Synthesis and application of a bifunctional fluorescent probe for simultaneous distinguishing detection of superoxide anion and viscosity

Abstract

The application discloses a bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anion and viscosity, and a chemical structural formula of the bifunctional fluorescent probe is as follows: The bifunctional fluorescent probe combines a hydroxyl derivative of phthalic anhydride with an indole derivative through piperazine, and detects superoxide anion through a double (trifluoromethyl) benzene sulfonic acid group as a recognition site and detects viscosity through a molecular rotor strategy. When the probe reacts with superoxide anion, green fluorescence of 510 nm is emitted under an excitation wavelength of 405 nm; when the probe is in high viscosity, red fluorescence of 630 nm is emitted under an excitation wavelength of 560 nm. In addition, the probe has the advantages of fast response speed and good selectivity in detecting superoxide anion and viscosity, and has a great application prospect in technical fields such as analytical chemistry, life science and biological medicine.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to the synthesis and application of a bifunctional fluorescent probe that simultaneously distinguishes between the detection of superoxide anions and viscosity. This probe connects two fluorophores via piperazine and can rapidly and selectively detect superoxide anions and viscosity from various bioactive substances. The green fluorescent channel selectively detects superoxide anions, while the red fluorescent channel selectively responds to viscosity. It possesses advantages such as a large Stokes shift, high detection sensitivity, and visual detection capabilities. Background Technology

[0002] Superoxide anion (O2) •- As a single-electron reduction product of oxygen, it is the first reactive oxygen species (ROS) produced in cells and a major precursor to other reactive oxygen species. Anal. Chem. , 2024, 96, 4632−4638. It plays a crucial role in a wide range of physiological and pathological processes and is considered a potential messenger regulating cell signaling networks under physiological conditions. Nature Reviews Molecular Cell Biology (2020, 21, 363–383). Maintaining normal O2 concentrations. •- O2 is crucial for intracellular redox homeostasis. •- Excessive accumulation of these substances can disrupt intracellular homeostasis, leading to oxidative damage to proteins and lipids. This process can further damage organelles and the cytoskeleton, ultimately inducing various pathological states and diseases, including ischemia-reperfusion injury, liver and kidney damage, and depression. Chem. Sci. , 2024, 15, 1969). On the other hand, cell viscosity, as a key microenvironment parameter, significantly affects a variety of physiological processes at the cellular level, such as enzyme activity regulation, intercellular signal transduction, protein aggregation state, and the diffusion behavior of biomolecules. Sensors&Actuators: B. Chemical ,2023, 381, 133470). Abnormal changes in viscosity can directly or indirectly interfere with these processes, thus having a profound impact on the occurrence and development of diseases. Sensors&Actuators: B. Chemical , 2025, 435, 137644).

[0003] Fluorescence imaging, as an emerging non-invasive imaging technique, has become one of the fastest-growing and most widely used imaging technologies in the biomedical field due to its significant advantages such as high spatial and temporal resolution, high specificity, and high sensitivity. Coordination Chemistry Reviews , 2026, 553, 217568). This technology enables real-time, in-situ, and non-invasive monitoring of biological processes, overcoming the limitations of traditional detection methods in dynamic analysis. Currently, many single-function fluorescent probes for detecting superoxide anions or viscosity have been reported ( , 2026, 553, 217568). Chemosphere, 2024, 356, 141829; Anal. Chem. (2025, 97, 21679-21687). However, compared to single-analyte sensors, sensors capable of simultaneously detecting two or more analytes can provide richer pathological information and improve the accuracy of early diagnosis. Therefore, the development of sensors capable of simultaneously distinguishing and detecting O2 is crucial. •- Bifunctional fluorescent probes with varying viscosity are more challenging and of greater value. 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 anions and viscosity. This probe can rapidly and selectively detect superoxide anions and viscosity 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 and preparation of a bifunctional fluorescent probe that can simultaneously distinguish and detect superoxide anions and viscosity, and the application of a device for quantitatively analyzing superoxide anions and viscosity in the environment and simultaneously distinguishing and imaging superoxide anions and viscosity in living cells. The chemical structural formula of the bifunctional probe is as follows: .

[0007] The synthesis of a bifunctional fluorescent probe capable of simultaneously distinguishing and detecting superoxide anions and viscosity, characterized in that the preparation method of the bifunctional fluorescent probe includes the following steps: Step 1. Synthesis of 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester 4-Hydroxyisobenzofuran-1,3-dione was added to ethanol, followed by the addition of 4-(2-aminoethyl)piperazine-1-carboxylic acid tert-butyl ester. The mixture was stirred overnight at 90°C. After the reaction was complete, the reaction system was evaporated to dryness, and the product was purified by column chromatography to obtain 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester. Step 2. Synthesis of 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester was added to anhydrous dichloromethane, followed by the addition of 3,5-ditrifluoromethylbenzenesulfonyl chloride. Triethylamine was then slowly added dropwise until the solution changed color. The reaction mixture was stirred at -5°C for 15 minutes and then allowed to react at room temperature for 20 minutes. After the reaction was completed, the mixture was poured into water and extracted with ethyl acetate. The organic layer was collected, the solvent was concentrated under vacuum, and then petroleum ether was added for recrystallization. The solid was filtered and evaporated to dryness to obtain 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester. Step 3. Synthesis of 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate) 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester was added to anhydrous dichloromethane, followed by the addition of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction system was evaporated to dryness, and ethanol was added. The mixture was then filtered to obtain 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate. Step 4. Synthesize the bifunctional fluorescent probe. (E)-5-carboxy-1-ethyl-3,3-dimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-1-iodide was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine (DMAP). The reaction was carried out at room temperature for 5 minutes. Subsequently, 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate) was added and stirred for 5 minutes. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added and stirred at room temperature for 1 hour. After the reaction was completed, the bifunctional fluorescent probe was purified by column chromatography.

