Synthesis and Application of a Bifunctional Fluorescent Probe for Simultaneous Differentiation and Detection of Superoxide Anion and Hydrogen Sulfide

Abstract

This invention discloses a bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anion and hydrogen sulfide. The chemical structure of the bifunctional probe is as follows: This bifunctional fluorescent probe achieves simultaneous detection by reacting with superoxide anion and hydrogen sulfide to form two coumarin derivatives with different fluorescent properties. The trifluorosulfonate group serves as the recognition site for superoxide anion, while the azide group responds to hydrogen sulfide. When the probe reacts with hydrogen sulfide, it emits blue fluorescence at 460 nm under an excitation wavelength of 380 nm; when detecting superoxide anion, it emits green fluorescence at 540 nm under an excitation wavelength of 470 nm. Furthermore, this probe exhibits high sensitivity and excellent selectivity in detecting superoxide anion and hydrogen sulfide, showing great promise for applications in analytical chemistry, life sciences, and biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, specifically relating to the synthesis and application of a bifunctional fluorescent probe that simultaneously distinguishes between superoxide anion and hydrogen sulfide. This probe links two coumarin derivatives with different fluorescent properties via piperazine, enabling rapid and selective detection of superoxide anion and hydrogen sulfide from various bioactive substances. The yellow fluorescent channel selectively detects superoxide anion, while the blue fluorescent channel selectively responds to hydrogen sulfide, offering advantages such as high detection sensitivity and visual detection. Background Technology

[0002] Redox homeostasis is crucial for maintaining normal cellular physiological functions. Long-term redox imbalance is associated with the development of various diseases, such as neurological disorders, cardiovascular diseases, liver and kidney damage, diabetes, and cancer. Biomolecules 2021, 11:44; Nature Reviews Nephrology 2024, 20, 101-119). Therefore, monitoring intracellular redox status is of great significance for understanding cellular physiological and pathological processes. Superoxide anion (O2) •- Reactive oxygen species (ROS) are produced during the metabolism of organisms and play a wide role in the regulation of cell function. Excessive production of ROS can lead to redox imbalance. Analytical Chemistry 2024, 96, 4632-4638. Hydrogen sulfide (H2S) is a highly reducing and nucleophilic reactive sulfur species capable of scavenging oxidants and intracellular electrophilic agents. Chemical Reviews 2024, 124, 7, 4124–4257). Real-time monitoring of changes in these two substances within cells helps elucidate their interactions in live-cell signal transduction and redox homeostasis regulation.

[0003] Although various methods have been developed for detecting O2 •- While fluorescent probes can detect H2S, existing detection technologies may suffer from insufficient sensitivity, poor selectivity, and the inability to monitor in real time. In recent years, fluorescent probes have been widely used to track biological events, signal transduction, changes in the levels of key biological molecules, or metabolic tracing, becoming an indispensable tool for the real-time tracking of trace metabolites and key biomolecules in living cells. Chemical Society Reviews 2021, 50, 1219-1250). However, many currently reported methods for detecting O2... •- Most of the fluorescent probes for / H2S detect only one of the components, while also distinguishing between O2. •- H2S remains a huge challenge. Summary of the Invention

[0004] In view of the above, and to overcome some shortcomings of existing technologies, the purpose of this invention is to provide a bifunctional fluorescent probe that can simultaneously distinguish and detect superoxide anion and hydrogen sulfide. This probe can rapidly and selectively detect superoxide anion and hydrogen sulfide from various bioactive substances under specific detection conditions.

[0005] The present invention also aims to provide a method for synthesizing and applying the above-mentioned bifunctional fluorescent probe, which is simple to prepare, highly sensitive, has a low detection limit, and is low in cost.

[0006] The specific technical solution adopted by this invention to solve the problem is the synthesis of a bifunctional fluorescent probe that can simultaneously distinguish and detect superoxide anion and hydrogen sulfide, and the application of a device for quantitative analysis of superoxide anion and hydrogen sulfide in the environment and simultaneous imaging of superoxide anion and hydrogen sulfide in living cells. The chemical structural formula of the bifunctional probe is as follows:

[0007] .

