CN110563689A - Long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells and preparation method and application thereof - Google Patents
Long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of fluorescent probes, and provides a long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells, and a preparation method and application thereof, wherein the molecular formula of the fluorescent probe is C29H26NO5 +3 the 3 probe 3 compound 3 is 3 named 3 as 32 3- 3 ( 3 4 3- 3 ( 3 acryloyloxy 3) 3 phenyl 3) 3- 3 4 3- 3 ( 32 3- 3 carboxyphenyl 3) 3- 3 7 3- 3 ( 3 diethylamino 3) 3 benzopyran 3, 3 PA 3- 3 A 3 for 3 short 3, 3 and 3 the 3 fluorescent 3 probe 3 which 3 is 3 constructed 3 by 3 taking 3 an 3 anthocyanidin 3 derivative 3 as 3 a 3 fluorophore 3 and 3 taking 3 acrylate 3 as 3 a 3 recognition 3 unit 3 and 3 can 3 specifically 3 recognize 3 cysteine 3. 3 The Michael addition-cleavage reaction of cysteine and a probe is utilized for high-selectivity detection. The invention belongs to the technical field of organic small molecule fluorescent probes, and the probe has obvious color change, good water solubility and can be arranged in a water-soluble ringEfficient cysteine recognition in environmental, organic and cellular environments. The method has the advantages of simple operation, high sensitivity, good selectivity and stable property, and can be stored and used for a long time.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells, a preparation method and application thereof, which can sensitively identify high selectivity of cysteine and respond to water solubility, organic environment and cell environment.
Background
In recent years, thiol-containing amino acids have attracted much attention because they play a crucial role in the regulation of physiological and pathological processes, including cysteine (Cys), homocysteine (Hcy) and Glutathione (GSH). Among them, cysteine is involved in protein synthesis, detoxification and metabolism, is an important biological thiol, and is involved in many diseases. For example, cysteine deficiency can lead to slow growth, edema, lethargy, liver damage, muscle and fat damage, skin lesions in children. Excess cysteine is also a significant cause of cardiovascular disease and alzheimer's disease.
Electrochemical analysis, mass spectrometry, and High Performance Liquid Chromatography (HPLC) can be used to detect cysteine. However, these methods are complicated to operate and require expensive equipment. Compared with the prior art, the fluorescence detection method has the advantages of simple operation, high sensitivity, good selectivity and real-time monitoring. In addition, the fluorescent probe can image cysteine in living cells by a non-invasive confocal method, and provides possibility for further application of the fluorescent probe in the future. However, cysteine, homocysteine and glutathione have similar structural and response properties and are present in almost all mammalian cells, and therefore, selective differentiation of cysteine, homocysteine and glutathione remains a significant challenge in this field.
Currently, many small molecule fluorescent probes for selective detection of cysteine have been based on specific chemical reactions, such as michael addition, cyclization with aldehydes, disulfide exchange and cleavage reactions, and the like. In 2011, Strongin et al constructed benzothiazole derivatives for the first time using acryloyl as a reactive site for specific recognition of cysteine. Since then, fluorescent probes based on this design concept have been widely developed. After reaction of the acryloyl group with cysteine, the acrylate unit of the probe is cleaved by cysteine to release the fluorescent signal unit, and highly selective detection of cysteine is achieved by fluorescent signal enhancement.
Disclosure of Invention
The invention provides a long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells, a preparation method and application thereof, a novel fluorescent dye is synthesized, the cysteine can be specifically identified by the novel probe generated after the novel fluorescent dye is combined with an acryloyl group at an identification site, and the novel probe is simple and convenient in synthesis method, strong in operability, good in selectivity, high in sensitivity and capable of being identified by naked eyes.
