Double-lock cascade activated fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a double-lock cascade activated fluorescent probe as well as a preparation method and application thereof. The probe BDP-PLN takes a BODIPY (boron dipyrromethene) derivative as a fluorophore and is integrated with a leucine peptide unit and a p-nitrobenzene glyoxylic acid unit to form a double-lock recognition module. According to the double-lock probe BDP-PLN constructed by the invention, through the ingenious AND logic gating molecular design, the sequential and cascade specific response to two pathological markers, namely peroxynitroso (ONOO-) and leucine aminopeptidase (LAP), is realized. The probe is stable under physiological conditions, and generates obvious fluorescence opening only in a pathological environment in which ONOO <-> triggers and then activates LAP, so that the problems that a single signal probe is insufficient in selectivity and is easy to generate false positive are effectively solved. Due to the high signal-to-noise ratio and cascade activation characteristic, an innovative tool is provided for early and accurate molecular imaging of oxidative stress related diseases such as acute liver injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical imaging and molecular probe technology, specifically relating to a dual-lock cascade activated fluorescent probe, its preparation method, and its application. Background Technology

[0002] With social development and changing lifestyles, the incidence of acute liver injury (ALI) continues to rise, becoming the second leading cause of liver injury in clinical practice after viral hepatitis. ALI is a rapid injury to hepatocytes caused by various factors such as drugs, toxins, and ischemia. It has a rapid onset and progression, and if not diagnosed and intervened in a timely manner, it can easily develop into liver failure or even death, seriously threatening patients' lives and health. Therefore, developing technologies that can achieve accurate and visual diagnosis of ALI is crucial for improving cure rates and patient prognosis. Currently, clinical diagnosis mainly relies on serological tests (such as ALT and AST), but these indicators lack tissue specificity and cannot achieve real-time, in-situ visualization of the injury process.

[0003] Fluorescence imaging technology boasts advantages such as high sensitivity, ease of operation, and real-time dynamic monitoring, demonstrating great potential in the early diagnosis of diseases. However, most currently reported fluorescent probes for the detection of acute liver injury are designed to target only a single biomarker (such as reactive oxygen species, specific enzymes, or pH). In the complex pathological microenvironment of the liver, such single biomarkers are often expressed in multiple liver disease states (such as hepatitis, liver fibrosis, drug-induced injury) and even in normal physiological processes. This leads to a lack of specificity in probe activation, a high likelihood of generating false positive signals, strong background interference, and a low signal-to-noise ratio, making it difficult to accurately differentiate acute liver injury from other liver diseases.

[0004] Therefore, in order to overcome the technical defects of existing single-target fluorescent probes in acute liver injury imaging, such as insufficient specificity and easy false positives, it is urgent to construct a logic "AND" gate probe that responds to two or more pathological factors simultaneously, so as to significantly reduce non-specific activation and improve signal-to-noise ratio and diagnostic accuracy. Summary of the Invention

[0005] The purpose of this invention is to provide a novel double-locked cascade activated fluorescent probe (BDP-PLN) with high specificity and high signal-to-noise ratio, its preparation method, and its application in precise imaging and differential diagnosis of acute liver injury.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a dual-lock cascade activated fluorescent probe BDP-PLN, which uses a fluoroboron dipyrrole derivative as the fluorescent group and integrates a dual-lock recognition module composed of a leucine peptide unit and a p-nitrophenylglyoxylic acid unit. Its chemical structure is shown below: .

[0007] A method for preparing the above-mentioned dual-locked cascade activated fluorescent probe BDP-PLN, wherein the probe is specifically prepared from compound BDP-P and compound Br-Ph-LN, and the structural formulas of BDP-P and Br-Ph-LN are as follows: ; .

[0008] Furthermore, the specific steps for synthesizing compound BDP-P are as follows: (1) 3.5 mmol of 4-pyridinecarboxaldehyde and 7.0 mmol of 2,4-dimethylpyrrole were dissolved in dichloromethane, and 0.08 mmol of trifluoroacetic acid was added. The mixture was stirred for 3 days under a nitrogen atmosphere. Then, 1.2 mmol of 2,3-dichloro-5,6-dicyanobenzoquinone was added and the mixture was stirred for 3-5 h. The mixture was then transferred to an ice bath, and 10 mL of triethylamine and 10 mL of boron trifluoride ether were added dropwise. The mixture was stirred at room temperature for 10-14 h. After filtration, rotary evaporation, extraction, drying and silica gel column chromatography purification, compound 1 was obtained. (2) Add 11.74 mmol piperidine, 17.4 mmol glacial acetic acid and 0.03 mmol anhydrous magnesium perchlorate to toluene containing 1 mmol of compound 1 and 8 mmol of 4-[2-[2-(2-methoxyethoxy)ethoxy]benzaldehyde, heat to 140~160℃ and react for 1~3 h. After the reaction is completed, concentrate the reaction solution, add saturated sodium bicarbonate solution, and extract with dichloromethane and water. Collect the organic phase, and obtain compound BDP-P after drying and purification by rotary evaporation silica gel column chromatography.

[0009] Furthermore, the specific steps for synthesizing Br-Ph-LN are as follows: (1) An equimolar amount of dichloromethane containing Boc-L-leucine and dichloromethane containing N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) were mixed and stirred in an ice bath for 0.5 h. An equimolar amount of dichloromethane containing 4-aminobenzyl alcohol was added, and 2 times the molar amount of DIPEA was added dropwise. The mixture was heated to room temperature and reacted for 24 h. The reaction solution was extracted with a mixture of dichloromethane and saturated sodium bicarbonate aqueous solution. The organic phases were combined, dried, filtered under reduced pressure, evaporated to dryness, and purified by silica gel column chromatography to obtain compound 2. (2) Under nitrogen protection, 4 mL of trifluoroacetic acid was added to anhydrous dichloromethane containing 3.0 mmol of compound 2. After stirring at room temperature for 3 h, the mixture was concentrated under vacuum. The residue was extracted with a mixture of dichloromethane and saturated sodium bicarbonate aqueous solution. The organic phases were combined, dried, filtered under reduced pressure, evaporated to dryness, and purified by silica gel column chromatography to obtain compound 3. (3) Under ice bath conditions, p-nitrophenylacetaldehyde acid and equimolar amounts of HATU were stirred and dissolved in anhydrous dichloromethane. Equimolar amounts of compound 3 and 2 times the molar amount of N,N-diisopropylethylamine (DIPEA) were added. The mixture was heated to room temperature and reacted for 1-3 h. The reaction solution was extracted with a mixture of dichloromethane and saturated sodium bicarbonate aqueous solution. The organic phases were combined, dried, filtered under reduced pressure, evaporated to dryness, and purified by silica gel column chromatography to obtain compound 4. (4) Add an equimolar amount of phosphorus tribromide to anhydrous dichloromethane containing compound 4, stir under ice bath conditions for 0.5-1 h, then concentrate under vacuum, extract the residue with a mixture of dichloromethane and saturated sodium bicarbonate aqueous solution, combine the organic phases, dry, filter under reduced pressure, evaporate to dryness, and purify by silica gel column chromatography to obtain Br-Ph-LN.

[0010] The structural formulas of compounds 2-4 are shown below: , , .

[0011] Furthermore, equimolar amounts of BDP-P and Br-Ph-LN were stirred at 80-90°C for 24-48 hours, followed by vacuum concentration. The residue was extracted with a mixed solution of dichloromethane and saturated sodium bicarbonate aqueous solution. The organic phases were combined, dried, filtered under reduced pressure, evaporated to dryness, and purified by silica gel column chromatography to obtain the double-locked cascade activated fluorescent probe BDP-PLN.

