Fluorescent probe for detecting cysteine as well as preparation method and detection method of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting cysteine as well as a preparation method and a detection method of the fluorescent probe. The specific chemical structural formula of the fluorescent probe is as follows: # imgabs0 #. The fluorescent probe provided by the invention is simple to prepare, low in cost, large in Stokes shift, high in detection speed and good in selectivity; the fluorescent probe can quickly recognize cysteine in dimethyl sulfoxide: PBS (1: 1); the detection method provided by the invention is simple to operate, the adopted solvent is low in price, and post-treatment is convenient.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, in particular to a fluorescent probe for detecting cysteine ​​and a preparation and detection method thereof. Background Art

[0002] Cysteine, a key sulfur-containing amino acid, plays a wide range of physiological roles in the body. It is not only a precursor of glutathione, but also plays a key role in maintaining intracellular redox homeostasis, participating in anti-inflammatory processes, and regulating immune function. Cysteine, through its sulfhydryl (-SH) group, participates in the formation of disulfide bonds in proteins, which is crucial for protein structural stability and function. Furthermore, cysteine ​​plays a central role in the synthesis of intracellular glutathione. As the primary cellular antioxidant, glutathione plays an irreplaceable role in protecting cells from oxidative stress. The liver regulates cysteine ​​metabolism and glutathione synthesis, precisely controlling intracellular cysteine ​​levels to meet normal metabolic needs and prevent various diseases, particularly liver damage. However, other effects include slowed growth in children, hair loss, edema, lethargy, liver damage, muscle and fat loss, skin lesions, Alzheimer's disease, folate and cobalamin (vitamin B12) deficiencies, and cardiovascular disease (CVD) risk factors. Therefore, cysteine ​​plays an important role in maintaining the health and function of organisms. Therefore, in the fields of medical diagnosis, physiological research and nutritional science, it is urgent to develop simple, sensitive and efficient detection and analysis technologies for Cys.

[0003] Currently, conventional methods for analyzing cysteine ​​(Cys) include high-performance liquid chromatography, colorimetry, mass spectrometry, electrochemical analysis, capillary electrophoresis, and fluorescence analysis. These traditional detection methods suffer from cumbersome procedures, long detection times, poor selectivity, high cost-effectiveness, the need for sample pretreatment, and dependence on specific environmental conditions (such as pH). In contrast, since the advent of fluorescent probes, they have been successfully applied in a wide range of research fields due to their advantages, including high sensitivity, low background interference, high selectivity, and good biocompatibility. They can also be combined with laser confocal microscopy for non-invasive, real-time, in situ imaging of biological cells and tissues, offering irreplaceable advantages and providing a powerful analytical technique for predicting and diagnosing human health. Therefore, the specific identification of Cys using fluorescent probes is a method worthy of promotion. Summary of the Invention

[0004] To address the above problems, the present invention provides a fluorescent probe for detecting cysteine ​​and its preparation and detection method. Through molecular design, a structure that can specifically recognize Cys fluorescent probe is obtained, making it have the advantages of specific selectivity, high sensitivity, and a wide detection concentration range.

[0005] According to one object of the present invention, the present invention provides a fluorescent probe for detecting cysteine, the specific chemical structure of which is shown below: .

