A conjugated extended bodipy near-infrared hypochlorite fluorescent probe and a preparation method and application thereof

Abstract

This invention discloses a conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe, its preparation method, and its application, belonging to the field of fluorescent probe technology. The molecular formula of the probe molecule is: C 41 H 42 The BF2N5O2S probe has the following structural formula: [Insert structural formula here]. The probe uses N-ethylphenothiazine as the recognition unit, and morpholinamide modification enhances its water solubility. Fluorescence is quenched by TICT charge transfer; HClO specifically oxidizes the phenothiazine thioether to block the ICT effect, resulting in significant enhancement of near-infrared fluorescence at 610 nm. The probe can achieve quantitative detection in water and soil samples, and when used with fluorescent test strips and chitosan hydrogels, it enables on-site naked-eye visual screening. It also possesses lysosomal targeting capabilities and has been successfully applied to HepG2 live cell and zebrafish live fluorescence imaging. This invention features a simple synthetic route, excellent biocompatibility, and near-infrared emission that avoids biological background interference. A single probe system covers environmental monitoring and live biological imaging, showing broad application prospects in pollution control and biological pathology research.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe, its preparation method, and its application. Background Technology

[0002] Hypochlorous acid (HClO) is a key reactive oxygen species (ROS) in living organisms, widely involved in physiological and pathological processes such as human immunity, inflammation, and apoptosis. Simultaneously, as a strong oxidant, HClO is extensively used in drinking water disinfection, food processing, textile bleaching, and industrial circulating water treatment. Excessive residual HClO in water and soil can damage ecosystems, induce oxidative damage in organisms, and long-term exposure can lead to arthritis, kidney disease, cardiovascular disease, and even cancer. Intracellular HClO imbalance can cause oxidative stress in organelles and cell necrosis. Therefore, developing sensing materials that can simultaneously and accurately detect HClO in both environmental samples and living organisms is of significant practical importance.

[0003] Current methods for HClO detection mainly fall into four categories: liquid chromatography-mass spectrometry (LC-MS), electrochemistry, spectrophotometry, and conventional fluorescent probes. LC-MS equipment is expensive, the operation process is complex, and rapid on-site detection is not possible; electrochemical detection has poor reproducibility and is easily affected by coexisting ions; traditional spectrophotometry involves cumbersome sample pretreatment and has relatively high detection limits. Fluorescent probes, with their advantages of high sensitivity, in-situ non-destructive imaging, ease of operation, and low cost, have become the mainstream detection solution.

[0004] Currently reported HClO fluorescent probe recognition units include amides, aryl dihydrazides, borate esters, selenyl groups, oximes, and phenol derivatives. Most of them suffer from defects such as small Stokes shift, emission wavelength concentrated in the visible light region, insufficient photostability, poor water solubility, and weak penetration into biological tissues. Visible light probes are easily affected by the autofluorescence of biological matrices, have low signal-to-noise ratios in live imaging, and most probes can only be used for water sample detection, making them unsuitable for simultaneous detection in multiple scenarios such as soil, cells, and live model organisms.

[0005] The BODIPY (fluoroboron dipyrrole methylene) fluorescent backbone possesses advantages such as high molar absorptivity, excellent fluorescence quantum yield, good photostability, easy molecular modification, and tunable spectrum, making it a classic matrix for constructing high-performance fluorescent probes. Through π-conjugation extension, BODIPY emission can be redshifted to the near-infrared band, significantly reducing biological background fluorescence interference and improving deep tissue imaging capabilities. However, existing BODIPY-based HClO probes generally lack organelle targeting, have insufficient water solubility, and lack supporting application solutions with solid-state visualization sensing carriers (test strips, hydrogels), limiting their integrated environmental and biological detection performance. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by using a conjugated extended BODIPY as a fluorescent backbone and introducing N-ethylphenothiazine as a specific recognition group for HClO. Morpholinamide side chains improve water solubility and lysosomal targeting, constructing a near-infrared activated fluorescent probe BDP-FL. Based on the intramolecular charge transfer (ICT) quenching mechanism, it achieves high specificity recognition of HClO, and can be applied in multiple dimensions such as water samples, soil, test strips, chitosan hydrogel solid-phase sensing, live cells, and zebrafish, making up for the shortcomings of traditional probes in terms of single application scenarios and poor biological imaging effects.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe, the molecular formula of which is: C 41 H 42 BF2N5O2S, structural formula: In this article, BDP-FL refers to the conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe.

