83%. 1H NMR (600MHz, DMSO-d) 6 )δ8.37(s， 1H)，8.16(d，J＝8.5Hz，1H)，7.89(d，J＝8.6Hz，1H)，7.57(dd，J＝9.4，2.8Hz，1H)， 6.93(d，J＝2.8Hz，1H)，4.34(q，J＝7.1Hz，2H)，3.12(s，6H)，4.34(t，J＝7.1Hz， 3H).13C NMR(150MHz，DMSO-d 6 )δ(ppm)162.80，153.71，150.44，144.67， 141.85，134.14，131.23，130.97，124.77，121.04，115.94，103.88，103.14，62.76，40.43， 14.50.HRMS(ESI-TOF)m/z：[M+H]+Calcd for C 17 H 18 N 3 O 2 296.13，Found 296.1410.
The property and activity of the fluorescent molecular compound are tested by applying experiments, the fluorescent molecular probe prepared in the first embodiment is tested in the second to tenth embodiments, and specific data and analysis are as follows:
the second embodiment:
FIG. 1: in PBS solution, the ultraviolet absorption spectrum of the fluorescent molecular probe
After 10. mu.M of fluorescent molecular probe, whose UV-visible absorption spectrum is shown in FIG. 1, was dissolved in PBS (pH7.4, 10mM, 5% DMSO) and incubated at 37 ℃, the probe was detected on Shimadzu UV-2550 apparatus.
Example three:
FIG. 2: in PBS solution, the fluorescent molecular probe is associated with SO 3 2- Fluorescence spectrum and fluorescence change curve chart of concentration change
mu.M fluorescent molecular probes were dissolved in PBS (pH7.4, 10mM, 5% DMSO), incubated at 37 ℃ for 1h, and then separately incubated in different SO 3 2- Measuring the fluorescence spectral feature, SO, at that concentration 3 2- The concentration range is 0-1000 μ M, the detection is carried out on a Hitachi F-7000 instrument, the excitation wavelength is 364nm, the slit width is 5nm, and the voltage of a photomultiplier is 500V.
The results show that SO is converted at 483nm 3 2- Increasing the concentration from 0 to 1000. mu.M (corresponding to 100 equivalents of probe) gave a standard curve; SO at 483nm 3 2- The concentration of the nano-particles shows a strong linear relation between 0 and 15 mu M, and the correlation coefficient is 0.9862. It can be seen that, with SO 3 2- Increasing concentration, increasing fluorescence intensity, SO 3 2- When the concentration reaches about 15 equivalent, the fluorescence intensity reaches the maximum and keeps stable.
Example four:
FIG. 3: selective experiment chart of fluorescent molecular probe in PBS (phosphate buffer solution)
Dissolving 10 μ M fluorescent molecular probe in PBS (pH7.4, 10mM, 5% DMSO), incubating at 37 deg.C for 1h, and detecting its selectivity, SO, with different analytes 3 2- Concentration of (C) and HSO 3 2- The concentration of the substance (D) is 100 mu M, the concentration of other tested substances is 1mM, the detection is carried out on a Hitachi F-7000 instrument, the excitation wavelength is 364nm, the slit width is 5nm, and the voltage of a photomultiplier is 500V.
The fluorescent molecular probe can be specific to SO 3 2- And HSO 3 2- In response, other substances tested were cysteine, glutathione, cysteine, thiophenol and other anions. The fluorescent molecular probe is directed against SO compared to cysteine, glutathione, cysteine and thiophenol 3 2- And HSO 3 2- Has good selectivity. The fluorescent molecule can be used as SO 3 2- And HSO 3 2- The probe of (1).
Example five:
FIG. 4: in PBS solution, the fluorescence spectrum of the fluorescent molecular probe responding to pH
After 10. mu.M of fluorescent molecular probe was dissolved in PBS (pH7.4, 10mM, 5% DMSO) and incubated at 37 ℃ for 1 hour, the performance was measured at different pH values, respectively, in the range of: 3-12. The detection is carried out on a Hitachi F-7000 instrument, the excitation wavelength is 364nm, the slit width is 5nm, and the voltage of a photomultiplier is 500V.
As can be seen from the figure, the fluorescent molecular probe itself is hardly affected by pH. The fluorescent molecular probe and 100 mu M SO 3 2- Upon co-incubation, the fluorescence intensity of the molecule was seen to decrease continuously at pH 1-5 and 8-12, whereas at pH 5-8 the fluorescence properties were stable, a stable fraction being sufficient for in vivo experiments.