[0012] This invention also provides the above-mentioned dual-lock cascade activated fluorescent probe BDP-PLN for the preparation of qualitative or quantitative detection of ONOO. - Applications of leucine aminopeptidase reagent.

[0013] The present invention also provides the application of the above-mentioned dual-lock cascade activated fluorescent probe BDP-PLN in the preparation of reagents for the detection of acute liver injury.

[0014] The present invention also provides the application of the above-mentioned dual-lock cascade activated fluorescent probe BDP-PLN in the preparation of reagents for detecting drug-induced liver injury.

[0015] This invention also provides the application of the above-mentioned dual-lock cascade activated fluorescent probe BDP-PLN in the preparation of fluorescent recognition and imaging reagents for acute liver injury.

[0016] The cascade activation mechanism of the probe BDP-PLN in this invention is as follows: Under normal physiological conditions, the probe fluorescence is in the "off" state. However, upon entering the microenvironment of acute liver injury, high concentrations of peroxynitrite (ONOO) are detected. - First, the probe specifically recognizes and cleaves the responsive chemical bond (α-ketoamide group) in the p-nitrophenylglyoxylic acid unit, releasing its binding to the leucine amino group; this step is the first-stage unlocking. Subsequently, the aberrantly expressed leucine aminopeptidase (LAP) recognizes and cleaves the exposed leucine amide bond; this step is the second-stage unlocking. After these two cascaded chemical and enzymatic cleavage reactions, the amino group is exposed, the self-eliminating group departs, and the fluorophore (BDP-P) in the probe molecule is completely released. The photoinduced electron transfer (PET) effect is quenched, resulting in a strong near-infrared fluorescence signal. This process is strictly dependent on ONOO. - The cascaded response with LAP forms a logical "AND" gate, ensuring that the probe is specifically activated only in the acute liver injury region.

[0017] Compared with the prior art, the present invention has the following advantages: (1) Ultra-high specificity and extremely low background: This invention is the first to use ONOO - The dual-lock cascade activation mechanism with LAP acts like setting two "molecular locks" for the probe. The probe only activates in the acute liver injury region when ONOO... - When the two markers, LAP and PP, are present and act sequentially, the fluorescence signal will be fully activated, effectively avoiding false positives caused by the non-specific expression of a single marker in other liver diseases or physiological processes. The intramolecular PET effect is significant, the fluorescence is completely quenched, and the background signal is extremely low.

[0018] (2) High signal-to-noise ratio and sensitivity: At the site of acute liver injury, the dual response leads to the concentrated release of fluorophores, generating strong local signals. The imaging contrast and signal-to-noise ratio are significantly higher than those of traditional single-target response probes, greatly improving the detection sensitivity.

[0019] (3) Precise molecular design and superior performance: The probe is designed based on the BODIPY fluorophore, and its absorption and emission wavelengths are located in the near-infrared region. It has strong tissue penetration ability and low autofluorescence interference. Through precise molecular structure modification, it achieves efficient and sequential response to two specific biomarker molecules. The probe has good stability and high biocompatibility.

[0020] (4) The preparation method is simple and easy to promote: the synthesis route is clear, the raw materials are readily available, the reaction conditions are mild, the post-processing is simple, and the yield is good, which is conducive to the large-scale preparation of the probe and its subsequent clinical translation application.

[0021] (5) This invention focuses on two closely related and pathologically specific key biomarkers in the occurrence and development of acute liver injury: peroxynitrite (ONOO).- ) and leucine aminopeptidase (LAP). ONOO - As a reactive nitrogen species, LAP is produced in large quantities during oxidative stress and inflammatory responses, and is a key biomarker of oxidative stress in the early stages of acute liver injury. LAP, on the other hand, is abnormally highly expressed after hepatocellular injury, and its activity is closely related to the degree of liver dysfunction. The development of two biomarkers specific to acute liver injury, ONOO, is also planned. - Fluorescent probes activated by LAP hold promise for achieving highly specific imaging of acute liver injury, thus meeting the needs for precise diagnosis and treatment of acute liver injury. This invention is the first to design and synthesize a novel double-locked cascade activated fluorescent probe. This probe uses a leucine peptide group as the recognition unit for LAP and a p-nitrophenylacetaldehyde group as the ONOO... - The response units are covalently linked to the BODIPY fluorophore. Activation of the probe strictly follows the "ONOO" principle. - The cascade logic of "prioritizing response, followed by LAP cleavage" ensures complete activation and strong fluorescence signals only within the microenvironment of acute liver injury. This design not only significantly improves the probe's specificity and resistance to interference but also provides a powerful tool for real-time, visual analysis of the pathological progression of acute liver injury at the molecular level. Attached Figure Description

[0022] Figure 1 The images show the UV-Vis absorption spectra of BDP-P at different concentrations in DMSO. The inset shows the linear fit between absorbance at the point of maximum absorption and concentration.

[0023] Figure 2 The images show the UV-Vis absorption spectra of BDP-PLN at different concentrations in DMSO. The inset shows the linear fit between absorbance at the point of maximum absorption and concentration.

[0024] Figure 3 The fluorescence emission spectra of BDP-P (10 μM) and BDP-PLN (10 μM) in DMSO are shown.

[0025] Figure 4 The following are the responsiveness measurements of BDP-PLN: (a) Electronic absorption spectra of BDP-PLN in PBS in different treatment groups; (b) Fluorescence emission spectra of BDP-PLN in PBS in different treatment groups; (c) Visual photographs of BDP-PLN in different treatment groups under a 365 nm UV lamp: 1. BDP-PLN + PBS; 2. BDP-PLN + ONOO - ; 3. BDP-PLN+LAP;4. BDP-PLN+LAP+ONOO - (d) Fluorescence ratio of BDP-PLN at 676 nm in different treatment groups.

[0026] Figure 5 The graph shows the detection limits of BDP-PLN; (a) BDP-PLN at LAP (0-100 U / L) and ONOO. - (b) Fluorescence emission spectrum of BDP-PLN under the action of LAP (100 U / L) and ONOO - (c) Fluorescence emission spectra of BDP-PLN under LAP (0-25 μM) and ONOO - Fluorescence emission spectra under (0-25 μM) treatment; (df) represents the fluorescence intensity of BDP-PLN at 676 nm in different treatment groups compared with different concentrations of LAP and ONOO. - The linear relationship.

[0027] Figure 6 This is a time response curve for BDP-PLN.

[0028] Figure 7 This is a selective test plot for BDP-PLN.

[0029] Figure 8 Figure 1 shows the experimental results of BDP-PLN inhibitor testing; 1 represents BDP-PLN + PBS; 2 represents BDP-PLN + LAP + ONOO. - ;3 is BDP-PLN+LAP+ONOO - +UA; 4 is BDP-PLN+LAP+ONOO - +BESTATIN; 5 is BDP-PLN+LAP+ ONOO - +BESTATIN+UA.

[0030] Figure 9 This is a graph showing the pH stability of BDP-PLN.

[0031] Figure 10 The graph shows the temperature stability of BDP-PLN.

[0032] Figure 11 The graph shows the lipid-water partition coefficient of BDP-PLN; (a) UV-Vis absorption spectra of BDP-PLN at different concentrations in saturated water-octanol solution, with an inset showing the linear fit between absorbance at maximum absorption and concentration; (b) UV-Vis absorption spectra of BDP-PLN at different concentrations in saturated water-octanol aqueous solution, with an inset showing the linear fit between absorbance at maximum absorption and concentration; (c) Linear relationship of concentration of BDP-PLN at different concentrations in oil and aqueous phases after shaking for 24 hours.

[0033] Figure 12 This is a graph showing the cytotoxicity test results for BDP-PLN.

[0034] Figure 13 This is a graph showing the cytotoxicity test results for APAP.

[0035] Figure 14 This is a graph showing the optimal cellular response time for BDP-PLN.