[0006] According to the second object of the present invention, the present invention provides a method for preparing the fluorescent probe for detecting cysteine, comprising the following steps: S1. 5-methylsalicylaldehyde (5-MSA) and 2-aminothiophenol (2-ABT) were subjected to three cycles of evacuation and nitrogen filling to ensure an inert atmosphere. The mixture was then dissolved in 60 ml of anhydrous ethanol. 4.84 ml of concentrated hydrochloric acid was slowly added dropwise in an ice-salt bath. The reaction mixture was stirred for 10 minutes, followed by the slow addition of 10 ml of 30% hydrogen peroxide. The reaction was allowed to proceed at room temperature for 2 hours. After completion of the reaction, the product was isolated by washing with ethanol to obtain GuS-01 as a white solid. S2. Weigh the intermediate GuS-01 and hexamethylenetetramine and dissolve them in 80 ml of trifluoroacetic acid. Stir thoroughly to dissolve the raw materials. After the reaction is complete, return the mixture to room temperature and add a saturated aqueous KOH solution to neutralize the acid until the reaction mixture becomes neutral. Extract the mixture with dichloromethane, dry it over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain a crude product. Purify the product by column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 20:1 to obtain the product GuS-02. S3. Weigh the intermediate GuS-02 and (formylmethylene)triphenylphosphine and evacuate and refill with nitrogen three times to ensure an inert atmosphere. Then, dissolve the mixture in 50 mL of anhydrous tetrahydrofuran (THF). After complete dissolution, heat the reaction mixture to 66°C and reflux for 24 hours. Purify the mixture by column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 20:1 to obtain GuS-03 as a light yellow solid. S4. Weigh the intermediate GuS-02 and acryloyl chloride and dissolve them in 20 ml of anhydrous dichloromethane. Add triethylamine dropwise in an ice-water bath while maintaining the temperature at 0°C. React for 12 hours. After the reaction is complete, wash the product with water to obtain a white solid, GuS-02-B. S5. Weigh the intermediate GuS-03 and acryloyl chloride and dissolve them in 20 ml of anhydrous dichloromethane. Add triethylamine dropwise in an ice-water bath while maintaining the temperature at 0°C for 12 hours. Extract the product with dichloromethane, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure. Purify the crude product by flash column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 5:1 to obtain GuS-03-B as a pale yellow solid. S6. Weigh the intermediate GuS-02, 2,4-dinitrofluorobenzene, and potassium carbonate in 8 ml of dimethylformamide (DMF), stir at room temperature for 1 hour, then pour the reaction mixture into ice water and centrifuge to separate the crude product; purify the crude product by flash column chromatography with petroleum ether (PE) and ethyl acetate (EA) in a ratio of 10:1 to obtain the product GuS-02-242; S7. Add GuS-03, 2,4-dinitrofluorobenzene, and potassium carbonate to 8 ml of dimethylformamide (DMF); pour the reaction mixture into ice water and centrifuge to separate the crude product; purify the crude product by flash column chromatography with petroleum ether (PE) and ethyl acetate (EA) in a ratio of 10:1 to obtain the product GuS-03-242.

[0007] Furthermore, in step S1, the molar ratio of 5-methylsalicylaldehyde (5-MSA) to 2-aminothiophenol (2-ABT) is 1:1.

[0008] Furthermore, in step S2, the volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate=20:1, and the molar ratio of GuS-01:hexamethylenetetramine is 1:1.2.

[0009] Furthermore, in step S3, the volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate=20:1, and the molar ratio of GuS-02:(formylmethylene)triphenylphosphine is 1:1.

[0010] Furthermore, in step S4, the molar ratio of GuS-02: acryloyl chloride is 1:1.2; in step S5, the volume ratio of the elution phase for column chromatography purification is petroleum ether: ethyl acetate = 5:1, and the molar ratio of GuS-03: acryloyl chloride is 1:1.2.

[0011] Furthermore, in step S6, the volume ratio of the elution phase for column chromatography purification is petroleum ether: ethyl acetate = 10:1, and the molar ratio of GuS-02: 2,4-dinitrofluorobenzene is 1:1.2; in step S7, the volume ratio of the elution phase for column chromatography purification is petroleum ether: ethyl acetate = 10:1, and the molar ratio of GuS-03: 2,4-dinitrofluorobenzene is 1:1.2.

[0012] Furthermore, the reactions in step S1 and step S3 are both carried out under nitrogen protection.

[0013] According to the third object of the present invention, the present invention provides a method for detecting the above-mentioned fluorescent probe for detecting cysteine, comprising the following steps: The fluorescent probe was dissolved in an appropriate amount of chromatographic grade dimethyl sulfoxide to prepare a concentrated probe solution. The probe solution and cysteine ​​solution were then added to a centrifuge tube in sequence. After reacting for a period of time, a certain volume of dimethyl sulfoxide:PBS (1:1) was added to dilute the solution and perform fluorescence testing.

[0014] Furthermore, the molar ratio of fluorescent probe to cysteine ​​is 1:1-16.

