Preparation and application of hydrogen peroxide fluorescent probe based on furannitrile-xanthene

Abstract

The invention designs synthesis and application of a hydrogen peroxide fluorescent probe based on furannitrile-xanthene. The structural formula of the probe is shown in the specification. The invention provides a preparation method for synthesizing the fluorescent probe by taking a compound FX and diphenyl phosphoryl chloride as raw materials. The fluorescent probe is a hydrogen peroxide (H2O2) fluorescent probe with high sensitivity, high selectivity and low cytotoxicity; firstly, the fluorescent probe has good sensitivity to fluorescent response of system H2O2 change, and the linear range is 1-70 [mu] M; secondly, the fluorescent probe is not influenced by other various cations, anions, amino acids, biological mercaptan and active oxygen, and only shows good selectivity to H2O2; in addition, the fluorescent probe can quickly act with H2O2, and the response time is within 10 minutes; when the pH value is 7.0-8.0, the fluorescence response signal of the probe is the strongest, which indicates that the probe can detect H2O2 in a complex physiological environment; in addition, the fluorescent probe is low in cytotoxicity and can be applied to detection of H2O2 in living cells.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the preparation and application of a hydrogen peroxide fluorescent probe based on furanone-oxanthracene. Background Technology

[0002] Hydrogen peroxide (H2O2) is widely present in most oxidation processes in biological systems (Wang S, Yao J, Wang B, et al. A ratiometric and two-photon fluorescent probe for imaging hydrogen peroxide in living cells [J]. Luminescence 2022, 37, 1037–1043.), and is closely related to many physiological and pathological processes. As an important reactive oxygen species and intracellular second messenger, H2O2 is not only a marker of oxidative stress, but also plays a key role in redox signaling pathways and immune responses, participating in the regulation of cell proliferation, differentiation, migration and disease development (Peng Z, Cui M, Chu J, et al. A novel AIE fluorescent probe for the detection and imaging of hydrogen peroxide in living tumor cells and in vivo[J]. Bioorganic Chemistry 2024, 150, 107592; Ren M, Deng B, Zhou K, et al. Single fluorescent probe for dual-imaging viscosity and H2O2 in mitochondria with different fluorescence signals in living cells[J]. Analytical Chemistry2017, 89, 552–555.).Numerous studies have shown that cellular H2O2 levels are closely related to various diseases, including cardiovascular disease, neurodegenerative diseases, and liver injury (Qiu X, Xin C, Qin W, et al. A novel pyrimidine based deep-red fluorogenic probe for detecting hydrogen peroxide in Parkinson's disease models [J]. Talanta 2019, 199, 628–633; Fan X, Sun Y, Fu J, et al. MRI-responsive nanoprobes for visualizing hydrogen peroxide in diabetic liver injury [J]. Biomaterials, 2025, 321, 123292.). Given the significant physiological and clinical importance of hydrogen peroxide, designing effective methods for its accurate detection in complex biological systems is crucial.