The invention is realized by the following technical scheme: a long wavelength emission fluorescent probe for specifically detecting cysteine in living cells, the molecular formula of the fluorescent probe is C29H26NO5 + 3 the 3 name 3 of 3 the 3 probe 3 compound 3 is 32 3- 3 ( 3 4 3- 3 ( 3 acryloyloxy 3) 3 phenyl 3) 3- 3 4 3- 3 ( 32 3- 3 carboxyphenyl 3) 3- 3 7 3- 3 ( 3 diethylamino 3) 3 benzopyran 3, 3 PA 3- 3 A 3 for 3 short 3, 3 and 3 the 3 structural 3 formula 3 of 3 the 3 fluorescent 3 probe 3 is 3 as 3 follows 3: 3。
The method for preparing the long-wavelength emission fluorescent probe for specifically detecting cysteine in living cells comprises the following steps:
(1) Dissolving m-N-diethylaminophenol and phthalic anhydride in benzene, and reacting at 130 ℃ for 12 h to obtain a white solid;
(2) Adding the white solid obtained in the step (1) and p-hydroxyacetophenone into a methanesulfonic acid solution, reacting for 8 h at 90 ℃, treating the obtained solid with ice water and perchloric acid, extracting with dichloromethane, and finally performing column chromatography separation to obtain a compound 1, wherein the structural formula is as follows:；
(3) 3 reacting 3 the 3 compound 3 1 3 with 3 acryloyl 3 chloride 3 in 3 alkaline 3 environment 3 triethylamine 3 for 3 16 3 h 3 at 3 normal 3 temperature 3, 3 and 3 separating 3 and 3 purifying 3 to 3 obtain 3 the 3 fluorescent 3 probe 3 compound 3 PA 3- 3 A 3. 3
The molar ratio of the m-N-diethylaminophenol to the phthalic anhydride in the step (1) is 1: 1; every millimole of m-diethylaminoaminophenol was dissolved in 10-70 mL of benzene.
The molar ratio of the white solid to the p-hydroxyacetophenone in the step (2) is 1: 1-1.5; the dosage of the methanesulfonic acid is 5-10 mL per millimole of p-hydroxyacetophenone; the dosage of the ice water is 50-100mL of ice water for each millimole of p-hydroxyacetophenone; the perchloric acid is used in an amount of 3 to 5 mL per millimole of p-hydroxyacetophenone.
In the step (3), the molar ratio of the compound 1 to the acryloyl chloride is 1: 1-4; the molar ratio of triethylamine to the compound 1 in the alkaline environment is 1: 1-4.
the specific method for separating and purifying in the step (3) comprises the following steps: and (3) diluting the solution after the reaction is finished with dichloromethane, washing with water for three times, drying, performing rotary evaporation to remove the solvent, dissolving the solid with dichloromethane, and performing column chromatography separation by using a mixed solvent of dichloromethane and methanol to obtain the fluorescent probe compound.
Use of a long wavelength emitting fluorescent probe for the specific detection of cysteine in living cells for the detection of cysteine in a water soluble environment, an organic environment or a cellular tissue environment.
After the fluorescent probe reacts with cysteine, the color of the solution is changed from light pink to light purple, and the color change is obvious.
The invention also provides a preparation method of the cysteine fluorescent probe, which is synthesized by stirring the corresponding anthocyanidin derivative corresponding to the cysteine fluorescent probe and acryloyl chloride in a dichloromethane solution for 16 h at room temperature.
the synthetic route of the cysteine fluorescent probe is shown in figure 1.
The fluorescent probe can act with cysteine to generate the change of a fluorescence spectrum, thereby realizing the quantitative detection of the cysteine.
The cysteine fluorescent probe disclosed by the invention respectively acts with homocysteine, glutathione, arginine, aspartic acid, histidine, lysine, glutamic acid, tryptophan, threonine, serine, alanine, tyrosine, sulfide ion, hypochlorite ion, hydrosulfite, sulfite ion, chloride ion, carbonate ion, bromide ion, acetate ion, thiosulfate radical, sulfate radical, magnesium ion, lead ion, copper ion, barium ion, manganese ion, zinc ion and other substances, so that the fluorescence spectrum of the probe cannot be obviously changed; after cysteine was added to these solutions, the fluorescence spectra of the solutions changed significantly and closely to the changes produced by the direct addition of cysteine, indicating that the reactants in the experiment had no effect on the detection of cysteine.
The reaction mechanism of the fluorescent probe is clarified through the nuclear magnetic hydrogen spectrum titration reaction, and the double bond peak disappears gradually after the reaction of the acrylate of the probe and the cysteine, thereby proving that the reaction does occur.
the fluorescent probes of the present invention successfully imaged cysteine in living cells.