[0008] The fluorescence emission mechanism of the fluorescent probe designed in this invention is as follows: .

[0009] A method for preparing a conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe includes the following steps: (1) Dissolve 1 mmol of pyrrole, 1 mmol of methyl 4-(chloroformyl)benzoate, 1 mmol of triethylamine, and 2 mmol of boron trifluoride diethyl ether in 1 mL of dry dichloromethane. Reflux the solution at 40 °C under nitrogen atmosphere. Monitor the reaction progress by TLC to see if any new compounds are formed. After the reaction is complete, dilute the mixture with 15 mL of CH2Cl2, wash the organic phase with 15 mL of water, dry, filter, and concentrate. Purify by silica gel column chromatography. Dry under high vacuum to obtain a deep red solid, BODIPY. Further hydrolysis of the carboxylic acid ester yields compound 1. The structural formula of compound 1 is: ; (2) 1 mmol of compound 1 and 1.5 mmol of 4-(2-aminoethyl)morpholine were dissolved in anhydrous dichloromethane and underwent an amide condensation reaction at room temperature in the presence of EDCI. After the reaction was completed as monitored by TLC, the organic phase was washed and concentrated. The crude product was purified by column chromatography to obtain the target amide product compound 2. The structural formula of compound 2 is as follows:

[0010] (3) Dissolve 1 mmol of the above compound 2 and 1.2 mmol of 4-ethyl-2-vinylphenthiazide in toluene, add 0.1 mmol of p-toluenesulfonic acid as a catalyst, reflux at 110 °C, remove the water generated by the reaction using a Dean-Stark water separator to drive the condensation; monitor the reaction endpoint by TLC, cool to room temperature, wash the organic phase with water and saturated brine, dry with anhydrous sodium sulfate, and evaporate the crude product under reduced pressure; purify by silica gel column chromatography to obtain the final product probe.

[0011] The synthetic route for the fluorescent probe is as follows:

[0012] An application of a conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe, wherein the probe is used for sensing and monitoring HClO content in aquatic and soil environments.

[0013] Furthermore, the sensing detection includes one or more of fluorescence detection, visual detection, and confocal imaging detection.

[0014] Beneficial effects: (1) The probe can freely penetrate the cell membrane. There is no obvious apoptosis or biological malformation during the incubation of cells and zebrafish. It is suitable for long-term in vivo fluorescence tracing. The molecular structure is easy to modify and can be expanded to other target groups, with a large application expansion space. It has a built-in morpholine target group and is specifically enriched in cell lysosomes to achieve precise detection at the organelle level. (2) It has no obvious fluorescence response to common metal ions, reactive oxygen species, amino acids and inorganic anions, and the coexisting interfering substances do not affect the probe's recognition of HClO; it has a stable working pH range of 4.0~10.0, covering natural water bodies, soil leachate and physiological body fluid environments; the fluorescence signal reaches equilibrium after 30 min, and the spectrum does not drift after long-term storage. (3) The fluorescence intensity in the concentration range of 0~120 μM showed a good linear relationship with the HClO concentration, with a correlation coefficient R²=0.986; the detection limit calculated by the 3σ / k method was as low as 0.46 μM, which meets the requirements for quantitative detection of trace residues; (4) A single probe can realize quantitative analysis of liquid water samples, solid phase extraction and detection of soil, rapid on-site screening with test strips, long-term adsorption monitoring of hydrogels, and fluorescence imaging of live cells and live animals. One system covers two major fields: environmental monitoring and biomedical research. (5) Under sunlight, the solution changes from light brown to reddish-brown as the HClO concentration increases; under 365 nm handheld UV light irradiation, it exhibits an orange-yellow near-infrared fluorescence gradient, allowing for on-site qualitative screening without the need for large instruments; the water sample spike recovery rate is 90%~100%, RSD < 3%, and the detection results are accurate and reliable. Therefore, this invention is a simple, rapid, and sensitive HClO molecule-specific detection reagent with broad application prospects in the field of biomolecular detection. Attached Figure Description

