A triazine-based fluorescent probe compound YJA, its preparation method and application

Abstract

This invention discloses a triazine-based fluorescent probe compound YJA, its preparation method, and its applications. The chemical name is 4'-(4,6-diphenyl-1,3,5-triazine-2-yl)-[1,1'-biphenyl]-4-yldiphenylphosphine ester, and the molecular formula is C2. 39 H 28 N3O2P. This invention prepares a novel triazine ring-based fluorescent probe, YJA, which exhibits advantages such as ultra-low detection limit (0.73 nM), rapid response, excellent selectivity, good pH stability (pH 4-11), and low cytotoxicity. It has been successfully applied to exogenous ONOO in HeLa cells. ‑ Fluorescence imaging and ONOO based on test strips ‑ Simple, visual, and rapid detection. The preparation method of this fluorescent probe is simple, uses readily available raw materials, and yields a white solid product that is easy to store. It has broad application prospects and can be mass-produced and applied.

Description

Technical Field

[0001] This invention belongs to the fields of analytical chemistry, biosensing and fluorescent probe technology, specifically relating to a triazine-based fluorescent probe compound YJA, its preparation method and application. Background Technology

[0002] peroxynitrite (ONOO) - ) is composed of nitric oxide (NO) and superoxide anion (O2·) - ONOO is a highly reactive nitrogen species (RNS) generated at a near-diffusion-controlled rate. It possesses extremely strong oxidizing and nitrifying capabilities and participates in key processes such as cell signal transduction and immune defense at physiological concentrations. However, its excessive production is closely related to oxidative stress and has been proven to be an important pathological factor in many major diseases, including neurodegenerative diseases (such as Alzheimer's and Parkinson's), inflammatory diseases, atherosclerosis, diabetic complications, and the occurrence and development of various cancers. Most importantly, ONOO is a significant component of tumor microenvironments. - The concentration of [a specific substance] is much higher than in normal tissues, making it a potential biomarker for tumor identification and diagnosis. Therefore, achieving [a specific biomarker] in vivo is crucial. - Real-time, in-situ, and highly sensitive horizontal monitoring is of great significance for a deeper understanding of its role in physiological and pathological processes and for developing diagnostic strategies for related diseases.

[0003] Due to ONOO - Fluorescent probes, characterized by extremely low concentrations (nanomolar levels), extremely short half-lives (approximately 10 milliseconds), and extremely high chemical activity in living organisms, present significant challenges for detection. Traditional detection methods, such as high-performance liquid chromatography (HPLC) and electrochemical analysis, typically require complex sample pretreatment, making real-time, in-situ, and non-invasive in vivo detection difficult. In contrast, small-molecule-based fluorescent probe technology, with its unique advantages of high sensitivity, high spatiotemporal resolution, ease of operation, real-time in-situ imaging, and minimal interference with biological samples, has become a promising approach for research on ONOO. - One of the most powerful tools for biological function.

[0004] Currently, there are multiple [targets / measures] for ONOO. - Fluorescent probes have been developed, and their design strategies are mainly based on ONOO. - Strong nucleophilicity, strong oxidizing properties, and specific reactions with specific functional groups. Common recognition groups include: α,β-unsaturated ketones, borates (esters), hydrazones, organoselenium / tellurium compounds, aryl oxime ethers, etc. Despite significant progress, existing probes still generally have some limitations, such as: (1) insufficient selectivity: susceptible to other reactive oxygen / nitrogen species (such as H2O2, ClO2, etc.). - NO2 -(2) Slow response speed: difficult to capture ONOO - (3) Instantaneous dynamic changes; (4) Background fluorescence interference or insufficient detection limit, affecting the detection of low concentrations of ONOO. - (4) The synthesis steps are complex or the photostability is poor, which limits its practical application.

