Synthesis and application of bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide

Abstract

The invention discloses a bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide. The chemical structural formula of the bifunctional probe is as follows: # imgabs0 #. After the bifunctional fluorescent probe reacts with superoxide anions and hydrogen sulfide, two coumarin derivatives with different fluorescent properties are formed to achieve the purpose of simultaneous distinguishing detection, the superoxide anions are detected by taking a trifluorosulfonate group as a recognition site, and the hydrogen sulfide is responded by an azide group. When the probe reacts with hydrogen sulfide, blue fluorescence of 460 nm is emitted under the excitation wavelength of 380 nm; when the probe detects superoxide anions, green fluorescence of 540 nm is emitted under the excitation wavelength of 470 nm. In addition, the probe has the advantages of high sensitivity, excellent selectivity and the like when being used for detecting superoxide anions and hydrogen sulfide, and has a huge application prospect in the technical fields of analytical chemistry, life science, biomedical treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and specifically relates to the synthesis and application of a dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide. The probe connects two coumarin derivatives with different fluorescent properties through piperazine, and can rapidly and selectively detect superoxide anions and hydrogen sulfide from various biologically active substances. The yellow fluorescent channel selectively detects superoxide anions, and the blue fluorescent channel selectively responds to hydrogen sulfide, and has the advantages of high detection sensitivity and visual detection. Background Art

[0002] Redox homeostasis is the key to maintaining normal physiological functions of cells. Long-term redox imbalance is associated with the occurrence of many diseases, such as neurological diseases, cardiovascular diseases, liver and kidney damage, diabetes, and cancer ( Biomolecules 2021, 11, 1144; Nature Reviews Nephrology 2024, 20, 101-119). Therefore, monitoring the redox state in cells is of great significance for understanding cellular physiological and pathological processes. Superoxide anion (O2 •- ) is a type of reactive oxygen species (ROS) produced during the metabolism of organisms. It plays a wide range of roles in regulating cell functions. Its excessive production can lead to redox imbalance ( Analytical Chemistry 2024, 96, 4632-4638). Hydrogen sulfide (H2S) is a highly reducing and nucleophilic reactive sulfur species that can scavenge oxidants and intracellular electrophiles ( Chemical Reviews 2024, 124, 7, 4124–4257). Real-time monitoring of the changes of these two substances in cells will help to elucidate their interactive roles in signal transduction and redox homeostasis regulation in living cells.

[0003] Although there are many methods for detecting O2 •- and H2S, but existing detection technologies may have problems such as insufficient sensitivity, poor selectivity, and inability to monitor in real time. In recent years, fluorescent probes have been widely used to track biological events, signal transduction, changes in levels of key biological molecules, or metabolic tracing, and have become an indispensable tool for real-time tracking of trace metabolites and key biological molecules in living cells ( Chemical Society Reviews 2021, 50, 1219-1250). However, many of the currently reported methods for detecting O2 •- Most fluorescent probes for H2S / O2 detect only one of them, and can also detect O2 •- and H2S remain huge challenges. Summary of the invention

[0004] In view of the above situation, in order to overcome some deficiencies of the prior art, the present invention aims to provide a dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide. The probe can rapidly and selectively detect superoxide anions and hydrogen sulfide from various biologically active 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 with simple preparation method, high sensitivity, low detection limit and low cost.

[0006] The specific technical solution adopted by the present invention to solve the problem is to synthesize a bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide, and to prepare a device for quantitatively analyzing superoxide anions and hydrogen sulfide in the environment and simultaneously distinguishing and imaging superoxide anions and hydrogen sulfide in living cells. The chemical structure of the bifunctional probe is as follows: .

[0007] A synthesis of a bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide, characterized in that the preparation method of the bifunctional fluorescent probe comprises the following steps: Step 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid 5-(Diethylamino)-2-formylphenyl trifluoromethanesulfonate and 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid are added to ethanol, and the mixture is reacted at 50 degrees Celsius overnight. After the reaction is complete, the mixture is filtered, and the solid is spin-dried to obtain (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid; Step 2. Synthesis of the bifunctional fluorescent probe (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, 4-(7-azido-2-oxo-2H-chromene-3-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate is added and stirred for 5 minutes. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the reaction is stirred at room temperature overnight. After the reaction is complete, the reaction system is dried by rotary evaporation, and the bifunctional fluorescent probe is separated by column chromatography.

[0008] The present invention provides a method for using a bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anions 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 where the volume ratio of the organic phase to the aqueous phase is 5:5, the organic phase is dimethyl sulfoxide, and the aqueous phase is a phosphate buffer solution (PBS) with a pH of 7.4.

[0009] The specific characteristics of the dual-function fluorescent probe of the present invention for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide are as follows: the dual-function fluorescent probe is dissolved in DMSO, and the dual-function probe is dissolved in an organic phase and an aqueous phase (5:5, v / v) solution. After reacting with superoxide anions for 30 minutes, it emits 540 nm yellow fluorescence at an excitation wavelength of 470 nm; after reacting with hydrogen sulfide for 30 minutes, the probe emits 460 nm blue fluorescence at an excitation wavelength of 380 nm. Therefore, it is possible to detect specific analytes with specific excitation and fluorescence emission signals, and when detecting simultaneously, the two can be well distinguished by using different excitation and fluorescence emission signals. The above dual-function fluorescent probe realizes the simultaneous distinguishing and detecting of superoxide anions and hydrogen sulfide under different detection conditions, and has no obvious response to other active oxygen, active sulfur, common amino acids, metal ions and active nitrogen. The detection limits of superoxide anions and hydrogen sulfide are as low as 14.8 nM and 207.1 nM, respectively. Therefore, the dual-function fluorescent probe disclosed in the present invention can realize highly sensitive distinguishing detection of the two. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the bifunctional fluorescent probe of the present invention.