[0003] In recent years, researchers have reported various analytical methods for detecting hydrogen peroxide, including chromatography, electrochemical methods, and spectrophotometry. However, these methods involve cumbersome sample pretreatment and cannot effectively reflect the dynamic changes of hydrogen peroxide in vivo (Zhou Y, Yang X, Lee H, et al. Small-molecule fluorescent probes for detecting hydrogen peroxide in biological systems [J]. Coordination Chemistry Review, 2025, 541: 216785). Therefore, adopting effective methods for detecting hydrogen peroxide is crucial. Fluorescence imaging technology has attracted widespread attention from researchers due to its advantages such as high sensitivity, high resolution, simplicity, and real-time detection capabilities (Zhou R, Sha H, Fu S, et al. Hyperspectral Fluorescence Imaging with a NewPolarity-Ultrasensitive Fluorescent Probe[J]. Advanced Science, 2025,e08792.). It has gradually become an important research tool in the fields of chemistry, biology and medicine (Wang F, Zhong Y, Bruns O, et al. In vivo NIR-II fluorescence imaging for biology and medicine[J].Nature Photonics, 2024, 18, 535-547.).To date, numerous fluorescent probes have been developed for the detection of H2O2 (Li W, Fu T, Zheng M, et al. Discovery of a highly selective fluorescent probe for hydrogen peroxide and its biocompatibility evaluation and bioimaging applications in cells and zebrafish [J]. Bioorganic Chemistry, 2024, 150, 107552; Peng Z, Cui M, Chu J, et al. A novel AIE fluorescent probe for the detection and imaging of hydrogen peroxide in living tumor cells and in vivo [J]. Bioorganic Chemistry, 2024, 150, 107592.). However, these probes suffer from several drawbacks: short fluorescence emission wavelengths and weak tissue penetration. Therefore, designing and synthesizing near-infrared fluorescent probes with long-wavelength emission and high sensitivity for the detection of hydrogen peroxide is of great significance.

[0004] Furanitrile-oxanthracene, as a novel fluorescent dye, possesses a long emission wavelength, thus exhibiting greater tissue penetration depth, which is more advantageous for bioimaging. Currently, there are no probes based on furanitrile-oxanthracene dyes for detecting H2O2. Therefore, this invention designs and synthesizes a fluorescent probe based on furanitrile-oxanthracene for detecting hydrogen peroxide. Summary of the Invention

[0005] Based on the requirements, the inventors conducted in-depth research and, after a great deal of creative work, provided a hydrogen peroxide fluorescent probe based on furanone-oxanthracene.

[0006] The technical solution of this invention is a hydrogen peroxide fluorescent probe based on furanone-oxanthracene, the structure of which is as follows:

[0007]

[0008] A method for preparing a hydrogen peroxide fluorescent probe based on furanone-oxanthracene, comprising the following steps:

[0009] At -4 °C, 1.2–1.5 equivalents of compound FX and 1.0 equivalent of 4-dimethylaminopyridine were added sequentially to a 50 mL round-bottom flask. Then, 20–30 mL of tetrahydrofuran was added, followed by 100 μL of triethylamine under nitrogen protection. After stirring for 10–30 min, 1.0 equivalent of diphenylphosphochloride was added. The reaction was allowed to proceed for 1–3 h, and then gradually raised to room temperature for 8–12 h. After the reaction was complete, the crude product was desolventized under reduced pressure and purified by column chromatography using dichloromethane / methanol eluent at a volume ratio of 100:1–50:1 to obtain a deep purple solid FX-P, which is the fluorescent probe.

[0010] The beneficial effect of this invention is the excellent performance of a hydrogen peroxide fluorescent probe based on furanyl nitrile-oxanthracene. First, the fluorescence response of probe FX-P to H2O2 was investigated. FX-P itself showed almost no fluorescence signal, but the fluorescence emission intensity at 820 nm significantly increased with increasing H2O2 concentration. Further analysis showed that within the H2O2 concentration range of 1.0 μM to 70 μM, the fluorescence intensity at 820 nm exhibited a good linear relationship with the H2O2 concentration. These results indicate that FX-P can be used for highly sensitive H2O2 detection. Next, the absorption spectrum of FX-P in the presence of H2O2 was measured. FX-P has a low absorption peak at 780 nm. The intensity of the absorption peak at 780 nm increased upon the addition of H2O2. Then, the selectivity of the probe was investigated by measuring the fluorescence response of FX-P to cations, anions, amino acids, biothiols, and reactive oxygen species to evaluate the selectivity of FX-P. The results showed that FX-P fluorescence was only activated by H2O2, while the influence of other analytes on fluorescence intensity was negligible, indicating that FX-P can detect H2O2 in complex biological systems. Finally, the effect of pH on the determination of H2O2 by the fluorescent probe was investigated; a pH between 7.0 and 8.0 did not affect the determination of H2O2 by the fluorescent probe. Furthermore, the fluorescent probe exhibited a rapid response, with a response time within 10 minutes.

