A ratio-type hydrogen peroxide fluorescent probe based on bis-naphthalimide derivatives and a preparation method and application thereof

Abstract

This invention discloses a ratiometric hydrogen peroxide fluorescent probe based on a bis(naphthyl)imide derivative, its preparation method, and its application. The chemical structure of the ratiometric fluorescent probe is as follows: The probe uses bis(naphthyl)imide as the fluorescent parent compound and arylboronic ester as the hydrogen peroxide recognition group. A ratiometric detection system is constructed using the emission spectral differences between methoxy and hydroxyl substitution states. After hydrogen peroxide treatment, the arylboronic ester undergoes oxidation and hydrolysis to generate phenolic hydroxyl groups, leading to changes in intramolecular charge transfer effects and thus altering the fluorescence emission spectrum. Under 380 nm excitation, the fluorescence intensity decreases at 460 nm and increases at 550 nm, which can be detected by I... 550 / I 460 This probe enables the quantitative analysis of hydrogen peroxide. It exhibits good selectivity and sensitivity, and can be used for the detection of hydrogen peroxide in environmental samples and for fluorescence imaging in biological systems.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry and fluorescence analysis detection technology, specifically relating to a ratiometric hydrogen peroxide fluorescent probe based on bis(naphthalimide) derivatives, its preparation method and application; more specifically, it relates to a fluorescent probe that uses naphthalimide as the fluorescent parent compound and arylboronic ester as the hydrogen peroxide recognition group, and utilizes the difference in emission spectra before and after substituent conversion to achieve ratio detection, and its application in quantitative analysis of hydrogen peroxide and fluorescence imaging. Background Technology

[0002] Maintaining redox homeostasis is crucial for normal cellular physiological function. Prolonged imbalance between the oxidative and antioxidant systems can easily lead to cellular dysfunction and is closely related to the development of various diseases. Hydrogen peroxide (H2O2), as an important reactive oxygen species (ROS), can be produced in vivo through the mitochondrial respiratory chain and various enzymatic reactions, participating in multiple physiological processes such as cell proliferation, apoptosis, and signal transduction. (Journal of Biological Chemistry, 2004, 279, 48742-48750) An abnormally high concentration of H2O2 can disrupt the intracellular redox balance, leading to oxidative damage. (Antioxidants, 2025, 14, 656; Cells, 2025, 14, 511) Therefore, establishing a sensitive, selective detection method that can monitor H2O2 changes in real time is of great significance for studying its physiological and pathological functions.

[0003] Currently, methods for detecting H2O2 include electrochemical methods, colorimetric methods, chemiluminescence methods, and fluorescence analysis methods. Among these, fluorescent probe methods have received widespread attention due to their advantages such as ease of operation, high sensitivity, good spatiotemporal resolution, and suitability for biological sample detection. (ChemistryOpen, 2018, 7, 262-265; Chinese Chemical Letters, 2019, 30, 1834-1842) However, most of the reported H2O2 fluorescent probes are single-wavelength intensity-responsive probes, whose fluorescence signals are easily affected by factors such as probe concentration, excitation light intensity, environmental polarity, pH value, and instrument conditions, thus limiting the accuracy of quantitative detection. (Dyes and Pigments, 2026, 244, 113125) Although ratiometric fluorescent probes can achieve self-calibration through the ratio of dual emission signals, thereby improving the reliability of detection results, they still suffer from problems such as complex synthesis routes, insufficient spectral separation, and difficulty in achieving both selectivity and sensitivity. Therefore, developing a fluorescent probe with a relatively simple synthesis method, good selectivity and sensitivity to hydrogen peroxide, and the ability to achieve ratiometric quantitative fluorescence detection remains of significant research importance and application value. Summary of the Invention

[0004] In view of the above, and to overcome some shortcomings of the prior art, the present invention aims to provide a ratiometric hydrogen peroxide fluorescent probe based on a bis(naphthyl)imide derivative. This probe enables rapid and selective fluorescent detection of hydrogen peroxide 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 fluorescent probe that 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 ratiometric hydrogen peroxide fluorescent probe based on a bis(naphthyl)imide derivative for the quantitative analysis of hydrogen peroxide in the environment and for imaging hydrogen peroxide in living cells. The chemical structural formula of the fluorescent probe is as follows: .