[0015] Figure 1 This is the proton spectrum of the probe BDP-FL of this invention; Figure 2 The carbon spectrum of the probe BDP-FL of this invention; Figure 3 The images show the UV absorption and fluorescence emission spectra of the probe BDP-FL in response to HClO, where (a) is the UV absorption spectrum of the probe BDP-FL solution (20 μg / mL), and the inset is a photograph of the probe solution under natural light; (b) is the fluorescence spectrum of the probe BDP-FL aqueous solution at 610 nm dependent on HClO concentration, and the inset is a photograph of the fluorescence color change of the solution under 365 nm UV light; (c) is the fluorescence spectrum of BDP-FL (10 μM) with different concentrations of HClO (0-120 μM); and (d) is the linear relationship between fluorescence intensity at 610 nm and HClO concentration (0-120 μM). Figure 4 The stability of the probe BDP-FL in response to HClO and the response to HClO under different pH conditions are shown in the following figures: (a) the fluorescence emission intensity changes of probe BDP-FL (20 μg / mL) with 80 μM HClO aqueous solution and BDP-FL (20 μg / mL) itself in PBS buffer solutions at different pH values; (b) the response time of probe BDP-FL (20 μg / mL) at 610 nm when 100 μM HClO aqueous solution is added to PBS buffer (pH=7.2) and the response time at 610 nm when no HClO aqueous solution is added. Figure 5 The selective spectral data of the probe BDP-FL solution of the present invention and the changes in fluorescence intensity before and after the addition of HClO are shown in the figure. (a) Fluorescence changes of probe BDP-FL (20 μg / mL) in the presence of various analytes (80 μM) and HClO (80 μM). (b) Fluorescence emission intensity changes of BDP-FL (20 μg / mL) in the presence of various analytes (80 μM) and HClO (80 μM). Figure 6The following are experimental results of the probe BDP-FL of this invention applied to the detection of HClO in different water samples: (a) Fluorescence intensity changes of probe BDP-FL (20 μg / mL) with different concentrations of HClO (100 μM) added to different water samples; (b) Analysis results of BDP-FL (20 μg / mL) with HClO (100 μM) in different water samples. Figure 7 The following are experimental images of the BDP-FL probe of this invention applied to the detection of HClO in different soils: (a) soil sample preparation process; (b) image of soil sample extract under a 365 nm UV lamp; (c) images of soil samples infiltrated with HClO (100 μM) taken under a fluorescent lamp and images of soil samples infiltrated with HClO (100 μM) taken under a UV lamp. Figure 8 The following are experimental results of the application of the probe BDP-FL of this invention to the adsorption detection of HClO solution by hydrogel: (a) Schematic diagram of the preparation process of BDP-FL functionalized chitosan fluorescent hydrogel for visual detection of hypochlorous acid; (b) Photographs of hydrogels treated with gradient concentrations of hypochlorous acid (0–120 μmol / L) under natural light and ultraviolet light; (c) The change of fluorescence intensity of hydrogel with soaking time during immersion in low concentration hypochlorous acid solution. Figure 9 The figures show the experimental results of applying the probe BDP-FL of this invention to the detection of HClO solution in cells and zebrafish. (A) is the fluorescence imaging of zebrafish. Zebrafish were pretreated with different concentrations of HClO for 30 minutes, then treated with BDP-FL (10 μM) for 15 minutes. (a1-b1) are bright-field images of zebrafish, (a2-b2) are green channel fluorescence images of zebrafish, and (a3-c3) are superimposed images of the bright-field and green channels. Scale bar: 100 μm. (B) is the fluorescence imaging of HepG2 cells. HepG2 cells were pretreated with different concentrations of HClO for 30 minutes, then treated with BDP-FL (10 μM) for 30 minutes. (a1 and c1) are bright-field images of HepG2 cells, (a2 and c2) are green channel fluorescence images of HepG2 cells, and (a3 and c3) are superimposed images of the bright-field and green channels. Scale bar: 10 μm. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0017] Example 1 Probe synthesis: 1) Dissolve 1 mmol of pyrrole, 1 mmol of methyl 4-(chloroformyl)benzoate, 1 mmol of triethylamine, and 2 mmol of boron trifluoride diethyl ether in 1 mL of dry dichloromethane. Reflux the solution at 40 °C under nitrogen atmosphere. Monitor the reaction progress by TLC to observe the formation of new compounds. After the reaction is complete, dilute the mixture with 15 mL of CH2Cl2, wash the organic phase with 15 mL of water, dry, filter, and concentrate. Purify by silica gel column chromatography. Dry under high vacuum to obtain a deep red solid, BODIPY. Further hydrolysis of the carboxylic acid ester yields compound 1. 1 H NMR (400 MHz, DMSO-d6) δ 8.15 –8.10 (m, 2H), 7.60 – 7.54 (m, 2H), 6.20 (s, 2H), 3.90 (d, J = 1.1 Hz, 3H), 2.46 (s, 6H), 1.32 (s, 6H). 2) 1 mmol of compound 1 and 1.5 mmol of 4-(2-aminoethyl)morpholine were dissolved in anhydrous dichloromethane and underwent an amide condensation reaction at room temperature under EDCI conditions. After the reaction was monitored by TLC until complete, the organic phase was washed and concentrated. The crude product was purified by column chromatography to obtain the target amide product, compound 2. 1 H NMR (400 MHz, DMSO-D6) δ 8.59 (s,1H), 8.01 (d, J = 7.5, 4.4, 2.2 Hz, 2H), 7.52 – 7.37 (m, 2H), 6.08 (s, 1H),6.04 (s, 1H), 3.59 (q, J = 4.7 Hz, 6H), 3.32 (d, J = 2.2 Hz, 6H), 2.45 (d, J= 15.2 Hz, 9H), 0.98 – 0.83 (m, 3H).