[0005] Triazine rings, as aromatic heterocycles with excellent optical properties, possess conjugated structures that endow them with good fluorescence characteristics. Their structures are also easily modified, making them ideal matrixes for constructing fluorescent probes. However, currently, there are no known methods for developing ONOO probes that use triazine rings as a fluorescent core combined with specific recognition units. - Related reports on fluorescent probes. Therefore, the development of a novel ONOO probe with higher selectivity, faster response speed, lower detection limit, good biocompatibility, and simple synthesis is desired. - Fluorescent probes remain a pressing need in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing peroxynitrite fluorescent probes, such as insufficient selectivity, slow response speed, susceptibility to in vivo interference, and high detection limits. This invention provides a triazine-based fluorescent probe compound YJA with high selectivity, rapid response, high sensitivity, wide pH applicability, and good biocompatibility, along with its preparation method and applications, enabling the detection of peroxynitrite (ONOO). - This method provides a specific, real-time, and visual detection of nitrite, and has been successfully applied to fluorescence imaging of endogenous / exogenous peroxynitrite in living cells. Its application is particularly significant in complex biological systems (such as living cells) and environmental samples. It offers a simple, efficient, and reliable detection method for the study of physiological and pathological processes related to peroxynitrite and the detection of disease biomarkers. The method has a simple route, readily available raw materials, mild reaction conditions, and good yield, making it suitable for large-scale preparation.

[0007] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0008] A triazine-based fluorescent probe compound, YJA, has the chemical name 4'-(4,6-diphenyl-1,3,5-triazine-2-yl)-[1,1'-biphenyl]-4-yldiphenylphosphine ester and the molecular formula C2. 39 H 28 N3O2P, with the following structure The molecular structure features a 4,6-diphenyl-1,3,5-triazine group as its fluorescent core, linked by a biphenyl bridge to a phenolic hydroxyl group protected by a diphenylphosphonate ester. This design cleverly combines the triazine group (acting as a strong electron acceptor, providing stable fluorescence) with the diphenylphosphonate ester (acting as a counter-fluorescent group). -Highly specific reaction sites are integrated into one.

[0009] The preparation method of the above-mentioned triazine-based fluorescent probe compound YJA includes the following steps:

[0010] Step 1, Synthesize intermediate 1:

[0011] 4-(4,6-diphenyl-1,3,5-triazin-2-yl)phenylboronic acid, 4-hydroxybromobenzene, potassium carbonate, and tetra(triphenylphosphine)palladium (0) were dissolved in a tetrahydrofuran / water mixed solvent in a molar ratio of 1:1.2:3:0.03. The mixture was refluxed at 65-75°C for 12 hours under a nitrogen atmosphere. After the reaction was completed, the solvent was removed by vacuum distillation at 45-55°C. The crude product was purified by silica gel column chromatography to obtain a white solid intermediate 1, namely 4'-(4,6-diphenyl-1,3,5-triazin-2-yl)-[1,1'-biphenyl]-4-ol.

[0012] Step 2, Synthesis of compound YJA: Intermediate 1 and diphenylphosphonic chloride were dissolved in anhydrous haloalkane solvent at a molar ratio of 1:1.2. Then an organic base was added, and the reaction was stirred at 20-30℃ for 8 hours. The reaction was monitored by thin-layer chromatography until the end of the reaction. The solvent was removed by vacuum distillation at 45-55℃. The crude solid obtained was purified by silica gel column chromatography to obtain white solid YJA.

[0013] Preferably, in step 1, the volume ratio of the tetrahydrofuran / water mixed solvent is 2-4:1; and the reaction temperature is 70°C.

[0014] Preferably, in step 1, the volume ratio of the tetrahydrofuran / water mixed solvent is 3:1.

[0015] Preferably, the organic base is triethylamine or N,N-diisopropylethylamine; and the anhydrous halocarbon solvent is dichloromethane or chloroform.

[0016] Preferably, in step 2, the organic base is triethylamine; and the anhydrous halocarbon solvent is dichloromethane.

[0017] More preferably, the organic base in step 2 is triethylamine, and the anhydrous halocarbon solvent is anhydrous dichloromethane.

[0018] The above-mentioned triazine-based fluorescent probe compound YJA is used as a probe for detecting peroxynitrite ions or exogenous peroxynitrite ions in water samples. The water sample is a mixture of dimethyl sulfoxide and water at a volume ratio of 5:5-7:3, and adjusted with phosphate buffer at pH 7.4.

