CN111253356A - Coumarin-benzopyrylium salt derivative and synthesis method and application thereof - Google Patents
Coumarin-benzopyrylium salt derivative and synthesis method and application thereof Download PDFInfo
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- 238000001308 synthesis method Methods 0.000 title abstract description 7
- RAHZWNYVWXNFOC-UHFFFAOYSA-N Sulphur dioxide Chemical compound O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 claims abstract description 120
- 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 claims abstract description 97
- 229960003180 glutathione Drugs 0.000 claims abstract description 47
- 108010024636 Glutathione Proteins 0.000 claims abstract description 46
- QXAMGWKESXGGNV-UHFFFAOYSA-N 7-(diethylamino)-1-benzopyran-2-one Chemical compound C1=CC(=O)OC2=CC(N(CC)CC)=CC=C21 QXAMGWKESXGGNV-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 6
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 claims abstract description 4
- 229910052804 chromium Inorganic materials 0.000 claims abstract description 4
- 239000011651 chromium Substances 0.000 claims abstract description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 claims abstract description 4
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- 239000000243 solution Substances 0.000 claims description 78
- 239000008055 phosphate buffer solution Substances 0.000 claims description 37
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
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- 238000006243 chemical reaction Methods 0.000 claims description 15
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 8
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 7
- 238000010898 silica gel chromatography Methods 0.000 claims description 7
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 6
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 6
- 239000005457 ice water Substances 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- XFVZSRRZZNLWBW-UHFFFAOYSA-N 4-(Diethylamino)salicylaldehyde Chemical compound CCN(CC)C1=CC=C(C=O)C(O)=C1 XFVZSRRZZNLWBW-UHFFFAOYSA-N 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000007850 fluorescent dye Substances 0.000 claims description 5
- -1 piperazine-1-yl Chemical group 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 4
- 230000004224 protection Effects 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- KTACZRWRTSQVNO-UHFFFAOYSA-N 1-phenyl-2-piperazin-1-ylethanone Chemical compound C=1C=CC=CC=1C(=O)CN1CCNCC1 KTACZRWRTSQVNO-UHFFFAOYSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000011261 inert gas Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- KPXVKKBJROCIJB-UHFFFAOYSA-N 1-(4-piperazin-1-ylphenyl)ethanone Chemical compound C1=CC(C(=O)C)=CC=C1N1CCNCC1 KPXVKKBJROCIJB-UHFFFAOYSA-N 0.000 claims description 2
- 239000003054 catalyst Substances 0.000 claims description 2
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- 230000002194 synthesizing effect Effects 0.000 claims 4
- 238000003384 imaging method Methods 0.000 abstract description 4
- 125000004194 piperazin-1-yl group Chemical group [H]N1C([H])([H])C([H])([H])N(*)C([H])([H])C1([H])[H] 0.000 abstract description 3
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 108010027912 Sulfite Oxidase Proteins 0.000 description 1
- 102000043440 Sulfite oxidase Human genes 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
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- 150000003384 small molecules Chemical class 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 1
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- 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
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a coumarin-benzopyrylium salt derivative and a synthesis method and application thereof, the derivatives have the Chinese name of (E) -2- (4- (4- (2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acryloyl) piperazin-1-yl) phenyl) -7- (diethylamino) chromium perchlorate and the English name of (E) -2- (4- (2-cyano-3- (7- (diazepamino) -2-oxo-2H-chromen-3-yl) aryloyl) piperazin-1-yl) phenyl) -7- (diazepamino) chromenylperchlorate, and the name of CM-BP. The invention also provides a synthesis method of the coumarin-benzopyrylium salt derivative, a method for distinguishing, identifying and detecting glutathione and sulfur dioxide and application of the coumarin-benzopyrylium salt derivative in sulfur dioxide metabolic imaging. The content of glutathione and sulfur dioxide can be quantitatively detected by a fluorescence spectrophotometer in PBS solution with pH 7.4. The detection process is simple, sensitive and quick, and the detection result has high accuracy.
Description
Technical Field
The invention relates to fluorescent probe synthesis and glutathione and sulfur dioxide detection, in particular to a coumarin-benzopyrylium salt derivative, a synthesis method thereof and application thereof in distinguishing and detecting glutathione and sulfur dioxide.