[0011] An application of a hydrogen peroxide fluorescent probe based on furanyl nitrile-oxanthracene was investigated using RAW 264.7 cells. After adding the fluorescent probe to the cells, almost no fluorescence was observed, indicating low H2O2 levels. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) followed by probe staining resulted in significantly enhanced fluorescence; treatment with N-acetyl-L-cysteine ​​(NAC) to inhibit intracellular H2O2 production resulted in a significant decrease in fluorescence. These results demonstrate that the fluorescent probe can monitor changes in intracellular H2O2 levels, providing a reliable method for monitoring changes in hydrogen peroxide levels related to pathological processes in the human body. Attached Figure Description

[0012] Figure 1 This is the synthetic route for the fluorescent probe.

[0013] Figure 2 The fluorescence spectra are shown after the fluorescent probe reacts with different concentrations of H2O2.

[0014] The x-axis represents wavelength, and the y-axis represents fluorescence intensity. The concentration of the fluorescent probe was 10 μM, and the H2O2 concentrations were 0, 1, 5, 10, 20, 30, 40, 50, 60, and 70 μM, respectively. The excitation wavelength was 780 nm, and the emission wavelength was 800–900 nm.

[0015] Figure 3 The graph shows the linear fluorescence response of the fluorescent probe to different H2O2 concentrations.

[0016] Figure 4 The image shows the UV-Vis absorption spectra of the fluorescent probe before and after its interaction with H2O2.

[0017] The x-axis represents wavelength, and the y-axis represents absorbance. The concentration of the fluorescent probe is 10 μM, and the concentration of H2O2 is 70 μM.

[0018] Figure 5 This is a selectivity diagram of the fluorescent probe.

[0019] The concentrations of fluorescent probes were all 10 μM, H2O2 concentrations were 70 μM, and the concentrations of other analytes were all 200 μM.

[0020] Figure 6 This is a graph showing the effect of pH on fluorescent probes.

[0021] Figure 7 The graph shows the relationship between fluorescence intensity and time after the fluorescent probe reacts with H2O2. The H2O2 concentrations are 0, 10, 20, 30, 50, and 70 μM.

[0022] Figure 8 This is a cytotoxicity assay. The horizontal axis represents the concentration of the fluorescent probe, and the vertical axis represents the cell viability.

[0023] Figure 9 This is a cell imaging image after the fluorescent probe reacts with H2O2.

[0024] Figure 10 This is a graph showing the relative fluorescence intensity of cells. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

[0026] Example 1:

[0027] Synthesis of fluorescent probes.

[0028] Synthetic routes such as Figure 1 At -4 °C, 1.2 equivalents of compound FX and 1.0 equivalent of 4-dimethylaminopyridine were added sequentially to a 50 mL round-bottom flask. Then, 20 mL of tetrahydrofuran was added, followed by 100 μL of triethylamine under nitrogen protection. After stirring for 20 min, 1.0 equivalent of diphenylphosphochloride was added, and the reaction was allowed to proceed for 2 h. The mixture was then gradually brought to room temperature and reacted for 10 h. After the reaction was complete, the crude product was purified by removing the solvent under reduced pressure and eluent by column chromatography with a 100:1 volume ratio of dichloromethane / methanol to obtain a deep purple solid FX-P, which is the fluorescent probe. 1 H NMR (400 MHz, DMSO-d6)δ 8.61-8.58 (d, J = 11.6 Hz, 1H), 8.39-8.36 (d, J =11.6 Hz, 1H), 7.83-7.78(dd, J1= 11.6 Hz, J2= 6.8 Hz, 4H), 7.58-7.51 (m, 6H), 7.48-7.29 (m, 4H), 7.15-7.05 (m, 4H), 6.99-6.97 (m, 2H), 6.59-6.56 (m, 2H), 6.13-6.10 (d, J =12.4 Hz, 1H), 5.74 (s, 1H), 2.63-2.60 (t, J= 9.2 Hz, 2H), 1.74-1.70 (t, J =6.4 Hz, 2H), 1.56 (s, 6H), 1.21 (s, 2H).