[0036] Figure 15 This is a graph showing the extracellular response of BDP-PLN.

[0037] Figure 16 This is a graph showing the intracellular response of BDP-PLN. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0039] Example 1: Preparation of probe BDP-PLN

[0040] (1) Synthesis of compound 1: 4-Pyridinecarboxaldehyde (0.38 g, 3.5 mmol) and 2,4-dimethylpyrrole (0.68 g, 7.0 mmol) were dissolved in 200 mL of dichloromethane and stirred for 0.5 h. Trifluoroacetic acid (0.01 g, 0.08 mmol) was then added dropwise under nitrogen protection and stirred at room temperature for three days. Next, 50 mL of dichloromethane containing 2,3-dichloro-5,6-dicyanobenzoquinone (1.0 g, 1.2 mmol) was slowly added, and the mixture was stirred at room temperature for 2 h. The reaction mixture was then placed in an ice bath, and 10 mL of triethylamine and 10 mL of boron trifluoride diethyl ether were added dropwise. After the addition was complete, the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was filtered, the filtrate was collected, and the solvent was evaporated to dryness. The filtrate was extracted with a 1:1 mixture of dichloromethane (100 mL) and water (100 mL), the organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness. The crude product was dissolved in dichloromethane and purified by silica gel column chromatography (eluent: CH2Cl2 / MeOH = 50 / 1, v / v) to give an orange-red solid 1 (0.50 g, yield 43.5%).

[0041] Structural characterization of compound 1: 1 H NMR (500MHz, CDCl3): δ (ppm) = 8.78 (dd, J1 = 4.5Hz, J 2 = 1.5 Hz, 2H), 7.30 (dd, J 1 = 4.5 Hz, J 2 = 1.5 Hz, 2H), 6.01 (s, 2H), 2.56 (s, 6H), 1.41 (s, 6H).

[0042] (2) Synthesis of compound BDP-P: Compound 1 (0.20 g, 1 mmol) and 4-[2-[2-(2-methoxyethoxy)ethoxy]benzaldehyde (0.74 g, 8 mmol, CAS: 153364-63-7) were dissolved in toluene (40 mL). Piperidine (1.0 g, 11.74 mmol), glacial acetic acid (1.0 g, 17.4 mmol), and anhydrous magnesium perchlorate (0.01 g, 0.03 mmol) were added sequentially. The mixture was heated to 150 °C and refluxed for 2 h. Water generated during the reaction was removed using a water separator. The reaction solution was concentrated to 2 mL under vacuum, and 50 mL of saturated sodium bicarbonate aqueous solution was added. The solution was extracted with dichloromethane (50 mL × 3). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the crude product. The crude product was dissolved in dichloromethane and purified by silica gel column chromatography (eluent: CH2Cl2 / MeOH = 30 / 1, v / v) to give a green solid BDP-P (0.20 g, yield 39.4%).

[0043] Structural characterization of compound BDP-P: 1 H NMR (500MHz, CDCl3): δ (ppm) = 8.79 (d, J =6.0 Hz, 2H), 7.61-7.56 (m, J = 15.5 Hz, J = 9.0 Hz, 6H), 7.35 (d, J = 6.0 Hz, 2H), 7.23 (d, J = 16.0 Hz, 2H), 6.95 (d, J = 8.5 Hz, 4H), 6.63 (s, 2H), 4.20-4.18 (m, 4H), 3.90-3.88 (m, 4H), 3.77-3.75 (m, 4H), 3.71-3.69 (m, 4H), 3.68-3.66 (m, 4H), 3.57-3.55 (m, 4H), 3.39 (s, 6H), 1.46 (s, 6H); 13 C NMR (126 MHz, CDCl3): δ (ppm) = 159.83, 153.33, 150.52, 143.91,141.07, 136.53, 133.81, 132.09, 129.50, 129.14, 123.96, 118.05, 116.94,114.97, 71.94, 70.87, 70.67, 70.59, 69.67, 67.53, 59.05, 14.85; HRMS-ESI (m / z): calculated for C 46 H 54 BF2N3O8 [M+K] + : 864.3604; found864.3595.

[0044]

[0045] (3) Synthesis of compound 2: Boc-L-leucine (1.0 g, 4.3 mmol) was dissolved in dichloromethane (20 mL), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) (1.6 g, 4.3 mmol) was added. The mixture was stirred in an ice bath for 0.5 h. Subsequently, 4-aminobenzyl alcohol (0.50 g, 4.3 mmol) was added, followed by dropwise addition of DIPEA (0.60 g, 8.6 mmol). The ice bath was removed and the mixture was stirred for 24 h. The reaction solution was extracted three times with a mixture of dichloromethane (60 mL) and saturated sodium bicarbonate aqueous solution (60 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered under reduced pressure, and evaporated to dryness to obtain the crude product. The crude product was dissolved in dichloromethane and purified by silica gel column chromatography. The product was eluted with a dichloromethane:methanol (60:1, v / v) mixture to give a white solid 2 (1.2 g, yield 84.1%).

[0046] Structural characterization of compound 2: 1 H NMR (500 MHz, CDCl3): δ (ppm) = 8.71 (s, 1H),7.45 (d, J =8.0 Hz, 2H), 7.22 (d, J =8.0 Hz, 2H), 5.14 (d, J=8.5 Hz, 1H),4.60 (s, 2H), 4.31 (m, 1H), 2.05-1.74 (m, 2H), 1.64-1.52 (m, 1H), 1.45 (s,12H), 0.97 (d, J =6.5 Hz, 3H), 0.95 (d, J =6.0 Hz, 3H).

[0047] (4) Synthesis of compound 3: Compound 2 (1.0 g, 3.0 mmol) was dissolved in anhydrous dichloromethane (40 mL) under nitrogen protection. 4 mL of trifluoroacetic acid was slowly added, and the resulting mixture was stirred at room temperature for 3 h and then concentrated under vacuum. The residue was extracted three times with a mixture of dichloromethane (60 mL) and saturated sodium bicarbonate aqueous solution (60 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered under reduced pressure, and evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography in dichloromethane, and eluted with a dichloromethane:methanol (20:1, v / v) mixture to give white solid 3 (0.43 g, yield 61.4%).

[0048] Structural characterization of compound 3: 1 H NMR (600 MHz, CDCl3): δ (ppm) = 9.51 (s, 1H),7.55 (d, J =7.0 Hz, 2H), 7.29 (d, J =7.0 Hz, 2H), 4.61 (s, 2H), 3.47 (m, 1H), 1.91 (m, 2H), 1.79-1.75 (m, 1H), 0.98 (d, J =5.5 Hz, 3H), 0.95 (d, J =5.0 Hz, 3H).

[0049] (5) Synthesis of compound 4: p-Nitrophenylacetaldehyde (0.35 g, 1.8 mmol) was dissolved in anhydrous dichloromethane (20 mL), and HATU (0.68 g, 1.8 mmol) was added. The mixture was stirred in an ice bath for 0.5 h. Then, compound 3 (0.43 g, 1.8 mmol) and DIPEA (0.46 g, 3.6 mmol) dissolved in 20 mL of anhydrous dichloromethane were added. The ice bath was removed, and the mixture was stirred for 2 h. The solid residue was removed by filtration. The filtrate was concentrated under vacuum and extracted with a mixture of dichloromethane (60 mL) and saturated brine (60 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered under reduced pressure, and evaporated to dryness to obtain the crude product. The crude product was dissolved in dichloromethane and purified by silica gel column chromatography. The elution with a dichloromethane:methanol (30:1, v / v) mixture gave a pale yellow solid 4 (0.61 g, yield 80.6%).