Background
Studies have shown that glutathione is the most abundant non-protein thiol in cells and plays an important role in maintaining the intracellular redox balance, and the concentration imbalance is associated with a range of diseases: including immune response, cancer, and Alzheimer's disease. There is no question that intracellular metabolic abnormalities of glutathione are a direct factor causing an imbalance in its concentration. Glutathione and sodium thiosulfate in mitochondria generate sulfur dioxide under the action of thiosulfate transferase. Sulfur dioxide is an important small molecule active sulfide, has various pathological activities such as antibacterial, antihypertensive, antioxidant and protection to myocardial ischemia reperfusion injury, and endogenously generated sulfur dioxide can enhance the cell antioxidant effect and plays an important role in the signal transmission process. However, the lack of sulfite oxidase in cells leads to abnormal sulfur dioxide metabolism, and the accumulation of sulfite in human body damages important organs such as brain, nervous system and even causes death. Therefore, the development of an effective method for monitoring the metabolic processes of glutathione and sulfur dioxide in cells plays an important role in biological research and clinical diagnosis.
In response to the requirement of sulfur dioxide level detection in biological systems, designing fluorescent probes with good selectivity, high accuracy and low cytotoxicity has become one of the leading challenges in biomedical development.
Disclosure of Invention
The invention aims to provide a coumarin-benzopyrylium salt derivative, a synthesis method thereof, and application of the derivative as a probe for distinguishing, identifying and detecting glutathione and sulfur dioxide.
The invention provides a coumarin-benzopyrylium salt derivative, wherein the derivative is named as (E) -2- (4- (4- (2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acryloyl) piperazine-1-yl) phenyl) -7- (diethylamino) chromium perchlorate in the Chinese language, is named as (E) -2- (4- (4- (2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) aryloyl) piperazin-1-yl) phenyl) -7- (diethyleneimine) chromenyl ammonium perchlorate in the English language, is named as CM-BP, and has the structural formula as follows:
the invention provides a synthesis method of coumarin-benzopyrylium salt derivative CM-BP, which comprises the following steps:
(1) according to the mol ratio of 1: 1-1.5 dissolving 4-aldehyde-7-diethylamino coumarin and cyanoacetic acid in absolute ethyl alcohol, adding a small amount of piperidine as a catalyst, and heating the mixture at 85 ℃ for reaction for 3-4 hours; after the reaction is finished, the solvent is decompressed and dried in a spinning way, and the obtained orange solid is separated by silica gel column chromatography by using methanol and dichloromethane with the volume ratio of 1:15 as eluent to obtain orange solid powder, namely (E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acrylic acid;
(2) according to the mol ratio of 1: 1-1.5 dissolving 4-diethylamino salicylaldehyde and 4-piperazino acetophenone in concentrated sulfuric acid, stirring and refluxing the mixture at 90 ℃ for 6-8 hours, cooling to room temperature, pouring the reaction liquid into ice water, continuously stirring, filtering, washing and vacuum-drying the obtained precipitate to obtain a black solid, namely 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate;
(3) mixing and dissolving (E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acrylic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole in anhydrous N, N-dimethylformamide, reacting the mixture at 0 ℃ for 0.5 to 1 hour under the protection of inert gas, then adding 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate and triethylamine to the reaction system, and reacting the mixture at room temperature for 24 to 30 hours; pouring the mixture into ice water after the reaction is finished, filtering, washing and drying the obtained precipitate in vacuum to obtain a crude product, and separating the crude product by silica gel column chromatography by using dichloromethane and methanol with the volume ratio of 15:1 as an eluent to obtain a purple powdery target product (E) -2- (4- (4- (2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acryloyl) piperazine-1-yl) phenyl) -7- (diethylamino) chromium perchlorate, wherein E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acrylic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate and triethylamine in a molar ratio of 1: 3-5: 3-5: 0.8-1: 3-5.
Preferably, the method comprises the following steps:
the molar ratio of the 4-aldehyde-7 diethylaminocoumarin to the cyanoacetic acid in the step (1) is 1: 1.5.
the molar ratio of the 4-diethylamino salicylaldehyde to the 4-piperazinylacetophenone in the step (2) is 1: 1.
the molar ratio of E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromen-3-yl) acrylic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate and triethylamine in step (3) is 1: 4: 4: 0.8: 4.
the fluorescent probe can be used for detecting glutathione and/or sulfur dioxide.
