A fluorescent probe for real-time imaging of mitochondrial h2s which can be photoactivated on demand and preparation and application thereof

Abstract

This invention belongs to the field of biochemistry, specifically disclosing a novel photoactivated fluorescent probe for real-time high-fidelity imaging of mitochondrial H2S, its preparation, and its application. The organic fluorescent probe has the structure shown in Formula I. The probe designed in this invention has excellent water solubility, exhibits yellow fluorescence, and can monitor its entry into the mitochondria in real time. It shows no fluorescence response to H2S during cell transfer, thus providing accurate information on the accumulation time in the mitochondria. Subsequently, the probe can be photoactivated on demand at specific mitochondrial locations, resulting in significant aggregation enhancement and emission responses to H2S, and activating near-infrared fluorescence emission. This invention has considerable application prospects in fluorescence imaging in the fields of biochemistry and medicine, and its synthesis method is simple and relatively inexpensive. It is hoped that this invention can promote research progress on mitochondrial H2S fluctuations in various physiological and pathological processes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochemistry, and relates to a fluorescent probe and its synthesis and application, which can be used for real-time monitoring of the process of the probe itself into mitochondria, and through the time and space control of the reaction activity of the probe to realize the high-fidelity imaging of mitochondrial H2S. Specifically, the present application relates to a synthesis method of an organic fluorescent probe and a cell imaging application. BACKGROUND

[0002] Mitochondria is a very important organelle, which exists in most eukaryotic cells, and it can provide energy for cells and participate in many other physiological processes such as signal transmission, cell differentiation, growth and apoptosis. Various signal molecules are involved in the complex physiological processes of mitochondria, including metal ions and active oxygen / nitrogen / sulfur species. In order to understand the role of these active species in mitochondria, scientists have developed many fluorescent probes that can target mitochondria. Although these probes will eventually be enriched in mitochondria and show bright fluorescence, the analytes in the cytosol may also react with these probes before entering the mitochondria, which may lead to false studies on the biological functions of mitochondrial analytes. Although there are a few examples that intend to solve these problems by the "accumulation first and activation later" strategy, real-time monitoring of the process of the probe into the mitochondria is still a challenge. Because the nature of the analytes in the mitochondria is changing, real-time monitoring of the process of the probe into the mitochondria cannot determine the best time to activate the probe, which will affect the specificity and accuracy of the probe.

[0003] Hydrogen sulfide (H2S) is an indispensable gas transmitter in the body, which is involved in many biological processes. In particular, the physiological and pathological functions of mitochondria are closely related to the content of H2S therein. H2S in mitochondria can prevent damage caused by oxidative stress and also provide additional energy for cells. There is evidence that abnormal production of H2S in mitochondria will lead to mitochondrial dysfunction. Therefore, it is very meaningful to real-time monitor mitochondrial H2S in living cells to understand the physiological and pathological consequences it may trigger. For many years, people have developed many fluorescent probes that can detect H2S, however, most of the existing probes do not have specific reactions to mitochondrial H2S, so they cannot specifically detect H2S in mitochondria without the interference of H2S outside the mitochondria.

[0004] This invention proposes an on-demand photoactivation method for molecularly designed probes to identify mitochondrial H2S fluctuations in real time with high fidelity. The probe's entry into the mitochondria can be monitored in real time based on its yellow fluorescence, providing an accurate timeline for H2S accumulation. The probe does not fluoresce in response to H2S during cell transfer; only subsequent photoactivation at the desired mitochondrial location elicits a significant aggregation enhancement and emission response, illuminating with red fluorescence at 690 nm. By using this photocontrolled fluorescence response to H2S, the probe successfully and accurately identifies mitochondrial H2S fluctuations. The novel on-demand photoactivatable fluorescent probe for real-time high-fidelity imaging of mitochondrial H2S provided by this invention can advance research on mitochondrial H2S in various physiological and pathological processes. Summary of the Invention

[0005] The first objective of this invention is to provide a novel fluorescent probe that can be photoactivated on demand for real-time high-fidelity imaging of mitochondrial H2S.

[0006] The second objective of this invention is to provide a method for preparing a novel fluorescent probe for real-time high-fidelity imaging of mitochondrial H2S that can be photoactivated on demand.

[0007] The third objective of this invention is to provide a novel fluorescent probe that can be photoactivated on demand for real-time high-fidelity imaging of mitochondrial H2S, using near-infrared fluorescence analysis, for the detection of endogenous hydrogen sulfide in human colon cancer HCT-116 cells in vitro.

[0008] The fourth objective of this invention is to provide a novel fluorescent probe for real-time high-fidelity imaging of mitochondrial H2S, which can be photoactivated on demand, using near-infrared fluorescence analysis, for the detection of mitochondrial H2S fluctuations under in vitro cellular inflammation and oxidative stress conditions.

[0009] Technical solution of the present invention:

[0010] A novel photoactivated fluorescent probe, BOD-PyNO, for real-time high-fidelity imaging of mitochondrial H2S, and its contrasting probe, BOD-PyNH, are shown in Formula I:

[0011]

[0012] The probe BOD-PyNO and its comparative probe BOD-PyNH described in this invention can be used in water content ratios (f w Tris:CH3CN buffer solution (f) greater than 70% w =70%, 80%, 90%; pH=7.4) self-assembled into nanoparticles (BOD-PyNO: f w =70%, 65±4nm; fw =80%, 83±5nm; f w =90%, 240±7nm; BOD-PyNH: f w =70%, 77±4nm; f w =80%, 107±5nm; f w =90%, 116±3nm), breaking the water solubility limitation of traditional hydrophobic organic probes.

[0013] The probe described in this invention can be illuminated (white light, 10mW / cm²). -2 (10 minutes) triggers the dissociation of nitric oxide, thereby generating its contrast probe BOD-PyNH.