[0050] Structural characterization of compound 4: 1 H NMR (500 MHz, CDCl3): δ (ppm) = 8.51 (d, J =8.5Hz, 2H), 8.32 (d, J =8.5 Hz, 2H), 7.94 (s, 1H), 7.63 (d, J =8.5 Hz, 1H), 7.51(d, J =8.5 Hz, 2H), 7.33 (d, J =8.5 Hz, 2H), 4.66 (s, 2H), 4.63-4.60 (m, 1H), 1.93-1.88 (m, 1H), 1.82-1.72 (m, 2H), 1.02 (t, J =6.0 Hz, 6H); 13 C NMR (126 MHz, CDCl3): δ (ppm) = 185.24, 168.96, 160.89, 150.90,137.52, 136.73, 136.32, 132.38, 127.90, 123.62, 120.25, 64.89, 52.98, 40.84,24.97, 22.93, 22.21; HRMS-ESI (m / z): calculated for C 21 H 25 N3O6 [M+K] + : 454.1375; found454.1402.

[0051] (6) Synthesis of compound Br-Ph-LN: Compound 4 (0.6 g, 1.4 mmol) was dissolved in anhydrous dichloromethane (20 mL), and phosphorus tribromide (0.39 g, 1.4 mmol) was added dropwise. The mixture was stirred in an ice bath for 30 min, followed by vacuum concentration. The residue was extracted with a mixture of dichloromethane (60 mL) and saturated sodium bicarbonate aqueous solution (60 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered under reduced pressure, and evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography in dichloromethane, and eluted with a dichloromethane:methanol (100:1, v / v) mixture to give a pale yellow oil Br-Ph-LN (0.68 g, 98.6% yield).

[0052] Structural characterization of compound Br-Ph-LN: 1 H NMR (500 MHz, CDCl3): δ (ppm) = 8.49 (d, J =9.0 Hz, 2H), 8.30 (d, J =9.0 Hz, 2H), 8.14 (s, 1H), 7.69 (d, J =8.5 Hz, 1H), 7.50 (d, J =8.5 Hz, 2H), 7.34 (d, J =8.5 Hz, 2H), 4.66-4.62 (m, 1H), 4.46 (s,2H), 1.90-1.88 (m, 1H), 1.83-1.72 (m, 2H), 1.01 (t, J =6.5 Hz, 6H); 13 C NMR (126 MHz, CDCl3): δ (ppm) = 185.15, 168.97, 160.89, 151.04,137.67, 137.43, 134.33, 132.41, 130.00, 123.63, 120.21, 53.03, 40.67, 33.13,24.96, 22.91, 22.20; HRMS-ESI (m / z): calculated for C 21 H 22 BrN3O5 [M+H] + : 476.0816; found476.0771.

[0053] (7) Synthesis of the dual-lock probe compound BDP-PLN:

[0054] Compound BDP-P (0.2 g, 0.24 mmol) was dissolved in anhydrous acetonitrile (20 mL), and compound Br-Ph-LN (0.12 g, 0.24 mmol) was added. The mixture was stirred and refluxed at 85 °C for 24 h, concentrated under vacuum, and the residue was extracted with a mixture of dichloromethane (60 mL) and saturated brine (60 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered under reduced pressure, and evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography in dichloromethane, and eluted with a dichloromethane:methanol (20:1, v / v) mixture to obtain the green solid BDP-PLN (0.15 g, yield 46.9%).

[0055] Structural characterization of compound BDP-PLN: 1 H NMR (500 MHz, CDCl3): δ (ppm)=10.37 (s,1H), 9.52 (s, 2H), 8.40 (s, 1H), 8.25 (d, J =8.0 Hz, 2H), 8.10 (d, J =8.5 Hz,2H), 7.92 (s, 2H), 7.72 (s, 2H), 7.53 (d, J =8.5 Hz, 4H), 7.46 (d, J =16.0 Hz,2H), 7.36 (s, 2H), 7.22 (d, J =16.5 Hz, 2H), 6.90 (d, J =8.5 Hz, 4H), 6.52 (s,2H), 6.09 (s, 2H), 4.88 (s, 1H), 4.13 (m, 4H), 3.86 (m, 4H), 3.74 (m, 4H),3.68 (m, 4H), 3.65 (m, 4H), 3.55 (m, 4H), 3.37 (s, 6H), 1.84 (m, 2H), 1.72 (m, 1H), 1.23 (s, 6H), 0.94 (s, 6H); 13C NMR (151 MHz, CDCl3) δ (ppm) = 170.75, 160.57, 154.03, 151.82,147.65, 145.89, 143.66, 142.69, 140.89, 140.43, 136.19, 130.83, 130.49,129.86, 129.61, 129.39, 129.11, 127.87, 127.66, 125.51, 124.13, 121.20,115.99, 115.52, 115.18, 71.91, 70.83, 70.54, 69.65, 67.61, 59.03, 42.11, 32.72, 29.70, 27.41, 25.13, 23.24, 22.51, 18.66.

[0056] HRMS-ESI (m / z): calculated for C 67 H 76 BF2N6O 13 + [M] + : 1220.5591; found1220.5562.

[0057] Example 2 Performance Testing (1) The UV-Vis absorption spectra of BDP-PLN and BDP-P in DMSO were measured to preliminarily evaluate the probe's ability to diagnose liver injury. DMSO solutions of BDP-PLN and BDP-P at concentrations of 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM were prepared, using DMSO solvent as a reference. The results are as follows: Figure 1 and 2 As shown, BDP-P and BDP-PLN exhibit sharp Q-band absorption peaks at wavelengths of 654 nm and 670 nm, respectively, and there is a strong linear relationship between the absorbance values ​​and the concentrations of the compounds, indicating that both compounds exist as monomers in DMSO. The molar extinction coefficients (ε) of BDP-PLN and BDP-P are 79310 and 68620 L / mol·cm, respectively. -1 By comparing the UV-Vis absorption spectra of BDP-PLN and BDP-P, it can be seen that the maximum absorption peak of BDP-PLN is red-shifted by 16 nm compared to BDP-P, which is consistent with the molecular design concept, that is, intramolecular PET occurs, which causes the molecular absorption to red-shift.

[0058] (2) Fluorescence emission spectroscopy study DMSO solutions of BDP-PLN and BDP-P at concentrations of 10 μM were prepared, and the fluorescence emission spectra of the compounds in the 670-800 nm range were measured with an excitation wavelength of 650 nm. The results are as follows: Figure 3 As shown, BDP-P exhibits a strong fluorescence characteristic peak of BODIPY at 685 nm. In contrast, the fluorescence of BDP-PLN formed by quaternization is significantly reduced, with a fluorescence intensity of only 3% of that of BDP-P. This fluorescence quenching phenomenon perfectly matches the molecular design expectation, namely, by designing an intramolecular photoinduced electron transfer (PET) effect, the pyridine site at the meso site of the BODIPY core undergoes a quaternization reaction through the insertion of a double-locked responsive group to form a nitrogen cation, thereby achieving the intramolecular PET effect and successfully quenching the fluorescence.

[0059] (3) BDP-PLN in vitro detection of ONOO - Performance analysis of LAP 1) Detection: Four sets of samples were prepared, namely BDP-PLN, BDP-PLN+ONOO. - BDP-PLN+LAP, BDP-PLN+ONOO - +LAP. The concentration of BDP-PLN in each group was 10 μM, ONOO - The concentration of leucine aminopeptidase (LAP) was 25 μM, the concentration was 100 U / L, the solution system was water, and the reaction was carried out at 37℃ for 2 h. After that, the UV-Vis absorption spectrum and fluorescence emission spectrum (excitation wavelength λ) were measured. ex =650 nm).