[0008] The present invention discloses a method for using a bifunctional fluorescent probe that can simultaneously distinguish and detect superoxide anions and viscosity: unless otherwise specified, the bifunctional probe is usually dissolved in dimethyl sulfoxide (DMSO) at room temperature, and the analysis and detection are performed in an environment where the volume ratio of organic phase to aqueous phase is 5:5. The organic phase is dimethyl sulfoxide, and the aqueous phase is phosphate buffer solution (PBS) with pH = 7.4.

[0009] The specific features of the bifunctional fluorescent probe of this invention for simultaneously distinguishing and detecting superoxide anion and viscosity 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 510 nm green fluorescence at an excitation wavelength of 405 nm. The probe's response viscosity was tested in glycerol-methanol systems with different ratios, emitting 630 nm red fluorescence at an excitation wavelength of 560 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 effectively distinguish between the two. The above-mentioned bifunctional fluorescent probe achieves simultaneous distinguishing and detection of superoxide anion and viscosity 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 viscosity are as low as 33.16 nM and 0.27 cP, respectively. Therefore, the bifunctional fluorescent probe disclosed in this invention can achieve highly sensitive distinguishing and detection of both. Attached Figure Description

[0010] Figure 1 The proton NMR spectrum of the bifunctional fluorescent probe described in this invention.

[0011] Figure 2 The fluorescence spectra of the bifunctional fluorescent probe described in this invention in response to superoxide anion and viscosity.

[0012] Figure 3 The fluorescence quantitative analysis diagram of the bifunctional fluorescent probe of the present invention in response to superoxide anion and viscosity.

[0013] Figure 4 The selective spectra of the bifunctional fluorescent probe described in this invention in response to superoxide anion and viscosity.

[0014] Figure 5 The bifunctional fluorescent probe described in this invention can simultaneously distinguish between intracellular superoxide anion and viscous cell images. Detailed Implementation

[0015] The invention will be further explained with reference to the following figures.

[0016] The synthesis route of the bifunctional fluorescent probe described in this invention is shown in the figure below:

[0017] Example 1. Synthesis of 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester 600.0 mg (3.66 mmol) of 4-hydroxyisobenzofuran-1,3-dione was added to 16 mL of ethanol, followed by 1.01 g (4.39 mmol) of 4-(2-aminoethyl)piperazine-1-carboxylic acid tert-butyl ester. The mixture was stirred overnight at 90°C. After the reaction was complete, the reaction mixture was evaporated to dryness, and the product was purified by column chromatography to obtain 1.24 g of 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester, with a yield of 90.50%. Example 2. Synthesis of tert-butyl 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid 1.03 g (2.74 mmol) of 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester was added to 16 mL of anhydrous dichloromethane, followed by 1.29 g (4.12 mmol) of 3,5-ditrifluoromethylbenzenesulfonyl chloride. Triethylamine was then slowly added dropwise until the solution changed color. The reaction mixture was stirred at -5°C for 15 minutes, then allowed to react at room temperature for 20 minutes. After the reaction was complete, the mixture was poured into water and extracted with ethyl acetate. The organic layer was collected, the solvent was concentrated under vacuum, and then recrystallized in petroleum ether. The solid was filtered and evaporated to dryness to give 1.65 g of 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester, with a yield of 91.95%. Example 3. Synthesis of 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate) 0.80 g (1.23 mmol) of 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester was added to 16 mL of anhydrous dichloromethane, followed by 3 mL of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was evaporated to dryness, and ethanol was added for recrystallization. The mixture was filtered, and the solid was evaporated to dryness to give 612.9 mg of 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate, with a yield of 75.01%. Example 4. Synthesis of the bifunctional fluorescent probe. 80.0 mg (147.48 μmol) of (E)-5-carboxy-1-ethyl-3,3-dimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-1-iodide was added to 6 mL of anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine (DMAP). The mixture was reacted at room temperature for 5 minutes, and then 98.14 mg (147.48 μmol) of (E)-5-carboxy-1-ethyl-3,3-dimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-1-iodide was added. After stirring for 5 minutes, 1-ethyl-(3-dioxomethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the bifunctional fluorescent probe was purified by column chromatography to obtain 130.4 mg of the probe, with a yield of 82.18%.