[0011] Figure 2 The dual-function fluorescent probe of the present invention responds to the fluorescence spectrum of superoxide anion and hydrogen sulfide.

[0012] Figure 3 The dual-function fluorescent probe of the present invention responds to the fluorescence quantitative spectrum of superoxide anion and hydrogen sulfide.

[0013] Figure 4 The dual-function fluorescent probe of the present invention responds to the selective spectrum of superoxide anion and hydrogen sulfide. DETAILED DESCRIPTION

[0014] The present invention is further described in conjunction with the following formula.

[0015] The synthesis route of the bifunctional fluorescent probe of the present invention is shown in the following formula:

[0016] Example 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid 300.0 mg (922.22 µmol) of 5-(diethylamino)-2-formylphenyl trifluoromethanesulfonate and 201.3 mg (922.22 µmol) of 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid were added to 12 mL of ethanol and reacted at 50 degrees Celsius overnight. After the reaction was complete, the mixture was filtered and the solid was spin-dried to obtain 390.0 mg of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid with a yield of 80.47%; Example 2. Synthesis of the bifunctional fluorescent probe 70.0 mg (133.20 µmol) (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid was added to 6 mL of anhydrous dichloromethane, and then 2 mg of 4-dimethylaminopyridine (DMAP) was added, and the mixture was reacted at room temperature for 5 min. Subsequently, 55.1 mg (133.20 µmol) of 4-(7-azido-2-oxo-2H-chromene-3-carbonyl)piperazin-1-ium 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 stirred at room temperature overnight. After the reaction was complete, the reaction system was dried by rotary evaporation, and 36.0 mg of the bifunctional fluorescent probe was obtained by column chromatography purification and separation, with a yield of 33.5%.

[0017] Example 3. Dual-functional fluorescent probe for simultaneous detection of superoxide anions and hydrogen sulfide in vitro The spectral property experiment of the bifunctional fluorescent probe described in the present invention is as follows: the bifunctional probe is dissolved in DMSO to prepare a probe solution with a concentration of 1 mM, and an analytical solution of superoxide anion and hydrogen sulfide with a concentration of 10 mM is prepared. The specific test method is: take 20 μL of 1 mM probe solution, add 20 μL of 10 mM superoxide anion solution, and finally add 980 μL DMSO and 980 μL PBS. For all tests, the volume ratio of the organic phase to the aqueous phase is kept at 5:5 (the total volume of each test sample is 2 mL). For example, when the fluorescence intensity of superoxide anion with a concentration of 100 μM is required to be tested, the sample preparation is as follows: take 20 μL of 1 mM probe solution, 20 μL of 10 mM superoxide anion solution, and then add 980 μL DMSO and 980 μL PBS in a 2 mL sample tube. After shaking at room temperature for 30 minutes, the fluorescence emission intensity can be measured with an excitation wavelength of 470 nm. This bifunctional probe can distinguish and detect superoxide anion and hydrogen sulfide, two biologically active substances, using different excitation wavelengths and fluorescence emission signals. It has high sensitivity and detection limits as low as 14.8 nM and 207.1 nM, respectively, making it very suitable for imaging analysis of superoxide anion and hydrogen sulfide in living cells.

[0018] The present invention provides a bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide. Two coumarin derivatives with different fluorescent properties are connected by piperazine, and superoxide anions are detected by trifluorosulfonate groups as recognition sites, and hydrogen sulfide is responded to by azide. When reacting with superoxide anions, a strong yellow fluorescence of 550nm is emitted at an excitation wavelength of 470nm; when responding to hydrogen sulfide, a blue fluorescence of 460nm is emitted at an excitation wavelength of 380nm, and the fluorescence phenomenon is obvious. And the product after the reaction has good water solubility, fast response speed, large Stokes shift and other advantages. It has great practical application value in the fields of biochemistry, fluorescence imaging analysis, etc. Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be regarded as a limitation of the present invention. After reading the above content, various modifications and substitutions of the present invention will be obvious to those skilled in the art. Therefore, fluorescent probes with similar technical features described herein all fall within the scope of protection of this patent.

Claims

1. A dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and hydrogen sulfide, characterized in that: The chemical structure of the bifunctional fluorescent probe is as follows: 。 2. The synthesis of the bifunctional fluorescent probe according to claim 1, characterized in that: The synthesis method of the bifunctional fluorescent probe comprises the following steps: Step 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid 5-(Diethylamino)-2-formylphenyl trifluoromethanesulfonate and 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid are added to ethanol, and the mixture is reacted at 50 degrees Celsius overnight. After the reaction is complete, the mixture is filtered, and the solid is spin-dried to obtain (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid; Step 2. Synthesis of the bifunctional fluorescent probe (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, 4-(7-azido-2-oxo-2H-chromene-3-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate is added and stirred for 5 minutes. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the reaction is stirred at room temperature overnight. After the reaction is complete, the reaction system is dried by rotary evaporation, and the bifunctional fluorescent probe is separated by column chromatography.

3. The method for synthesizing a bifunctional probe according to claim 2, wherein: The molar ratio of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid and 4-(7-azido-2-oxo-2H-chromene-3-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate in step 5 is 1:

1.

4. The use of the bifunctional fluorescent probe according to claim 1 for preparing a device, characterized in that: The prepared device is capable of quantitatively analyzing superoxide anions and hydrogen sulfide in the environment, as well as simultaneously distinguishing and imaging superoxide anions and hydrogen sulfide in cells, tissues, and living bodies.

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