Example six:
FIG. 5: in PBS solution, the fluorescence spectrum of the fluorescent molecular probe responding with time
10 μ M of fluorescent molecular probe was dissolved in PBS (pH7.4,10mM, 5% DMSO), 100. mu.M SO was added 3 2- The performance was measured at 37 ℃ and different incubation times, respectively, in the following ranges: 0-12 h. The detection is carried out on an F-7000 instrument, the excitation wavelength is 364nm, the emission wavelength is 483nm, the slit width is 5nm, and the voltage of a photomultiplier is 500V.
The fluorescent molecular probe shows strong fluorescence increase (more than half of fluorescence intensity increase) within 1min, and the fluorescence performance is continuously stable after the time reaches 1 h. By precisely controlling the response time, the detection system can be used in a shorter incubation time (e.g. 10min) while ensuring that it lasts more than 12h, which indicates the stability of the fluorescent molecular probe detection system.
Example seven:
FIG. 6: experimental chart of the fluorescent molecular probe on four different cytotoxicity
The cytotoxicity of the fluorescent molecular probe is evaluated by an MTT method. The test cells were HeLa cells (tumor cells), HEK293T cells (human embryonic kidney cell line), a549 cells (human alveolar epithelial AL cell line) and LO2 cells (human embryonic liver cell line). The cell culture medium is complete medium (DMEM) containing 10% FBS and 0.1% double antibody, and the cells are planted in 96-well plate and cultured at 37 deg.C and 5% CO 2 In the incubator, the cell density is 5X 10 5 one/mL.
From the figure, it can be concluded that the fluorescent molecular probe has less cytotoxicity, even at higher probe concentrations, which demonstrates that the fluorescent molecular probe can be further clinically tested.
Example eight:
FIG. 7: in living cells, the fluorescent molecular probe is used for time-dependent experimental fluorescence confocal picture of entering cells
Mixing 10 μ M of the fluorescent molecular probe with HeLa cells at 37 deg.C and 5% CO 2 Co-incubation in incubator, cells were cultured in complete medium containing 10% fetal bovine serum FBS, 0.1% double antibody, co-incubation time of each group with probe was set to 1, 1, 2, 10, 30 and 30min, respectively, and then 150 μ M SO 3 2- Incubate with cells for 30 min. Analysis of HeLa cell fluorescence Image (lambda) ex ＝364nm，λ em 425 and 475nm), scale bar: 25 μ M.
As can be seen from the figure, the fluorescent molecular probe has completely entered into HeLa cells at the incubation time of 2min, and the rapid cell permeation process is very excellent and important, which can shorten the whole detection period.
Example nine:
FIG. 8: in living cells, the exogenous SO of the fluorescent molecular probe along with the cells is detected 3 2- Fluorescence confocal mapping of concentration changes
HeLa cells were cultured in complete medium (DMEM, high sugar) containing 10% FBS, 0.1% double antibody, cells at 37 ℃, 5% CO 2 After culturing in an incubator for 12h, adding 10 μ M of the fluorescent molecular probe into the cells, and adding SO with different concentrations into the cells after 2min 3 2- The solution (50. mu.M, 100. mu.M, 150. mu.M) was incubated for 30min before taking fluorescence confocal pictures.
The image analysis can obtain that the fluorescence intensity of the fluorescent molecular probe is in positive correlation with the increase of the sulfite concentration, and no obvious fluorescence intensity can be observed after the cells and the fluorescent molecules are incubated together. The result shows that the fluorescent molecular probe can be used as an open fluorescent sensor to detect sulfite of an overproof exogenous source, and has better performance in the aspects of cell permeation and live thin imaging.
Example ten:
FIG. 9: in living cells, detecting the endogenous SO of the fluorescent molecular probe to the cells under different conditions 3 2- Fluorescence confocal mapping of concentration changes
The intracellular imaging performance of the fluorescent molecular probe is further detected subsequently. HeLa cells were cultured in complete medium (DMEM, high sugar) containing 10% FBS, 0.1% double antibody, cells at 37 ℃, 5% CO 2 After culturing in an incubator for 12h, 10 μ M of the fluorescent molecular probe was added to the cells, and after 2min, 200 μ M SO was added to the cells 2 After further incubation of the donor (noradrenaline bitartrate) for 30min, significant fluorescence was observed under confocal fluorescence microscopyThe result of light enhancement can also show that the fluorescent molecular probe can effectively detect the endogenous SO of the cells 3 2- . After incubation of the probes, 2mM NEM (N-ethylmaleimide, a thiol scavenger) and 200. mu.M SO were added to the cells 2 The donor (noradrenaline bitartrate) incubated for 30min had no significant increase in fluorescence, and therefore the increase in fluorescence was with endogenous SO in living cells 3 2- Is relevant to the generation of (2). Subsequently, 150. mu.M of exogenous SO was added to the cells 3 2- Incubation was carried out for 30min, at which time a significant fluorescence increase was again seen. The experimental results show that the fluorescent molecular probe can detect exogenous SO and SO in endogenous biological cells 3 2- . The result shows that the compound can be used as a molecular sensor and can be sensitive. The detection mechanism is not influenced by sulfide and sulfhydryl compounds, has the characteristic of high selectivity, and more remarkably, the fluorescent molecular probe is almost non-toxic to cells and has very good cell membrane permeation, and can penetrate through the cell membrane within 2min to start the fluorescence reaction. The method provides a new method for detecting the endogenous sulfite of the living cells, and has very important practical significance.
