Hypochlorous acid fluorescent probe with large Stokes shift as well as preparation method and application of hypochlorous acid fluorescent probe

Abstract

The invention provides a hypochlorous acid fluorescent probe with large Stokes shift and a preparation method and application thereof, and belongs to the technical field of hypochlorous acid fluorescent probes, the hypochlorous acid fluorescent probe is a cationic iridium (III) complex and contains antianions; the Stokes shift of the hypochlorous acid fluorescent probe is larger than or equal to 170 nm, background interference caused by overlapping of exciting light and an emission spectrum is reduced, and the detection signal-to-noise ratio is increased; the hypochlorous acid fluorescent probe disclosed by the invention has the characteristic of quick response to HOCl, and shows relatively high detection sensitivity and excellent selectivity; the hypochlorous acid fluorescent probe can trigger detectable fluorescence signal change after acting with HOCl in cells, so as to realize in-situ detection and imaging of HOCl in cells; the fluorescent probe can be applied to a lambda-carrageenan induced mouse arthritis model, in-vivo fluorescence imaging of endogenous HOCl signals in an inflammation joint area is achieved, and a sensitive and reliable imaging tool is provided for research of inflammation-related disease related HOCl.

Description

Technical Field

[0001] This invention belongs to the field of hypochlorous acid fluorescent probe detection technology, specifically relating to a hypochlorous acid fluorescent probe with a large Stokes shift, its preparation method, and its application. Background Technology

[0002] Hypochlorous acid (HOCl) is one of the important reactive oxygen species in organisms, mainly produced by the myeloperoxidase system, and plays a key role in immune defense and inflammatory responses. Studies have shown that abnormally elevated HOCl levels are closely related to inflammatory pathological processes such as inflammatory joint diseases. Therefore, developing analytical tools capable of rapid and accurate detection and visualization of HOCl in complex biological environments is of great significance for the study of the mechanisms and early assessment of related diseases.

[0003] Currently, fluorescent probe detection technology is widely used for the detection and imaging of bioactive small molecules due to its advantages such as high sensitivity, rapid response, real-time in-situ imaging, and relatively simple operation. In the field of HOCl detection, small organic molecule probes based on fluorophores such as coumarin, 1,8-naphthalimide, fluorescein, rhodamine, and difluoroboron dipyrrolemethane (BODIPY) have been reported and have shown certain application value in HOCl imaging at the cellular and even animal levels.

[0004] However, many existing small organic molecule HOCl fluorescent probes generally suffer from small Stokes shifts, which easily lead to overlap between excitation and emission spectra. This, in turn, causes background interference, signal crosstalk, and fluorescence self-absorption / quenching, resulting in a decreased signal-to-noise ratio and limited quantitative accuracy, especially in deep tissues or complex biological systems. Therefore, developing an HOCl-responsive fluorescent probe that simultaneously possesses a large Stokes shift and high selectivity, and is suitable for complex biological environments, remains a pressing technical challenge in this field. Summary of the Invention

[0005] In view of this, the purpose of this invention is to address the problems existing in the prior art by providing a hypochlorous acid fluorescent probe with a large Stokes shift, its preparation method, and its application. This hypochlorous acid fluorescent probe can achieve rapid response and visual detection of HOCl in complex biological environments, and has the advantages of fast response, high sensitivity, good selectivity, and high biosafety. This overcomes the problems of small Stokes shift, significant spectral overlap and background interference, and insufficient signal-to-noise ratio of existing small organic molecule HOCl probes, thereby improving the detection and imaging effect of HOCl.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: One objective of this invention is to provide a hypochlorous acid fluorescent probe with a large Stokes shift, wherein the hypochlorous acid fluorescent probe has the structure shown in Formula I: Formula I This hypochlorous acid fluorescent probe is a cationic iridium(III) complex and contains anion-resistant components; The anti-anion is selected from one or more of PF6⁻, BF4⁻, Cl⁻, ClO4⁻, OTf⁻ or BPh4⁻.

