A self-anchoring fluorescent probe reversibly responsive to naclO / gsh, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biochemistry, and relates to a self-anchoring fluorescent probe reversibly responding to NaClO / GSH and a preparation method and application thereof. The preparation method of the self-anchoring fluorescent probe reversibly responding to NaClO / GSH comprises the following steps: (1) performing first reaction on compound (I), compound (II) and tetrakis(triphenylphosphine)palladium(0) to obtain compound (III); and (2) performing second reaction on compound (III), compound (IV), acetic acid and piperidine to obtain the probe. The fluorescent probe provided by the application has high selectivity and sensitivity; after responding to NaClO, the fluorescence is enhanced by about 10 times, and the probe has the characteristics of obvious opening type response; the probe has good stability under physiological pH conditions, and is conducive to real-time in-situ imaging of NaClO / GSH; the probe itself has strong self-anchoring ability, and is conducive to prolonging the residence time of the probe in a lesion site.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a self-anchored fluorescent probe that is reversibly responsive to NaClO / GSH, its preparation method, and its application. Background Technology

[0002] Ferroprelation is a novel form of cell death distinct from apoptosis and necrosis, with its core mechanism being the abnormal accumulation of intracellular iron-dependent lipid peroxides. Studies have shown that ferroptosis is closely related to the pathological evolution of tumors, neurodegenerative diseases, and urinary system diseases. During ferroptosis, myeloperoxidase (MPO)-mediated oxidative stress is considered a key factor in inducing cell damage: MPO catalyzes the reaction of hydrogen peroxide (H₂O₂) with chloride ions (Cl₂). - The reaction produces hypochlorous acid (HClO), a highly oxidizing acid. The large-scale production of HClO not only depletes glutathione (GSH), a key intracellular antioxidant, leading to redox imbalance, but also further accelerates lipid peroxidation, ultimately inducing cell death. Therefore, developing tools capable of in-situ, real-time monitoring of intracellular HClO dynamics is of great significance for elucidating the biological function of ferroptosis and the clinical diagnosis of related diseases.

[0003] In recent years, fluorescence imaging technology has become the mainstream method for monitoring reactive oxygen species (ROS) in ferroptosis due to its high sensitivity and high resolution. Researchers have developed a variety of fluorescent probes targeting HClO and have made significant progress in cellular and superficial tissue imaging. For example, existing technologies have developed "on" fluorescent probes that respond to HClO, enabling real-time imaging of endogenous hypochlorous acid in pathological processes such as epilepsy models. However, most existing HClO probes are irreversibly responsive, meaning that the probe cannot return to its initial state after reacting with the target. This irreversibility not only limits the dynamic monitoring of HClO fluctuations but also allows probe molecules to easily diffuse freely within cells, leading to interference from "false positive" signals.

[0004] To address the reversibility issue, previous studies have utilized organoselenium functional groups to construct reversible fluorescent probes (such as MPhSe-BOD). In these probes, selenium atoms can be oxidized to selenium sulfone by HClO and reduced back to their original state by GSH, thus enabling reciprocating monitoring capabilities. However, these small molecule probes still face challenges in complex biological microenvironments, such as short retention time at lesion sites and easy metabolic loss, making it difficult to achieve long-term, accurate monitoring of ferroptosis regions. Although recent studies have demonstrated that introducing benzylfluoroglycoside groups can... β-Galactosidase hydrolyzes to produce quinone methyl intermediates, which are used to enhance the enrichment of probes at target sites by bioorthogonal covalent binding with proteins. However, in MPO-mediated ferroptosis monitoring tasks, how to balance the dynamic response capability and spatial positioning accuracy of probes still faces the following challenges: (1) the ability to capture dynamic changes in real time; (2) the signal sensitivity and stability in complex environments; and (3) the contradiction between spatiotemporal resolution and precise positioning. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned problems in the prior art by providing a self-anchored fluorescent probe that responds reversibly to NaClO / GSH, its preparation method, and its applications. The probe of this invention possesses both self-anchoring ability to ferroptosis lesions and a real-time reversible response to NaClO / GSH. Combined with live-cell fluorescence imaging technology, this probe has the potential for dynamic tracking of NaClO / GSH in live cells, providing a reliable research tool for elucidating the regulatory and intervention mechanisms of NaClO / GSH on ferroptosis and for the diagnosis and treatment of related diseases.