[0014] The probe illumination (white light, 10mW / cm²) described in this invention -2 The probe's response to H2S can be controlled temporally and spatially (within 10 minutes) to achieve precise detection of H2S in mitochondria.

[0015] The probe described in this invention can monitor its entry into the mitochondria in real time using its own yellow fluorescence, thereby determining the optimal time for subsequent photoactivation.

[0016] This invention provides a novel fluorescent probe, BOD-PyNO, for real-time high-fidelity imaging of mitochondrial H2S that can be photoactivated on demand, and a method for preparing its contrasting probe, BOD-PyNH.

[0017]

[0018] The specific method is as follows:

[0019] Synthesis of Compound 2

[0020] 1 (purchased from a commercial company, no further purification required) was dissolved with di-tert-butyl dicarbonate in 1,4-dioxane and heated to 85°C. After the reaction was complete, the mixture was cooled to room temperature, extracted with CH2Cl2, washed with deionized water, and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation. The solid was then separated by column chromatography.

[0021] Synthesis of Compound 3

[0022] Under an argon atmosphere, sodium hydride and sodium hydride were dissolved in DMF in an ice bath and stirred for 30 minutes. Then, iodomethane was injected into the above system and the reaction was carried out at room temperature. After the reaction was completed, deionized water was added dropwise at 0°C to quench the reaction. The mixture was extracted with ethyl acetate, washed with saturated NaCl solution, dried with anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation. The solution was separated by column chromatography to obtain a pale yellow viscous liquid.

[0023] Synthesis of Compound 4

[0024] Dissolve 3 in ultra-dry dichloromethane, and slowly add trifluoroacetic acid dropwise to the system. React at room temperature. Monitor the reaction by TLC. After the reaction is complete, remove the solvent by vacuum distillation, dissolve in a small amount of dichloromethane, and repeat the above operation three times to obtain a brown oily sample.

[0025] Synthesis of Compound 5

[0026] Dissolve sodium nitrite in an ice bath in a mixed solvent of DCM:AcOH = 9:1. After stirring for 5 minutes, add sodium nitrite to the system and react at room temperature. After the reaction is complete, quench the AcOH with an appropriate amount of saturated NaHCO3 solution, extract with dichloromethane, wash with saturated NaCl solution, dry with anhydrous Na2SO4, filter, and remove the solvent by vacuum distillation to obtain a grayish-brown liquid.

[0027] Synthesis of Compound 6

[0028] Add an appropriate amount of iodomethane dissolved in acetonitrile to a 5% acetonitrile solution, and react at 60°C in the dark, monitored by TLC. After the reaction is complete, cool the reaction system to room temperature and recrystallize by adding diethyl ether. Filter the above system using a Buchner funnel, and wash the filter cake with a small amount of diethyl ether to obtain a milky white solid powder.

[0029] Synthesis of Compound 7

[0030] The synthesis of compound 7 is referenced in the patent: Zhao Chunchang, Zhang Xiuli, Zhang Lili, Wang Feiyi, Jiang Haifeng, An Jiancai, Zhang Fan, A fluorescent probe for detecting endogenous H2S and its preparation method and application, Chinese Patent No.: ZL 201410766204.6, 2015-03-11, Certificate No.: 2250092.

[0031] Synthesis of the fluorescent probe BOD-PyNO

[0032] Dissolve substances 6 and 7 in ethanol, add a small amount of acetic acid and ammonium acetate to the system, and react at 60°C in the dark, monitoring the reaction by TLC. After the reaction is complete, cool the system to room temperature, extract with dichloromethane, wash with saturated NaCl solution, dry with anhydrous Na2SO4, filter, remove the solvent by vacuum distillation, and separate by column chromatography to obtain a blackish-red solid, which is a novel fluorescent probe for real-time high-fidelity imaging of mitochondrial H2S that can be photoactivated on demand.

[0033] Synthesis of Compound 8

[0034] The synthesis steps are the same as those in step 6.

[0035] Synthesis of Compound 9

[0036] Dissolve 7 and 8 in 20 mL of toluene. Add a small amount of acetic acid and ammonium acetate to the system and react at 95 °C. Detect by TLC. After the reaction is complete, cool the system to room temperature, extract with dichloromethane, wash with saturated NaCl solution, dry with anhydrous Na₂SO₄, filter, remove the solvent by vacuum distillation, and separate by column chromatography to obtain a blackish-red solid.

[0037] Synthesis of the comparative probe BOD-PyNH

[0038] Trifluoroacetic acid was added to 15 mL of dichloromethane solution of 9, and the reaction was carried out at room temperature. The reaction was then detected by TLC. After the reaction was complete, the solvent was removed by vacuum distillation, and the solid obtained by column chromatography was a purplish-black solid, which is the novel fluorescent probe BOD-PyNO that can be photoactivated on demand for real-time high-fidelity imaging of mitochondrial H2S, and its contrast probe BOD-PyNH.

[0039] The present invention also provides the application of the novel fluorescent probe described above, which can be photoactivated on demand for real-time high-fidelity imaging of mitochondrial H2S, in detecting endogenous H2S in human colon cancer HCT-116 cells in vitro.

[0040] The present invention also provides an application of the novel fluorescent probe described above, which can be photoactivated on demand for real-time high-fidelity imaging of mitochondrial H2S, in monitoring H2S fluctuations in in vitro cellular inflammation.

[0041] The present invention also provides an application of the novel fluorescent probe described above, which can be photoactivated on demand for real-time high-fidelity imaging of mitochondrial H2S, in monitoring fluctuations of H2S under in vitro cellular oxidative stress conditions.