[0007] Furthermore, the Stokes shift of this hypochlorous acid fluorescent probe is ≥170 nm.

[0008] A second objective of this invention is to provide a method for preparing a hypochlorous acid fluorescent probe with a large Stokes shift as described above, comprising the following synthetic route: S1: Under an inert atmosphere, tetra(triphenylphosphine)palladium catalyst was added to a reaction system containing 6-chlorophenanthridine, benzo[b]thiophene-2-boronic acid, solvent and base. The reaction was heated. After the reaction was completed, the mixture was extracted with organic solvent, dried and concentrated, and purified by silica gel column chromatography to obtain ligand intermediate A. S2: Add IrCl3·xH2O and ligand intermediate A obtained in step S1 into a mixed solvent and heat to reflux. After the reaction is completed and cooled, filter to obtain the precipitate, wash with water and petroleum ether in sequence, and dry under vacuum to obtain chlorinated bridged iridium intermediate B; the obtained intermediate does not need to be purified and can be used directly in the next step of the reaction. S3: The chlorobridged iridium intermediate B obtained in step S2 and 4-carboxy-4′-methyl-2,2′-bipyridine were added to a reaction vessel, a solvent was added, and the mixture was heated under reflux in an inert atmosphere. Subsequently, an anion exchange was carried out by adding a salt containing an anion inhibitor, and the mixture was stirred at room temperature to obtain compound I.

[0009] In the above preparation method, further, in step S1, the equivalent ratio of tetrakis(triphenylphosphine)palladium to 6-chlorophenanthridine is 1:(20~50); the solvent is selected from one or more of dioxane, tetrahydrofuran, toluene, dimethylformamide, and ethanol / water mixed solvent; the base is selected from one or more of potassium carbonate, cesium carbonate, potassium phosphate, and sodium tert-butoxide; the extraction organic solvent is selected from one or more of dichloromethane, ethyl acetate, and toluene.

[0010] In the above preparation method, further, in step S2, the molar ratio of IrCl3·xH2O to the ligand intermediate A obtained in step S1 is 1:(1~10); the mixed solvent is selected from ethylene glycol ether / water system or alcohol / water system.

[0011] In the above preparation method, further, in step S3, the molar ratio of chlorobridged diiridium intermediate B to 4-carboxy-4′-methyl-2,2′-bipyridine is 1:(1~10); the solvent is selected from one or more of dichloromethane, chloroform, methanol, ethanol, and acetonitrile; the salt containing the anti-anion is selected from one or more of ammonium hexafluorophosphate, ammonium tetrafluoroborate, potassium hexafluorophosphate, and tetraphenylborate.

[0012] The third objective of this invention is to provide the application of the above-mentioned hypochlorous acid fluorescent probe with a large Stokes shift in the selective detection of hypochlorous acid.

[0013] Furthermore, the detection mechanism of this hypochlorous acid fluorescent probe is as follows: When a compound with the structure of Formula I reacts with HOCl, the sulfur atom in the structure of the Formula I compound is oxidized to sulfoxide, causing a detectable change in its fluorescence signal, which can then be used to achieve the qualitative or quantitative detection of HOCl.

[0014] The fourth objective of this invention is to provide the application of a hypochlorous acid fluorescent probe with a large Stokes shift in the in-situ detection and imaging analysis of HOCl in inflammatory disease models.

[0015] Furthermore, the inflammatory disease model includes an inflammatory joint disease-related model, which is a λ-carrageenan-induced mouse arthritis model.