[0060] The results are as follows Figure 4 As shown in a, in the UV-Vis absorption spectrum, only when ONOO - When coexisting with LAP, the maximum absorption peak of BDP-PLN exhibits a blue shift (Δλ = 15 nm), while in the presence of a single component (ONOO)... - No significant spectral shift was observed under LAP treatment conditions. Fluorescence emission spectroscopy further validated the probe's dual-lock response characteristics. Figure 4 b and Figure 4 c) In the case of only ONOO - Under conditions of LAP stimulation alone, the fluorescence intensity of BDP-PLN remained essentially unchanged; however, when ONOO - When present in synergy with LAP, the probe's fluorescence signal is specifically activated, with an activation degree as high as 71 times ( Figure 4 d) Explicitly verify that it has an "AND" logic gate double-lock response.

[0061] 2) Detection limit determination: 24 portions of 10 μM BDP-PLN aqueous solution were prepared and divided into three groups for different treatments.

[0062] (a) With a fixed LAP concentration of 100 U / L, ONOO was added in a gradient. - The final concentrations were 0, 1, 2, 3, 5, 10, 20, and 25 μM, respectively. (b) Fixed ONOO - The concentration of LAP was 25 μM, and LAP was added in a gradient to achieve final concentrations of 0, 5, 10, 15, 25, 50, 75, and 100 μM. (c) Simultaneously increment LAP and ONOO - The solution was prepared according to the ratios of the first two groups, with a LAP concentration of 0-100 U / L, ONOO. - The concentration ranges from 0 to 25 μM, with a total of 8 corresponding concentration groups (e.g., ONOO). - The concentration of 1 μM corresponds to a LAP concentration of 5 μM.

[0063] After reacting at 37℃ for 2 hours, the fluorescence emission spectra (λ) of each group of solutions were measured. ex =650 nm). Based on fluorescence intensity data, BDP-PLN fluorescence intensity and LAP concentration, ONOO were respectively constructed. - The concentration-response curve and its linear relationship. The experiment was repeated three times under the same conditions. Figure 5 As shown in a, in fixed ONOO - In this case, as the concentration of LAP enzyme increases, the fluorescence intensity gradually increases; such as Figure 5 As shown in b, with a fixed LAP enzyme concentration, as ONOO - As the concentration increases, the fluorescence intensity gradually increases; for example... Figure 5 As shown in c, with proportional increases in LAP enzyme and ONOO - Under these conditions, the fluorescence intensity increased proportionally. These experimental results indicate that when a particular responder reaches saturation, increasing the concentration of that responder alone can enhance fluorescence, revealing the dynamic response mechanism of BDP-PLN's dual-substrate synergistic effect. Further quantitative analysis showed that BDP-PLN has effects on LAP enzymes and ONOO... - The responses of both exhibited good linearity, with detection limits as low as 0.77 U / L (LAP) and 0.415 μM (ONOO), respectively. - This indicates that the probe has a small but sensitive response to both responders.

[0064] 3) Response time measurement: BDP-PLN (10 μM) and ONOO -(25 μM) and LAP (100 U / L) were rapidly mixed in PBS (pH=7.4), and the fluorescence intensity at 670 nm (excitation wavelength 650 nm) was recorded at different time points (0, 1, 3, 5, 7, 10, 15, 20, 30, 60, 90, 120, 240, 360 min) at 37℃. The experiment was repeated three times under the same conditions. The results are as follows: Figure 6 As shown, the fluorescence intensity of the solution continuously increases with time, rising sharply within 30 minutes and reaching a saturation point at 30 minutes. After this point, the fluorescence intensity no longer shows significant enhancement, indicating that the optimal response time for the probe in in vitro solution testing is 30 minutes. Furthermore, the probe exhibits good stability; the fluorescence intensity did not show significant changes within 3 hours after the response, remaining at a stable level.

[0065] 4) Selective assay: Common biologically relevant molecules were selected as interfering factors for assay. Each interfering substance was prepared and diluted with pure water, as follows: 1 (blank group), 2 (K... + ), 3 (Na + ), 4 (Zn 2+ ), 5 (Mg 2+ ), 6 (Mn 2+ ), 7(Cu 2+ ), 8 (Fe 3+ ), 9 (Fe 2+ ), 10 (Hg) 2+ ), 11 (Ca 2+ ), 12 (I - ), 13 (S 2- ), 14 (SO4) 2- ), 15(SO3) 2- ), 16 (HSO3) - ), 17 (S2O3) 2- ), 18 (CO3) 2- ), 19 (HCO3) - ), 20 (Cl - ), 21 (ClO) - ), 22 (NO2) - ), 23 (NO3) - ), 24 (NaVC), 25 (GSH), 26 (Cys), 27 (Leu), 28 (Lys), 29 (His), 30 (Ala), 31 (H2O2), 32 (ONOO - ), 33 (LAP), 34 (ONOO) -+LAP). All interfering substances were added to the BDP-PLN (10 μM) PBS solution via stock solution (1 mM), with the added volume controlled to be less than 1% of the total system volume to ensure the consistency of the experimental system. After the reaction system was reacted at 37℃ for 2 h, the fluorescence emission spectra of each group of solutions (excitation wavelength λ) were measured. ex =650 nm). The results are as follows: Figure 7 As shown, BDP-PLN exhibits good anti-interference capabilities, showing no response to common interfering substances such as metal ions, oxidizing agents, reducing agents, some enzymes, and anions, except for LAP and ONOO. - Under the condition that they coexist, the fluorescence intensity changes significantly.

[0066] 5) Inhibitor experiment: Five groups of samples were prepared in parallel, namely (1) BDP-PLN+PBS; (2) BDP-PLN+LAP+ONOO - (3) BDP-PLN+LAP+ONOO - +UA; (4) BDP-PLN+LAP+ONOO - +BESTATIN; (5) BDP-PLN+LAP+ONOO - +BESTATIN+UA. BDP-PL final concentrations were all 10 μM, ONOO - The final concentration of all samples was 25 μM, and the final concentration of LAP was 100 U / L. The final concentrations of BESTATIN (LAP enzyme inhibitor) and UA (uric acid, ONOO⁻ scavenger) added were both 100 μM. All groups used PBS as the reaction system. After reacting at 37℃ for 2 h, the fluorescence emission spectra (excitation wavelength λ) of each sample were measured. ex =650nm). The experiment was repeated three times under the same conditions. The targeting of the probe response was verified by specific inhibition experiments with the addition of BESTATIN and UA, respectively. From Figure 8 It can be seen that only in LAP and ONOO - Significant changes in solution fluorescence intensity only occur when both conditions are present and not suppressed.

[0067] 6) pH stability determination: PBS solutions (10 μM) containing BDP-PLN were prepared and divided into 11 groups. The pH was precisely adjusted to 4.0, 4.5, 5.0, 6.0, 6.5, 7.0, 7.4, 8.0, 8.5, and 9.0 using either 1 M HCl or 1 M NaOH stock solution. After adjustment, the solutions were allowed to stand for 10 min to ensure pH stability. Then, LAP and ONOO were added to each group of solutions. -The final concentrations were adjusted to 100 U / 25 μM. PBS was added if necessary to ensure a consistent total reaction volume. After reacting at 37℃ for 2 hours, the fluorescence emission spectra (excitation wavelength λ) of each group of samples were measured. ex =650 nm). The experiment was repeated three times under the same conditions. The results are as follows. Figure 9 As shown, the probe exhibits good pH stability, demonstrating its resistance to LAP enzyme and ONOO within the pH range of 6.5-8.0. - All of them have good response performance and can be applied to the requirements of complex biological microenvironments.

