A fluorescent probe for detecting thiophenol, its preparation method and application

Abstract

This invention relates to the field of fluorescence detection technology, and more particularly to a fluorescent probe for detecting thiophene, its preparation method, and its application. In this invention, intermediate 1 is synthesized from 2,4-dinitrobenzenesulfonyl chloride and p-phenylenediamine, and then intermediate 1 is reacted with compound 2 to synthesize the probe MC-PHSH, which is used to detect thiophene. This probe uses a 2,4-dinitrobenzenesulfonyl group as the recognition group. In neutral aqueous solution, the fluorescence of this probe is very weak. The 2,4-dinitrobenzenesulfonyl group can quench the fluorescence of the probe, while thiophene can cleave the 2,4-dinitrobenzenesulfonyl group, significantly enhancing its fluorescence intensity. Therefore, the probe MC-PHSH can detect thiophene without interference from other components.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology, and particularly relates to a fluorescent probe for detecting thiophenol, its preparation method and application. Background Technology

[0002] Thiols are an important class of molecules in biological systems and chemical science. Aliphatic thiols are present in several biologically important molecules, including cysteine, homocysteine, and glutathione, which are associated with a wide range of biological functions. While thiophenols have broad synthetic utility, they are highly toxic and polluting compounds. Thiols are highly toxic pollutants; studies have shown that the median lethal dose (LC50) of thiophenols in rodents is... 50 The concentration of thiophene is between 0.01 mM and 0.4 mM. Prolonged exposure to air containing thiophene can lead to a range of serious health consequences, including damage to the central nervous system, shortness of breath, muscle weakness, limb paralysis, coma, and even death. Despite its high toxicity, thiophene is an essential and widely used chemical, primarily in the production of agricultural chemicals, pharmaceuticals, and various pharmaceutical industrial products. Its main sources include oil refineries, the plastics and rubber industries, and landfills. Therefore, it is crucial to develop simple, rapid, and sensitive methods for detecting thiophene in environmental and biological samples.

[0003] In recent years, various analytical methods, such as high-performance liquid chromatography, gas chromatography-mass spectrometry, and ultraviolet-visible spectroscopy, have been developed for the detection of thiophenols. However, these methods typically require expensive equipment and highly skilled professionals. Compared to traditional detection methods, fluorescence chemiluminescence sensing technology offers significant advantages, such as non-destructive testing, high sensitivity, good selectivity, ease of operation, and low cost, making it a recent research hotspot. Fluorescence chemiluminescence sensing technology is not only helpful in detecting environmental pollution but also plays a crucial role in the diagnosis and treatment of diseases, thus its widespread application is noteworthy. Therefore, researchers have devoted considerable effort to developing fluorescence detection methods for various biologically related substances.

[0004] However, existing probe studies show that most probes exhibit poor selectivity for aliphatic thiols and thiophenes. Since aliphatic thiols and thiophenes share similar chemical characteristics, there is an urgent need for fluorescent reagents capable of selectively differentiating between them. Designing such fluorescent reagents is a challenging task. At neutral pH, aromatic thiols exist primarily as thiolates, while aliphatic thiols are protonated. When added to a neutral solution, they undergo a selective nucleophilic reaction with aromatic thiols, resulting in enhanced selective fluorescence. However, at alkaline pH, aliphatic thiols also exist as thiolates, showing enhanced fluorescence. Therefore, a new strategy is to create sensors that facilitate the selective detection of aromatic thiols over a wide pH range. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a fluorescent probe for detecting thiophene and its preparation method, which addresses the shortcomings of the prior art. The probe MC-PHSH is synthesized by reacting intermediate 1 with 2,4-dinitrobenzenesulfonyl chloride and p-phenylenediamine, and then reacting intermediate 1 with compound 2 to synthesize the probe for detecting thiophene.

[0006] This invention provides a fluorescent probe for detecting thiophene, wherein the fluorescent probe for detecting thiophene is probe MC-PHSH, and the structural formula of probe MC-PHSH is:

[0007] .

