Near-infrared fluorescent probe for detecting thiophenol as well as preparation and application of near-infrared fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe for detecting thiophenol as well as preparation and application of the near-infrared fluorescent probe. The prepared near-infrared fluorescent probe shown in the formula I is used for detecting thiophenol when the pH value is 6.0, and has the advantages of good sensitivity, good selectivity and the like. Along with the increase of the concentration of thiophenol, the fluorescence intensity of a detection system at 650 nm is obviously increased. Therefore, the fluorescent probe shown in the formula I can be used for detecting the content of thiophenol in a water body. In addition, the fluorescent probe can be used for carrying out fluorescence imaging on the content of thiophenol in cells.

Description

Technical Field

[0001] This invention relates to a near-infrared fluorescent probe for detecting thiophene, its preparation and application, belonging to the field of small organic molecule fluorescent probes. Background Technology

[0002] Thiol is a colorless liquid with a distinctive odor, poorly soluble in water, and widely used in pharmaceuticals, pesticides, polymer materials, and as an auxiliary in organic synthesis. Currently, thiophenol is mainly found in chemical and pharmaceutical wastewater, and can enter the body through various routes, causing poisoning. Inhalation of large amounts of thiophenol in a short period can be fatal, and contact with open flames, high heat, or oxidizers may pose a risk of combustion or explosion. Therefore, developing highly selective and sensitive detection methods for thiophenol is of great significance.

[0003] Currently, various methods exist for detecting thiophene, including high-performance liquid chromatography (HPLC), gas chromatography (GC), and ultraviolet spectrophotometry (UV spectrophotometry). However, these methods share some common limitations: cumbersome sample pretreatment, low sensitivity, susceptibility to interference, expensive equipment, and the need for specialized technical support, limiting their application to laboratory settings. Among these analytical methods, fluorescent probe methods are highly favored and anticipated due to their advantages of high efficiency, sensitivity, and specificity, leading to their widespread use in the detection of thiophene.

[0004] In recent years, an increasing number of fluorescent probes for thiophenol have been developed. However, only a few chemical sensors have been proposed that can distinguish between aromatic thiols and aliphatic thiols. All of these sensors are based on thiolate anions (-S... - The nucleophilic reaction of aromatic thiols. K a The pH value (approximately 6.5) is lower than that of aliphatic thiols (approximately 8.5). Therefore, at pH 7.4, approximately 88.8% of aromatic thiols exist as thioglycolic anions, while only 7.4% of aliphatic thiols exist as thioglycolic anions. At the weakly acidic pH 6.0, 24.0% of aromatic thiols exist as thioglycolic anions, while only 0.3% of aliphatic thiols exist as thioglycolic anions. Therefore, developing fluorescent probes for detecting thiophenol under weakly acidic conditions would help improve its selectivity. Based on this difference, researchers have developed many fluorescent probes for detecting thiophenol. However, most fluorescent probes only show fluorescence response in the visible light range, and usually only exhibit fluorescence response without obvious color change. Therefore, this greatly limits the application of these probes. Therefore, developing a thiophenol fluorescent probe with good selectivity, high sensitivity, ease of operation, strong anti-interference ability, visualization, and wide applicability is of great significance for the detection of thiophenol content. Summary of the Invention

[0005] To address some problems existing in the fluorescent probes for detecting thiophene in the prior art, such as short fluorescence emission wavelength and poor selectivity, the present invention aims to provide a near-infrared fluorescent probe for detecting thiophene under weakly acidic conditions.

[0006] Another objective of this invention is to provide a method for preparing the above-mentioned fluorescent probe for detecting thiophene, which uses readily available raw materials, has simple synthesis steps, and achieves high yield.

[0007] Another object of the present invention is to provide the use of the above-described fluorescent probe for detecting thiophene in the preparation of reagents for detecting the content of thiophene in water and cells.

[0008] To achieve the above objectives, the present invention adopts the following technical solution.

[0009] A near-infrared fluorescent probe for detecting thiophene has the structural formula shown in Formula I.

[0010]

[0011] A method for preparing the above-mentioned near-infrared fluorescent probe for detecting thiophenol includes the following steps.

[0012] (1) Compound 1 and 2,4-dinitrobenzenesulfonyl chloride were dissolved in dichloromethane at 0 °C, and a small amount of triethylamine was added dropwise as a catalyst. After the reaction proceeded for 1 hour, the temperature was gradually raised to room temperature and the reaction continued for 10 hours. After the reaction was completed, the reaction solution was added to water and extracted with dichloromethane. The organic phases were combined and the solvent was evaporated. Finally, compound 2 was obtained by column chromatography for separation and purification.