[0006] To achieve the above objectives, a first aspect of the present invention provides a self-anchored fluorescent probe that responds reversibly to NaClO / GSH, the probe having the following structural formula:

[0007] .

[0008] A second aspect of the present invention provides a method for preparing the self-anchored fluorescent probe that is reversibly responsive to NaClO / GSH, comprising the following steps:

[0009] (1) Compound (I), compound (II), and tetrakis(triphenylphosphine)palladium (0) were added to the first solvent to carry out the first reaction, yielding compound (III);

[0010] (2) Compound (III), compound (IV), acetic acid and piperidine are added to a second solvent to carry out a second reaction to obtain the probe;

[0011] The reaction process is as follows:

[0012] .

[0013] A third aspect of the invention provides the application of the self-anchored fluorescent probe that is reversibly responsive to NaClO / GSH in the preparation of an MPO-mediated iron death oxidative stress imaging agent.

[0014] The present invention has the following beneficial effects:

[0015] 1. The fluorescent probe provided by this invention has high selectivity and sensitivity; after responding to NaClO, the emission wavelength of the complex formed is blue-shifted by about 25 nm and the fluorescence is enhanced by about 10 times, showing obvious characteristics of an on-response; it has good stability under physiological pH conditions, which is helpful for real-time in-situ imaging of NaClO / GSH; the probe itself has strong self-anchoring ability, which is beneficial to prolonging the residence time of the probe at the lesion site.

[0016] 2. The method for preparing the fluorescent probe provided by this invention uses readily available raw materials, has mild and easily controllable reaction conditions, saves reaction costs, and ensures the yield of the target product.

[0017] 3. The fluorescent probe Gal-F-SeBDP provided by this invention possesses highly efficient self-anchoring ability and can be used for dynamic tracing of NaClO / GSH in ferroptosis. Combined with live-cell fluorescence imaging technology, the probe Gal-F-SeBDP can dynamically image the NaClO / GSH dynamics in ferroptosis, providing a new detection method for NaClO / GSH and a promising tool for MPO-mediated ferroptosis oxidative stress disease models.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0019] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the invention.

[0020] Figure 1 The nuclear magnetic resonance spectrum of Gal-F-SeBDP in this invention is shown.

[0021] Figure 2 The following are the spectra of Gal-F-SeBDP in PBS buffer solution before and after the response to NaClO in this invention. Figure 2 In the diagram, 'a' shows the ultraviolet absorption spectrum. Figure 2 b in the figure shows the fluorescence spectrum.

[0022] Figure 3 The fluorescence imaging and fluorescence intensity diagrams of the present invention are shown, wherein, Figure 3 Figure 'a' shows live-cell fluorescence imaging after the probe Gal-F-SeBDP responded to the control group, NaClO, and NaClO+GSH group. Figure 3 Figure b shows live-cell fluorescence imaging after the probe Gal-F-SeBDP responded to the control group, LPS+PMA, and LPS+PMA+NAC groups. Figure 3c in the figure shows live cell fluorescence imaging after the probe Gal-F-SeBDP responded to the control group, Erastin, and Fer-1 group. Detailed Implementation

[0023] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0024] A first aspect of the present invention provides a self-anchored fluorescent probe that is reversibly responsive to NaClO / GSH, the probe having the following structural formula:

[0025] .

[0026] A second aspect of the present invention provides a method for preparing the self-anchored fluorescent probe that is reversibly responsive to NaClO / GSH, comprising the following steps:

[0027] (1) Compound (I), compound (II), and tetrakis(triphenylphosphine)palladium (0) were added to the first solvent to carry out the first reaction, yielding compound (III);

[0028] (2) Compound (III), compound (IV), acetic acid and piperidine are added to a second solvent to carry out a second reaction to obtain the probe;

[0029] The reaction process is as follows:

[0030] .