[0008] This invention provides a method for preparing the above-mentioned fluorescent probe for detecting thiophenol, comprising the following steps:

[0009] S1. Add p-phenylenediamine to dichloromethane containing 2,4-dinitrobenzenesulfonyl chloride, then add triethylamine, stir at room temperature to react fully to obtain a reaction solution, and distill the reaction solution under reduced pressure to obtain crude product 1. Perform column chromatography to separate and purify crude product 1 to obtain intermediate 1.

[0010] S2. Take intermediate 1 obtained from S1 and dissolve it in anhydrous ethanol. Then add compound 2 and react fully. After vacuum distillation, crude product 2 is obtained. Crude product 2 is separated and purified by column chromatography to obtain probe MC-PHSH.

[0011] The structural formula of compound 2 is:

[0012] .

[0013] According to the preparation method provided by the present invention, the ratio of p-phenylenediamine, 2,4-dinitrobenzenesulfonyl chloride, dichloromethane and triethylamine in S1 is 2 mmol: 2 mmol: 10 mL: 0.06 mL.

[0014] According to the preparation method provided by the present invention, the stirring time in S1 is 4 h, and the eluent used for purification is dichloromethane / methanol with a volume ratio of 20 / 1.

[0015] According to the preparation method provided by the present invention, the ratio of intermediate 1, anhydrous ethanol and compound 2 in S2 is 1 mmol: 10 mL: 1 mmol, and the reaction time is 2 h.

[0016] According to the preparation method provided by the present invention, the eluent used for purification in S2 is dichloromethane / methanol with a volume ratio of 50 / 1.

[0017] The present invention also provides an application of the above-mentioned fluorescent probe for detecting thiophene, wherein the probe MC-PHSH can be used to detect thiophene.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] This invention provides a fluorescent probe for detecting thiophene, its preparation method, and its application. In this invention, intermediate 1 is synthesized using 2,4-dinitrobenzenesulfonyl chloride and p-phenylenediamine. Intermediate 1 is then reacted with compound 2 to synthesize probe MC-PHSH, which is used for detecting thiophene. This probe uses a 2,4-dinitrobenzenesulfonyl group as the recognition group. In neutral solution, the probe exhibits very weak fluorescence. The 2,4-dinitrobenzenesulfonyl group quenches the fluorescence of the probe, while thiophene can cleave the 2,4-dinitrobenzenesulfonyl group, significantly enhancing its fluorescence intensity. Therefore, probe MC-PHSH can detect thiophene without interference from other components. Attached Figure Description

[0020] Figure 1 The mass spectrum of the probe MC-PHSH;

[0021] Figure 2 The fluorescence spectra of the probe MC-PHSH at different pH values ​​are shown.

[0022] Figure 3 The UV absorption spectra of the probe MC-PHSH at different pH values;

[0023] Figure 4 UV absorption spectra of probe MC-PHSH with different concentrations of thiophenol added;

[0024] Figure 5 Fluorescence spectra of probe MC-PHSH with different concentrations of thiophenol added. Detailed Implementation

[0025] In the following examples, the purity of p-phenylenediamine was 97%, the purity of p-toluene was 98%, the purity of N,N-dimethylformamide was 99.5%, the purity of acetonitrile was chromatographic grade, and all other reagents were analytical grade.

[0026] Example 1

[0027] This embodiment provides a method for preparing a fluorescent probe for detecting thiophenol, the specific steps of which are as follows;

[0028] S1. 2 mmol of p-phenylenediamine was added to 10 mL of dichloromethane containing 2 mmol of 2,4-dinitrobenzenesulfonyl chloride, followed by 0.06 mL of triethylamine. The mixture was stirred rapidly at room temperature with a magnetic stirrer. The color of the reaction solution changed from colorless to orange-red, and finally to yellow. The reaction was monitored by thin-layer chromatography until the reaction was completed, which took 4 h. The reaction solution was then treated by rotary evaporation (reduced pressure distillation) to remove the solvent. The crude product was separated by column chromatography using CH2Cl2:MeOH (dichloromethane:methanol) at a volume ratio of 20:1 to obtain a yellow solid intermediate 1.