[0015] Beneficial effects: The fluorescent probe provided by the present invention is simple to prepare, low in cost, has a large Stokes shift, fast detection speed and good selectivity; the fluorescent probe can quickly identify cysteine ​​in dimethyl sulfoxide:PBS (1:1); the detection method provided by the present invention is simple to operate, uses a low-cost solvent, and is convenient for post-processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the synthetic route of the fluorescent probe GuS-03-B of the present invention; Figure 2 is the hydrogen nuclear magnetic resonance spectrum of GuS-03-B of the present invention; Figure 3 Time titration graphs of GuS-02-B, GuS-02-242, GuS-03-B and GuS-03-242 of the present invention; Figure 4 This is a graph showing the fluorescence intensity reaching the highest value when the fluorescent probe GuS-03-B of the present invention interacts with cysteine ​​at different concentrations and the cysteine ​​equivalent is 16eq; Figure 5 is the Stokes shift diagram of the fluorescent probe GuS-03-B of the present invention; Figure 6 Figure a is the ion selectivity of the fluorescent probe GuS-03-B of the present invention; Figure 7 Figure b is the ion selectivity of the fluorescent probe GuS-03-B of the present invention; Figure 8 Figure a is the pH tolerance of the fluorescent probe GuS-03-B of the present invention; Figure 9 Figure b is the pH tolerance of the fluorescent probe GuS-03-B of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] Example 1 A fluorescent probe for detecting cysteine, the specific chemical structure is shown below: .

[0019] Example 2 like Figure 1-Figure 2 As shown, a method for preparing a fluorescent probe for detecting cysteine ​​comprises the following steps: S1. 5-Methylsalicylaldehyde (5-MSA) (2.5 g, 20.0 mmol, 1 eq) and 2-aminothiophenol (2-ABT) (2.72 g, 20.0 mmol, 1 eq) were evacuated and nitrogen-filled three times to ensure an inert atmosphere. The mixture was then dissolved in 60 mL of anhydrous ethanol. Concentrated hydrochloric acid (4.84 mL, 60 mmol, 3 eq.) was slowly added dropwise in an ice-salt bath. The reaction mixture was stirred for 10 minutes, and then 30% hydrogen peroxide (13.6 mL, 120 mmol, 6 eq) was slowly added. The reaction was carried out at room temperature for 2 hours. After completion of the reaction, the product was isolated by washing with ethanol to obtain GuS-01 as a white solid. S2. The intermediate GuS-01 (1.11 g, 4.6 mmol, 1 eq) and hexamethylenetetramine (773 mg, 5.52 mmol, 1.2 eq) were weighed and dissolved in 80 ml of trifluoroacetic acid. The raw materials were thoroughly stirred to dissolve. After the reaction was completed, the temperature was returned to room temperature. A saturated aqueous solution of KOH was added to the reaction mixture to neutralize the acid until the reaction mixture became neutral. The mixture was then extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 20:1 to obtain the product GuS-02. S3. Weigh the intermediate GuS-02 (269 mg, 1 mmol, 1 eq) and (formylmethylene)triphenylphosphine (304 mg, 1 mmol, 1 eq) and evacuate and refill with nitrogen three times to ensure an inert atmosphere. Then, dissolve the mixture in 50 mL of anhydrous tetrahydrofuran (THF). After complete dissolution, heat the reaction mixture to 66°C and reflux for 24 hours. Purify the mixture by column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 20:1 to obtain GuS-03 as a light yellow solid. S4. Weigh the intermediate GuS-02 (269 mg, 1 mmol, 1 eq) and acryloyl chloride (109 mg, 1.2 mmol, 1.2 eq) and dissolve them in 20 ml of anhydrous dichloromethane. Add triethylamine dropwise in an ice-water bath while maintaining the temperature at 0°C. React for 12 hours. After the reaction is complete, wash the product with water to obtain a white solid, GuS-02-B. S5. Weigh the intermediate GuS-03 (296 mg, 1 mmol, 1 eq) and acryloyl chloride (109 mg, 1.2 mmol, 1.2 eq) and dissolve them in 20 ml of anhydrous dichloromethane. Add triethylamine dropwise in an ice-water bath while maintaining the temperature at 0°C for 12 hours. Extract the product with dichloromethane, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure. Purify the crude product by flash column chromatography with petroleum ether (PE) and ethyl acetate (EA) in a ratio of 5:1 to obtain GuS-03-B as a light yellow solid. S6. The intermediate GuS-02 (269 mg, 1 mmol, 1 eq), 2,4-dinitrofluorobenzene (205 mg, 1.1 mmol, 1.1 eq), and potassium carbonate (274 mg, 2 mmol, 2 eq) were weighed and mixed in 8 ml of dimethylformamide (DMF). The mixture was stirred at room temperature for 1 hour, and then the reaction mixture was poured into ice water and centrifuged to separate the crude product. The crude product was purified by flash column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 10:1 to obtain the product GuS-02-242. S7. Weigh the intermediate GuS-03 (296 mg, 1 mmol, 1 eq), 2,4-dinitrofluorobenzene (205 mg, 1.1 mmol, 1.1 eq), and potassium carbonate (274 mg, 2 mmol, 2 eq) in 8 ml of dimethylformamide (DMF), stir at room temperature for 1 hour, then pour the reaction mixture into ice water and centrifuge to separate the crude product; purify the crude product by flash column chromatography with petroleum ether (PE) and ethyl acetate (EA) in a ratio of 10:1 to obtain the product GuS-02-242.