[0042] To accurately monitor mitochondrial H2S without interference from extramitochondrial H2S, this invention designs and synthesizes a probe with on-demand photoactivation for real-time, high-fidelity identification of mitochondrial H2S fluctuations. The probe's monochlorofluoroboron dipyrrole core serves as the H2S response unit, equipped with a photoionized nitric oxide 2-methyl-5-methylamino-N-methylpyridine cation as the mitochondrial targeting group. The probe molecule designed in this invention exhibits amphiphilic characteristics. This amphiphilic structure allows the probe to self-assemble into a highly water-soluble nanoprobe, thus overcoming the limitations of traditional lipophilic probes in terms of water solubility. Notably, the assembled probe shows poor reactivity to H2S and almost no fluorescence response under physiological conditions. In stark contrast, white light (10 mW / cm²) provides excellent fluorescence response. -2The introduction of H2S (within 10 minutes) triggers the release of nitric oxide, resulting in a significant increase in the probe's aggregation and luminescence response to H2S. Correspondingly, the probe maintains inactive fluorescence reactivity during transport to the mitochondria. Furthermore, the probe's own yellow fluorescence allows for real-time monitoring of its entry into the mitochondria, enabling precise determination of the probe's spatial location and the timing of light-induced nitric oxide removal. After light irradiation, the probe reacts in situ with H2S within the mitochondria, illuminating with red fluorescence at 690 nm, thus achieving independence from interference from external H2S. All these characteristics of the probe make it suitable for high-fidelity tracking of mitochondrial H2S in biological systems. By utilizing these photophysical characteristics of "light control" and "H2S triggering," the probe designed in this invention also achieves precise and controllable identification of mitochondrial H2S fluctuations under cellular inflammation and oxidative stress conditions, both temporally and spatially.

[0043] Beneficial effects

[0044] The fluorescent probe synthesis method described in this invention is simple and relatively inexpensive. Due to the subtle adjustment of its amphiphilic structure, it can self-assemble into nanoparticles (f) under physiological conditions. w =70%, 65±4nm; f w =80%, 83±5nm; f w =90%, 240±7nm), which solves the problems of poor water solubility and easy precipitation in physiological environment of most traditional probes.

[0045] The fluorescent probe BOD-PyNO provided by this invention exhibits a very weak reaction with H2S during cell delivery and shows no significant fluorescence response. However, upon entering the mitochondria, it can be induced by light to leave nitric oxide, and the resulting product, BOD-PyNH, restores its responsiveness to H2S, emitting bright red fluorescence at 690 nm upon H2S detection. This allows for temporal and spatial control of its H2S response activity through light irradiation, ensuring accurate detection of H2S within mitochondria.

[0046] The fluorescent probe BOD-PyNO described in this invention only produces bright red fluorescence when it first undergoes illumination to generate BOD-PyNH and then reacts with H2S to generate BOD-PyNH-SH. However, if it first undergoes a weak reaction with H2S and then is illuminated, it will not induce the departure of nitric oxide to produce red emission because the "-SH" bond in the BOD-PyNO-SH formed with H2S will be photo-oxidized, losing its ability to produce red fluorescence (see appendix). Figure 6In this way, even if a small amount of BOD-PyNO reacts with H2S outside the mitochondria, it will not emit red fluorescence after entering the mitochondria and being exposed to light, further ensuring the accuracy of its activation by H2S after entering the mitochondria first.

[0047] The fluorescent probe described in this invention can monitor its entry into mitochondria through its own yellow fluorescence, thereby determining the optimal time for on-demand photoactivation.

[0048] The fluorescent probe described in this invention has good biocompatibility.

[0049] The fluorescent probe described in this invention can visualize mitochondrial H2S fluctuations under various physiological processes, and has considerable application prospects in fluorescence imaging in the fields of biochemistry and medicine. Attached Figure Description

[0050] Figure 1 The diagram shows the synthetic route of the fluorescent probe BOD-PyNO and its contrast probe BOD-PyNH, as shown in Formula I.

[0051] Figure 2 The fluorescent probe BOD-PyNO in d6-DMSO as shown in Formula I 1 HNMR spectrum.

[0052] Figure 3 The contrast probe BOD-PyNO in d6-DMSO is shown in Equation I. 13 CNMR spectrum.

[0053] Figure 4 The contrast probe BOD-PyNH in d6-DMSO is shown in Equation I. 1 HNMR spectrum.

[0054] Figure 5 The fluorescent probe BOD-PyNH in d6-DMSO as shown in Formula I 13 CNMR spectrum.

[0055] Figure 6 The response mechanism of the fluorescent probe BOD-PyNO (as shown in Equation I) and its contrast probe BOD-PyNH to H2S and light is described.

[0056] Figure 7 This is a schematic diagram illustrating the mechanism of action of the fluorescent probe BOD-PyNO, as shown in Formula I, as a novel fluorescent probe that can be photoactivated on demand for real-time high-fidelity imaging of mitochondrial H2S.

[0057] Figure 8 The absorption and luminescence of the fluorescent probe BOD-PyNO shown in Formula I are illustrated.

[0058] Figure 9 The high-performance liquid chromatography (HPLC) chromatogram shows the formation of BOD-PyNH by the departure of nitric oxide from the fluorescent probe BOD-PyNO (as shown in Formula I) after illumination.

[0059] Figure 10 The high-resolution mass spectrometry diagram is a proof of the formation of BOD-PyNH by nitric oxide removal after illumination of the fluorescent probe BOD-PyNO shown in Formula I.

[0060] Figure 11 The fluorescence response of the fluorescent probe BOD-PyNO (5 μM) shown in Formula I to H2S in a Tris / CH3CN buffer system with fw = 90% is shown.

[0061] Figure 12 The comparative probe BOD-PyNH (5 μM) shown in Formula I exhibited enhanced fluorescence in response to H2S in Tris / CH3CN buffer systems with different volume fractions of water (fw). Data were recorded 60 minutes after the addition of NaHS (100 μM).