[0019] As an improvement, the detection of compound YJA is based on a fluorescence quenching mechanism, with a detection limit of 0.5 nM to 1.0 nM in a 0.2 mM PBS buffer system with a dimethyl sulfoxide to water volume ratio of 6:4 and pH 7.4.

[0020] As an improvement, a triazine-based fluorescent probe is used to detect ONOO. - The detection limit was 0.73 nM.

[0021] A test strip comprising a porous substrate material and a triazine-based fluorescent probe compound YJA as described in claim 1, loaded on the porous substrate material, for use in ONOO. - Rapid, visual, and semi-quantitative detection.

[0022] The reaction formula is shown below:

[0023] principle:

[0024] This invention uses a triazine ring as the fluorescent signal core and diphenylphosphine ester as the ONOO. - A novel triazine-based fluorescent probe, YJA, was designed and synthesized using a specific recognition unit (the recognition site being the P–O–C bond between the diphenylphosphine ester and the benzene ring in the probe molecule). The ether bond of YJA itself serves as the reaction site. - This reaction can specifically induce the cleavage of the ether bond, leading to the decomposition of the probe molecule. At the electronic structure level, the energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the probe decreases after the reaction, weakening the intramolecular charge transfer process and ultimately triggering rapid fluorescence quenching. This significant decrease in fluorescence intensity achieves the quenching of ONOO... - Specific detection. Based on this specific reaction, the probe targets ONOO. - It exhibits excellent selectivity and significant fluorescence quenching effect. Figure 10 The fluorescent probes YJA and ONOO prepared in Example 1 - ONOO's response mechanism. - The specificity induces the breakage of ether bonds in the YJA molecule, leading to the decomposition of the probe molecule structure. Based on density functional theory calculations, the HOMO-LUMO band gap of the probe decreases from 4.0307 eV to 3.7793 eV after the reaction. The intramolecular charge transfer process is weakened, ultimately triggering rapid fluorescence quenching and achieving the desired effect for ONOO. - The specificity of this reaction for ONOO. - It exhibits high specificity; common interfering substances in biological systems were not observed to induce this structural change under these experimental conditions, which is also why YJA showed no significant difference in ONOO. - The reason for its high selectivity. Beneficial effects

[0025] Compared with existing technologies, the present invention provides a triazine-based fluorescent probe compound YJA, its preparation method, and its application, which have the following advantages:

[0026] 1. Extremely high sensitivity: for ONOO - The detection limit (LOD) reaches 0.73 nM, enabling nanomolar-level ONOO detection in biological systems. - Accurate detection.

[0027] 2. Excellent selectivity and anti-interference capability: The probe is compatible with ONOO. - Its response signal far exceeds that of other common interfering substances, including H2O2, ·OH, and ClO. - NO2 - Biothiols (Cys, GSH) and various metal ions (Fe) 2+ / 3+ Cr 3+ Hg 2+ Cd 2+ (etc.). Even under conditions where multiple interfering factors coexist, it can still maintain control over ONOO. - Specific identification.

[0028] 3. Rapid Response: With ONOO - The reaction reaches equilibrium within 1 minute, satisfying the ONOO requirement. - This necessitates real-time, dynamic monitoring of transient molecules.

[0029] 4. Wide pH adaptability: It maintains stable sensing performance over a wide pH range of 4.0-11.0, and performs best near the physiological pH of 7.4, making it ideal for complex biological systems.

[0030] 5. Excellent biocompatibility and practicality: Cytotoxicity experiments show that the probe exhibits extremely low cytotoxicity at effective working concentrations. It has been successfully applied to exogenous fluorescence imaging in HeLa live cells, demonstrating its excellent cell membrane permeability and potential for biological applications.

[0031] 6. Multi-mode applications: In addition to solution detection and cell imaging, it can be easily made into fluorescent test strips. These strips exhibit a visible fluorescence color change (from blue to colorless / yellow) under ultraviolet light in the presence of UV light, enabling on-site, rapid, and visual semi-quantitative detection without the need for complex instruments, thus expanding its application scenarios.