Drawings
FIG. 1 is a schematic diagram of the synthesis of the fluorescent probe for detecting biological thiols in an aqueous environment according to the invention;
FIG. 2 is the change of fluorescence intensity of the fluorescent probe under the condition of different concentrations of cysteine in the environment of PBS buffer solution with pH =7.4 in example 3; wherein the lowest curve is a fluorescence intensity curve when no biological thiol is added, the concentrations of the biological thiols in the curves sequentially increase from bottom to top, and the fluorescence curve of the biological thiol is when the concentration of the uppermost curve is 500 mu M (50 equivalent);
FIG. 3 is a fluorescence curve of fluorescence intensity of the fluorescent probe at 575 nm with respect to the ratio of bio-thiol to probe at pH =7.4 in example 3 of the present invention, and it can be seen that the saturation equivalent of cysteine reacted with the fluorescent probe is 15 equivalents (150. mu.M);
FIG. 4 is a linear relationship of cysteine in a concentration range of 0 to 36 μ M when reacted with a fluorescent probe at pH =7.4 in example 3 of the present invention;
FIG. 5 is a graph showing the change of fluorescence intensity with time of the reaction between the fluorescent probe and cysteine at pH =7.4 in example 3 of the present invention, and it can be seen that the optimal reaction time of the fluorescent probe and cysteine is 30 min;
FIG. 6 shows the change of fluorescence intensity at 575 nm between a fluorescent probe and cysteine at pH = 2-12 in example 3 of the present invention;
FIG. 7 shows the reaction of fluorescent probes with different equivalents of cysteine, homocysteine and glutathione;
FIGS. 8(a), 8(b) are graphs comparing the change in fluorescence intensity of fluorescent probes after the addition of different amino acids and ions, respectively, under the same conditions in example 5 of the present invention;
FIG. 9 is a nuclear magnetic titration reaction hydrogen spectrum of the probe in the deuterated reagent and cysteine in example 6 of the present invention, wherein the double bond hydrogen disappears after the reaction, which indicates that the probe actually reacts with cysteine;
FIG. 10 is an image of cysteine in cells imaged by the probe of example 7 of the present invention, which is a confocal image of the red channel, white channel and overlay when the probe is added, the thiol depleting reagent maleimide (NEM) and the probe are added, and the NEM and the probe are added and cysteine is added, wherein: a is an image added with the probe, B is an image added with the NEM and the probe, C is an image when the NEM, the probe and the added cysteine are added, 1 is a red light confocal image, 2 is a white light channel confocal image, and 3 is a confocal image when the NEM, the probe and the added cysteine are superposed.
Detailed Description
Example 1: preparation of compound 1: reacting N-diethylaminophenol (1.8 g, 10.9 mmol) with phthalic anhydride (1.8 g, 12.2 mmol) in 200 mL of benzene at 130 ℃ for 12 hours, suction-filtering to obtain a white solid, adding the white solid (0.626 g, 2 mmol) to 10 mL of methanesulfonic acid containing p-hydroxyacetophenone (0.272 g, 2 mmol), reacting at 90 ℃ for 8 hours, cooling, adding to 200 mL of ice water, adding 10 mL of perchloric acid, extracting with dichloromethane (50 mL. times.3), drying, rotary-evaporating the dried solvent, and separating by column chromatography using a mixed solvent of dichloromethane and glacial acetic acid (volume ratio: 10: 1) to obtain compound 1, yield: 33 percent. The synthetic route is shown in figure 1.
example 2: the preparation of the fluorescent probe for detecting the biological mercaptan in the water-soluble environment comprises the following steps: compound 1(600 mg, 1.45 mmol) was stirred with acryloyl chloride (468. mu.L, 5.8 mmol) and triethylamine (804. mu.L, 5.8 mmol) in 30mL redistilled dichloromethane in ice bath for 1 h and then returned to room temperature for 16 h. 3 adding 3 30 3 mL 3 of 3 dichloromethane 3 for 3 dilution 3, 3 washing 3 with 3 water 3 ( 3 50 3 mL 3 multiplied 3 by 33 3) 3, 3 drying 3, 3 then 3 rotationally 3 evaporating 3 the 3 solvent 3, 3 and 3 performing 3 column 3 chromatography 3 separation 3 by 3 using 3 a 3 mixed 3 solvent 3 of 3 dichloromethane 3 and 3 methanol 3 ( 3 the 3 volume 3 ratio 3 is 3 30 3: 3 1 3) 3 to 3 obtain 3 the 3 target 3 compound 3 PA 3- 3 A 3, 3 wherein 3 the 3 yield 3 is 3 as 3 follows 3: 3 50 percent.