[0068] 7) Temperature stability test: Prepare PBS solution (10 μM) of BDP-PLN, and divide it into 10 groups. Set the temperature to 30, 32, 34, 36, 37, 38, 40, 42, 44, and 46℃ respectively. Then add LAP and ONOO to each group of solution. - The final concentrations were adjusted to 100 U / 25 μM. PBS was added if necessary to ensure a consistent total reaction volume. After 2 hours of reaction, the fluorescence emission spectra (excitation wavelength λ) of each group of samples were measured. ex =650 nm). The experiment was repeated three times under the same conditions. The results are as follows. Figure 10 As shown, the probe BDP-PLN exhibits good temperature stability and demonstrates excellent response within the 30-46℃ range.

[0069] 8) Determination of lipid-water partition coefficient: First, add equal volumes of 5 mL n-octanol and 5 mL ultrapure water to an Erlenmeyer flask, seal the flask with plastic wrap, and fix the flask in a constant temperature incubator shaker at 37℃, 100 rpm / min, and in the dark for 24 h to allow the water and n-octanol to become mutually saturated. After the reaction is complete and the phases are allowed to separate, the two phases are separated for later use. The absorbance (gradient concentration) of BDP-PLN in saturated n-octanol and water is measured separately. The maximum absorption wavelength λ is used as the reference wavelength. max The absorbance measured at the concentration was plotted against the concentration to obtain a linear regression equation. Next, equal volumes of probe standard solutions (concentrations within the established linear range) were prepared using n-octanol and water, respectively, and mixed in sample vials. The solutions were then shaken again at 100 rpm / min at 37°C in the dark for 24 h. After shaking, the n-octanol and aqueous phases were separated, and the absorbance at the maximum absorption wavelength was measured separately. The concentrations of the probe in n-octanol and water were then calculated using the linear regression equation, and the Log P value was calculated. The formula for calculating the lipid-water partition coefficient is as follows: Log P = C O / C W C o : Equilibrium concentration of solute in the oil phase, C w : The equilibrium concentration of the solute in the aqueous phase.

[0070] The results are as follows Figure 11 As shown, BDP-PLN has a lipid-water partition coefficient of 0.307, indicating it is a compound with good membrane permeability and water solubility, which basically meets the requirements for LAP and ONOO in living cells under physiological conditions. - The detection.

[0071] Example 3: Analysis of BDP-PLN's Response Performance at the Cellular Level (1) Biosafety assessment of probe BDP-PLN Select healthy AML-12 (normal mouse hepatocytes) and HepG2 (human hepatocellular carcinoma cells) cells, respectively. Discard the old culture medium in the culture flasks, wash twice with PBS, add 1 mL of trypsin, and incubate for 2 min. Remove the flasks, add 2 mL of cell culture medium to stop the digestion, and carefully pipette the cells onto the flask walls until the flask walls become clear. Continue pipetting to mix the cell suspension thoroughly, then transfer equal portions to three 1 mL centrifuge tubes and centrifuge. Discard the old culture medium, add 1 mL of culture medium to resuspend the cells, and count them. Dilute the cells to a density of 8 × 10⁻⁶. 4 Cells were added evenly to a 96-well plate at a concentration of 100 μL / mL. Each concentration was set up in 6 replicates. 100 μL of cell suspension was added to each well and incubated in a 37°C incubator for 24 h. DMSO stock solutions (containing 10% polyoxyethylene castor oil) with BDP-PLN concentrations of 10 μM, 100 μM, 200 μM, 500 μM, 1 mM, 2 mM, 4 mM, and 5 mM were prepared. 10 μL of the stock solution was diluted to 1 mL of culture medium, resulting in concentrations of 0.1 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 50 μM, respectively. The old culture medium was removed from the 96-well plates, and the plates were washed twice with PBS. Then, 100 μL of culture medium containing the corresponding concentration of BDP-PLN was added to each well. The 96-well plates were incubated for 24 h. After incubation, the old culture medium was removed with a pipette, and the plates were washed twice with PBS. Then, 10 μL of LCK-8 solution was added to each well, and the 96-well plates were incubated for another 1 h. The OD value of the solution at 450 nm was measured using a microplate reader. The cytotoxicity assay results of the BDP-PLN probe are shown below. Figure 12 As shown, the probe only exerts a killing effect on cells at high concentrations. At 40 μM, cell viability decreases to 90%. In actual experiments, the probe concentration is usually maintained around 10 μM. Therefore, the probe has good biosafety and can be considered a safe and effective probe.

[0072] (2) Construction of APAP-induced liver injury model at the cellular level A liver injury model was established by incubating hepatocytes with the classic antipyretic and analgesic drug acetaminophen (APAP) for 12 h. Due to the significant differences in the metabolic sensitivity of different cell lines to acetaminophen, the dose-response relationship of APAP was systematically assessed using the CCK-8 assay. The half-maximal inhibitory concentration (IC50) of APAP obtained by the CCK-8 assay was used as the concentration for subsequent modeling.

[0073] The specific experimental steps are as follows: The steps for cell plating are the same as above, diluting the cells to a density of 8 × 10⁻⁶. 4 Cells were added evenly to a 96-well plate at a concentration of 100 μL / mL. Each concentration was set up in 6 replicates. 100 μL of cell suspension was added to each well and incubated in a 37°C incubator for 24 h. Culture media containing different concentrations of APAP were prepared, with final APAP concentrations of 20 mM, 10 mM, 5 mM, 2.5 mM, 1.5 mM, 0.5 mM, and 0 mM. After removing the old culture medium from the 96-well plates and washing twice with PBS, 100 μL of the APAP-containing medium was added to each well, and the plates were incubated at 37°C for 12 h. After incubation, the old culture medium was removed by pipetting and the plates were washed twice with PBS. Then, 10 μL of CCK-8 solution was added to each well, and the 96-well plates were incubated for another 1 h. The OD value of the solution at 450 nm was then measured using a microplate reader. The cytotoxicity of APAP to hepatocytes is shown in the figure below. Figure 13 As shown. The IC50 of APAP on AML-12 and HepG2 hepatocytes. 50 The values ​​were 5.46 mM and 10.14 mM, respectively. Based on this, subsequent cell experiments used the corresponding IC50 values ​​for the cell lines. 50 Damage modeling is performed based on concentration.

[0074] (3) Confirmation of the optimal response time of probe BDP-PLN To clarify the cellular response kinetics of the probe BDP-PLN to acute liver injury and determine its optimal detection time window, based on a successful hepatocyte injury model induced by acetaminophen (APAP), the changes in fluorescence signal at different time points after probe addition were systematically investigated. Real-time monitoring was conducted to determine the response of the probe to endogenous peroxynitrite (ONOO) after internalization into cells. - The experiment revealed the dynamic process of the activation of the probe signal by the cascade of leucine aminopeptidase (LAP), thus revealing the temporal characteristics of the probe signal activation. This experiment directly determined the key time point at which the probe generates the strongest specific signal-to-noise ratio at the cellular level.

[0075] 1) The cell plating experiment was the same as above. Based on the modeling concentration obtained above, culture media containing different concentrations of APAP were prepared (5.46 mM and 10.14 mM, respectively). The old culture medium was aspirated with a pipette, washed twice with PBS, and then the culture medium containing APAP was added. The medium was then incubated in a 37°C incubator for 12 h. 2) Prepare a culture medium containing 10 μM BDP-PLN (containing 0.1% polyoxyethylene castor oil). Use a pipette to remove the old culture medium, wash twice with PBS, and then add the above culture medium. Set different uptake times of 24 h, 3 h, 2 h, 1 h, 0.5 h, and 0 h, and incubate in a constant temperature incubator at 37℃. 3) After taking the old culture medium and washing it three times with PBS, add 100 μL of fresh culture medium and take a high content scanning photograph (BDP-PLN probe: excitation wavelength 633 nm, detection wavelength 650-760 nm).