[0018] Example 5. Bifunctional fluorescent probes simultaneously distinguish between superoxide anion and viscosity in an in vitro environment. The bifunctional fluorescent probe of this invention was used for spectral property experiments. The bifunctional probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM probe solution. Solutions with 10 mM superoxide anion concentration and different viscosities (different ratios of methanol:glycerol) were also prepared. Specifically, 20 μL of the 1 mM probe solution was taken, followed by 20 μL of the 10 mM superoxide anion solution, and finally 980 μL of DMSO and 980 μL of PBS were added. All tests maintained a 5:5 volume ratio of organic to aqueous phase (total volume of each test sample was 2 mL). For example, when testing the fluorescence intensity of superoxide anion at a concentration of 100 μM, 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 of DMSO and 980 μL of PBS to a 2 mL sample tube. After shaking and mixing at room temperature for 30 minutes, the fluorescence emission intensity can be measured using an excitation wavelength of 405 nm. When detecting viscosity, the specific testing method is as follows: take 20 μL of 1 mM probe solution, add 1980 μL of solutions prepared with different viscosities, shake and mix at room temperature for 30 minutes, and then measure the fluorescence emission intensity using an excitation wavelength of 560 nm. This bifunctional probe enables the differentiation and detection of two bioactive substances, superoxide anion and viscosity, using different excitation wavelengths and fluorescence emission signals. It has high sensitivity, with detection limits as low as 33.16 nM and 0.27 cP, respectively, making it very suitable for imaging / quantitative analysis of superoxide anion and viscosity in live cells.

[0019] Example 6. Dual-channel fluorescence imaging analysis of endogenous superoxide anion and viscosity in HEK293T (embryonic kidney epithelial cells) cells. HEK293T cells were passaged into confocal cell culture medium and cultured under standard growth conditions for 24 hours. Then, an appropriate amount of probe (5 μM) was added and cultured under standard growth conditions for another 30 minutes. The cells were then photographed under a confocal fluorescence microscope, and superoxide anion and viscosity in HEK293T cells were imaged using the 450-550 nm green channel and the 600-700 nm red channel, respectively. The bifunctional fluorescent probe of this invention can emit two different fluorescences in cells to simultaneously distinguish and detect superoxide anion and viscosity in cells, successfully realizing dual-channel fluorescence imaging analysis of superoxide anion and viscosity in cells.

[0020] This invention provides a bifunctional fluorescent probe that simultaneously distinguishes between superoxide anion and viscosity. It combines a hydroxyl derivative of phthalic anhydride with an indole derivative via piperazine, and detects superoxide anion using the bis(trifluoromethyl)benzenesulfonic acid group as the recognition site. Viscosity is responded to via a molecular rotor strategy. When reacting with superoxide anion, it emits 510 nm green fluorescence at an excitation wavelength of 405 nm; when reacting with viscosity, it emits 630 nm red fluorescence at an excitation wavelength of 560 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 biochemistry, analytical detection, and other fields. 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 limiting 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 between superoxide anion and viscosity, 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 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester 4-Hydroxyisobenzofuran-1,3-dione was added to ethanol, followed by the addition of 4-(2-aminoethyl)piperazine-1-carboxylic acid tert-butyl ester. The mixture was stirred overnight at 90°C. After the reaction was complete, the reaction system was evaporated to dryness, and the product was purified by column chromatography to obtain 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester. Step 2. Synthesis of 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester 4-(2-(4-hydroxy-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester was added to anhydrous dichloromethane, followed by the addition of 3,5-ditrifluoromethylbenzenesulfonyl chloride. Triethylamine was then slowly added dropwise until the solution changed color. The reaction mixture was stirred at -5°C for 15 minutes and then allowed to react at room temperature for 20 minutes. After the reaction was completed, the mixture was poured into water and extracted with ethyl acetate. The organic layer was collected, the solvent was concentrated under vacuum, and then petroleum ether was added for recrystallization. The solid was filtered and evaporated to dryness to obtain 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester. Step 3. Synthesis of 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate) 4-(2-(4-((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-carboxylic acid tert-butyl ester was added to anhydrous dichloromethane, followed by the addition of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction system was evaporated to dryness, and ethanol was added for recrystallization. The mixture was filtered, and the solid was evaporated to dryness to obtain 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate. Step 4. Synthesize the bifunctional fluorescent probe. (E)-5-carboxy-1-ethyl-3,3-dimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-1-iodide was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine (DMAP). The reaction was carried out at room temperature for 5 minutes. Subsequently, 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate) was added and stirred for 5 minutes. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added and stirred at room temperature for 1 hour. After the reaction was completed, the bifunctional fluorescent probe was purified by column chromatography.

3. The method for synthesizing a bifunctional probe as described in claim 2, characterized in that, The molar ratio of (E)-5-carboxy-1-ethyl-3,3-dimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-1-iodide and 4-(2-(4-(((3,5-bis(trifluoromethyl)phenyl)sulfonyl)oxy)-1,3-dioxoisoindoline-2-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate in step 4 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 viscosity in the environment, and simultaneously distinguishing and imaging superoxide anions and viscosity in cells and tissues.

Images