[0018] 3) Dissolve 1 mol of compound 2 and 1.2 mol of 4-ethyl-2-vinylphenthiazide in toluene, add 0.1 mmol of p-toluenesulfonic acid as a catalyst, reflux at 110 °C, remove the water generated by the reaction using a Dean-Stark water separator to drive the condensation; monitor the reaction endpoint by TLC, cool to room temperature, wash the organic phase with water and saturated brine, dry with anhydrous sodium sulfate, and evaporate the crude product under reduced pressure; purify by silica gel column chromatography to obtain the blue solid probe BDP-FL, with a total yield of 47%. Figure 1-2As shown, ¹H NMR (400 MHz, DMSO-D6) δ 8.62 (s, 1H), 8.04–7.97 (m, 2H), 7.48 (s, 3H), 7.24–7.18 (m, 3H), 7.15 (d, J = 7.7, 1.5 Hz, 2H), 7.07–7.03 (m, 3H), 6.96 (t, J = 7.4 Hz, 2H), 4.03–3.86 (m, 4H), 3.58 (d, J = 4.8 Hz, 4H), 2.43 (s, 6H), 1.35–1.28 (m, 12H). 13 C NMR (151 MHz, DMSO-D6) δ 165.59, 151.11, 144.17, 143.65,142.74, 139.60, 138.21, 136.07, 134.40, 131.19, 129.28, 127.76, 127.64,127.03, 126.83, 124.95, 123.14, 122.63, 122.20, 118.62, 115.51, 112.45,66.21, 57.43, 53.35, 41.31, 36.65, 23.40, 19.38, 14.59, 13.78, 12.60. HRMS(ESI) m / z calcd for C 41 H 42 BF2N5O2S [M]:668.3120; found 717.3857.

[0019] Example 2 UV absorption and fluorescence emission spectra of probe BDP-FL in response to HClO Weigh the BDP-FL prepared in Example 1 and dissolve it in acetonitrile to prepare a 1 mg / mL stock solution. Take 20 μL of the stock solution and add it to 2 mL of PBS buffer (pH=7.2). Add different volumes of HClO aqueous solution to prepare HClO aqueous solutions with different concentration gradients (0-120 μM). Test the UV absorption and fluorescence emission properties of the probe BDP-FL. Figure 3 As shown in Figure a, after the addition of HClO, the blank probe showed a characteristic BODIPY absorption peak at 580 nm. As the concentration of HClO increased, the absorption at 580 nm continued to decrease, and a new absorption band appeared at 300~350 nm. Figure 3c shows that with increasing HClO concentration, the fluorescence emission of the probe BDP-FL solution at 610 nm continuously increases with a good linear relationship. Under UV light, the color of the probe BDP-FL solution changes from colorless to orange-yellow, as... Figure 3 As shown in e.

[0020] Example 3 The stability of the probe BDP-FL in response to HClO and its response to HClO under different pH conditions; Take 20 μL of the fluorescent probe stock solution from Implementation Case 2 and add it to 2 mL of PBS buffer solution (containing) at different pH values. One group serves as a control group without HClO aqueous solution, while the other group is treated with 100 μM HClO solution. Test the probe's response to HClO under different pH conditions at room temperature. Figure 4 As shown in Figure a, the mixture of probe BDP-FL and HClO exhibits good fluorescence emission across an acidic to alkaline range. 20 μL of the fluorescent probe stock solution from Example 2 was added to 2 mL of PBS buffer (pH=7.2), followed by the addition of 100 μM HClO aqueous solution. The time-dependent spectrum of the probe was then measured at room temperature. Figure 4 As shown in b, the maximum fluorescence emission of the probe tends to stabilize within 30 minutes.