[0076] The optimal response time of BDP-PLN in the liver injury model is as follows: Figure 14 As shown, the fluorescence intensity of BDP-PLN gradually increases with time, and does not significantly decrease within 24 hours, indicating that the probe has good stability. The quantification plot of fluorescence intensity versus time shows that the optimal response time of BDP-PLN in the liver injury model is 2 hours, indicating that the probe has good sensitivity and can achieve a good response effect in a short time. Therefore, 2 hours was selected as the response imaging time in subsequent tests.

[0077] (4) Validation of the response of the cell-level probe BDP-PLN to exogenous biomarkers To verify the core activation mechanism and specificity of the probe BDP-PLN under controlled conditions, different types and concentrations of key biomarkers (such as peroxynitrite ONOO) were directly introduced into the culture system. - (Leucine aminopeptidase LAP and related biological inhibitors) to simulate and analyze the response behavior of probes in complex intracellular environments.

[0078] The steps for cell plating are the same as above, diluting the cells to a density of 8 × 10⁻⁶. 4 Cells were added evenly to 96-well plates at a concentration of 100 μL / mL, with 6 replicates for each concentration. The plates were then incubated at 37°C for 24 h. Following the experimental design below, each group of cells underwent the following pretreatments (2 h each): 1) Group 1 (blank control): Normal culture, no irritants added; 2) Second group (ONOO) -Stimulation group): Add SIN-1 (ONOO) - Donor), final concentration 100 µM; 3) Group 3 (ONOO) - + LAP stimulation group): SIN-1 (100 µM) and exogenous LAP (1 U / L) were added sequentially. 4) Group 4 (Inhibitor Control I): Added UA (uric acid, ONOO) - (⁻ Scavenger, 100 µM) and BESRATIN (LAP inhibitor, 100 µM); 5) Group 5 (Inhibitor Control II): Added UA (uric acid, ONOO) - Cleaner, 100 µM); 6) Group 6 (Inhibitor Control III): BESTATIN (LAP inhibitor, 100 µM) was added.

[0079] All operations were performed in complete culture medium, and the cells were gently washed twice with PBS after treatment.

[0080] Complete culture medium containing 10 µM BDP-PLN (containing 0.1% polyoxyethylene castor oil) was prepared. After removing the liquid from each well, the cells were washed twice with PBS, and then the complete culture medium containing BDP-PLN was added. The cells were incubated at 37°C with 5% CO2 for 2 hours to allow for probe uptake and response. After incubation, the culture medium was discarded, and the cells were washed three times with PBS to remove any probes that had not entered the cells. 100 µL of fresh complete culture medium was added to each well, and high-content imaging was immediately performed. Imaging conditions were: excitation wavelength 633 nm, detection wavelength 650-760 nm. After image acquisition, ImageJ was used to quantitatively analyze the average fluorescence intensity of each group, comparing the differences in probe response under different treatment conditions. Results are as follows: Figure 15 As shown, after pretreatment with different exogenous stimuli, the fluorescence response of the probe BDP-PLN in AML-12 and HepG2 cells exhibited clear cascade activation characteristics and dose dependence.

[0081] Quantitative analysis showed: The second group only adds exogenous ONOO - Under the condition of donor SIN-1, the probe is subjected to exogenous ONOO. - In conjunction with endogenous LAP, it can trigger the activation of fluorescence signals, significantly enhancing the average fluorescence intensity. The third group, while simultaneously providing exogenous ONOO... -With the addition of exogenous LAP, the probe was dually activated by endogenous and exogenous LAP, resulting in further enhanced fluorescence intensity, and the average fluorescence intensity was significantly higher than that of the second group. Although the fourth group had exogenous ONOO... - The virus was present, but because BESTATIN effectively inhibited the activity of endogenous LAP in cells, the probe could not complete the cascade response, and the fluorescence signal was significantly suppressed, with an intensity similar to the background group. Groups five and six cleared endogenous ONOO through UA, respectively. - Alternatively, by inhibiting endogenous LAP through BESTATIN, the probes lack the necessary conditions for cascade activation, and the fluorescence signal remains at the background level.

[0082] The fluorescence images visually presented the following results: only the second and third groups of cells showed obvious red fluorescence, and the fluorescence intensity of the third group was higher than that of the second group; the fluorescence of the cells in the other groups was weak and indistinguishable from the background.

[0083] This result clearly confirms that the activation of the probe BDP-PLN at the cellular level depends on ONOO. - The probe interacts sequentially with LAP. Exogenous supplementation enhances the response, while the scavenging or inhibition of any key molecule effectively blocks signal output. This, from both positive and negative perspectives, comprehensively validates the probe "ONOO" at the cellular level. - → The specificity and necessity of the “LAP” cascade activation path provide direct evidence for its low background and high signal-to-noise ratio imaging performance.

[0084] (5) Endogenous response: Response test of BDP-PLN in APAP-induced liver injury model To verify whether the probe BDP-PLN can be absorbed by endogenous ONOO in a real acute liver injury pathological environment. - Activated in conjunction with the LAP cascade, and further clarifying its response specificity and pathological relevance, this study investigated the response behavior of probes to damage-induced endogenous biomarkers by selectively inhibiting endogenous targets in a drug-induced liver injury (DILI) cell model.

[0085] AML-12 and HepG2 cells were mixed at a ratio of 8 × 10⁻⁶. 4 100 μL of APAP was seeded at a density of 1 / mL in 96-well plates and cultured for 24 h until adherence. The old culture medium was then discarded. Except for the first group (blank control), which received PBS, all other groups were replaced with complete culture medium containing the modeling concentration of APAP and incubated for another 12 h to establish the acute liver injury model. After APAP treatment, the culture medium was discarded, and the plates were washed twice with PBS. The following groups underwent inhibitor pretreatment for 2 h (all in complete culture medium): 1) Group 1 (blank control): Continue normal culture; 2) Group 2 (APAP injury group): No inhibitor treatment was given; 3) Group 3 (APAP+UA group): Add UA (uric acid, ONOO) - Cleaner, 100 μM); 4) Group 4 (APAP+BESTATIN group): BESTATIN (LAP inhibitor, 100 μM) was added. 5) Group 5 (APAP + 100 μM NAC pretreatment group): The APAP model was first established after pretreatment with 100 μM NAC (N-acetylcysteine, 10 mM) for 2 h. 6) Group 6 (APAP + 200 μM NAC pretreatment group): The APAP model was first established after pretreatment with 200 μM NAC (N-acetylcysteine, 10 mM) for 2 h.

[0086] After pretreatment, the cells were washed twice with PBS and then incubated for another 2 hours with medium containing 10 μM BDP-PLN (containing 0.1% polyoxyethylene castor oil). The medium was then discarded, the cells were washed three times with PBS, and 100 μL of medium was added to each well. A high-intrinsicity imaging system was used to scan (excitation wavelength 633 nm, detection wavelength 650-760 nm) to acquire fluorescence images and quantitatively analyze the average fluorescence intensity. The endogenous response of BDP-PLN was as follows: Figure 16 As shown, after appropriate treatment, the intracellular BDP-PLN probe fluorescence signal was detected using a high-content imaging system. The quantitative analysis of the mean fluorescence intensity (MFI) data and the statistical analysis results are as follows: In the first group, both AML-12 and HepG2 cells showed only extremely weak basal fluorescence signals, with no significant difference in average fluorescence intensity compared to background fluorescence levels. This result indicates that under normal physiological conditions, endogenous ONOO in cells... - At extremely low levels, the BDP-PLN probe remained in a stable "off" state. Compared to the first group, both cell types showed significant fluorescence activation after APAP treatment. This result confirms that APAP successfully induced an acute liver injury model, triggering intracellular ONOO. - The sharp increase in levels and effective activation of downstream LAP enzymes successfully activated the BDP-PLN cascade response. The third group added UA (ONOO). - After removal of the scavenger, the strong fluorescence signal induced by APAP was effectively blocked. The average fluorescence intensity was significantly lower than that of the second group (damaged group), and there was no significant difference compared to the first group. This result directly proves that removing endogenous ONOO... - This is sufficient to interrupt the entire fluorescence initiation cascade reaction, verifying ONOO. -This is the crucial first trigger signal in the activation process of the probe. After treatment with BESTATIN (a specific inhibitor of LAP), the fluorescence signal in the fourth group also showed a significant decrease, with the average fluorescence intensity significantly lower than that of the damaged group. However, its signal intensity was still slightly higher than that of the UA group and the first group. This result indicates that inhibiting LAP enzyme activity can effectively block ONOO. - The downstream probe activation step, but due to a small amount of endogenous ONOO - The fluorescence itself was not eliminated, and its upstream signals remained, partially explaining the residual weak fluorescence. The results of groups five and six showed that cells pre-protected with NAC exhibited significantly lower fluorescence intensity compared to the damaged group. This indicates that NAC, through its antioxidant effects, partially alleviated APAP-induced oxidative stress, thereby inhibiting ONOO. - The generation and subsequent reactions of [the virus / organism]. The fluorescence signal attenuation in group 6 was significantly better than that in the low-dose NAC group. This result indicates a dose-dependent hepatoprotective effect of NAC. High-dose NAC can more effectively maintain intracellular redox balance and almost completely prevent APAP-triggered ONOO. - And LAP cascade activation.