[0029] ;

[0030] S2. Dissolve 1 mmol of intermediate 1 in a round-bottom flask containing 10 mL of anhydrous ethanol, then add 1 mmol of compound 2 to the flask and react for 2 h.

[0031] After the reaction was completed, the solvent was removed by vacuum distillation to obtain the crude product. The crude product was then separated by column chromatography. The eluent used for column chromatography was CH2Cl2:MeOH with a volume ratio of 50:1, which yielded a dark blue solid compound, namely the probe MC-PHSH.

[0032] .

[0033] Example 2

[0034] The probe MC-PHSH prepared in Example 1 was subjected to mass spectrometry analysis.

[0035] Take a small amount of the solid product probe MC-PHSH and place it in a 1.5 mL centrifuge tube. Add 40 μL of acetonitrile solution to dissolve it as a sample. Then, take another centrifuge tube, add 200 μL of acetonitrile solution, and then add 20 μL of sample. Shake well and repeat the previous step until the liquid is colorless. Then, start the mass spectrometry measurement.

[0036] The mass spectrum of the probe MC-PHSH is as follows Figure 1 As shown.

[0037] Example 3

[0038] The fluorescence spectrum of the probe MC-PHSH prepared in Example 1 was measured.

[0039] Preparation of buffer solutions:

[0040] Accurately weigh 17.805 g of disodium hydrogen phosphate using an electronic balance and place it in a small beaker. Add a small amount of distilled water, stir with a glass rod to dissolve it, transfer it to a 250 mL volumetric flask and make up to volume, then shake well.

[0041] Weigh 21.014 g of citric acid into a small beaker, add a small amount of distilled water, stir with a glass rod to dissolve it, transfer to a 250 mL volumetric flask, and dilute to 250 mL. Shake well. Then mix the two in a certain proportion to prepare disodium hydrogen phosphate-citric acid buffer solutions with different pH values.

[0042] Fluorescence spectroscopy measurement:

[0043] Weigh 29.4 mg of the synthesized fluorescent probe MC-PHSH and dissolve it in 5 mL of acetone to prepare a stock solution of a certain concentration for spectral testing. All the following spectra were performed in a disodium hydrogen phosphate-citrate buffer system.

[0044] The parameters of the fluorescence spectrophotometer are as follows: λ ex =510 nm, and the slit widths of Ex and Em are both 10 nm.

[0045] Fluorescence spectroscopy determination at different pH values: Take multiple colorimetric tubes and place them on a test tube rack. Use a pipette to add 500 μL of buffer solutions with pH values ​​of 2.2, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 into each colorimetric tube. Then add 20 μL of stock solution to each tube, dilute with distilled water to 3 mL, shake well, and let stand at room temperature for 3 min before starting the measurement.

[0046] The fluorescence spectra of the probe MC-PHSH at different pH values ​​are shown below. Figure 2 As shown, the pH ranges from 2.2 to 11.0. From... Figure 2 It can be concluded that the fluorescence intensity gradually decreases as the pH of the solution increases, reaching its maximum at a pH of 2.2, and becoming very weak at pH values ​​greater than or equal to 6.

[0047] Example 4

[0048] The probe MC-PHSH prepared in Example 1 was subjected to ultraviolet absorption spectroscopy.

[0049] Preparation of reference solution: Place one colorimetric tube on a test tube rack, use a pipette to add 500 μL of disodium hydrogen phosphate-citric acid buffer solution with pH 7.4 into the colorimetric tube, then add 40 μL of acetone solution, and then dilute with distilled water to 10 mL for baseline scanning.

[0050] Preparation of the test solution: Place multiple colorimetric tubes on a test tube rack. Use a pipette to add 500 μL of buffer solutions with pH values ​​of 2.2, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 to the colorimetric tubes. Add 20 μL of stock solution to each tube, then dilute with distilled water to 3 mL. Shake well and let stand at room temperature for 3 min before starting the measurement.