[0032] 7. Simple synthesis: The preparation method involves only two steps, the raw materials are readily available, the reaction conditions are mild, the yield of intermediate 1 is 90%, the yield of the target product YJA is 40%, the overall yield is high, and it has the potential for large-scale preparation, which is conducive to the further promotion and application of this probe. Attached Figure Description

[0033] Figure 1 Different solvents were used to treat YJA-ONOO in Example 1. - The graph shows the effect of the mixture on fluorescence intensity, where the horizontal axis represents the solvent type and the vertical axis represents the fluorescence intensity. DMSO / H2O=6:4 (v / v) is the optimal detection solvent system.

[0034] Figure 2 The fluorescent probe YJA prepared in Example 1 was reacted with ONOO in a DMSO / H2O (v / v=6:4) solution. - Comparison of UV-Vis absorption spectra after treatment;

[0035] Figure 3 The fluorescent probe YJA prepared in Example 1 exhibits selective fluorescence spectra for different reactive oxygen species, metal ions, and biological reducing agents in DMSO / H2O (v / v=6:4) solution.

[0036] Figure 4 The fluorescent probe YJA prepared in Example 1 was used to target ONOO in a DMSO / H2O (v / v=6:4) solution. - Anti-interference fluorescence response diagram;

[0037] Figure 5 The fluorescent probe YJA prepared in Example 1 was used to target ONOO in a DMSO / H2O (v / v=6:4) solution. - The fluorescence titration spectrum (interpolated as a linear relationship between concentration and fluorescence intensity);

[0038] Figure 6 The fluorescent probe YJA prepared in Example 1 was used to detect the activity of ONOO at different pH values. - The response effect;

[0039] Figure 7 The fluorescent probe YJA prepared in Example 1 was used to target ONOO in a DMSO / H2O (v / v=6:4) solution. - Response time versus fluorescence intensity variation graph;

[0040] Figure 8 The image shows fluorescence imaging of the fluorescent probe YJA in HeLa cells in Example 1. A is a bright-field image, B is the fluorescence image of the YJA-incubated group, and C is the fluorescence image of YJA+ONOO. - Fluorescence image of the incubation group;

[0041] Figure 9 This is a test of the sensitivity of the fluorescent probe YJA prepared in Example 1 on paper.

[0042] Figure 10 The fluorescent probes YJA and ONOO prepared in Example 1 - Response mechanism;

[0043] Figure 11 The MS mass spectrum of compound 1 obtained in Example 1;

[0044] Figure 12 The nuclear magnetic resonance of the fluorescent probe YJA prepared in Example 1 in CDCl3. 1 H-NMR spectrum;

[0045] Figure 13 The nuclear magnetic resonance of the fluorescent probe YJA prepared in Example 1 in CDCl3. 13 C-NMR spectrum;

[0046] Figure 14 The images show the 1H-NMR, 13C-NMR, and HRMS spectra of the fluorescent probe YJA prepared in Example 1.

[0047] Figure 15 High-resolution mass spectra of the fluorescent probe YJA prepared in Example 1;

[0048] Figure 16 The fluorescent probes YJA and ONOO prepared in Example 1 - High-resolution mass spectrometry of the mixture. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the experiments are commercially available. All reagents used in the embodiments are commercially available analytical grade or chemically pure.

[0050] In the embodiments, the various active oxygen solutions and metal ion solutions were prepared according to conventional methods. The active oxygen solutions were prepared and used immediately, and the metal ion solutions were prepared by dissolving chloride salts with a purity of 99% or higher in deionized water and adjusting the volume.

[0051] Example 1: Preparation of the triazine ring parent fluorescent probe YJA:

[0052] Preparation of intermediate 1:

[0053] 4-(4,6-diphenyl-1,3,5-triazin-2-yl)phenylboronic acid (400 mg, 1.03 mmol), 4-hydroxybromobenzene (272 mg, 1.24 mmol), potassium carbonate (426 mg, 3.09 mmol), and tetra(triphenylphosphine)palladium(0) (54 mg, 0.031 mmol) were dissolved in a tetrahydrofuran / water mixture (THF / H2O = 3:1, v / v, 24 mL) and refluxed at 70 °C for 12 hours under a nitrogen atmosphere. After the reaction was completed, the solvent was removed by vacuum distillation at 45 °C, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate v / v = 5:1) to give a white solid intermediate 1. The name is 4'-(4,6-diphenyl-1,3,5-triazin-2-yl)-[1,1'-biphenyl]-4-ol, with a yield of 90%.