The invention provides a method for detecting glutathione, which comprises the following steps:
(1) preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 0.2M glutathione solution, and dissolving CM-BP in DMSO to prepare a 2mM solution;
(2) adding 2mL of PBS (phosphate buffer solution) containing 10% DMSO and having pH of 7.4 and 10 mu L of DMSO solution of CM-BP into a fluorescence cuvette, adding glutathione into the fluorescence cuvette, and gradually increasing the fluorescence intensity at 638nm with the addition of glutathione to be detected;
(3) 2mL of a 10% DMSO-containing PBS solution having a pH of 7.4 and 10. mu.L of a CM-BP solution were added to each of 10 cuvettes, and glutathione solutions were added to each cuvette in a volume of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20. mu.L, respectively, and then the fluorescence intensity at 638nm was measured on a fluorescence spectrometer at 402, 469, 525, 588, 648, 713, 777, 826, 879, 925, 953. Plotting and drawing by taking the concentration of glutathione as an abscissa and fluorescence intensity as an ordinate to obtain a valleyWorking curve of cystine peptide concentration; the linear regression equation is: f638283.9c +416.5, c has a unit of 10-3mol/L；
(4) And when the sample solution is measured, substituting the measured fluorescence intensity into a linear regression equation to obtain the concentration of the glutathione.
The invention provides a method for detecting sulfur dioxide, which comprises the following steps:
(1) preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 2mM sulfur dioxide solution, and dissolving CM-BP in DMSO to prepare a 2mM solution;
(2) adding 2mL of PBS (phosphate buffer solution) containing 10% DMSO and having pH of 7.4 and 10 mu L of DMSO solution of CM-BP into a fluorescence cuvette, adding a sulfur dioxide aqueous solution into the fluorescence cuvette, and gradually reducing the fluorescence intensity at 638nm along with the addition of sulfur dioxide to be detected;
(3) 2mL of a 10% DMSO-containing PBS solution having a pH of 7.4 and 10. mu.L of a CM-BP solution were added to each of 8 cuvettes, and sulfur dioxide solutions were added to each cuvette in a volume of 0, 5, 10, 15, 20, 25, 30, and 35. mu.L, respectively, and then fluorescence intensity at 638nm was measured on a fluorescence spectrometer as 268, 230, 189, 145, 114, 90, 62, and 44. Plotting a chart by taking the sulfur dioxide concentration as an abscissa and the fluorescence intensity as an ordinate to obtain a working curve of the sulfur dioxide concentration; the linear regression equation is: f638-6.51c +256.7, c having a unit of 10-6mol/L；
(4) And when the sample solution is measured, substituting the measured fluorescence intensity into a linear regression equation to obtain the concentration of the sulfur dioxide.
The invention provides a method for simultaneously detecting glutathione and sulfur dioxide, which comprises the following steps:
(1) preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 0.2M glutathione solution, preparing a 2mM sulfur dioxide solution, and dissolving CM-BP in DMSO to prepare a 2mM solution;
(2) taking 2mL of 10% DMSO-containing PBS solution with the pH value of 7.4 and 10 mu L of DMSO solution of CM-BP, adding 20 mu L of glutathione solution into a fluorescence cuvette, and observing the fluorescence intensity increase at 638nm after 5 minutes; on the basis, adding a sulfur dioxide aqueous solution into a reaction system, and gradually reducing the fluorescence intensity at 638nm along with the addition of sulfur dioxide to be detected; the fluorescence intensity at 494nm is gradually increased.
Compared with the prior art, the invention has the following advantages and effects:
1. the preparation method of the coumarin-benzopyrylium salt derivative CM-BP is simple;
2. the coumarin-benzopyrylium salt derivative CM-BP can be used as a fluorescent probe to realize accurate detection of mitochondria glutathione and sulfur dioxide, and has high sensitivity and excellent selectivity;
3. the detection method is simple and can be realized only by means of a fluorescence spectrometer;
4. the invention can realize the visual real-time imaging of the metabolic process of sulfur dioxide at the cellular level.