[0062] Figure 13 The fluorescent probe BOD-PyNO (5 μM) of Formula I was subjected to light (white light, 10 mW / cm²) in Tris / CH₃CN buffer systems with different volume fractions of water (fw). -2 After irradiation (10 minutes), the sample showed enhanced fluorescence in response to H2S in a photoinduced aggregation response. Data were recorded 60 minutes after the addition of NaHS (100 μM).

[0063] Figure 14 The high-resolution mass spectrometry diagram shows that the fluorescent probe BOD-PyNO, as shown in Formula I, is first treated with light and then with NaHS to generate the final product BOD-PyNH-SH.

[0064] Figure 15 The fluorescent probe BOD-PyNO (5 μM) shown in Formula I was first incubated with NaHS (100 μM) for 60 minutes in a Tris / CH3CN buffer system with different volume fractions of water (fw), and then exposed to light (white light, 10 mW cm⁻¹). -2 Fluorescence response diagram (10 minutes).

[0065] Figure 16 The fluorescent probe BOD-PyNO, as shown in Formula I, was exposed to light (white light, 10 mW / cm²) for 10 minutes, or not, in buffer solutions with different water content ratios (fw). -2 A comparison of fluorescence intensity after the reaction with H2S. Data were recorded 60 minutes after the addition of NaHS.

[0066] Figure 17The DLS size curves of the fluorescent probe BOD-PyNO (5μM) and the control probe BOD-PyNH (5μM) shown in Equation I are presented at different fw.

[0067] Figure 18 The fluorescent probe BOD-PyNO shown in Formula I is used to demonstrate the cytotoxicity of HCT-116 cells in vitro.

[0068] Figure 19 This is a schematic diagram showing the mitochondrial colocalization ability of the fluorescent probe shown in Formula I before and after reacting with H2S in human colon cancer HCT-116 cells in vitro.

[0069] Figure 20 This is a comparison of the intensity of the red fluorescence channel in HCT-116 cells in vitro with and without light exposure, showing the fluorescent probe shown in Formula I.

[0070] Figure 21 This is a schematic diagram illustrating the imaging ability of the fluorescent probe shown in Formula I on endogenous and exogenous H2S in HCT-116 cells in vitro.

[0071] Figure 22 This is a schematic diagram illustrating the imaging capability of the fluorescent probe shown in Formula I in vitro cells, where... Figure 22 In Figure A, the fluorescent probe shown in Formula I is used to image the fluctuations in H2S caused by LPS-induced cellular inflammation in human breast cancer MCF-7 cells in vitro. Figure 22 In Figure B, the fluorescent probe shown in Formula I is used to image the fluctuations in H2S caused by endogenous and exogenous H2O2-induced cellular oxidative stress in human ovarian cancer HeLa cells in vitro. Figure 22 C and Figure 22 D in the middle are respectively Figure 22 China A and Figure 22 The relative fluorescence intensity quantized in image B. Detailed Implementation

[0072] The present invention will be further described below with reference to embodiments. Those skilled in the art should understand that the embodiments are for illustrative purposes only and do not constitute any limitation on the present invention.

[0073] Formula I illustrates a method for synthesizing a novel fluorescent probe for real-time high-fidelity imaging of mitochondrial H2S that can be photoactivated on demand.

[0074]

[0075] Preparation Example 1: Synthesis of Compound 2

[0076] 1 (2 g, 18.25 mmol, purchased from a commercial company, no further purification required) was dissolved in 50 mL of 1,4-dioxane with di-tert-butyl dicarbonate (4.85 g, 22.3 mmol). The mixture was heated to 85 °C and reacted for 14 hours, monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, extracted with CH2Cl2, washed with deionized water, and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation. The residue was separated by column chromatography to give 1.98 g of a white solid, yield 52%. 1 HNMR (400MHz, CDCl3, ppm): δ8.32-8.31(d,1H),7.88(s,1H),7.11-7.09(d,1H),6.58(s,1H),2.50(s,3H),1.51(s,9H). 13 CNMR(151MHz,CDCl3,ppm):δ152.95,152.67,132.79,126.65,123.08,28.28,23.55.HRMS(ESI,m / z):calculated forC 11 H 17 N₂O₂[M+H] + :209.1285,found:209.1292.

[0077] Preparation Example 2: Synthesis of Compound 3

[0078] Under an argon atmosphere, 2 (1 g, 4.8 mmol) and sodium hydride (173 mg, 7.2 mmol) were dissolved in 20 mL of LDM under ice bath conditions and stirred for 30 minutes. Then, iodomethane (1.36 g, 9.6 mmol) was injected into the above system, and the reaction was carried out at room temperature for 3 hours. After the reaction was completed, the reaction was quenched dropwise with deionized water at 0 °C, extracted with ethyl acetate, washed with saturated NaCl solution, dried over anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation. The solution was separated by column chromatography to obtain 955 mg of a pale yellow viscous liquid, with a yield of 89.6%. 1 H NMR (400MHz, CDCl3, ppm): δ8.39(s,1H),7.49(d,1H),7.13(d,1H),3.26(s,3H),2.55(s,3H),1.45(s,9H). 13 C NMR (151MHz, CDCl3, ppm): δ153.99,153.47,144.97,136.77,132.05,121.72,79.82,36.12,28.67,27.25,22.83.HRMS (ESI, m / z): calculated for C 12 H 19N₂O₂[M+H] + :223.1441,found:223.1448.

[0079] Preparation Example 3: Synthesis of Compound 4

[0080] Dissolve 200 mg (0.9 mmol) of 3 in 20 mL of ultra-dry dichloromethane. Slowly add 2 mL of trifluoroacetic acid to the system and react at room temperature for 2 hours. Monitor the reaction by TLC. After the reaction is complete, remove the solvent by vacuum distillation, dissolve in a small amount of dichloromethane, and repeat the above operation 3 times to obtain 96 mg of brown oily sample, with a yield of 87.4%. 1 HNMR (400MHz, CDCl3, ppm): δ8.11(s,1H),7.37(s,2H),6.98(s,1H),2.92(s,3H),2.64(s,3H). 13 CNMR(151MHz, CDCl3,ppm): δ146.20,139.69,127.90,127.39,123.15,29.76,18.11.HRMS(ESI,m / z):calculated forC7H 11 N2[M+H] + :123.0917,found:123.0923.