[0029] Example 2:

[0030] Fluorescent probe and H2O2 solution were prepared.

[0031] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in dimethyl sulfoxide to prepare a 1.0 × 10⁻⁶ solution. -4 A mol / L probe buffer solution is prepared. Preparation of H₂O₂ solution: Measure a certain amount of 30% H₂O₂ solution, then dilute to 50 mL with distilled water in a volumetric flask to prepare a 1.0 × 10⁻⁶ mol / L solution. -2 A detection solution of 1.0 × 10⁻⁶ mol / L. -2 The H₂O₂ solution was gradually diluted to obtain a concentration of 1.0 × 10⁻⁶ mol / L. -5 -7.0×10 -4A mol / L H₂O₂ solution. Add 1.0 mL of the probe's prepared solution and 1.0 mL of H₂O₂ solution to a 10 mL volumetric flask, and dilute to volume with buffer solution to obtain a concentration of 1×10⁻⁶ mol / L H₂O₂ solution. -5 mol / L fluorescent probe and 1.0×10 -6 -7.0×10 -5 The test solution is a mixture of mol / L H2O2.

[0032] Example 3:

[0033] Determination of fluorescence spectra of the interaction between fluorescent probes and H2O2.

[0034] Figure 2 The fluorescence spectrum is shown for the interaction between the fluorescent probe and H₂O₂. The concentration of the fluorescent probe was 10 μM, and the concentrations of H₂O₂ were 0, 1, 5, 10, 20, 30, 40, 50, 60, and 70 μM, respectively. The excitation wavelength used in the experiment was 780 nm, and the emission wavelength range was 800–900 nm. The slit width was 5.0 nm / 5.0 nm, and the fluorescence measurement instrument used was a Hitachi F4600 fluorescence spectrophotometer. Figure 2 As can be seen, before the addition of H2O2, the fluorescent probe had almost no fluorescence emission peak; after the addition of H2O2, fluorescence emission appeared in the near-infrared region (820 nm). Furthermore, the fluorescence intensity of the probe continuously increased with the increase of H2O2 concentration. Figure 3 The graph shows the linear response of the probe to different H2O2 concentrations. The fluorescence intensity is linearly related to the H2O2 concentration, with a linear range of 1 μM to 70 μM, indicating that the probe can detect H2O2 with high sensitivity.

[0035] Example 4:

[0036] Determination of the UV-Vis absorption spectrum of the reaction between the fluorescent probe and H2O2.

[0037] Figure 4 The image shows the UV-Vis absorption spectrum of the fluorescent probe after reaction with H₂O₂. The concentration of the fluorescent probe was 10 μM, and the concentration of H₂O₂ was 70 μM. The UV-Vis absorption spectroscopy was performed using an Agilent Cary 60 UV-Vis spectrophotometer. Figure 4 It can be seen that FX-P has a low absorption peak at 780 nm. When H2O2 is added, the intensity of the absorption peak at 780 nm increases.

[0038] Example 5:

[0039] Selectivity of fluorescent probes for H2O2 determination.

[0040] Figure 5This is a selectivity curve for the fluorescent probe to determine H2O2. The determination was investigated by adding H2O2 and cations (K+) to a 10 μM fluorescent probe solution. + Mg 2+ Ca 2+ Na + Fe 3+ Fe 2+ Cu 2+ NH4 + ), anion (HS) - ,Br - NO - NO2 - SO3 2- HSO3 - NO3 - HPO4 2- CH3COO - ), amino acids (Lys, Try, Leu, Phe, Met, Thr, lle, Val), and biothiols (Cys, Hcy, GSH) reactive oxygen species (ONOO) - The fluorescence response of (HClO). From Figure 5 As can be seen, only H2O2 can cause a significant enhancement of fluorescence, while other analytes have no significant effect on the fluorescence of the probe. These results indicate that the fluorescent probe has good selectivity for H2O2.