Drawings
FIG. 1 nuclear magnetic hydrogen spectrum of CM-BP derivative prepared in example 1
FIG. 2 nuclear magnetic carbon spectrum of CM-BP derivative prepared in example 1
FIG. 3 Mass Spectroscopy of CM-BP derivative prepared in example 1
FIG. 4 fluorescence emission diagram of the effect of CM-BP derivative and glutathione in example 2
FIG. 5 working curve of glutathione measurement of CM-BP, a derivative of example 3
FIG. 6 fluorescent histogram of the effect of example 4 derivative CM-BP on sulfur dioxide
FIG. 7 working curve of sulfur dioxide determination of the derivative CM-BP of example 5
FIG. 8 fluorescence histograms of the derivative CM-BP and various analytes of example 6
FIG. 9 fluorescence histogram of the effect of the derivative CM-BP of example 7 on glutathione and sulfur dioxide
FIG. 10 imaging of CM-BP and glutathione derivatives of example 8
FIG. 11 photograph of CM-BP and sulfur dioxide cells as a derivative of example 9
FIG. 12 imaging of sulfur dioxide metabolism cells monitored by CM-BP as a derivative of example 10
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1
Preparation and characterization of coumarin-benzopyrylium salt derivative CM-BP
(1) 4-aldehyde-7 diethylaminocoumarin (0.49g,2mmol) and cyanoacetic acid (0.255g,3mmol) were dissolved in 100mL absolute ethanol, and the mixture was reacted with heating at 87 ℃ for 4 hours; after completion of the reaction, the solution was dried by evaporation under reduced pressure, and the resulting orange solid was subjected to silica gel column chromatography using methanol and methylene chloride as an eluent in a volume ratio of 1:15 to obtain an orange solid powder (0.303g, yield: 48.7%);
(2) dissolving 4-diethylamino salicylaldehyde (0.386g,2mmol) and 4-piperazinylacetophenone (0.408g,2mmol) in 10mL concentrated sulfuric acid, stirring and refluxing the mixture at 90 ℃ for 8 hours, cooling to room temperature, pouring the reaction solution into ice water, continuing stirring, filtering, washing and vacuum-drying the obtained precipitate to obtain a black solid, and separating the obtained orange solid by silica gel column chromatography with methanol and dichloromethane serving as eluent in a volume ratio of 1:10 to obtain orange solid powder (0.62g, yield: 67.3%);
(3) mixing and dissolving (E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acrylic acid (0.156g,0.5mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.26g,2mmol) and 1-hydroxybenzotriazole (0.38g,2mmol) in 10mL of anhydrous N, N-dimethylformamide, reacting the mixture at 0 ℃ for 0.5 hour under the protection of an inert gas, then 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate (0.184g,0.4mmol) and triethylamine (0.202g,2mmol) were added to the reaction system, respectively, and the mixture was reacted at room temperature for 24 hours; after the reaction, the mixture was poured into ice water, and the obtained precipitate was filtered, washed, and vacuum-dried to obtain a crude product, which was subjected to silica gel column chromatography using dichloromethane and methanol at a volume ratio of 15:1 as eluents to obtain the objective compound (0.105g, yield 34.3%) as a purple powder.1H NMR(600MHz,DMSO)δ8.69(s,1H),8.63(d,J＝8.4Hz,1H),8.28(d,J＝8.7Hz,2H),7.98(d, J ═ 8.5Hz,1H),7.90(d, J ═ 9.2Hz,1H),7.77(s,1H),7.60(d, J ═ 8.8Hz,1H),7.35(d, J ═ 9.7Hz,1H),7.30(s,1H),7.18(d, J ═ 8.9Hz,2H),6.83(d, J ═ 8.7Hz,1H),6.64(s,1H),3.79(s,4H),3.73(s,4H),3.67(d, J ═ 6.9Hz,4H),3.52(d, J ═ 6.7Hz,4H),1.23(t, J ═ 6.7Hz,6H),1.16(t, J ═ 8, 6H), fig. 1H)13C NMR (151MHz, DMSO) delta 148.39(s),143.66(s),132.24(s),131.26(s),117.29(s), 117.25-116.77 (M),114.20(s),110.94(d, J ═ 7.2Hz),108.67(s),108.14(s),102.93(s),97.05(s),96.47(s),45.65(s),44.99(s),12.87(s) (FIG. 2) ESI-MS: [ M + H ] S]+Calcd. For 656.3231, Foundation 656.3234 (FIG. 3)
Example 2
Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 0.2M glutathione solution, and dissolving CM-BP in DMSO to prepare a 2mM solution; adding 2mL of 10% DMSO-containing PBS solution with the pH value of 7.4 and 10 mu L of CM-BP solution into a fluorescence cuvette, adding glutathione into the fluorescence cuvette, and gradually increasing the fluorescence intensity at 638nm with the addition of glutathione to be detected; the fluorescence emission pattern is shown in FIG. 4.