[0016] Beneficial effects of this invention: This invention provides and prepares a novel hypochlorous acid fluorescent probe based on a transition metal iridium(III) complex. This probe has a significant large Stokes shift, which can reach about 170 nm. This is beneficial for reducing background interference caused by the overlap of excitation light and emission spectrum and improving the detection signal-to-noise ratio. (2) The hypochlorous acid fluorescent probe of the present invention has a rapid response to HOCl and exhibits high detection sensitivity and excellent selectivity, making it suitable for the identification and analysis of HOCl in complex biological systems; (3) The hypochlorous acid fluorescent probe of the present invention has low cytotoxicity and has a biocompatibility basis for imaging and detection at the cellular level; (4) The hypochlorous acid fluorescent probe of the present invention can induce a detectable change in fluorescence signal (fluorescence quenching) after interacting with intracellular HOCl, thereby realizing in situ detection and imaging of intracellular HOCl. (5) The hypochlorous acid fluorescent probe of the present invention can be applied to the λ-carrageenan-induced mouse arthritis model to realize in vivo fluorescence imaging of local HOCl signals in the inflamed joint region, providing a sensitive and reliable imaging tool for the study of HOCl related to inflammation-related diseases. Attached Figure Description

[0017] Figure 1 For compounds with the structure of Formula I in DMSO-d6 1 H NMR spectrum; Figure 2 The HRMS spectrum of the compound having the structure of Formula I; Figure 3 The absorption and emission spectra of the compound with and without HOCl are shown; wherein the concentration of the compound with the formula I structure is 5 μM and the concentration of HOCl is 50 μM. Figure 4 The emission spectra of the compound with different concentrations of HOCl are shown below. The concentration of the compound with the formula I structure is 5 μM, the excitation wavelength λex is 550 nm, and the concentrations of HOCl are 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 20 μM, 30 μM, and 50 μM. Figure 5 The emission change of the compound with structure I of formula I over time after the addition of HOCl is shown in the figure; where the concentration of the compound with structure I of formula I is 5 μM, the concentration of HOCl is 20 μM, and the excitation wavelength is 710 nm. Figure 6 The graph shows the changes in emission intensity of the compound with structure I before and after the addition of HOCl at different pH values; where the concentration of the compound with structure I is 5 μM, the concentration of HOCl is 50 μM, and the excitation wavelength is 710 nm. Figure 7 The emission intensity diagrams are for compounds of Formula I after the addition of various reactive oxygen species; wherein the concentration of the compound of Formula I is 5 μM, the concentration of reactive oxygen species is 50 μM, the excitation wavelength is 710 nm, and the reactive oxygen species AJ is blank, H2O2. • OH, TBHP, ROO • O2 − , t-BuOO • ONOO − , NO, HOCl; Figure 8 The cell viability is shown in the graph after co-incubation of the compound of formula I with ATDC5 cells for 24 h. Figure 9Confocal imaging of immobilized ATDC5 cells; where A: only compound of formula I was added; B: HOCl (50 μM, 15 min) was added. Figure 10 The image shows the time-dependent in vivo fluorescence imaging of HOCl for compound I (50 μL, 440 μM) in a mouse model of arthritis; where A: 25 μL of λ-carrageenan (5 mg / mL aqueous solution) was injected into the right joint, λex = 540 nm, λem = 710 nm. Detailed Implementation

[0018] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0019] In the following examples, the reagents not specifically mentioned are conventional reagents, all of which can be purchased from conventional reagent manufacturing and sales companies. Unless otherwise specified, the methods used are all prior art.

[0020] Example 1: A method for preparing a compound with structure I, comprising the following steps: (1) Tetra(triphenylphosphine)palladium (0.21 g, 0.18 mmol) was added to a mixture containing 6-chlorophenanthridine (1.28 g, 6 mmol), benzo[b]thiophene-2-boric acid (1.28 g, 7.2 mmol), toluene (10 mL), ethanol (5 mL), and 2 M sodium carbonate aqueous solution (10 mL), and refluxed under nitrogen protection for 5 hours. After the reaction was complete, the solvent was removed by vacuum evaporation, and the residue was dissolved in dichloromethane (50 mL). The reaction mixture was then poured into water (200 mL), and the product was extracted with dichloromethane (50 mL × 3). The organic layer was washed with water (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated under vacuum. The product was purified by silica gel column chromatography using a dichloromethane / petroleum ether (1 / 1) mixture as the eluent to give a white powder of ligand intermediate A (955 mg, yield 51.2%).