[0081] Preparation Example 4: Synthesis of Compound 5

[0082] 4 (244 mg, 2.0 mmol) was dissolved in a mixed solvent of DCM:AcOH = 9:1 in an ice bath. After stirring for 5 minutes, sodium nitrite (414 mg, 6.0 mmol) was added to the system, and the reaction was allowed to proceed at room temperature for 1 hour. After the reaction was complete, an appropriate amount of saturated NaHCO3 solution was added to quench AcOH, followed by extraction with dichloromethane, washing with saturated NaCl solution, drying with anhydrous Na2SO4, filtration, and distillation under reduced pressure to remove the solvent, yielding 286 mg of a grayish-brown liquid, with a yield of 94.6%. 1 HNMR (400MHz, CDCl3, ppm): δ8.69(s,1H),7.84(d,1H),7.30(d,1H),3.47(s,3H),2.63(s,3H). 13 C NMR (151MHz, CDCl3, ppm): δ157.60, 139.49, 136.49, 126.84, 123.57, 31.25, 23.97. HRMS (ESI, m / z): calculated for C7H 10 N3O[M+H] + :152.0818,found:152.0825.

[0083] Preparation Example 5: Synthesis of Compound 6

[0084] An appropriate amount of iodomethane dissolved in acetonitrile (705 mg, 4.97 mmol) was added to a 500 mg (3.3 mmol) acetonitrile solution. The reaction was carried out at 60 °C in the dark for 6 hours, monitored by TLC. After the reaction was complete, the reaction system was cooled to room temperature and recrystallized in diethyl ether. The above system was filtered through a Buchner funnel, and the filter cake was washed with a small amount of diethyl ether to give 653 mg of milky white solid powder, with a yield of 67.5%. 1 HNMR (400MHz, CD3OD, ppm): δ9.34(d,1H),8.78(dd,1H),8.12(d,1H),4.42(s,3H),3.52(s,3H),2.91(s,3H). 13 CNMR(151MHz,CD3OD,ppm):δ153.68,140.67,135.55,133.37,129.74,46.04,29.31,18.93.HRMS(ESI,m / z):calculated for C8H 12 N3O[MI] + :166.0975,found:166.0981.

[0085] Preparation Example 6: Synthesis of Compound 7

[0086] The synthesis of compound 7 is referenced in the patent: Zhao Chunchang, Zhang Xiuli, Zhang Lili, Wang Feiyi, Jiang Haifeng, An Jiancai, Zhang Fan, A fluorescent probe for detecting endogenous H2S and its preparation method and application, Chinese Patent No.: ZL 201410766204.6, 2015-03-11, Certificate No.: 2250092.

[0087] Preparation Example 7: Synthesis of Fluorescent Probe BOD-PyNO

[0088] Dissolve 6 (120 mg, 0.41 mmol) and 7 (100 mg, 0.26 mmol) in 20 mL of ethanol. Add a small amount of acetic acid and ammonium acetate to the system, and react at 60 °C for 30 min. Detect by TLC. After the reaction is complete, cool the system to room temperature, extract with dichloromethane, wash with saturated NaCl solution, dry with anhydrous Na₂SO₄, filter, remove the solvent by vacuum distillation, and separate by column chromatography to obtain 32 mg of a dark red solid, yield 18.6%. 1HNMR (400MHz, d6-DMSO, ppm): δ9.29(s,1H),8.72(d,1H),8.66(d,1H),7.67(m,1H),7.64(m,2H),7.62(m,1H),7.52(d ,2H),7.35(d,1H),6.94(s,1H),4.34(s,3H),3.48(s,3H),2.65(s,3H),2.43-2.39(dd,2H),1.42(s,3H),1.00(t,3H). 13 C NMR (151MHz, d6-DMSO, ppm): δ167.27,150.21,143.56,139.80,138.60,138.40,135.79,135.20,134.48,133.10,132.91,132.08,131 .89,129.93,128.84,128.76,125.03,122.98,120.75,115.92,46.30,30.85,16.37,13.65,13.44,12.02.HRMS(ESI,m / z):calculated for C 28 H 28 BClF2N5O[MI] + :534.2038,found:534.2048.

[0089] Preparation Example 8: Synthesis of Compound 8

[0090] The synthesis steps are the same as in step 6, with a yield of 68%. 1 H NMR (400MHz, d6-DMSO, ppm): δ9.08(d,1H),8.49-8.46(dd,1H),7.99-7.97(d,1H),4.22(s,3H),3.29(s,3H),2.74(s,3H),1.46(s,9H). 13 CNMR(151MHz,d6-DMSO,ppm):δ153.15,151.89,141.96,140.82,128.81,82.37,46.33,36.68,28.16,19.76.HRMS(ESI,m / z):calculatedforC 13 H 21 N2O2[MI] + :237.1598,found:237.1604.