[0021] Example 4 Selectivity spectral data of probe BDP-FL solution and changes in fluorescence intensity before and after the addition of HClO Take 20 μL of the fluorescent probe stock solution from Example 2 and add it to 2 mL of PBS buffer solution (pH = 7.2). Add an equal volume of 10 mM competing substance to each solution. For the latter, add 100 μM HClO aqueous solution and then detect the change in the fluorescence emission spectrum of the solution. Figure 5 As shown, the fluorescence intensity of the probe BDP-FL at 610 nm significantly increased after the addition of HClO, while the fluorescence intensity of the probe BDP-FL at 610 nm remained almost unchanged after the addition of other interfering substances; however, when HClO and these interfering substances were present simultaneously, the fluorescence intensity of the probe BDP-FL at 610 nm significantly increased. This indicates that BDP-FL has good selectivity for HClO.

[0022] Example 5 The probe BDP-FL was used to detect HClO in different water samples; PBS was prepared from three types of surface water: lake water, river water, and tap water. Subsequently, 0, 30, 60, and 90 μM HClO were added to the water samples, and the HClO concentration was determined using the standard addition method. After reacting the BDP-FL solution with the sample for 5 min, fluorescence images were captured and analyzed using a smartphone under a handheld UV lamp (365 nm). The accuracy of the determination was determined by the recovery rate, which is the ratio of the amount of HClO calculated based on the test results to the amount of added HClO. Experimental results are as follows: Figure 6 As shown, the recovery rate ranges from 90% to 100%, and the relative standard deviation (RSD) is less than 3%, indicating that the test results are reliable.

[0023] Example 6 The probe BDP-FL was used to detect HClO in different soils. 20 μL of the fluorescent probe stock solution from Case 2 was added to 2 mL solutions of different aqueous fractions for fluorescence measurement. The experimental procedure and results are as follows: Figure 7 As shown, soil samples were first placed in petri dishes and divided into two groups: one group was directly mixed with BDP-FL solution, and the other group was first sprayed with HClO solution before being mixed with BDP-FL solution. The soil samples from each group were then extracted using acetonitrile:PBS (1:1, v:v, pH 7.2). The supernatant was then collected by centrifugation and placed under a 365 nm UV lamp. Under 365 nm UV illumination, the HClO-rich soil extract exhibited significant green fluorescence. In contrast, only a weak fluorescence of the probe itself was observed in the HClO-free soil extract. Furthermore, the soil sample sprayed with HClO solution did not produce fluorescence under either visible or ultraviolet light. This demonstrates the ability of BDP-FL to identify HClO in soil sample detection.

[0024] Example 7 The probe BDP-FL was used to detect the adsorption of HClO solution onto hydrogels. Weigh 1.5 g of chitosan powder with a degree of deacetylation ≥85%, add 80 mL of 1.5% acetic acid aqueous solution (prepared from 1.25 mL of glacial acetic acid and deionized water), and magnetically stir at 200 r / min for 4 h until the powder is completely dissolved, obtaining a clear and transparent solution. Then, add approximately 2 mL of 0.1 mol / L sodium hydroxide solution and 10 mL of probe stock solution dropwise to adjust the pH of the system to 5.0–5.5. Separately, take 0.6 mL of 50% glutaraldehyde stock solution and dilute to 10 mL with deionized water to prepare the crosslinking agent. Maintain the system at a constant temperature of 30 ℃ and a stirring rate of 200 r / min. Slowly add the diluted glutaraldehyde crosslinking agent dropwise to the chitosan solution at a rate of 1 drop every 2 seconds. After the addition is complete, continue stirring for 30 min. Remove the stir bar, transfer the mixture to a petri dish, and let it stand at room temperature for 6 h to obtain a translucent, pale yellow, elastic, non-flowing chitosan hydrogel. The hydrogel was cut into 1 cm × 1 cm pieces and washed three times with deionized water for 1 hour each time to remove residual acetic acid and free glutaraldehyde. A 0.03 mol / L copper nitrate solution (pH 6.0–6.5) was prepared and used to completely submerge the hydrogel pieces at a solid-liquid ratio of 1:10. The mixture was shaken at 35 °C and 150 r / min for 4 hours to allow copper ions to coordinate with the chitosan functional groups. Finally, the hydrogel was repeatedly rinsed with deionized water; EDTA titration was used to confirm the absence of copper ion residue in the washing solution, indicating that the copper ion loading modification was complete. The experimental procedure and results are as follows. Figure 8 As shown, when the hypochlorous acid concentration increases from 0 to 120 μM, the hydrogel gradually changes from light brown to reddish-brown under natural light; under ultraviolet irradiation, the orange-yellow fluorescence increases with the concentration gradient, enabling semi-quantitative identification by the naked eye. The fluorescence intensity continues to rise within 4 hours of immersion, and the fluorescence color development gradually becomes more obvious, proving that this hydrogel sensor can be used for time-series visual monitoring of hypochlorous acid in the aquatic environment.