Example 3
Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 0.2M glutathione solution, and dissolving CM-BP in DMSO to prepare a 2mM solution; 2mL of a 10% DMSO-containing PBS solution having a pH of 7.4 and 10. mu.L of a CM-BP solution were added to each of 10 cuvettes, and glutathione solutions were added to each cuvette in a volume of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. mu.L, respectively, and then the fluorescence intensity at 638nm was measured on a fluorescence spectrometer at 402, 469, 525, 588, 648, 713, 777, 826, 879, 925, and 953. Plotting a chart by taking the concentration of the glutathione as an abscissa and the fluorescence intensity as an ordinate to obtain a working curve of the concentration of the glutathione; the linear regression equation is: f638283.9c +416.5, c has a unit of 10-3mol/L; (see FIG. 5).
Example 4
Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 2mM sulfur dioxide solution, and dissolving CM-BP in DMSO to prepare a 2mM solution; adding 2mL of 10% DMSO-containing PBS solution with the pH value of 7.4 and 10 mu L of CM-BP solution into a fluorescence cuvette, adding a sulfur dioxide aqueous solution into the fluorescence cuvette, and gradually reducing the fluorescence intensity at 638nm along with the addition of sulfur dioxide to be detected; the fluorescence emission pattern is shown in FIG. 6.
Example 5
Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 2mM sulfur dioxide solution, and dissolving CM-BP in DMSO to prepare a 2mM solution; 2mL of a 10% DMSO-containing PBS solution at pH 7.4 and 10. mu.L of CM-BP were added to each of 8 cuvettes, and sulfur dioxide solutions were added to each cuvette at a volume of 0, 5, 10, 15, 20, 25, 30, 35. mu.L, and the fluorescence intensity at 638nm was measured on a fluorescence spectrometer at 268, 230, 189, 145, 114, 90, 62, 44. Plotting a chart by taking the sulfur dioxide concentration as an abscissa and the fluorescence intensity as an ordinate to obtain a working curve of the sulfur dioxide concentration; the linear regression equation is: f638-6.51c +256.7, c having a unit of 10-6mol/L; (see FIG. 7).
Example 6
Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 2mM sulfur dioxide solution, preparing a 0.2M glutathione solution, and dissolving CM-BP in DMSO to prepare a 2mM solution; in the fluorescence cuvette, 2mL of 10% DMSO in PBS at pH 7.4, 10 μ L of CM-BP in DMSO, and physiological concentrations of other analytes and aqueous solutions of glutathione and sulfur dioxide were added: GSH, Cys, Hcy, Lys, Arg, Asp, ClO-,H2O2,SCN-,SO4 2-,S2O3 2-,HS-,SO3 2-The fluorescence intensity values at 638nm for the different analytes were plotted by detection on a fluorescence spectrophotometer (see FIG. 8). Glutathione increases the fluorescence intensity of the probe at 638nm, sulfur dioxide significantly reduces the fluorescence intensity of the detection system at 638nm, and other analytes cause substantially no change in the fluorescence intensity of the detection system.
Example 7
Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a 2mM sulfur dioxide solution, preparing a 0.2M glutathione solution, and dissolving CM-BP in DMSO to prepare a 2mM solution; adding 20. mu.L of glutathione solution to the fluorescence cuvette, and observing an increase in fluorescence intensity at 638nm after 5 minutes; on the basis, adding a sulfur dioxide aqueous solution into a reaction system, and gradually reducing the fluorescence intensity at 638nm along with the addition of sulfur dioxide to be detected; the fluorescence intensity at 494nm is gradually enhanced; (see FIG. 9).