[0091] Preparation Example 9: Synthesis of Compound 9

[0092] Dissolve 7 (50 mg, 0.13 mmol) and 8 (141 mg, 0.39 mmol) in 20 mL of toluene. Add a small amount of acetic acid and ammonium acetate to the system and react at 95 °C for 3 hours, monitoring by TLC. After the reaction is complete, cool the system to room temperature, extract with dichloromethane, wash with saturated NaCl solution, dry with anhydrous Na₂SO₄, filter, remove the solvent by vacuum distillation, and separate by column chromatography to obtain 36 mg of a dark red solid, yield 37.8%. 1 HNMR (400MHz, d6-DMSO, ppm): δ8.93(s,1H),8.47-8.41(q,2H),7.64-7.62(d,3H),7.55-7.48(m,3H),7.30-7.26(d,1H) ,6.91(s,1H),4.24(s,3H),3.30(s,3H),2.64(s,3H),2.43-2.38(dd,2H),1.47(s,9H),1.41(s,3H),1.02-0.98(t,3H). 13 CNMR (151MHz, d6-DMSO, ppm): δ167.64,153.12,148.42,144.07,141.21,140.56,140.41,139.59,138.87,135.52,134.96,133.58,132.68,131.4 2,130.48,129.36,129.32,124.48,123.55,121.27,116.72,82.51,46.63,36.54,28.16,16.94,14.23,13.99,12.57.HRMS(ESI,m / z):calculated for C 33 H 37 BClF2N4O2[MI] + :605.2661,found:605.2675.

[0093] Preparation Example 10: Synthesis of the comparative probe BOD-PyNH

[0094] 2 mL of trifluoroacetic acid was added to 15 mL of a solution of 9 (30 mg, 0.04 mmol) in dichloromethane, and the mixture was reacted at room temperature for 2 hours. The reaction was then monitored by TLC. After the reaction was complete, the solvent was removed by vacuum distillation, and the product was purified by column chromatography to obtain 13 mg of a purple-black solid, with a yield of 50.2%. 1HNMR (400MHz, d6-DMSO, ppm): δ8.14-8.12(d,1H),7.96(d,1H),7.66-7.58(m,2H),7.57-7.54(dd,1H),7.50-7.49(d,2H),7.25-7.21 (m,1H),7.20(s,2H),6.83(s,1H),4.16(s,3H),2.79-2.78(d,3H),2.62(s,3H),2.42-2.36(dd,2H),1.40(s,3H),1.01-0.97(t,3H). 13 C NMR (151MHz, d6-DMSO, ppm): δ166.22,146.58,143.19,139.89,138.96,137.90,134.32,134.04,132.79,132.21,129.85,128.79,128.7 5,126.83,126.02,125.53,124.71,123.48,120.35,117.12,45.81,29.18,16.36,13.70,13.33,11.97.HRMS(ESI,m / z):calculatedforC 28 H 29 BClF2N4[MI] + :505.2136,found:505.2144.

[0095] Example 1

[0096] Figure 6 The fluorescent probe BOD-PyNO and its contrast probe BOD-PyNH, as shown in Formula I, are used to evaluate the effects of H2S on light (white light, 10 mW / cm²). -2 The response mechanism (10 minutes) is demonstrated. This illustrates the response mechanism of the fluorescent probe provided by this invention to H2S and light, as well as the structure before and after the reaction. It also shows that the fluorescent probe provided by this invention can only be activated by light irradiation followed by a response to H2S, ensuring the accuracy of the probe in detecting mitochondrial H2S.

[0097] Example 2

[0098] Figure 7 This diagram illustrates the mechanism of action of the fluorescent probe BOD-PyNO (Formula I), a novel photoactivated fluorescent probe for real-time high-fidelity imaging of mitochondrial H2S. The diagram demonstrates the probe's response mechanism to H2S in the cell, indicating that this probe can perform high-fidelity imaging of mitochondrial H2S while avoiding signal interference from extramitochondrial H2S.

[0099] Example 3

[0100] Figure 8 The absorption and emission positions of the fluorescent probe BOD-PyNO shown in Equation I are given.

[0101] Example 4

[0102] Figure 9 The image shows a high-performance liquid chromatography (HPLC) chromatogram demonstrating that the fluorescent probe BOD-PyNO (as shown in Formula I) induces the departure of nitric oxide to generate BOD-PyNH upon illumination. This confirms that BOD-PyNO does indeed induce the departure of nitric oxide upon illumination, resulting in the formation of BOD-PyNH.

[0103] Example 5

[0104] Figure 10 The image shows a high-resolution mass spectrometry result demonstrating that the fluorescent probe BOD-PyNO (as shown in Formula I) induces the departure of nitric oxide to generate BOD-PyNH upon illumination. This proves that BOD-PyNO does indeed induce the departure of nitric oxide and generate BOD-PyNH upon illumination.

[0105] Example 6

[0106] Figure 11 The figure shows the fluorescence response of the fluorescent probe BOD-PyNO (5 μM) as shown in Formula I to H2S in a Tris / CH3CN buffer system with fw = 90%. This figure indicates that the direct reaction of BOD-PyNO with NaHS (100 μM) does not induce a significant fluorescence on-off phenomenon at 690 nm.

[0107] Example 7

[0108] Figure 12 The comparative probe BOD-PyNH (5 μM) shown in Formula I exhibited enhanced fluorescence in response to H2S in Tris / CH3CN buffer systems with different volume fractions of water (fw). Data were recorded 60 minutes after the addition of NaHS (100 μM). This figure illustrates that the direct reaction of the comparative probe BOD-PyNH with NaHS (100 μM) induces a significant fluorescence onset at 690 nm, and that it exhibits enhanced fluorescence in response to H2S in Tris / CH3CN buffer systems with different volume fractions of water (fw).

[0109] Example 8

[0110] Figure 13 The fluorescent probe BOD-PyNO (5 μM) of Formula I was subjected to light (white light, 10 mW / cm²) in Tris / CH₃CN buffer systems with different volume fractions of water (fw). -2After irradiation (10 minutes), the sample showed enhanced fluorescence in response to H2S in a light-induced aggregation reaction. Data were recorded 60 minutes after the addition of NaHS (100 μM). This figure shows that BOD-PyNO, upon light irradiation, induces the departure of nitric oxide to produce BOD-PyNH. The closer the response to H2S is to physiological conditions, the better the response, making it suitable for further biological applications.