[0021] (2) IrCl3·xH2O (317 mg, 1 mmol) and ligand intermediate A (2.2 mmol) were added to a mixed solution of water (10 mL) and 2-ethoxyethanol (30 mL). The mixture was refluxed overnight. After cooling, the precipitate was filtered, washed with water and petroleum ether respectively, and then dried under vacuum to obtain a solid product, namely chloro-bridged iridium dimer intermediate B. This product can be used without further purification.

[0022] (3) Chlorinated iridium dimer intermediate B (255 mg, 0.15 mmol) and 4-carboxy-4'-methyl-2,2'-bipyridine (65 mg, 0.3 mmol) were added to a 50 mL flask; then 10 mL of CH2Cl2 and 10 mL of CH3OH were added, and the mixture was refluxed overnight under nitrogen protection. KPF6 (167 mg, 0.9 mmol) was then added, and the mixture was stirred at room temperature for 1 h. After evaporating the solvent, the product was purified by silica gel chromatography using CH2Cl2 / CH3OH (20:1, v / v) as the eluent to obtain a deep red product with a yield of 52.6%. The target compound was characterized by 1H NMR and HRMS, as detailed below. Figure 1 and Figure 2 As shown.

[0023] Example 2: Experiment on the response behavior of the hypochlorous acid fluorescent probe obtained in Example 1 to HOCl. The specific test method is as follows: (1) Preparation of HOCl stock solution: According to the Lambert-Beer law, its absorbance at 290 nm was tested, and 1 mM HOCl stock solution was prepared with NaClO.

[0024] (2) Preparation of hypochlorous acid fluorescent probe stock solution: Weigh the hypochlorous acid fluorescent probe and prepare a 5 mM stock solution with N',N-dimethylformamide (DMF). Store in the dark and refrigerated for later use.

[0025] (3) Preparation of test solution: Prepare a test solution with a probe concentration of 5 μM. The specific preparation method is shown in Table 1. Table 1: Depend on Figure 3 It can be seen that the probe's maximum absorption is around 550 nm, its maximum emission intensity is around 710 nm, and its Stokes shift can reach approximately 170 nm; Figure 4 It can be seen that as the HOCl concentration gradually increases, the emission intensity of the probe at 710 nm exhibits a significant dose-dependent decay; when the HOCl concentration is 50 μM, the fluorescence intensity at 710 nm decreases by approximately 77.64% compared to the initial state; Figure 5 It can be seen that after the addition of HOCl, the fluorescence signal basically reached a stable state within about 300 seconds.

[0026] Example 3: pH stability test of the hypochlorous acid fluorescent probe obtained in Example 1 of this invention. The specific test method is as follows: (1) Preparation of HOCl stock solution: According to the Lambert-Beer law, its absorbance at 290 nm was tested, and 1 mM HOCl stock solution was prepared with NaClO.

[0027] (2) Preparation of probe stock solution: Weigh the hypochlorous acid fluorescent probe obtained in Example 1 of this invention, and prepare a stock solution with a concentration of 5 mM using N',N-dimethylformamide. Store it in the dark and refrigerated for later use.

[0028] (3) Preparation of test solution: Prepare a test solution with a hypochlorous acid fluorescent probe concentration of 5 μM. The specific preparation method is shown in Table 2. Table 2: The results are as follows Figure 6 As shown, the test results indicate that within the pH range of 5-8, the fluorescence signal of the hypochlorous acid fluorescent probe obtained in Example 1 of this invention remains basically stable in the absence of HOCl, while obvious fluorescence quenching can still be observed after the addition of HOCl. This indicates that the probe has good photostability under weakly acidic to neutral conditions and can reliably respond to HOCl, meeting the application requirements of most physiological environments.

[0029] Example 4: Reactive oxygen species response test of the hypochlorous acid fluorescent probe obtained in Example 1 of the present invention. The test method is as follows: (1) Preparation of ROS stock solution: Prepare 20mM of H2O2, TBHP, NO, FeSO4 and ROO − Mother liquor; prepare 10mM O2 − Mother liquor; prepare 1mM ONOO − And HOCl mother liquor. • OH and t-BuOO − The FeSO4 mother liquor was prepared by mixing H2O2 and TBHP at a 1:1 ratio and used immediately.