[0051] The UV absorption spectra of the probe MC-PHSH at different pH values ​​are shown below. Figure 3 As shown, the pH ranges from 2.0 to 11.0. From... Figure 3 It can be seen that the absorption peak is around 620 nm when the pH is between 2.2 and 6.0. When the pH increases, a new absorption peak appears at 490 nm and an isoabsorption point appears at around 550 nm. This indicates that the probe MC-PHSH has an equilibrium in different pH ranges, that is, it exists in two different forms in different pH ranges.

[0052] UV-Vis absorption spectra of probe MC-PHSH with different concentrations of thiophenol are shown below. Figure 4 As shown, a new absorption peak appears at around 500 nm, indicating that the probe reacted with thiophenol, and the absorption peak gradually increased with the increase of thiophenol concentration.

[0053] Fluorescence spectra of probe MC-PHSH with different concentrations of thiophenol are shown below. Figure 5 As shown, the fluorescence intensity increased sharply after the addition of thiophene. As the amount of thiophene gradually increased, the fluorescence intensity also increased. When the concentration of thiophene reached 80 μM, the fluorescence intensity almost reached saturation.

[0054] In summary, the present invention provides a hemicyanine fluorescent probe MC-PHSH, which uses 2,4-dinitrobenzenesulfonyl as a recognition group.

[0055] Fluorescence spectra of the probe MC-PHSH under different pH conditions revealed that its fluorescence was very weak in neutral solution. Fluorescence and UV absorption spectra of the probe against different concentrations of thiophene showed a new absorption peak at 500 nm, and both the fluorescence intensity and absorption peak increased with increasing thiophene concentration.

[0056] Therefore, it can be assumed that the 2,4-dinitrobenzenesulfonyl group can quench the fluorescence of the probe, while thiophenol can cleave the 2,4-dinitrobenzenesulfonyl group, thus significantly enhancing its fluorescence intensity.

[0057] Based on this phenomenon, the probe MC-PHSH can detect thiophene without interference from other components. Therefore, the fluorescent probe constructed in this study can provide a good method for detecting thiophene.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting thiophenol, characterized in that, The fluorescent probe used for detecting thiophene is probe MC-PHSH, and the structural formula of probe MC-PHSH is: 。 2. A method for preparing a fluorescent probe for detecting thiophenol as described in claim 1, characterized in that, Includes the following steps: S1. Add p-phenylenediamine to dichloromethane containing 2,4-dinitrobenzenesulfonyl chloride, then add triethylamine, stir at room temperature to react fully to obtain a reaction solution, and distill the reaction solution under reduced pressure to obtain crude product 1. Perform column chromatography to separate and purify crude product 1 to obtain intermediate 1. S2. Take intermediate 1 obtained from S1 and dissolve it in anhydrous ethanol. Then add compound 2 and react fully. After vacuum distillation, crude product 2 is obtained. Crude product 2 is separated and purified by column chromatography to obtain probe MC-PHSH. The structural formula of compound 2 is: 。 3. The preparation method according to claim 2, characterized in that, The ratio of p-phenylenediamine, 2,4-dinitrobenzenesulfonyl chloride, dichloromethane and triethylamine in S1 is 2 mmol: 2 mmol: 10-20 mL: 0.02-1 mL.

4. The preparation method according to claim 2, characterized in that, The stirring time for the reaction in S1 is 4 h, and the eluent used for purification is dichloromethane / methanol with a volume ratio of 20 / 1.

5. The preparation method according to claim 2, characterized in that, In S2, the ratio of intermediate 1, anhydrous ethanol, and compound 2 is 1 mmol: 10-15 mL: 1-1.5 mmol, and the reaction time is 2 h.

6. The preparation method according to claim 2, characterized in that, The eluent used for purification as described in S2 is a dichloromethane / methanol with a volume ratio of 50 / 1.

7. The application of a fluorescent probe for detecting thiophene as described in claim 1, characterized in that, The probe MC-PHSH can be used to detect thiophenol.

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