[0020] Example 3 A method for detecting cysteine ​​using a fluorescent probe comprises the following steps: The probe GuS-03-B was dissolved in an appropriate amount of chromatographic-grade dimethyl sulfoxide to prepare a 1 mM probe solution, and a 1-16 eq cysteine ​​solution was also prepared. Subsequently, 50 μl of the probe solution and 50 μl of the cysteine ​​solution were added to a centrifuge tube in sequence. The mixture was reacted for 10 minutes, and then 1.9 ml of dimethyl sulfoxide:PBS (v / v = 1:1) was added for dilution and fluorescence testing was performed. The fluorescence test parameters were: an excitation light slit of 10 nm, an emission light slit of 10 nm, and an excitation light wavelength of 486 nm.

[0021] like Figure 3 As shown, (a) is the time titration of GuS-02-B; (b) is the time titration of GuS-02-242; (c) is the time titration of GuS-03-B; (d) is the time titration of GuS-03-242; when the fluorescent probes GuS-02-B, GuS-03-B, GuS-02-242 and GuS-03-242 acted on cysteine ​​simultaneously, only GuS-03-B showed obvious fluorescence enhancement in a short time.

[0022] like Figure 4 As shown, when the fluorescent probe GuS-03-B is reacted with cysteine ​​at different concentrations, the fluorescence intensity reaches the highest value when the cysteine ​​equivalent is 16eq; like Figure 5 As shown, the Stokes shift of the fluorescent probe GuS-03-B; like Figure 6 and Figure 7 As shown, the ion selectivity of the fluorescent probe GuS-03-B; like Figure 8 and Figure 9 As shown, the pH tolerance of the fluorescent probe GuS-03-B; The fluorescent probe provided by the present invention is simple to prepare, low-cost, has a large Stokes shift, fast detection speed, and good selectivity; it can quickly identify cysteine. This fluorescent probe can quickly identify cysteine ​​in a 1:1 dimethyl sulfoxide:PBS mixture. The detection method provided by the present invention is simple to operate, uses inexpensive solvents, and allows for convenient post-processing.

[0023] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent probe for detecting cysteine, characterized in that The specific chemical structure is shown below: .