Example 3: titration experiment of pH =7.4 fluorescent probe with cysteine: in PBS buffer of pH =7.4, a fluorescent probe was added at an initial concentration of 2 mM so that the concentration of the fluorescent probe in the solution was 10 μ M. Then, different amounts of the initial concentration of 20 mM of biological thiol were added sequentially so that the concentrations of biological thiol in the solution were 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 10. mu.M, 12. mu.M, 14. mu.M, 16. mu.M, 18. mu.M, 20. mu.M, 24. mu.M, 28. mu.M, 32. mu.M, 36. mu.M, 40. mu.M, 45. mu.M, 50. mu.M, 60. mu.M, 80. mu.M, 100. mu.M, 120. mu.M, 140. mu.M, 160. mu.M, 180. mu.M, 200. mu.M, 250. mu.M, 300. mu.M, and 500. mu.M, respectively, and cysteine was allowed to react sufficiently with the fluorescent probe. Absorption spectra and fluorescence spectra of cysteine under different concentrations were measured with an absorption spectrometer and a fluorescence spectrometer, respectively, the excitation wavelength of the fluorescence spectra was 570 nm, the emission wavelength was 626 nm, and the detection wavelength was 575 nm, with the results shown in fig. 2-5.
As can be seen from FIG. 2, the fluorescence intensity at 626 nm gradually increased with the increase of cysteine concentration in a certain concentration range, indicating that the fluorescent probe can respond to cysteine. The increase in fluorescence intensity of cysteine in the range from 150. mu.M (15 equivalents) to 500. mu.M (50 equivalents) is no longer observed, indicating that the concentration of cysteine is saturated at 150. mu.M (15 equivalents). The fluorescence curve for cysteine at the uppermost curve concentration of 500. mu.M (50 equivalents).
FIG. 3 is a graph showing the change in fluorescence as a function of the ratio of cysteine to fluorescent probe, and it can be seen that the number of saturation equivalents of cysteine to fluorescent probe is 15 equivalents (150. mu.M).
FIG. 4 shows the change of fluorescence spectra with the ratio of cysteine to fluorescent probethe fluorescence spectrum of (1) is linearly changed. As can be seen from the curves, the concentration of cysteine showed a good linear relationship in the range of 0-36. mu.M. 3 the 3 detection 3 limit 3 of 3 PA 3- 3 A 3 for 3 cysteine 3 was 3 calculated 3 to 3 be 3 8.5 3 X 3 10 3 according 3 to 3 the 3 formula 3 for 3 calculation 3 of 3 detection 3 limit 3 defined 3 by 3 IUPAC 3 ( 3 CDL 3 = 33 3Sb 3 / 3 m 3, 3 where 3Sb 3 is 3 the 3 standard 3 deviation 3 and 3 m 3 is 3 the 3 slope 3) 3-8 mol / L。
pH =7.4 fluorescent probe reacted with cysteine as the change in fluorescence intensity over time is shown in fig. 5. It can be seen from the figure that the probe is able to respond to cysteine over time.
the reaction of fluorescent probes with cysteine at pH = 2-12 is shown in fig. 6. As can be seen from the figure, the probe showed a good reaction to cysteine in the range of pH = 7-11 under physiological conditions, and is expected to be used for in vivo detection.