[0087] The above experimental results show that: ① Probe activation is pathologically specific: The fluorescence activation signal of the BDP-PLN probe is strictly dependent on endogenous ONOO in the APAP liver injury model. - It is upregulated in the cascade with LAP and remains silent in normal cells.

[0088] ② The response mechanism was verified: Blocking experiments with a specific scavenger (UA) and an inhibitor (BESTATIN) confirmed that the probe activation follows the "ONOO" pattern. - The cascading mechanism of "trigger → LAP activation" is triggered.

[0089] ③ The effects of hepatoprotective drugs can be monitored: the antioxidant NAC can dose-dependently inhibit this fluorescence response, proving that this probe system can be used to intuitively and quantitatively evaluate the intervention effects of hepatoprotective drugs.

[0090] In conclusion, the BDP-PLN probe demonstrates high reliability and application potential in an APAP-induced acute liver injury model, and can serve as a visual indicator of endogenous ONOO. - The specific molecular tools of LAP provide strong support for the study of liver injury mechanisms and drug screening.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dual-lock cascade activation fluorescent probe BDP-PLN, characterized in that: The dual-lock cascade activated fluorescent probe uses a fluoroboron dipyrrole derivative as the fluorescent group and integrates a dual-lock recognition module composed of a leucine peptide unit and a p-nitrophenylglyoxylic acid unit. Its chemical structure is shown below: 。 2. A method for preparing the dual-locked cascade activated fluorescent probe BDP-PLN as described in claim 1, characterized in that: The probe was prepared from compounds BDP-P and Br-Ph-LN, and the structural formulas of BDP-P and Br-Ph-LN are as follows: ； 。 3. The method according to claim 2, characterized in that: The specific steps for compound BDP-P are as follows: (1) 3.5 mmol of 4-pyridinecarboxaldehyde and 7.0 mmol of 2,4-dimethylpyrrole were dissolved in dichloromethane, and 0.08 mmol of trifluoroacetic acid was added. The mixture was stirred for 3 days under a nitrogen atmosphere. Then, 1.2 mmol of 2,3-dichloro-5,6-dicyanobenzoquinone was added and the mixture was stirred for 3-5 h. The mixture was then transferred to an ice bath, and 10 mL of triethylamine and 10 mL of boron trifluoride ether were added dropwise. The mixture was stirred at room temperature for 10-14 h. After filtration, rotary evaporation, extraction, drying and silica gel column chromatography purification, compound 1 was obtained. (2) Add 11.74 mmol piperidine, 17.4 mmol glacial acetic acid and 0.03 mmol anhydrous magnesium perchlorate to toluene containing 1 mmol of compound 1 and 8 mmol of 4-[2-[2-(2-methoxyethoxy)ethoxy]benzaldehyde, heat to 140~160℃ and react for 1~3 h. After the reaction is completed, concentrate the reaction solution, add saturated sodium bicarbonate solution, and extract with dichloromethane and water. Collect the organic phase, and obtain compound BDP-P after drying and purification by rotary evaporation silica gel column chromatography.

4. The method according to claim 2, characterized in that: The specific steps for Br-Ph-LN are as follows: (1) An equimolar amount of dichloromethane containing Boc-L-leucine and dichloromethane containing N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea were mixed in an ice bath and stirred for 0.5 h. An equimolar amount of 4-aminobenzyl alcohol was added, and two times the molar amount of N,N-diisopropylethylamine was added dropwise. The mixture was heated to room temperature and reacted for 24 h. The reaction solution was extracted with a mixture of dichloromethane and saturated sodium bicarbonate aqueous solution. The organic phases were combined, dried, filtered under reduced pressure, evaporated to dryness, and purified by silica gel column chromatography to obtain compound 2. (2) Under nitrogen protection, 4 mL of trifluoroacetic acid was added to anhydrous dichloromethane containing 3.0 mmol of compound 2. After stirring at room temperature for 3 h, the mixture was concentrated under vacuum. The residue was extracted with a mixture of dichloromethane and saturated sodium bicarbonate aqueous solution. The organic phases were combined, dried, filtered under reduced pressure, evaporated to dryness, and purified by silica gel column chromatography to obtain compound 3. (3) Under ice bath conditions, p-nitrophenylacetaldehyde acid and an equimolar amount of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea were stirred and dissolved in anhydrous dichloromethane. An equimolar amount of compound 3 and two times the molar amount of N,N-diisopropylethylamine were added, and the mixture was heated to room temperature for 1-3 h. The reaction solution was extracted with a mixed solution of dichloromethane and saturated sodium bicarbonate aqueous solution. The organic phases were combined, dried, filtered under reduced pressure, evaporated to dryness, and purified by silica gel column chromatography to obtain compound 4. (4) Add an equimolar amount of phosphorus tribromide to anhydrous dichloromethane containing compound 4, stir for 0.5-1 h under ice bath conditions, then concentrate under vacuum, extract the residue with a mixture of dichloromethane and saturated sodium bicarbonate aqueous solution, combine the organic phases, dry, filter under reduced pressure, evaporate to dryness, and purify by silica gel column chromatography to obtain Br-Ph-LN.

5. The method according to claim 4, characterized in that: The structural formulas of compounds 2-4 are shown below: 、 、 。 6. The method according to claim 2, characterized in that: The specific steps are as follows: Equimolar amounts of BDP-P and Br-Ph-LN were stirred at 80~90℃ for 24~48 h, followed by vacuum concentration. The residue was extracted with a mixed solution of dichloromethane and saturated sodium bicarbonate aqueous solution. The organic phases were combined, dried, filtered under reduced pressure, evaporated to dryness, and purified by silica gel column chromatography to obtain the double-locked cascade activated fluorescent probe BDP-PLN.

7. The dual-locked cascade activated fluorescent probe BDP-PLN as described in claim 1, used in the preparation of qualitative or quantitative detection of ONOO - Applications of leucine aminopeptidase reagent.

8. The application of the dual-lock cascade activated fluorescent probe BDP-PLN as described in claim 1 in the preparation of reagents for detecting acute liver injury.

9. The application of the dual-locked cascade activated fluorescent probe BDP-PLN as described in claim 1 in the preparation of a reagent for detecting drug-induced liver injury.

10. The application of the dual-lock cascade activated fluorescent probe BDP-PLN as described in claim 1 in the preparation of a fluorescent recognition and imaging reagent for acute liver injury.

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