[0013]

[0014] (2) At room temperature, compound 2 was dissolved in a dichloromethane solution containing trifluoroacetic acid. After the reaction proceeded for 2.5 hours, the solvent was evaporated, and the crude product was separated and purified by column chromatography using a mixed eluent of dichloromethane and methanol (volume ratio of 20:1) to obtain a brownish-red solid compound 3.

[0015]

[0016] (3) At room temperature, compound 3, compound 4 and anhydrous sodium acetate were added to anhydrous ethanol and reacted for 1 hour. After the reaction was completed, the solvent was dried by rotary evaporation, and the product was separated and purified by column chromatography to obtain fluorescent probe formula I.

[0017]

[0018] The reaction in step (1) is carried out in the presence of an organic solvent, namely dichloromethane.

[0019] The reaction in step (1) is carried out under weakly alkaline conditions, wherein the weak base is triethylamine.

[0020] In step (1), at 0°C, compound 1 and 2,4-dinitrobenzenesulfonyl chloride are first dissolved in dichloromethane, a small amount of triethylamine is added dropwise, and after reacting for 0.5-1 hours, the temperature is raised to room temperature and the reaction continues for 10 hours.

[0021] The reaction in step (2) is carried out under strongly acidic conditions, wherein the strong acid is trifluoroacetic acid.

[0022] The reaction in step (2) is carried out in an organic solvent, namely dichloromethane.

[0023] The reaction in step (3) is carried out in an organic solvent, namely anhydrous ethanol.

[0024] The fluorescent probes described above are used to prepare reagents for detecting thiophenol in water and biological cells.

[0025] The mechanism by which the fluorescent probe of this invention responds to thiophenol is as follows: Under weakly acidic conditions, fluorophore 5 exhibits a strong fluorescence signal at 650 nm. Upon introduction of a 2,4-dinitrobenzenesulfonyl group, compound I is formed, and the fluorescence signal decreases significantly. For example... Figure 8 As shown, when it reacts with thiophenol in the system, the 2,4-dinitrobenzenesulfonyl group leaves, releasing the original fluorophore 5, and the fluorescence signal increases.

[0026] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects: After the fluorescent probe compound I reacts with thiophene, the fluorescence intensity at a wavelength of 650 nm gradually increases with increasing thiophene concentration. Compared with existing fluorescent probes for detecting thiophene, it has three main advantages: First, the fluorescence emission peak generated after reacting with thiophene is at 650 nm, which gives the probe better cell penetration and less damage to cells; second, the pH of the reaction between the probe and thiophene is controlled at 6.0, which can greatly improve the selective detection of thiophene; and third, the synthesis steps of this fluorescent probe are simple, with high yield and good stability. Attached Figure Description

[0027] Figure 1 This is a structural diagram of fluorescent probe type I.

[0028] Figure 2 This is a high-resolution mass spectrum of probe type I.

[0029] Figure 3 UV absorption spectra of probe I and fluorophore 5 at pH 6.0.

[0030] Figure 4 The fluorescence spectrum (λ) of probe I and fluorophore 5 at pH=6.0 is shown. ex = 580 nm).

[0031] Figure 5 The image shows the UV absorption spectra of different concentrations of thiophenol added to the Probe I solution (pH = 6.0).

[0032] Figure 6 The fluorescence spectra of probe I with different concentrations of thiophenol added (pH = 6.0, λ) ex = 580 nm).

[0033] Figure 7 This is a graph showing the fluorescence intensity of probe I at 650 nm as a function of thiophenol concentration (pH = 6.0, λ). ex =580 nm).

[0034] Figure 8 This is a schematic diagram of the reaction mechanism between fluorescent probe type I and thiophenol.

[0035] Figure 9 This is the mass spectrum of fluorophore 5 released after the reaction of fluorescent probe I with thiophenol.

[0036] Figure 10 This is a fluorescence imaging image of the thiophenol content in cells. Implementation

[0037] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0038] Example 1 Synthesis of Fluorescent Probe Formula I

[0039] (1) Compound 1 (0.188 g, 0.9 mmol) and 2,4-dinitrobenzenesulfonyl chloride (0.2133 g, 0.8 mmol) were added to a 25 mL round-bottom flask, and then 4 mL of dichloromethane was added to dissolve them. After adding 1 drop of triethylamine, the flask was placed in an ice-water bath and reacted for 1 hour. Then the temperature was adjusted to room temperature and the reaction was continued for 10 hours. After the reaction was completed, the reaction mixture was diluted with 6 mL of dichloromethane and then extracted three times with water. The organic phase was dried with anhydrous sodium sulfate and the solvent was evaporated. The crude product was separated and purified by column chromatography (eluent: dichloromethane: petroleum ether = 5:1) to obtain compound 2.