[0030] (2) Preparation of probe stock solution: Weigh the hypochlorous acid fluorescent probe obtained in Example 1 and prepare a 5 mM stock solution with N',N-dimethylformamide (DMF). Store the stock solution in the dark and refrigerated for later use.

[0031] (3) Preparation of test solution: Prepare a test solution with a probe concentration of 5 μM. See Table 3 for specific preparation methods. Table 3: The results are as follows Figure 7 As shown, the test results indicate that even at a high concentration of reactive oxygen species (50 μM), no significant fluorescence change was observed in the probe at the detection wavelength; only the presence of HOCl resulted in a noticeable decrease in fluorescence intensity. This demonstrates that the probe exhibits a highly specific response to HOCl.

[0032] Example 5: Cytotoxicity experiment of the hypochlorous acid fluorescent probe obtained in Example 1 of the present invention. The specific test method is as follows: Different concentrations of probe (0-150 μM) were co-incubated with ATDC5 cells for 24 h to observe the toxicity of the probe to the cells.

[0033] The results are as follows Figure 8 As shown, the test results indicate that even at relatively high concentrations (≤100 μM), the cell viability of the probe-treated group remained above 80%, indicating that the probe has almost no significant toxicity to ATDC5 cells near the working concentration and has good biocompatibility and application safety.

[0034] Example 6: Cell imaging experiment of the hypochlorous acid fluorescent probe obtained in Example 1 of the present invention. The specific test method is as follows: ATDC5 cells (5 × 10) 8 Cells were seeded at 14 mm² / mL onto 14 mm glass-bottomed culture dishes and allowed to adhere for 12 hours, then fixed with 4% paraformaldehyde (1.0 mL per dish) for 30 minutes. A stock solution of the plasmid prodrug (10 mM, dissolved in N,N-dimethylformamide (DMF)) was diluted to a final concentration of 5 μM with phosphate-buffered saline (PBS, pH 7.4). Cells were then incubated at 37°C for 1 hour, washed twice with PBS, and then imaged.

[0035] The results are as follows Figure 9 As shown, the test results indicate that after incubation with the probe, fixed ATDC5 cells exhibited uniform red / near-infrared emission in the corresponding channels, indicating good cell staining and preservation of emission characteristics. Subsequent treatment with HOCl (50 μM) significantly reduced cell emission, consistent with the shut-off response observed in solution. These results demonstrate that the probe can detect HOCl in the cellular environment through a significant decrease in emission intensity.

[0036] Example 7: In vivo imaging experiment of the hypochlorous acid fluorescent probe obtained in Example 1 of the present invention. The specific test method is as follows: A λ-carrageenan-induced arthritis model was established in 4-5 week old female BALB / c mice. Arthritis was induced by intra-articular injection of 25 μL of λ-carrageenan (5 mg / mL, aqueous solution) into the right joint cavity, while an equal volume of pure water was injected into the left joint as a control. Four hours after injection, an equal volume of hypochlorous acid fluorescent probe solution (50 μL, 440 μM) was injected into both joints, and in vivo fluorescence imaging was performed. Fluorescence was recorded in the red channel, where λex = 540 nm and λem = 710 nm.

[0037] The results are as follows Figure 10As shown, the test results indicate that a strong red / NIR signal was observed in the control group (water injection), suggesting that the probe maintained its emission in normal tissue. In contrast, the inflamed joints showed a significant signal drop approximately 5 minutes after probe injection and remained at a low level during the imaging window, consistent with the elevated HOCl levels in the inflammatory microenvironment leading to probe quenching.

[0038] The above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.