[0111] Example 9

[0112] Figure 14 The high-resolution mass spectrometry image shows the final product BOD-PyNH-SH generated by the fluorescent probe BOD-PyNO (as shown in Formula I) after phototreatment followed by NaHS treatment. This image confirms that the final product generated by BOD-PyNO after phototreatment followed by NaHS treatment is indeed BOD-PyNH-SH, which is the same as the product generated by the direct reaction of the control probe BOD-PyNH with NaHS. This explains the fluorescence activation at 690 nm.

[0113] Example 10

[0114] Figure 15 The fluorescent probe BOD-PyNO (5 μM) shown in Formula I was first incubated with NaHS (100 μM) for 60 minutes in a Tris / CH3CN buffer system with different volume fractions of water (fw), and then exposed to light (white light, 10 mW cm⁻¹). -2 The fluorescence response diagram (after 10 minutes) shows that BOD-PyNO treated with NaHS followed by light irradiation cannot produce BOD-PyNH-SH. This is because the "-SH" bond in the BOD-PyNO-SH formed with H2S is photo-oxidized, losing its ability to produce red fluorescence. Therefore, even if a small amount of BOD-PyNO reacts with H2S outside the mitochondria, it will not emit red fluorescence after entering the mitochondria and being exposed to light, further ensuring the precision of its activation by H2S after entering the mitochondria. Figure 15 In the middle a, fw = 80%; Figure 15 (b, fw = 90%)

[0115] Example 11

[0116] Figure 16 The fluorescent probe BOD-PyNO, as shown in Formula I, was exposed to light (white light, 10 mW / cm²) for 10 minutes, or not, in buffer solutions with different water content ratios (fw). -2The graph shows a comparison of fluorescence intensity after BOD-PyNO reacts with H2S following light irradiation. Data were recorded 60 minutes after the addition of NaHS. This graph indicates that the product generated by BOD-PyNO reacting with H2S after light irradiation exhibits a higher fluorescence quantum yield, making it more suitable for biofluorescence imaging than the unirradiated probe. Furthermore, the closer the reaction environment is to the physiological environment, the more pronounced the contrast becomes.

[0117] Example 12

[0118] Figure 17 In the figure, a represents the DLS size curves of the fluorescent probe BOD-PyNO shown in Formula I under different fw values: 65±4 nm when fw=70%, 83±5 nm when fw=80%, and 240±7 nm when fw=90%. Figure 17 Figure b shows the DLS size curves of the comparative probe BOD-PyNH under different fw values, as shown in Equation I: 77±4 nm for fw=70%, 107±5 nm for fw=80%, and 116±3 nm for fw=90%. This figure illustrates that BOD-PyNO and the comparative probe BOD-PyNH self-assemble into nanoparticles under physiological conditions, solving the problem of poor water solubility of traditional hydrophobic probes.

[0119] Example 13

[0120] Figure 18 The cytotoxicity of the fluorescent probe BOD-PyNO (Formula I) to HCT-116 cells in vitro was measured. At a probe concentration of 10 μM, HCT-116 cells maintained a cell viability of over 94%; even at a concentration of 25 μM, the cell viability remained above 90%, demonstrating the low biotoxicity and good biocompatibility of BOD-PyNO.

[0121] Example 14

[0122] Figure 19This diagram illustrates the mitochondrial colocalization ability of the fluorescent probe shown in Formula I in human colon cancer HCT-116 cells before and after reaction with H2S. The targeting of the probe was determined through colocalization experiments with commercially available Mito-Tracker Green (MTG). The entry of BOD-PyNO into the mitochondria was monitored in real-time using its yellow fluorescence at 585 nm. The Pearson's localization coefficient between the native yellow fluorescence of BOD-PyNO and the green fluorescence of MTG showed a time-dependent characteristic, gradually increasing to 0.83 as the staining time increased from 0 to 30 minutes. This indicates that BOD-PyNO is a good mitochondrial-targeting fluorescent probe, enriched in mitochondria for 30 minutes. Therefore, 30 minutes after the addition of BOD-PyNO to the cells is the optimal time for photoinduced BOD-PyNH production. Furthermore, no significant red fluorescence signal was observed in these co-staining experiments, indicating that BOD-PyNO did not fluoresce in response to H2S during transport to the mitochondria. After determining the good targeting and photoactivation time of BOD-PyNH generation, the activation of red fluorescence at 690 nm was observed to verify that photocontrolled BOD-PyNH can react in situ with H2S in mitochondria. Cells loaded with BOD-PyNO for 30 minutes were exposed to light (0.01 W / cm²). -2 After 10 minutes of incubation followed by 60 minutes, a bright red fluorescent signal was produced. This red fluorescence overlapped well with the green fluorescence of MTG, with a Pearson colocalization coefficient as high as 0.87. This figure illustrates that the optimal photo-activated time for the probe is 30 minutes, and that the probe is localized in the mitochondria both before and after the reaction with H2S. (Note: The green channel excitation wavelength is 488 nm, and the collection wavelength range is 500-530 nm; the yellow channel excitation wavelength is 514 nm, and the collection wavelength range is 560-600 nm; the red channel excitation wavelength is 633 nm, and the collection wavelength range is 650-750 nm. This figure illustrates that the optimal photo-activated time for the probe is 30 minutes, and that the probe is localized in the mitochondria both before and after the reaction with H2S.)

[0123] Example 15

[0124] Figure 20This is a comparison of the red fluorescence intensity of the fluorescent probe shown in Formula I in HCT-116 cells under light and without light exposure. The figure illustrates that when cells are incubated with BOD-PyNO without light, very little red fluorescence is produced because BOD-PyNO does not exhibit a fluorescent response to H2S. Conversely, HCT-116 cells treated with BOD-PyNO and light show a bright red fluorescence signal, indicating that light-induced BOD-PyNO is converted to BOD-PyNH, which then reacts with H2S to form BOD-PyNH-SH. (Note: Excitation wavelength is 633 nm, collection wavelength is 650-750 nm).