Example 8
Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a DMSO solution of 2mMCM-BP, and adding a 10 mu LCM-BP DMSO solution into 2mL PBS; adding the probe solution into a HepG2 cell culture solution to enable the concentration of the probe solution to be 10 mu M, incubating the probe solution with HeLa cells at 37 ℃, and enabling the probe to show red fluorescence under a fluorescence imager; the red fluorescence of the probe increased with increasing incubation time, see FIG. 10.
Example 9
Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, preparing a DMSO solution of 2mM CM-BP, and preparing an aqueous solution of 2mM sulfur dioxide; add 10. mu. LCM-BP in DMSO to 2mL PBS; adding the probe solution into a HepG2 cell culture solution to enable the concentration of the probe solution to be 10 mu M, and incubating the probe solution with HeLa cells for 30 minutes at 37 ℃, wherein the probe shows red fluorescence under a fluorescence imager; the probe system was added with 50 μ M aqueous solution of sulfur dioxide and the red fluorescence of the probe decreased with increasing incubation time, see FIG. 11.
Example 10
Preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, preparing a DMSO solution of 2mM CM-BP, and preparing a 2M sodium thiosulfate aqueous solution; add 10. mu. LCM-BP in DMSO to 2mL PBS; adding the probe solution into a HepG2 cell culture solution to enable the concentration of the probe solution to be 10 mu M, incubating the probe solution with HeLa cells at 37 ℃ for 30 minutes, and enabling the probe to show red fluorescence under a fluorescence imager; to the probe system was added a 500 μ M aqueous solution of sodium thiosulfate, and the red fluorescence of the probe decreased and the blue fluorescence increased with increasing incubation time, as shown in FIG. 12.
Claims (9)
1. A coumarin-benzopyrylium salt derivative CM-BP, characterized by the structural formula:
2. the method for synthesizing a coumarin-benzopyrylium salt derivative CM-BP according to claim 1, comprising the following steps:
(1) according to the mol ratio of 1: 1-1.5 dissolving 4-aldehyde-7-diethylamino coumarin and cyanoacetic acid in absolute ethyl alcohol, adding a small amount of piperidine as a catalyst, and heating the mixture at 85 ℃ for reaction for 3-4 hours; after the reaction is finished, the solvent is decompressed and dried in a spinning way, and the obtained orange solid is separated by silica gel column chromatography by using methanol and dichloromethane with the volume ratio of 1:15 as eluent to obtain orange solid powder, namely (E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acrylic acid;
(2) according to the mol ratio of 1: 1-1.5 dissolving 4-diethylamino salicylaldehyde and 4-piperazino acetophenone in concentrated sulfuric acid, stirring and refluxing the mixture at 90 ℃ for 6-8 hours, cooling to room temperature, pouring the reaction liquid into ice water, continuously stirring, filtering, washing and vacuum-drying the obtained precipitate to obtain a black solid, namely 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate;
(3) mixing and dissolving (E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acrylic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole in anhydrous N, N-dimethylformamide, reacting the mixture at 0 ℃ for 0.5 to 1 hour under the protection of inert gas, then adding 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate and triethylamine to the reaction system, and reacting the mixture at room temperature for 24 to 30 hours; pouring the mixture into ice water after the reaction is finished, filtering, washing and drying the obtained precipitate in vacuum to obtain a crude product, and separating the crude product by silica gel column chromatography by using dichloromethane and methanol with the volume ratio of 15:1 as an eluent to obtain a purple powdery target product (E) -2- (4- (4- (2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromium-3-yl) acryloyl) piperazine-1-yl) phenyl) -7- (diethylamino) chromium perchlorate; wherein the molar ratio of E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromen-3-yl) acrylic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate and triethylamine is 1: 3-5: 3-5: 0.8-1: 3-5.
3. The method for synthesizing CM-BP according to claim 2, wherein the molar ratio of 4-aldehyde-7 diethylaminocoumarin and cyanoacetic acid in step (1) is 1: 1.5.
4. the method for synthesizing CM-BP according to claim 2, wherein the molar ratio of 4-diethylamino salicylaldehyde to 4-piperazinylacetophenone in the step (2) is 1: 1.