Example 4: the fluorescent probe reacts with cysteine, homocysteine and glutathione which have the same sulfhydryl structure in the organism environment under the condition of different equivalent weights:
The fluorescent probes were reacted with 1, 5, 10, 15, 20, 30, 40, 50 equivalents (i.e., 10. mu.M, 50. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 300. mu.M, 400. mu.M, 500. mu.M) of cysteine, homocysteine, glutathione, respectively, under the same test conditions as described in example 3. The fluorescence intensity was measured after 30 min of reaction as shown in FIG. 7. As can be seen from the figure, the change of the reaction between the fluorescent probe and cysteine is larger than that between the fluorescent probe and homocysteine or glutathione under the same concentration, which indicates that the fluorescent probe has better selectivity for cysteine in thiol.
Example 5: selectivity test for detecting cysteine with fluorescent probe: the fluorescence spectra after addition of the different bioactive small molecules were tested as described in example 3, with an excitation wavelength of 570 nm, an emission wavelength of 626 nm and a detection wavelength of 575 nm, with an excess of the other bioactive small molecules added to the solution under the same test conditions.
the bioactive small molecules tested were 1, cysteine (Cys, 150 μ M); 2. homocysteine (Hcy, 150 μ M); 3. glutathione (GSH, 1 mM); 4. threonine (Thr, 150. mu.M); 5. arginine (Arg, 150. mu.M); 6. asparagine (Asp, 150. mu.M); 7. serine (Ser, 150. mu.M), 8, alanine (Ala, 150. mu.M); 9. lysine (Lys, 150. mu.M); 10. tryptophan (Trp, 150 μ M); 11. glutamic acid (Glu, 150. mu.M); 12. histidine (His, 150 μ M); 13. sodium hypochlorite (10 μ M); 14. hydrogen peroxide (10 μ M); 15. sulfate ion (10 μ M); 16. chloride (10 μ M); 17. bicarbonate ion (10 μ M); 18. bromide (10 μ M); 19. acetate ion (10 μ M); 20. sulfite ion (10 μ M); 21. thiosulfate ion (10 μ M); 22. sulfate ion (10 μ M); 23. disulfite ion (10 μ M); 24. magnesium ions (10 μ M); 25. lead ions (10 μ M); 26. copper ions (10 μ M); 27. barium ion (10 μ M); 28. manganese ions (10 μ M); 29. zinc ion (10. mu.M).
The result shows that the fluorescence intensity is only changed obviously by cysteine, other bioactive small molecules do not interfere the detection result, and the fluorescence probe can be proved to have higher selectivity to cysteine. When cysteine (150. mu.M) was added to these solutions, the fluorescence spectra of the solutions changed significantly and closely to the change produced by the direct addition of cysteine, and the results are shown in FIGS. 8(a) and 8(b), indicating that the reactants in the experiment had no effect on the detection of cysteine.
Example 6: the probe (5 mg) was dissolved in deuterated acetonitrile, 15 equivalents of cysteine dissolved in deuterated deuterium was added, and nuclear magnetic resonance spectroscopy (NMR) was performed at 600 Hz for 30 min before the reaction of the fluorescent probe with cysteine and 1 h after the reaction, respectively, as shown in FIG. 9. The box part in the figure shows that the double bond hydrogen peak disappears after the reaction of the acrylic ester bond of the probe and the cysteine, and the mechanism shows that the probe actually reacts with the cysteine.
example 7: imaging of intracellular cysteines with probes: as a result, as shown in FIG. 10, it was found that the cells were brightened by confocal imaging after imaging the cells with the probe (5. mu.M), which is a fluorescence reaction generated after the probe reacts with cysteine existing in the cells. Cells were treated with NEM (a thiol depleting agent, 200. mu.M), cleared of intracellular thiol and probed (5. mu.M) to reveal a fluorescent dark.
After adding NEM (200. mu.M) to the cells to consume the intracellular thiol, the added cysteine (400. mu.M) was added, and then the probe (5. mu.M) was added to find that the cells became brighter again, indicating that the probe can actually react with cysteine in the cells to generate fluorescence, and indicating that the probe can react with cysteine in the cells.