[0031] According to the present invention, preferably, both the first solvent and the second solvent are anhydrous toluene.

[0032] According to the present invention, preferably, in step (1), the molar ratio of compound (I) to compound (II) is 1:1.1-1.3.

[0033] According to the present invention, preferably, in step (1), the volume of the first solvent is 5-15 mL, with 1 mmol of compound (I) and the amount of tetra(triphenylphosphine)palladium(0) added is 30-80 mg.

[0034] According to the present invention, preferably, in step (1), the conditions of the first reaction include: a reaction temperature of 100-110°C and a reaction time of 1-2 h.

[0035] According to the present invention, preferably, in step (2), the amount of compound (IV) added is 350-450 mg, with 1 mmol of compound (III) as the basis, and the volume of the second solvent is 20-30 mL.

[0036] According to the present invention, preferably, in step (2), the conditions of the second reaction include: a reaction temperature of 90-100°C and a reaction time of 0.5-1 h.

[0037] According to the present invention, preferably, step (2) further includes: drying the reaction solution after the reaction is completed to obtain the probe.

[0038] A third aspect of the invention provides the application of the self-anchored fluorescent probe that is reversibly responsive to NaClO / GSH in the preparation of an MPO-mediated iron death oxidative stress imaging agent.

[0039] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0040] In this embodiment, the device used for laser confocal imaging is a Zeiss LSM-710 confocal microscope.

[0041] Example 1

[0042] Synthesis of Compound III: Under nitrogen protection, Compound I (200 mg, 1 mmol) and Compound II (440 mg, 1.3 mmol) were dissolved in 10 mL of anhydrous toluene and added to a 100 mL Schlenk tube. Tetra(triphenylphosphine)palladium(O) (40 mg, 0.05 mmol) was then added, and the reaction mixture was stirred at 100 °C for 2 h. The mixture was extracted with ethyl acetate, washed three times with saturated brine, and the organic phase was dried over anhydrous MgSO4. The mixture was filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 230 mg of a golden-purple oil (eluent: petroleum ether and dichloromethane, petroleum ether:dichloromethane = 4:1). v / v Yield: 70%.

[0043] Synthesis of compound Gal-F-SeBDP: Under nitrogen protection, compound III (200 mg, 0.40 mmol) and compound IV (150 mg, 0.48 mmol) were dissolved in 10 mL of anhydrous toluene and added to a 100 mL Schlenk tube. Then, 0.01 mL of piperidine and 0.03 mL of acetic acid were added. The reaction mixture was stirred at 90 °C for 1 h. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to give 85 mg of a blackish-green solid (eluting buffer: dichloromethane and methanol, dichloromethane:methanol = 10:1). v / v (Yield: 22%)

[0044] The NMR spectrum of compound Gal-F-SeBDP is as follows: Figure 1 As shown in the figure, 1 H NMR (600 MHz, DMSO) δ / ppm 8.07 (s, 1H), 7.85 (s, 2H), 7.71 – 7.60 (m, 6H), 7.50 – 7.39 (m, 7H),7.29 (d, J = 6.6 Hz, 1H), 6.96 (s, 1H), 5.65 – 5.5 (m, 2H), 5.30 (s, 1H), 4.92 (d, J = 7.2 Hz, 2H), 4.68 (s, 1H), 4.58 (s, 1H), 3.73 (s, 1H), 3.64 (m, J = 6.8 Hz, 2H), 3.55 (m, 2H), 3.44 (m, 1H). The structure of Gal-F-SeBDP can be verified.

[0045] The probe Gal-F-SeBDP was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The test solution was an ethanol / PBS buffer solution (50 / 50, 10 mM, pH 7.4), and the probe concentration was 10 μM. The absorption spectrum results are as follows. Figure 2 As shown in Figure a, the absorption peak of the probe Gal-F-SeBDP is around 655 nm, and the absorption intensity of the probe Gal-F-SeBDP does not change significantly after reacting with NaClO. Meanwhile, as... Figure 2 As shown in b, after adding NaClO to the probe solution, the fluorescence intensity of the probe at 677 nm gradually increased with the increase of NaClO concentration. When the NaClO content reached 5 equivalents, the fluorescence of the probe reached saturation, and the fluorescence intensity of Gal-F-SeBDP increased by about 10 times.