[0054] 1 H NMR (400 MHz, CDCl3)δ8.75 (d, J=7.8 Hz,4H), 8.02 (d,J=8.2 Hz, 2H), 7.76 (t, J=7.5 Hz, 2H), 7.65 (t, J=7.3 Hz, 1H), 7.58 (d, J=8.4 Hz, 2H), 7.01(d, J=8.4 Hz, 2H), 5.21 (s,1H,-OH); 13 C NMR (101 MHz, CDCl3)δ171.82, 156.94, 151.05, 144.21, 139.87,136.83, 132.68, 130.25, 129.53, 128.86, 128.73, 127.12, 121.30, 115.67; HRMS (ESI): calcd for [M+H] + 402.1606, found 402.1599.

[0055] Preparation of compound YJA:

[0056] Intermediate 1 (116 mg, 0.29 mmol) and diphenylphosphonic chloride (67 μL, 0.35 mmol) were dissolved in anhydrous dichloromethane (5 mL), followed by the addition of triethylamine (0.05 mL). The reaction was stirred at room temperature for 8 hours, and the reaction progress was monitored by silica gel thin-layer chromatography (TLC) until the reaction was complete. After the reaction was completed, the solvent was removed by vacuum distillation at 45 °C. The crude solid obtained was purified by silica gel column chromatography (petroleum ether / ethyl acetate v / v = 5:1) to give a white solid YJA, 4'-(4,6-diphenyl-1,3,5-triazin-2-yl)-[1,1'-biphenyl]-4-yldiphenylphosphonate. Yield: 40%.

[0057] 1 H NMR (400 MHz, CDCl3)δ8.76 (dd, J=8.3, 1.7 Hz, 6H), 7.96 (dd, J=12.6,6.8 Hz, 4H), 7.67 (d, J=8.4 Hz, 2H), 7.58 (t, J=8.7 Hz, 10H), 7.52 (dd,J=7.7,3.7 Hz, 4H), 7.34 (d,J=7.4 Hz,2H); 13 C NMR (101 MHz, CDCl3)δ171.66, 171.36, 151.07, 150.99, 144.19,136.85, 136.31, 135.14, 132.70, 132.62, 132.01, 131.91, 131.64, 130.27,129.55, 129.06, 128.88, 128.75, 128.63, 127.14, 121.32, 121.27; HRMS (ESI): calcd for [M+H] + 602.1997; found 602.1990.

[0058] The MS mass spectrum of intermediate 1 obtained in Example 1 is as follows: Figure 11 The triazine ring parent fluorescent probe YJA 1 H-NMR spectrum, 13 The C-NMR spectrum and HRMS mass spectrum are respectively Figure 12 , Figure 13 , Figure 15 Triazine ring parent fluorescent probe YJA and ONOO - The HRMS mass spectrum of the mixture is as follows Figure 16 .

[0059] Example 2

[0060] The triazine ring precursor fluorescent probe YJA prepared in Example 1 was subjected to a series of performance testing experiments to evaluate its activity against ONOO. - The sensitivity, selectivity, response time, pH stability, and anti-interference ability of the detection were all tested. All experiments were conducted at room temperature (25°C), and all solutions were prepared and used immediately.