Claims

1. A hypochlorous acid fluorescent probe with a large Stokes shift, characterized in that, The hypochlorous acid fluorescent probe has the structure shown in Formula I: Formula I This hypochlorous acid fluorescent probe is a cationic iridium(III) complex and contains anionic inhibitors; The anti-anion is selected from one or more of PF6⁻, BF4⁻, Cl⁻, ClO4⁻, OTf⁻ or BPh4⁻.

2. The hypochlorous acid fluorescent probe with a large Stokes shift according to claim 1, characterized in that, The Stokes shift of this hypochlorite fluorescent probe is ≥170 nm.

3. A method for preparing a hypochlorous acid fluorescent probe with a large Stokes shift according to any one of claims 1-2, characterized in that, The following synthetic routes are included: S1: Under an inert atmosphere, tetra(triphenylphosphine)palladium catalyst was added to a reaction system containing 6-chlorophenanthridine, benzo[b]thiophene-2-boronic acid, solvent and base. The reaction was heated. After the reaction was completed, the mixture was extracted with organic solvent, dried and concentrated, and purified by silica gel column chromatography to obtain ligand intermediate A. S2: Add IrCl3·xH2O and ligand intermediate A obtained in step S1 into a mixed solvent and heat to reflux. After the reaction is completed and cooled, filter to obtain the precipitate, wash with water and petroleum ether in sequence, and dry under vacuum to obtain chlorinated bridged iridium intermediate B; the obtained intermediate does not need to be purified and can be used directly in the next step of the reaction. S3: The chlorobridged iridium intermediate B obtained in step S2 and 4-carboxy-4′-methyl-2,2′-bipyridine were added to a reaction vessel, a solvent was added, and the mixture was heated under reflux in an inert atmosphere. Subsequently, an anion exchange was carried out by adding a salt containing an anion inhibitor, and the mixture was stirred at room temperature to obtain compound I.

4. The method for preparing a hypochlorous acid fluorescent probe with a large Stokes shift according to claim 3, characterized in that, In step S1, the equivalent ratio of tetra(triphenylphosphine)palladium to 6-chlorophenanthridine is 1:(20~50); the solvent is selected from one or more of dioxane, tetrahydrofuran, toluene, dimethylformamide, and ethanol / water mixed solvent; the base is selected from one or more of potassium carbonate, cesium carbonate, potassium phosphate, and sodium tert-butoxide; and the extraction organic solvent is selected from one or more of dichloromethane, ethyl acetate, and toluene.

5. The method for preparing a hypochlorous acid fluorescent probe with a large Stokes shift according to claim 3, characterized in that, In step S2, the molar ratio of IrCl3·xH2O to the ligand intermediate A obtained in step S1 is 1:(1~10); the mixed solvent is selected from ethylene glycol ether / water system or alcohol / water system.

6. The method for preparing a hypochlorous acid fluorescent probe with a large Stokes shift according to claim 3, characterized in that, In step S3, the molar ratio of chlorobridged iridium intermediate B to 4-carboxy-4′-methyl-2,2′-bipyridine is 1:(1~10); the solvent is selected from one or more of dichloromethane, chloroform, methanol, ethanol, and acetonitrile; the salt containing the anti-anion is selected from one or more of ammonium hexafluorophosphate, ammonium tetrafluoroborate, potassium hexafluorophosphate, and tetraphenylborate.

7. The application of the hypochlorous acid fluorescent probe with a large Stokes shift as described in claim 1 in the selective detection of hypochlorous acid.

8. The application according to claim 7, characterized in that, The detection mechanism of this hypochlorous acid fluorescent probe is as follows: When a compound with the structure of Formula I reacts with HOCl, the sulfur atom in the structure of the Formula I compound is oxidized to sulfoxide, causing a detectable change in its fluorescence signal, which can then be used to achieve the qualitative or quantitative detection of HOCl.

9. The application of the hypochlorous acid fluorescent probe with a large Stokes shift as described in claim 1 in the in-situ detection and imaging analysis of HOCl in an inflammatory disease model.

10. The application according to claim 9, characterized in that, The inflammatory disease model includes an inflammatory joint disease-related model, which is a λ-carrageenan-induced mouse arthritis model.

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