[0046] Example 2

[0047] The probe Gal-F-SeBDP exhibits reversible fluorescence imaging response to NaClO / GSH in live-cell fluorescence imaging experiments.

[0048] To further evaluate the performance of the probe Gal-F-SeBDP in live-cell fluorescence imaging, the probe was used to stain and image HepG2 cells pretreated with exogenous HClO / GSH, HepG2 cells pretreated with endogenous HClO / GSH, and HepG2 cells pretreated with ferroptosis inducers and inhibitors, respectively. Figure 3As shown in figure a, after the probe was co-incubated with the cells, the reversible response of the probe in the cells was monitored. Figure 3 As shown in b, the fluorescence of cells after the addition of exogenous NaClO was enhanced compared to the control group; however, the fluorescence gradually weakened upon the addition of GSH. To better verify the feasibility of the probe for in vivo imaging, as shown in... Figure 3 As shown in Figure c, confocal imaging was performed by co-incubating the probe with endogenous HClO / GSH. Similarly, the fluorescence of cells treated with LPS and PMA was enhanced, while the fluorescence was weakened after the addition of the inhibitor NAC. To observe the reversible response of the probe in the ferroptosis model, the fluctuation of endogenous HClO / GSH levels in ferroptosis-affected cells was also studied. When HepG2 cells were stimulated with Erastin and stained with the probe, the fluorescence intensity of the cells increased significantly. When the ferroptosis inhibitor Fer-1 was added to clear ROS in the live cells, the fluorescence intensity decreased significantly. These observations indicate that the probe Gal-F-SeBDP has the potential for in vivo imaging.

[0049] The above experimental results demonstrate that the probe Gal-F-SeBDP has a highly efficient and reversible response capability. The introduction of the benzyl fluoride group can prolong the retention time of the probe at the ferroptosis lesion site, effectively compensating for the defect of easy probe release in vivo, and has great application prospects in the biomedical field.

[0050] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A self-anchored fluorescent probe reversibly responsive to NaClO / GSH, characterized in that, The structural formula of the probe is: 。 2. The method for preparing the self-anchored fluorescent probe with reversible response to NaClO / GSH as described in claim 1, characterized in that, Includes the following steps: (1) Compound (I), compound (II), and tetrakis(triphenylphosphine)palladium (0) were added to the first solvent to carry out the first reaction, yielding compound (III); (2) Compound (III), compound (IV), acetic acid and piperidine are added to a second solvent to carry out a second reaction to obtain the probe; The reaction process is as follows: 。 3. The preparation method according to claim 2, characterized in that, Both the first solvent and the second solvent are anhydrous toluene.

4. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of compound (I) to compound (II) is 1:1.1-1.

3.

5. The preparation method according to claim 2, characterized in that, In step (1), the volume of the first solvent is 5-15 mL, with 1 mmol of compound (I) and 30-80 mg of tetra(triphenylphosphine)palladium(0) added.

6. The preparation method according to claim 2, characterized in that, In step (1), the conditions for the first reaction include: a reaction temperature of 100-110 ℃ and a reaction time of 1-2 h.

7. The preparation method according to claim 2, characterized in that, In step (2), with compound (III) as 1 mmol, the amount of compound (IV) added is 350-450 mg, the amount of piperidine added is 0.02-0.03 mL, the amount of acetic acid added is 0.06-0.08 mL, and the volume of the second solvent is 20-30 mL.

8. The preparation method according to claim 2, characterized in that, In step (2), the conditions for the second reaction include: a reaction temperature of 90-100 ℃ and a reaction time of 0.5-1 h.

9. The preparation method according to claim 2, characterized in that, Step (2) further includes: after the reaction is completed, the reaction solution is dried by rotary evaporation to obtain the probe.

10. The application of the self-anchored fluorescent probe with reversible response to NaClO / GSH as described in claim 1 in the preparation of an MPO-mediated iron death oxidative stress imaging agent.

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