[0040]

[0041] (2) At room temperature, compound 2 (0.2197 g, 0.5 mmol) was added to a mixed solution of 5 mL dichloromethane and 1 mL trifluoroacetic acid. After reacting for 2.5 hours, the mixture was evaporated to dryness. The crude product was purified by column chromatography (eluent: dichloromethane:methanol = 20:1) to obtain a brownish-red solid product, which was compound 3.

[0042]

[0043] (3) At room temperature, compound 3 (0.0509 g, 0.15 mmol), compound 4 (0.0359 g, 0.1 mmol), and anhydrous sodium acetate (0.0002 g, 0.022 mmol) were added to 5 mL of anhydrous ethanol. The reaction was then carried out at 80 °C for 1 hour. After the reaction was completed, the solvent was evaporated, and the product was separated and purified by column chromatography to obtain fluorescent probe formula I.

[0044]

[0045] Example 2 Detection of p-Thiophenol using Fluorescent Probe Type I

[0046] The probe was dissolved in acetone to prepare a 10 mL stock solution. The experiment was performed in phosphate-buffered saline (PBS). The absorption peaks of fluorophore 5 were located at 490 nm and 620 nm, while fluorescent probe I showed significant absorption at 620 nm (e.g., ...). Figure 3 (As shown). Fluorophore 5 exhibits a strong fluorescence emission peak at 650 nm, while probe I shows relatively weak fluorescence emission at 650 nm (as shown). Figure 4 (As shown). After adding different concentrations of thiophene, probe I reacted with thiophene, resulting in a decrease in the absorption peak at 490 nm and an increase in the absorption peak at 620 nm (as shown). Figure 5 As shown in the figure, the fluorescence intensity of the detection system at 650 nm also increases with increasing thiophene concentration (e.g., as shown in the figure). Figure 6 (As shown). The reaction mechanism of probe I with thiophenol is as follows: Figure 8 As shown, high-resolution mass spectrometry confirmed that the product generated by the reaction is fluorophore 5 (e.g., Figure 9 (As shown). This indicates that probe type I can be used for the detection of thiophenol in aqueous solution.

[0047] Example 3 Cell Imaging

[0048] In live-cell imaging experiments, three groups of HeLa cells were co-cultured with probe I for 0.5 hours. Subsequently, one group was treated with 10 μM thiophenol, another with 20 μM thiophenol, while the blank control group received no thiophenol. Figure 10As shown, only a weak fluorescence signal was observed in cells without added thiophene. Upon addition of 10 μM and 20 μM thiophene, the intracellular fluorescence signal increased sequentially, indicating that thiophene entered the cells and that the increased concentration led to the increased fluorescence signal. This demonstrates that probe I can be used to detect intracellular thiophene.

Claims

1. A near-infrared fluorescent probe for detecting thiophene, the structural formula of which is shown in Formula I. 。 2. A method for preparing a near-infrared fluorescent probe for detecting thiophenol as described in claim 1, characterized in that, Includes the following steps: (1) Compound 1 and 2,4-dinitrobenzenesulfonyl chloride were dissolved in an organic solvent under alkaline conditions and reacted at a certain reaction time and temperature. After the reaction was completed, the mixture was extracted and dried to obtain compound 2. ， (2) Dissolve compound 2 in an organic solvent and react it under acidic conditions for a period of time. After the reaction is complete, compound 3 is obtained. ， (3) Compound 3 and compound 4 are reacted in an organic solvent containing anhydrous sodium acetate for a period of time. After the reaction is completed, fluorescent probe formula I is obtained. 。 3. The preparation method according to claim 2, characterized in that, The preparation method has one or more of the following characteristics: The reaction in step (1) is carried out in the presence of an organic solvent, namely dichloromethane; The reaction in step (1) is carried out under alkaline conditions, wherein the base is triethylamine; The reaction in step (2) is carried out under acidic conditions, wherein the acid is trifluoroacetic acid; The reaction in step (2) is carried out in an organic solvent, namely dichloromethane; The reaction in step (3) is carried out in an organic solvent, namely anhydrous ethanol.

4. The preparation method according to claim 3, characterized in that, In step (1), at 0°C, compound 1 and 2,4-dinitrobenzenesulfonyl chloride are first dissolved in dichloromethane, a small amount of triethylamine is added dropwise, and after reacting for 0.5-1 hours, the temperature is raised to room temperature and the reaction continues for 10 hours.

5. The use of the near-infrared fluorescent probe for detecting thiophenol as described in claim 1 in the preparation of a reagent for detecting the content of thiophenol in water.

6. The use of the near-infrared fluorescent probe for detecting thiophene as described in claim 1 in the preparation of reagents for detecting intracellular thiophene content.

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