[0008] Synthesis of a bifunctional fluorescent probe capable of simultaneously distinguishing and detecting superoxide anion and hydrogen sulfide, characterized in that the preparation method of the bifunctional fluorescent probe includes the following steps:

[0009] Step 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid

[0010] 5-(diethylamino)-2-carboxyylphenyltrifluoromethanesulfonate and 2-(cyanomethyl)benzo[d]thiazol-6-carboxylic acid were added to ethanol and reacted overnight at 50°C. After the reaction was complete, the mixture was filtered, and the solid was evaporated to dryness to give (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid;

[0011] Step 2. Synthesize the bifunctional fluorescent probe.

[0012] (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine (DMAP). The reaction was carried out at room temperature for 5 min, followed by the addition of 4-(7-azido-2-oxo-2H-chromene-3-carbonyl)piperazine-1-onium 2,2,2-trifluoroacetate and stirring for 5 min. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred overnight at room temperature. After the reaction was completed, the reaction system was evaporated to dryness and purified by column chromatography to obtain the bifunctional fluorescent probe.

[0013] The present invention discloses a method for using a bifunctional fluorescent probe that can simultaneously distinguish and detect superoxide anion and hydrogen sulfide: unless otherwise specified, the bifunctional probe is usually dissolved in dimethyl sulfoxide (DMSO) at room temperature and used for analysis and detection in an environment with an organic phase and an aqueous phase volume ratio of 5:5. The organic phase is dimethyl sulfoxide and the aqueous phase is phosphate buffer solution (PBS) with pH = 7.4.

[0014] The specific characteristics of the bifunctional fluorescent probe of this invention for simultaneously distinguishing and detecting superoxide anion and hydrogen sulfide are as follows: The bifunctional fluorescent probe is dissolved in DMSO in an organic and aqueous (5:5, v / v) solution. After reacting with superoxide anion for 30 minutes, it emits 540 nm yellow fluorescence at an excitation wavelength of 470 nm; after reacting with hydrogen sulfide for 30 minutes, it emits 460 nm blue fluorescence at an excitation wavelength of 380 nm. Therefore, it enables the detection of specific analytes using specific excitation and fluorescence emission signals. When detecting simultaneously, different excitation and fluorescence emission signals can also effectively distinguish between the two. The above-mentioned bifunctional fluorescent probe achieves simultaneous distinguishing and detection of superoxide anion and hydrogen sulfide under different detection conditions, showing no significant response to other reactive oxygen species, reactive sulfur, common amino acids, metal ions, and reactive nitrogen. The detection limits for superoxide anion and hydrogen sulfide are as low as 14.8 nM and 207.1 nM, respectively. Therefore, the bifunctional fluorescent probe disclosed in this invention can achieve highly sensitive distinguishing and detection of both. Attached Figure Description

[0015] Figure 1 The proton NMR spectrum of the bifunctional fluorescent probe described in this invention.

[0016] Figure 2 The fluorescence spectra of the bifunctional fluorescent probe described in this invention in response to superoxide anion and hydrogen sulfide.

[0017] Figure 3 The bifunctional fluorescent probe of this invention displays the quantitative fluorescence spectra of superoxide anion and hydrogen sulfide.

[0018] Figure 4 The selective spectra of the bifunctional fluorescent probe described in this invention in response to superoxide anion and hydrogen sulfide. Detailed Implementation

[0019] The present invention will be further explained in conjunction with the following formula.

[0020] The synthetic route of the bifunctional fluorescent probe described in this invention is shown in the following formula:

[0021]

[0022] Example 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid

[0023] 300.0 mg (922.22 µmol) of 5-(diethylamino)-2-carboxyphenyl trifluoromethanesulfonate and 201.3 mg (922.22 µmol) of 2-(cyanomethyl)benzo[d]thiazol-6-carboxylic acid were added to 12 mL of ethanol and reacted overnight at 50°C. After the reaction was complete, the mixture was filtered, and the solid was evaporated to dryness to give 390.0 mg of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid, with a yield of 80.47%.

[0024] Example 2. Synthesis of the bifunctional fluorescent probe.

[0025] 70.0 mg (133.20 µmol) of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid was added to 6 mL of anhydrous dichloromethane, followed by 2 mg of 4-dimethylaminopyridine (DMAP). The mixture was reacted at room temperature for 5 min, then 55.1 mg (133.20 µmol) of 4-(7-azido-2-oxo-2H-chromene-3-carbonyl)piperazine-1-onium 2,2,2-trifluoroacetate was added and stirred for 5 min. Finally, 38.3 mg (199.80 µmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was evaporated to dryness and purified by column chromatography to obtain 36.0 mg of the bifunctional fluorescent probe, with a yield of 33.5%.