2. The method for preparing a fluorescent probe for detecting cysteine ​​according to claim 1, wherein The following steps are involved: S1. 5-methylsalicylaldehyde (5-MSA) and 2-aminothiophenol (2-ABT) were subjected to three cycles of evacuation and nitrogen filling to ensure an inert atmosphere. The mixture was then dissolved in 60 ml of anhydrous ethanol. 4.84 ml of concentrated hydrochloric acid was slowly added dropwise in an ice-salt bath. The reaction mixture was stirred for 10 minutes, followed by the slow addition of 10 ml of 30% hydrogen peroxide. The reaction was allowed to proceed at room temperature for 2 hours. After completion of the reaction, the product was isolated by washing with ethanol to obtain GuS-01 as a white solid. S2. Weigh the intermediate GuS-01 and hexamethylenetetramine and dissolve them in 80 ml of trifluoroacetic acid. Stir thoroughly to dissolve the raw materials. After the reaction is complete, return the mixture to room temperature and add a saturated aqueous KOH solution to neutralize the acid until the reaction mixture becomes neutral. Extract the mixture with dichloromethane, dry it over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain a crude product. Purify the product by column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 20:1 to obtain the product GuS-02. S3. Weigh the intermediate GuS-02 and (formylmethylene)triphenylphosphine and evacuate and refill with nitrogen three times to ensure an inert atmosphere. Then, dissolve the mixture in 50 mL of anhydrous tetrahydrofuran (THF). After complete dissolution, heat the reaction mixture to 66°C and reflux for 24 hours. Purify the mixture by column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 20:1 to obtain GuS-03 as a light yellow solid. S4. Weigh the intermediate GuS-02 and acryloyl chloride and dissolve them in 20 ml of anhydrous dichloromethane. Add triethylamine dropwise in an ice-water bath while maintaining the temperature at 0°C. React for 12 hours. After the reaction is complete, wash the product with water to obtain a white solid, GuS-02-B. S5. Weigh the intermediate GuS-03 and acryloyl chloride and dissolve them in 20 ml of anhydrous dichloromethane. Add triethylamine dropwise in an ice-water bath while maintaining the temperature at 0°C for 12 hours. Extract the product with dichloromethane, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure. Purify the crude product by flash column chromatography using petroleum ether (PE) and ethyl acetate (EA) in a ratio of 5:1 to obtain GuS-03-B as a pale yellow solid. S6. Weigh the intermediate GuS-02, 2,4-dinitrofluorobenzene, and potassium carbonate in 8 ml of dimethylformamide (DMF), stir at room temperature for 1 hour, then pour the reaction mixture into ice water and centrifuge to separate the crude product; purify the crude product by flash column chromatography with petroleum ether (PE) and ethyl acetate (EA) in a ratio of 10:1 to obtain the product GuS-02-242; S7. Add GuS-03, 2,4-dinitrofluorobenzene, and potassium carbonate to 8 ml of dimethylformamide (DMF); pour the reaction mixture into ice water and centrifuge to separate the crude product; purify the crude product by flash column chromatography with petroleum ether (PE) and ethyl acetate (EA) in a ratio of 10:1 to obtain the product GuS-03-242.

3. The method for preparing a fluorescent probe for detecting cysteine ​​according to claim 2, wherein In step S1, the molar ratio of 5-methylsalicylaldehyde (5-MSA) and 2-aminothiophenol (2-ABT) is 1:

1.

4. The method for preparing a fluorescent probe for detecting cysteine ​​according to claim 2, wherein In step S2, the volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate=20:1, and the molar ratio of GuS-01:hexamethylenetetramine is 1:1.

2.

5. The method for preparing a fluorescent probe for detecting cysteine ​​according to claim 2, wherein In step S3, the volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate=20:1, and the molar ratio of GuS-02:(formylmethylene)triphenylphosphine is 1:

1.

6. The method for preparing a fluorescent probe for detecting cysteine ​​according to claim 2, wherein: In step S4, the molar ratio of GuS-02: acryloyl chloride is 1:1.2; in step S5, the volume ratio of the elution phase for column chromatography purification is petroleum ether: ethyl acetate = 5:1, and the molar ratio of GuS-03: acryloyl chloride is 1:1.

2.

7. The method for preparing a fluorescent probe for detecting cysteine ​​according to claim 2, wherein: In step S6, the volume ratio of the elution phase for column chromatography purification is petroleum ether: ethyl acetate = 10:1, and the molar ratio of GuS-02: 2,4-dinitrofluorobenzene is 1:1.2; in step S7, the volume ratio of the elution phase for column chromatography purification is petroleum ether: ethyl acetate = 10:1, and the molar ratio of GuS-03: 2,4-dinitrofluorobenzene is 1:1.

2.

8. The method for preparing a fluorescent probe for detecting cysteine ​​according to claim 2, wherein: The reactions in step S1 and step S3 are both carried out under nitrogen protection.

9. The method for detecting cysteine ​​using a fluorescent probe according to claim 1, wherein: The following steps are involved: The fluorescent probe was dissolved in an appropriate amount of chromatographic grade dimethyl sulfoxide to prepare a concentrated probe solution. The probe solution and cysteine ​​solution were then added to a centrifuge tube in sequence. After reacting for a period of time, a certain volume of dimethyl sulfoxide:PBS (1:1) was added to dilute the solution and perform fluorescence testing.

10. The method for detecting cysteine ​​using a fluorescent probe according to claim 9, wherein: The molar ratio of fluorescent probe:cysteine ​​is 1:1-16.

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