5. the method for synthesizing CM-BP according to claim 2, wherein the molar ratio of E) -2-cyano-3- (7- (diethylamino) -2-oxo-2H-chromen-3-yl) acrylic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 7- (diethylamino) -2- (4- (piperazin-1-yl) phenyl) chromium perchlorate and triethylamine in the step (3) is 1: 4: 4: 0.8: 4.
6. the CM-BP of claim 1 as a fluorescent probe for the detection of glutathione and/or sulfur dioxide.
7. A method for detecting glutathione is characterized by comprising the following steps:
(1) preparing a 0.2M glutathione solution in a PBS buffer solution having a pH of 7.4 and a concentration of 10mM, and dissolving the CM-BP according to claim 1 in DMSO to prepare a 2mM solution;
(2) adding 2mL of PBS (phosphate buffer solution) containing 10% DMSO and having pH of 7.4 and 10 mu L of DMSO solution of CM-BP into a fluorescence cuvette, adding glutathione into the fluorescence cuvette, and gradually increasing the fluorescence intensity at 638nm with the addition of glutathione to be detected;
(3) 2mL of a 10% DMSO-containing PBS solution having a pH of 7.4 and 10. mu.L of a CM-BP solution were added to each of 10 cuvettes, and glutathione solutions were added to each cuvette in a volume of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20. mu.L, respectively, and then the fluorescence intensity at 638nm was measured on a fluorescence spectrometer at 402, 469, 525, 588, 648, 713, 777, 826, 879, 925, 953. Plotting a chart by taking the concentration of the glutathione as an abscissa and the fluorescence intensity as an ordinate to obtain a working curve of the concentration of the glutathione; the linear regression equation is: f638283.9c +416.5, c has a unit of 10-3mol/L；
(4) And when the sample solution is measured, substituting the measured fluorescence intensity into a linear regression equation to obtain the concentration of the glutathione.
8. A method for detecting sulfur dioxide is characterized by comprising the following steps:
(1) preparing a 2mM sulfur dioxide solution in a PBS buffer solution having a pH of 7.4 and a concentration of 10mM, and preparing a 2mM solution by dissolving the CM-BP according to claim 1 in DMSO;
(2) adding 2mL of PBS (phosphate buffer solution) containing 10% DMSO and having pH of 7.4 and 10 mu L of DMSO solution of CM-BP into a fluorescence cuvette, adding a sulfur dioxide aqueous solution into the fluorescence cuvette, and gradually reducing the fluorescence intensity at 638nm along with the addition of sulfur dioxide to be detected;
(3) 2mL of a 10% DMSO-containing PBS solution having a pH of 7.4 and 10. mu.L of a CM-BP solution were added to each of 8 cuvettes, and sulfur dioxide solutions were added to each cuvette in a volume of 0, 5, 10, 15, 20, 25, 30, and 35. mu.L, respectively, and then fluorescence intensity at 638nm was measured on a fluorescence spectrometer as 268, 230, 189, 145, 114, 90, 62, and 44. Plotting a chart by taking the sulfur dioxide concentration as an abscissa and the fluorescence intensity as an ordinate to obtain a working curve of the sulfur dioxide concentration; the linear regression equation is: f638-6.51c +256.7, c having a unit of 10-6mol/L；
(4) And when the sample solution is measured, substituting the measured fluorescence intensity into a linear regression equation to obtain the concentration of the sulfur dioxide.
9. A method for simultaneously detecting glutathione and sulfur dioxide is characterized by comprising the following steps:
(1) preparing a 10mM PBS buffer solution with a pH of 7.4, preparing a 0.2M glutathione solution, preparing a 2mM sulfur dioxide solution, and dissolving the CM-BP of claim 1 in DMSO to prepare a 2mM solution;
(2) taking 2mL of 10% DMSO-containing PBS solution with the pH value of 7.4 and 10 mu L of DMSO solution of CM-BP, adding 20 mu L of glutathione solution into a fluorescence cuvette, and observing the fluorescence intensity increase at 638nm after 5 minutes; on the basis, adding a sulfur dioxide aqueous solution into a reaction system, and gradually reducing the fluorescence intensity at 638nm along with the addition of sulfur dioxide to be detected; the fluorescence intensity at 494nm is gradually increased.
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Application publication date: 20200609 |