[0026] Example 3. Bifunctional fluorescent probes simultaneously distinguish and detect superoxide anion and hydrogen sulfide in an in vitro environment.

[0027] The bifunctional fluorescent probe of this invention is used for spectral property experiments. The bifunctional probe is dissolved in DMSO to prepare a 1 mM probe solution, and analytical solutions of 10 mM superoxide anion and hydrogen sulfide are prepared. Specifically, 20 μL of the 1 mM probe solution is taken, followed by 20 μL of 10 mM superoxide anion solution, and finally 980 μL of DMSO and 980 μL of PBS. The volume ratio of organic to aqueous phase is maintained at 5:5 for all tests (total volume of each test sample is 2 mL). For example, when testing the fluorescence intensity of 100 μM superoxide anion, the sample preparation is as follows: 20 μL of the 1 mM probe solution, 20 μL of 10 mM superoxide anion solution, 980 μL of DMSO and 980 μL of PBS are added to a 2 mL sample tube. After shaking at room temperature for 30 minutes, the fluorescence emission intensity can be measured using an excitation wavelength of 470 nm. This bifunctional probe enables the differentiation and detection of two bioactive substances, superoxide anion and hydrogen sulfide, using different excitation wavelengths and fluorescence emission signals. It exhibits high sensitivity with detection limits as low as 14.8 nM and 207.1 nM, respectively, making it ideal for imaging analysis of superoxide anion and hydrogen sulfide in live cells.

[0028] This invention provides a bifunctional fluorescent probe that simultaneously distinguishes and detects superoxide anion and hydrogen sulfide. It links two coumarin derivatives with different fluorescent properties via piperazine, using a trifluorosulfonate group as the recognition site to detect superoxide anion and an azide reaction to hydrogen sulfide. When reacting with superoxide anion, it emits a strong yellow fluorescence of 550 nm at an excitation wavelength of 470 nm; when responding with hydrogen sulfide, it emits a blue fluorescence of 460 nm at an excitation wavelength of 380 nm, exhibiting a clear fluorescence phenomenon. Furthermore, the reaction product has good water solubility, fast response speed, and a large Stokes shift. It has significant practical application value in fields such as biochemistry and fluorescence imaging analysis. Although the invention has been described in detail through the above preferred embodiments, it should be understood that the above description should not be considered as a limitation of the invention. Various modifications and substitutions to the invention will be obvious to those skilled in the art after reading the above content. Therefore, fluorescent probes with similar technical features as described herein fall within the protection scope of this patent.

Claims

1. A bifunctional fluorescent probe that simultaneously distinguishes and detects superoxide anion and hydrogen sulfide, characterized in that, The chemical structure of the bifunctional fluorescent probe is shown below: 。 2. The synthesis of the bifunctional fluorescent probe as described in claim 1, characterized in that, The method for synthesizing the bifunctional fluorescent probe includes the following steps: Step 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid 5-(diethylamino)-2-carboxyylphenyltrifluoromethanesulfonate and 2-(cyanomethyl)benzo[d]thiazol-6-carboxylic acid were added to ethanol and reacted overnight at 50°C. After the reaction was complete, the mixture was filtered, and the solid was evaporated to dryness to give (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid; Step 2. Synthesize the bifunctional fluorescent probe. (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine (DMAP). The reaction was carried out at room temperature for 5 min, followed by the addition of 4-(7-azido-2-oxo-2H-chromene-3-carbonyl)piperazine-1-onium 2,2,2-trifluoroacetate and stirring for 5 min. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred overnight at room temperature. After the reaction was completed, the reaction system was evaporated to dryness and purified by column chromatography to obtain the bifunctional fluorescent probe.

3. The method for synthesizing a bifunctional probe as described in claim 2, characterized in that, In step 5, the molar ratio of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazol-6-carboxylic acid and 4-(7-azido-2-oxo-2H-chromene-3-carbonyl)piperazine-1-onium 2,2,2-trifluoroacetate is 1:

1.

4. The application of the bifunctional fluorescent probe as described in claim 1 in device fabrication, characterized in that, The fabricated device is capable of quantitatively analyzing superoxide anions and hydrogen sulfide in the environment, and simultaneously distinguishing and imaging superoxide anions and hydrogen sulfide in cells, tissues, and living organisms.

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