[0007] A method for synthesizing a ratiometric hydrogen peroxide fluorescent probe, characterized by comprising the following steps: Step 1. Synthesis of tert-butyl 4-(2-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester 6-Bromo-1H,3H-benzo[de]isochrome-1,3-dione was added to ethanol, followed by the addition of tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylic acid ester, and the reaction was carried out at 80 degrees Celsius. After the reaction was completed, the reaction solution was added to water to precipitate the solid, which was then filtered and the solvent was removed under reduced pressure to obtain tert-butyl 4-(2-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester. Step 2. Synthesis of tert-butyl 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester The compound obtained in step 1 was added to methanol with potassium carbonate and reacted at 80 degrees Celsius. After the reaction was completed, the reaction solution was added to water to precipitate the solid. The solid was filtered and the solvent was removed under reduced pressure to obtain tert-butyl 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester. Step 3. Synthesis of 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate The compound obtained in step 2 was added to dichloromethane, followed by trifluoroacetic acid, and the reaction was stirred at room temperature. After the reaction was completed, the solvent was removed under reduced pressure, and the mixture was recrystallized in ethanol to give 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate. Step 4. Synthesize the ratiometric fluorescent probe. 3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)propionic acid was added to dry dichloromethane, followed by the sequential addition of N,N-dimethylpyridin-4-amine, 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazin-1-onium 2,2,2-trifluoroacetate and 3-(((ethylimino)methylene)amino)-N,N-dimethylpropane-1-amine. The reaction was stirred at room temperature. After the reaction was completed, the reaction system was extracted, concentrated, and purified by column chromatography to obtain the ratiometric hydrogen peroxide fluorescent probe.

[0008] The method of using a ratiometric hydrogen peroxide fluorescent probe of the present invention is as follows: Unless otherwise specified, the 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.

[0009] The specific features of the ratiometric fluorescent probe for hydrogen peroxide in this invention for accurate detection are as follows: The ratiometric fluorescent probe is dissolved in DMSO in an organic and aqueous (5:5, v / v) solution. After reacting with hydrogen peroxide for 30 minutes, under a single excitation wavelength of 380 nm, the blue fluorescence at 460 nm is significantly weakened, while the yellow fluorescence at 550 nm is greatly enhanced. Thus, quantitative detection of hydrogen peroxide is achieved through the change in the fluorescence intensity ratio of the two emission wavelengths. Even in complex biological matrices, the concentration changes of hydrogen peroxide can be accurately captured based on the signal differences of characteristic wavelengths. The above-mentioned ratiometric fluorescent probe achieves highly efficient detection of hydrogen peroxide in the same detection system, with no significant response to other reactive oxygen species, biothiols, common amino acids, metal ions, and reactive nitrogen, and a detection limit as low as 25.3 nM for hydrogen peroxide. Therefore, the ratiometric fluorescent probe disclosed in this invention can achieve highly sensitive and selective detection of hydrogen peroxide. Attached Figure Description

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

[0011] Figure 2 The ratiometric hydrogen peroxide fluorescent probe of the present invention shows the ultraviolet and fluorescence spectra of hydrogen peroxide in response.

[0012] Figure 3 The ratiometric hydrogen peroxide fluorescent probe of the present invention provides a quantitative fluorescence spectrum of hydrogen peroxide in response.

[0013] Figure 4 The selective spectrum of the ratiometric hydrogen peroxide fluorescent probe described in this invention in response to hydrogen peroxide. Detailed Implementation

[0014] The invention will be further explained with reference to the following figures.

[0015] The synthetic route of the ratiometric hydrogen peroxide fluorescent probe of the present invention is shown in the figure below:

[0016] Example 1. Synthesis of tert-butyl 4-(2-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester 1.00 g (3609 µmol) of 6-bromo-1H,3H-benzo[de]isochromene-1,3-dione was added to 20 mL of ethanol, followed by 978.32 mg (4266.18 µmol) of (tert-butyl4-(2-aminoethyl)piperazine-1-carboxylic acid ester). The reaction was carried out at 80 °C. After the reaction was completed, the reaction solution was added to water to precipitate the solid. The solid was filtered and the solvent was removed under reduced pressure to obtain 1.5329 g of tert-butyl4-(2-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester, with a yield of 87.1%. Example 2. Synthesis of tert-butyl 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester 1.45 g (3043.92 µmol) of the compound obtained in step 1 and 1.23 g of potassium carbonate were added to 25 mL of methanol and reacted at 80°C. After the reaction was completed, the reaction solution was added to water to precipitate the solid. The solid was filtered and the solvent was removed under reduced pressure to give 1.2127 g of tert-butyl 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester, with a yield of 93.28%. Example 3. Synthesis of 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate 1.16 g (2788.46 µmol) of the compound obtained in step 2 was added to 15 mL of dichloromethane, followed by 4 mL of trifluoroacetic acid. The mixture was stirred at room temperature. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was recrystallized from ethanol to give 0.9808 mg of 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate, with a yield of 80.88%. Example 4. Synthesis of the ratiometric fluorescent probe. 87.7 mg (206.71 µmol) of 3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)propionic acid was added to 7 mL of dry dichloromethane, followed by the addition of 16.0 mg (133.17 µmol) of N,N-dimethylpyridin-4-amine, 100 mg (213.93 µmol) of 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate, and 136.5 mg (695.36 µmol) of [amount missing]. µmol) 3-(((ethylimino)methylene)amino)-N,N-dimethylpropane-1-amine was stirred at room temperature. After the reaction was completed, the reaction system was extracted, concentrated, and purified by column chromatography to obtain 15.5 mg of the ratiometric hydrogen peroxide fluorescent probe, with a yield of 9.81%.