[0025] Example 8 The probe BDP-FL was used to detect HClO solution in cells and zebrafish. The ability of a probe to detect HClO in vivo was evaluated by using BDP-FL imaging in zebrafish. Imaging results are shown below. Figure 9 As shown in Figure A, a represents zebrafish stained only with the probe solution, while b and c represent zebrafish treated with the probe and then incubated with HClO. Zebrafish stained only with the BDP-FL probe did not show any fluorescence signal under a fluorescence field. However, zebrafish incubated with the BDP-FL probe solution for 30 minutes and then treated with HClO solution for 15 minutes exhibited an orange-yellow fluorescence signal under a fluorescence field. These results demonstrate that the BDP-FL probe prepared in this study can be effectively applied to in vivo biological detection experiments.

[0026] We stained HepG2 cells with only the probe BDP-FL solution and found no fluorescence signal. Then, we added solutions of different concentrations of HClO to the cells already containing the probe solution and incubated them for 30 minutes, resulting in the observation of a distinct orange-yellow fluorescence signal in the cells. The results are as follows... Figure 9 B indicates that BDP-FL can penetrate cells and interact with HClO inside the cell, enabling the detection of the biological value of HClO in living cells.

[0027] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe, characterized in that, The molecular formula of the probe molecule is: C 41 H 42 BF2N5O2S, structural formula: .

2. A method for preparing the conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe according to claim 1, characterized in that, Includes the following steps: (1) Dissolve 1 mmol of pyrrole, 1 mmol of methyl 4-(chloroformyl)benzoate, 1 mmol of triethylamine, and 2 mmol of boron trifluoride diethyl ether in 1 mL of dry dichloromethane. Reflux the solution at 40 °C under nitrogen atmosphere. Monitor the reaction progress by TLC to see if any new compounds are formed. After the reaction is complete, dilute the mixture with 15 mL of CH2Cl2, wash the organic phase with 15 mL of water, dry, filter, and concentrate. Purify by silica gel column chromatography. After high vacuum drying, obtain a deep red solid BODIPY. Further hydrolysis of the carboxylic acid ester yields compound 1. The structural formula of compound 1 is: ; (2) 1 mmol of compound 1 and 1.5 mmol of 4-(2-aminoethyl)morpholine were dissolved in anhydrous dichloromethane and underwent an amide condensation reaction at room temperature in the presence of EDCI. After the reaction was completed as monitored by TLC, the organic phase was washed and concentrated. The crude product was purified by column chromatography to obtain the target amide product compound 2. The structural formula of compound 2 is as follows: ; (3) Dissolve 1 mmol of compound 2 and 1.2 mmol of 4-ethyl-2-vinylphenthiazide in toluene, add 0.1 mmol of p-toluenesulfonic acid as a catalyst, reflux at 110 °C, remove the water generated by the reaction using a Dean-Stark water separator to drive the condensation; monitor the reaction endpoint by TLC, cool to room temperature, wash the organic phase with water and saturated brine, dry with anhydrous sodium sulfate, and evaporate the crude product under reduced pressure; purify by silica gel column chromatography to obtain the final product probe.

3. An application of the conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe according to claim 1, characterized in that, The probe is used for sensing and monitoring the HClO content in aquatic and soil environments.

4. The application of the conjugated extended BODIPY near-infrared hypochlorous acid fluorescent probe according to claim 3, characterized in that, The sensing detection includes one or more of fluorescence detection, visual detection, and confocal imaging detection.

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