FIG. 1: in PBS solution, the ultraviolet absorption spectrum of the fluorescent molecular probe
FIG. 2 is a schematic diagram: in PBS solution, the fluorescent molecular probe is associated with SO 2 Fluorescence spectrum and fluorescence change curve chart of concentration change
FIG. 3: selective experiment chart of fluorescent molecular probe in PBS (phosphate buffer solution)
FIG. 4: in PBS solution, the fluorescence spectrum of the fluorescent molecular probe responding to pH
FIG. 5: in PBS solution, the fluorescence spectrum of the fluorescent molecular probe responding with time
FIG. 6: toxicity test chart of the fluorescent molecular probe on four different cells
FIG. 7: in living cells, the fluorescent molecular probe is used for time-dependent experimental fluorescence confocal picture of entering cells
FIG. 8: in living cells, the exogenous SO of the fluorescent molecular probe along with the cells is detected 3 2- Fluorescence confocal mapping of concentration changes
FIG. 9: in living cells, detecting the endogenous SO of the fluorescent molecular probe to the cells under different conditions 3 2- Fluorescence confocal mapping of (1).
Claims (3)
1. A quinoline ring fluorescent probe is characterized by comprising the following structural formula:
2. a method for preparing the quinoline ring fluorescent probe of claim 1, which comprises the steps of:
step 1, adding 4-N, N-dimethylaniline into a hydrochloric acid solution to be fully dissolved, adding crotonaldehyde, carrying out magnetic stirring to uniformly mix, reacting for 1h at normal temperature, detecting the reaction progress degree by TLC (thin layer chromatography), wherein the mass ratio of the substances is 4-N, N-dimethylaniline to crotonaldehyde is 1: 2, adding toluene into a reaction solution, further refluxing at 115 ℃ overnight, cooling to room temperature, removing a toluene layer, neutralizing a water layer with a saturated sodium hydroxide solution, extracting the solution with dichloromethane, washing twice with a saturated sodium chloride solution, drying, filtering with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying a crude product by silica gel column chromatography to obtain a brown yellow solid, namely a first-step product N, N, 2-trimethylquinoline-6-amine;
Step 2, adding selenium dioxide into a solution of dioxane/water in a volume ratio of 10: 1, heating at 80 ℃ for 30min, adding the product of the first step, magnetically stirring to uniformly mix, reacting at 80 ℃ for 4h, cooling to room temperature, filtering through diatomite, washing filter residue with a small amount of dichloromethane, concentrating the filtrate under reduced pressure, and separating by silica gel column chromatography, wherein an eluent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 6: 1 to obtain a second-step product 6- (dimethylamino) quinoline-2-formaldehyde;
and 3, dissolving the product obtained in the step 2 and ethyl cyanoacetate in an ethanol solution, stirring at room temperature for 1h, washing the obtained ethanol mixture with cold ethanol for 3 times, and recrystallizing the obtained solid in a mixed solution of ethanol and acetone at a volume ratio of 9: 1 of ethanol to acetone to obtain the target compound.
3. Use of quinoline ring fluorescent probes according to claims 1 and 2 for detecting SO for non-diagnostic and therapeutic purposes 2 Or the use of sulphite.
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| A fluorescence probe acted on Site I binding for Human Serum Albumin;Yuqing Xu et al.;《Talanta》;20180410;第185卷;第568-572页 * |
| Imaging of formaldehyde in plants with a ratiometric fluorescent probe;Zhen Li et al.;《Chem. Sci.》;20170706;第8卷;左栏倒数第1段,第5617页方案2 * |
| 二氧化硫衍生物荧光探针的研究进展;李东钰等.;《中国科学：化学》;20171123;第48卷(第1期);第45-57页 * |
| 可逆荧光探针的设计、合成及其在药物研究中的应用;柳志学.;《中国博士学位论文全文数据库·医药卫生科技辑》;20171115(第11期);正文第18页合成路线，第70页最后1段，第10 * |
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