[0017] Example 5. Quantitative detection of hydrogen peroxide in vitro using a ratiometric fluorescent probe. The ratiometric fluorescent probe spectral property experiment of this invention involves dissolving the ratiometric probe in DMSO to prepare a 1 mM probe solution, and preparing an analytical solution of 10 mM hydrogen peroxide. The specific testing method is as follows: Take 20 μL of the 1 mM probe solution, add 20 μL of the 10 mM hydrogen peroxide solution, and finally add 980 μL of DMSO and 980 μL of PBS. For all tests, maintain a 5:5 volume ratio of organic phase to aqueous phase (total volume of each test sample is 2 mL). For example, when testing the fluorescence intensity of 100 μM hydrogen peroxide, the sample preparation is as follows: Take 20 μL of the 1 mM probe solution, 20 μL of the 10 mM hydrogen peroxide solution, and then add 980 μL of DMSO and 980 μL of PBS to a 2 mL sample tube. Shake well at room temperature for 30 minutes, and then measure the fluorescence emission intensity using an excitation wavelength of 380 nm. This ratiometric probe enables the quantitative detection of hydrogen peroxide by changing the ratio of fluorescence intensity at two emission wavelengths. It has high sensitivity and a detection limit as low as 25.3 nM, making it ideal for imaging and quantitative analysis of hydrogen peroxide in live cells.

[0018] This invention provides a fluorescent probe for ratio detection of hydrogen peroxide. It covalently binds a bis(naphthyl)imide fragment to a piperazine linker via an amidation condensation reaction, and detects hydrogen peroxide using an arylboronic ester group as the recognition site. Ratio detection is achieved by utilizing the oxidative hydrolysis of the boronic ester group by hydrogen peroxide. When the probe reacts with hydrogen peroxide, at an excitation wavelength of 380 nm, the blue fluorescence at 460 nm is significantly weakened, while the yellow fluorescence at 550 nm is greatly enhanced, showing a clear difference in fluorescence signal. Furthermore, the reaction product exhibits good water solubility and a fast response speed. It has significant practical application value in fields such as biochemistry, fluorescence imaging analysis, and biomedical detection.

[0019] Although the present invention has been described in detail through the preferred embodiments described above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, fluorescent probes having similar technical features as described herein fall within the protection scope of this patent.

Claims

1. A ratiometric hydrogen peroxide fluorescent probe based on a bis(naphthylimide) derivative, characterized in that, The chemical structural formula of the ratiometric hydrogen peroxide fluorescent probe is shown below: 。 2. The method for synthesizing the ratiometric hydrogen peroxide fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Step 1. Synthesis of tert-butyl 4-(2-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester 6-Bromo-1H,3H-benzo[de]isochrome-1,3-dione was added to ethanol, followed by the addition of tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylic acid ester, and the reaction was carried out at 80 degrees Celsius. After the reaction was completed, the reaction solution was added to water to precipitate the solid, which was then filtered and the solvent was removed under reduced pressure to obtain tert-butyl 4-(2-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester. Step 2. Synthesis of tert-butyl 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester The compound obtained in step 1 was added to methanol with potassium carbonate and reacted at 80 degrees Celsius. After the reaction was completed, the reaction solution was added to water to precipitate the solid. The solid was filtered and the solvent was removed under reduced pressure to obtain tert-butyl 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-carboxylic acid ester. Step 3. Synthesis of 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate The compound obtained in step 2 was added to dichloromethane, followed by trifluoroacetic acid, and the reaction was stirred at room temperature. After the reaction was completed, the solvent was removed under reduced pressure, and the mixture was recrystallized in ethanol to give 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate. Step 4. Synthesize the ratiometric fluorescent probe. 3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)propionic acid was added to dry dichloromethane, followed by the sequential addition of N,N-dimethylpyridin-4-amine, 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazin-1-onium 2,2,2-trifluoroacetate and 3-(((ethylimino)methylene)amino)-N,N-dimethylpropane-1-amine. The reaction was stirred at room temperature. After the reaction was completed, the reaction system was extracted, concentrated, and purified by column chromatography to obtain the ratiometric hydrogen peroxide fluorescent probe.

3. The synthesis method as described in claim 2, characterized in that, In step 4, the molar ratio of 4-(2-(6-methoxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)piperazine-1-onium 2,2,2-trifluoroacetate and 3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxoborane-2-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)propionic acid is 1:

1.

4. The application of the ratiometric hydrogen peroxide fluorescent probe as described in claim 1 in the detection of hydrogen peroxide, characterized in that, The fluorescent probe is used for the quantitative analysis of hydrogen peroxide in environmental samples and for fluorescence imaging of hydrogen peroxide in cells, tissues and living organisms.

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