[0125] Example 16

[0126] Figure 21 This diagram illustrates the imaging capability of the fluorescent probe shown in Formula I for endogenous and exogenous H2S in HCT-116 cells in vitro. Upregulation of cysteine-β-synthetase (CBS) promotes H2S levels in HCT-116 cells. The production of endogenous H2S was inhibited using the CBS inhibitor aminooxyacetic acid (AOAA), while the CBS agonist S-adenosyl-L-methionine (SAM) stimulated H2S production. Pretreatment of cells with AOAA before BOD-PyNO illumination significantly reduced red fluorescence intensity, while SAM treatment increased red fluorescence. Furthermore, treatment with exogenous NaHS also significantly increased red fluorescence. This figure demonstrates that on-demand photoactivation enables the probe BOD-PyNO to recognize fluctuations in endogenous and exogenous H2S with high fidelity in real time. (Note: Excitation wavelength was 633 nm, collection wavelength was 650-750 nm).

[0127] Example 17

[0128] Figure 22 In Figure A, the fluorescent probe shown in Formula I is used to image the fluctuations in H2S caused by LPS-induced cellular inflammation in human breast cancer MCF-7 cells in vitro. Figure 22 In Figure B, the fluorescent probe shown in Formula I is used to image the fluctuations in H2S caused by endogenous and exogenous H2O2-induced cellular oxidative stress in human ovarian cancer HeLa cells. Figure 22 C and Figure 22 D in the middle are respectively Figure 22 China A and Figure 22 The relative fluorescence intensity quantified in the B-mode image. Lipopolysaccharide (LPS) can induce inflammation in living cells, subsequently stimulating the production of endogenous H2S. For example... Figure 22 Figure 22As shown in Figure A, BOD-PyNO treatment alone resulted in weak red fluorescence, while BOD-PyNO-loaded cells exhibited a red fluorescence signal under white light stimulation. When MCF-7 cells were pretreated with LPS 10 minutes before BOD-PyNO light irradiation, a stronger red fluorescence was detected, with a 3-fold increase in fluorescence intensity compared to BOD-PyNO-treated cells without on-demand photoactivation. This indicates that these cells experienced inflammation, subsequently leading to an upregulation of intracellular H2S levels. H2O2, a typical ROS, can increase the activity of intracellular CBS, stimulating H2S production to counteract H2O2-induced acute oxidative stress. The presence of light significantly enhanced fluorescence intensity due to the conversion of BOD-PyNO (which does not fluoresce to H2S) to BOD-PyNH (which does fluoresce to H2S). In cells treated with exogenous H2O2 and those treated with PMA (which stimulates cells to produce endogenous H2O2), photoactivation following incubation with BOD-PyNO resulted in even stronger red fluorescence intensity. Conversely, stimulation with the CBS inhibitor AOAA reduced H2S production in cells, resulting in a significant decrease in fluorescence intensity. These imaging results indicate that both endogenous and exogenous H2O2 can induce high levels of H2S production in living cells, suggesting a correlation between elevated H2S levels and H2O2-induced oxidative stress. (Note: Excitation wavelength was 633 nm, collection wavelength was 650-750 nm, PMA: stimulates cells to produce endogenous H2O2).

Claims

1. A photoactivated fluorescent probe for real-time imaging of mitochondrial H2S, characterized in that, Their structural formulas are shown below: BOD-PyNO and BOD-PyNH: ， BOD-PyNO:R=NO BOD-PyNH:R=H.

2. The on-demand photoactivated fluorescent probe for real-time imaging of mitochondrial H2S according to claim 1, characterized in that, The probe BOD-PyNO or probe BOD-PyNH at a water content ratio (f) w They self-assembled into nanoparticles in a Tris:CH3CN buffer solution containing more than 70% Tris:CH3CN.

3. The on-demand photoactivated fluorescent probe for real-time imaging of mitochondrial H2S according to claim 2, characterized in that, The particle size of the nanoparticles is: BOD-PyNO:f w =80%, 83 ± 5 nm; f w =90%, 240 ± 7 nm; BOD-PyNH: f w =80%, 107 ± 5 nm; f w =90%, 116 ± 3 nm.

4. The on-demand photoactivated fluorescent probe BOD-PyNO for real-time imaging of mitochondrial H2S according to claim 1, characterized in that, Nitric oxide is dissociated by light irradiation to generate its contrast probe BOD-PyNH; the light irradiation is white light, 10 mW cm⁻¹. -2 10 minutes.

5. The on-demand photoactivated fluorescent probe for real-time imaging of mitochondrial H2S according to claim 1, characterized in that, The probe's response to H2S is controlled temporally and spatially by illumination, enabling precise detection of H2S within mitochondria; the illumination is white light, 10 mW cm⁻¹. -2 10 minutes.

6. The on-demand photoactivated fluorescent probe for real-time imaging of mitochondrial H2S according to claim 1, characterized in that, The probe's yellow fluorescence is used to monitor its entry into the mitochondria in real time, thereby determining the optimal time for subsequent photoactivation.

7. A method for preparing the on-demand photoactivated fluorescent probes BOD-PyNO and BOD-PyNH for real-time imaging of mitochondrial H2S as described in any one of claims 1-6, characterized in that, Its preparation method is as follows: ， Preparation route of BOD-PyNO ， Preparation route of BOD-PyNH.

8. The use of the fluorescent probe of claim 1 in detecting endogenous H2S in human colon cancer HCT-116 cells in vitro.

9. The application of the fluorescent probe of claim 1 in monitoring H2S fluctuations in in vitro cellular inflammation.

10. The application of the fluorescent probe of claim 1 in monitoring H2S fluctuations under in vitro cellular oxidative stress conditions.

Images