[0041] Example 6:

[0042] The effect of solution pH on the fluorescence properties of H2O2 measured by fluorescent probe.

[0043] The effect of pH on the fluorescence spectrum of H2O2 determined by a fluorescent probe was investigated, and the results are as follows: Figure 6 To verify the applicability of the probe in different environments, the effect of pH on the fluorescence performance of the FX-P probe was evaluated. Within the pH range of 2.0–10.0, the FX-P probe itself produced almost no fluorescence. However, upon the addition of H₂O₂ to the system, a significant enhancement in fluorescence was observed in the pH range of 7.0–8.0. This characteristic indicates that the FX-P probe can effectively detect H₂O₂ under physiological conditions (pH 7.4).

[0044] Example 7:

[0045] Determination of the response time of a fluorescent probe to H2O2.

[0046] The response time of the fluorescent probe to H2O2 was studied, and the results are as follows: Figure 7 As shown in the figure, the probe's response time to H2O2 is 10 minutes, which meets the requirements for real-time monitoring in actual samples. Figure 7It can also be seen that after the fluorescence intensity reaches its maximum value, the fluorescence intensity no longer changes over time, which indicates that this fluorescent probe has good photostability.

[0047] Example 8:

[0048] Application of fluorescent probes in living cells.

[0049] First, cytotoxicity experiments were conducted using RAW 264.7 cells, such as... Figure 8 As shown in the figure, when 0–30 μM FX-P probe was added, the cell viability was above 90%, indicating that the fluorescent probe has low toxicity and can be used to detect H2O2 in live cells. Then, the application of the fluorescent probe in live cells was investigated. RAW 264.7 cells were selected for confocal microscopy imaging, and the results are shown in the figure. Figure 9 and Figure 10 As shown in the figure, almost no fluorescence was observed after adding the fluorescent probe to the cells, indicating a low H2O2 content. Literature reports that phorbol-12-myristate-13-acetate (PMA) can stimulate H2O2 production in cells, while N-acetyl-L-cysteine ​​(NAC) can inhibit intracellular H2O2 production. Pretreatment of cells with PMA for 10 h followed by probe staining for 0.5 h resulted in significantly enhanced fluorescence; treatment with both PMA and NAC for 10 h followed by probe staining for 0.5 h resulted in significantly weakened fluorescence. These results demonstrate that fluorescent probes can monitor changes in intracellular H2O2 content, providing a reliable method for monitoring hydrogen peroxide-related diseases in humans.

Claims

1. A hydrogen peroxide fluorescent probe based on furanone-oxanthracene, namely FX-P, characterized in that, The structure is as follows: 。 2. The preparation of a hydrogen peroxide fluorescent probe based on furanone-oxanthracene according to claim 1, characterized in that, The reaction steps are as follows: At -4 °C, 1.2–1.5 equivalents of compound FX and 1.0 equivalent of 4-dimethylaminopyridine were added sequentially to a 50 mL round-bottom flask. Then, 20–30 mL of tetrahydrofuran was added, followed by 100 μL of triethylamine under nitrogen protection. After stirring for 10–30 min, 1.0 equivalent of diphenylphosphochloride was added. The reaction was allowed to proceed for 1–3 h, and then gradually raised to room temperature for 8–12 h. After the reaction was complete, the crude product was desolventized under reduced pressure and purified by column chromatography using dichloromethane / methanol eluent at a volume ratio of 100:1–50:1 to obtain a deep purple solid FX-P, which is the fluorescent probe.

3. The application of the hydrogen peroxide fluorescent probe based on furanone-oxanthracene according to claim 1, characterized in that, The fluorescent probe can be used to detect hydrogen peroxide in living cells.

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