Claims (7)
1. a long wavelength emitting fluorescent probe for specifically detecting cysteine in a living cell, comprising: the molecular formula of the fluorescent probe is C29H26NO5 + 3 the 3 name 3 of 3 the 3 probe 3 compound 3 is 32 3- 3 ( 3 4 3- 3 ( 3 acryloyloxy 3) 3 phenyl 3) 3- 3 4 3- 3 ( 32 3- 3 carboxyphenyl 3) 3- 3 7 3- 3 ( 3 diethylamino 3) 3 benzopyran 3, 3 PA 3- 3 A 3 for 3 short 3, 3 and 3 the 3 structural 3 formula 3 of 3 the 3 fluorescent 3 probe 3 is 3 as 3 follows 3: 3。
2. A method for preparing a long wavelength-emitting fluorescent probe for specifically detecting cysteine in a living cell according to claim 1, characterized in that: the method comprises the following steps:
(1) dissolving m-N-diethylaminophenol and phthalic anhydride in benzene, and reacting at 130 ℃ for 12 h to obtain a white solid;
(2) Adding the white solid obtained in the step (1) and p-hydroxyacetophenone into a methanesulfonic acid solution, reacting for 8 h at 90 ℃, treating the obtained solid with ice water and perchloric acid, extracting with dichloromethane, and finally performing column chromatography separation to obtain a compound 1, wherein the structural formula is as follows:；
(3) 3 reacting 3 the 3 compound 3 1 3 with 3 acryloyl 3 chloride 3 in 3 alkaline 3 environment 3 triethylamine 3 for 3 16 3 h 3 at 3 normal 3 temperature 3, 3 and 3 separating 3 and 3 purifying 3 to 3 obtain 3 the 3 fluorescent 3 probe 3 compound 3 PA 3- 3 A 3. 3
3. The method of preparing a long wavelength-emitting fluorescent probe specifically detecting cysteine in a living cell according to claim 2, characterized in that: the molar ratio of the m-N-diethylaminophenol to the phthalic anhydride in the step (1) is 1: 1; every millimole of m-diethylaminoaminophenol was dissolved in 10-70 mL of benzene.
4. The method of preparing a long wavelength-emitting fluorescent probe specifically detecting cysteine in a living cell according to claim 2, characterized in that: the molar ratio of the white solid to the p-hydroxyacetophenone in the step (2) is 1: 1-1.5; the dosage of the methanesulfonic acid is 5-10 mL per millimole of p-hydroxyacetophenone; the dosage of the ice water is 50-100mL of ice water for each millimole of p-hydroxyacetophenone; the perchloric acid is used in an amount of 3 to 5 mL per millimole of p-hydroxyacetophenone.
5. The method of preparing a long wavelength-emitting fluorescent probe specifically detecting cysteine in a living cell according to claim 2, characterized in that: in the step (3), the molar ratio of the compound 1 to the acryloyl chloride is 1: 1-4; the molar ratio of triethylamine to the compound 1 in the alkaline environment is 1: 1-4.
6. The method of preparing a long wavelength-emitting fluorescent probe specifically detecting cysteine in a living cell according to claim 2, characterized in that: the specific method for separating and purifying in the step (3) comprises the following steps: and (3) diluting the solution after the reaction is finished with dichloromethane, washing with water for three times, drying, performing rotary evaporation to remove the solvent, dissolving the solid with dichloromethane, and performing column chromatography separation by using a mixed solvent of dichloromethane and methanol to obtain the fluorescent probe compound.
7. Use of a long wavelength emitting fluorescent probe for the specific detection of cysteine in living cells according to claim 1, characterized in that: the fluorescent probe is applied to detection of cysteine in a water-soluble environment, an organic environment or a cell tissue environment.
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| CN110894193A (en) * | 2018-09-13 | 2020-03-20 | 南京大学 | Synthesis of novel fluorescent probe and application of novel fluorescent probe in cysteine detection |
| CN111518071A (en) * | 2020-05-20 | 2020-08-11 | 湘潭大学 | Preparation and application of cysteine near-infrared fluorescent probe |
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| CN113999218A (en) * | 2021-12-03 | 2022-02-01 | 德州学院 | Flavonol compound, preparation method and application thereof in detection of biological thiol |
| CN114163398A (en) * | 2021-10-31 | 2022-03-11 | 南京碳硅人工智能生物医药技术研究院有限公司 | Design synthesis of biosensing tool for monitoring cysteine fluctuation in oxidative stress of living cells based on ICT principle |
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