[0061] Testing the effects of different solvents on YJA-ONOO - To investigate the effect of the mixture's fluorescence intensity, six commonly used solvents were prepared: methanol (MeOH), ethanol (EtOH), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and acetonitrile (MeCN), along with a peroxynitrite stock solution (prepared by mixing 3 mL of 0.6 mol / L NaNO2 solution, 1.5 mL of 0.7 mol / L H2O2 solution, and 1.5 mL of 0.6 mol / L HCl solution under ice bath conditions, followed by the rapid addition of 3 mL of 1.5 mol / L NaOH solution to obtain the peroxynitrite stock solution). The peroxynitrite detection fluorescent probe prepared in Example 1 was prepared into a 1 mM probe stock solution using DMSO. 30 μL of the probe stock solution and 26 μL of peroxynitrite (ONOO) were added to every 3 mL of solvent. - The solution was prepared, and then the fluorescence intensity was measured under the same conditions (λex = 330 nm, λem = 408 nm). The results are as follows. Figure 1 As shown, the YJA fluorescent probe exhibits the strongest fluorescence intensity in DMSO, and the fluorescence quenching is most pronounced upon the addition of the detection ion peroxynitrite. Therefore, DMSO has been identified as the optimal solvent for effectively maintaining the fluorescence intensity of YJA.

[0062] For probe YJA and ONOO - The UV-Vis absorption spectra after treatment were compared. Two cuvettes were used, with 3 mL of blank solution DMSO and 30 μL of 1 mM probe stock solution added to one cuvette, and 30 μL of peroxynitrite stock solution added to the other cuvette. UV spectroscopy was then performed. The graph shows that the fluorescent probe has a characteristic absorption peak at 325 nm, similar to ONOO. - After treatment, a significant spectral change was observed: the absorption peak at 325 nm weakened, while a new absorption peak appeared at 475 nm, showing a significant red shift, proving that YJA interacts with ONOO. - A specific reaction occurred.

[0063] To obtain the selective fluorescence spectra of probe YJA for different reactive oxygen species, metal ions, and bioreducing agents, 30 μL of YJA probe stock solution (10 μM) and 50 μL of various reactive oxygen species, bioreducing agents, and metal ion stock solutions (·OH, H2O2, ClO) were added to 3 mL of blank solution DMSO-H2O (v / v=6:4). - reactive oxygen species such as O2, Fe 2+ Fe 3+ Cr 3+ Hg 2+ Cd 2+ (e.g., metal ions, bio-reducing agents such as Cys and Glu), results are as follows Figure 3 As shown in the figure. It can be seen from the figure that only adding ONOO... - At that time, the fluorescence intensity of YJA at 408 nm was significantly quenched, while the other interfering substances did not cause significant changes in fluorescence intensity, proving that YJA inhibits ONOO. - It has excellent selectivity.

[0064] To test probe YJA against ONOO - The fluorescence response map of the anti-interference test was obtained, and common interfering substances in the biological system were prepared: reactive oxygen species / reactive nitrogen species (·OH, H2O2, ClO). - , 1 O2, NO2 - t-BuOOH), small biomolecules (Cys, Glu), and metal ions (Fe) 2+ Fe 3+ Cr 3+ Hg 2+ Cd 2+ The probe stock solution had a concentration of 10 μM. The test procedure included a blank group and YJA+ONOO. - Group, YJA + single interfering agent group, YJA + ONOO - + Interference group. Add 30 μL of YJA probe stock solution and 50 μL of any other interfering substance stock solution to 3 mL of blank solution DMSO-H2O (v / v=6:4). Finally, add 50 μL of ONOO to the blank solution. - The stock solution was thoroughly mixed, and the fluorescence intensity was measured at λex = 330 nm and λem = 408 nm. The fluorescence changes of each group were compared to evaluate the anti-interference performance. Experimental results are only available at ONOO. - It can significantly quench the fluorescence of YJA, while other interfering substances cannot cause obvious fluorescence changes; under the condition of coexistence of interfering substances, YJA has a significant effect on ONOO. - The response is almost unaffected, indicating that the probe has excellent selectivity and anti-interference ability.

[0065] For probe YJA to ONOO - The fluorescence titration spectrum was obtained by dissolving the fluorescent probe YJA in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution, and adding peroxynitrite (ONOO). - Prepare freshly with distilled water. The test system is DMSO / H2O = 6:4 (v / v), containing 0.2 mM PBS buffer, and the pH is adjusted to 7.4. Add the YJA stock solution to the test system to prepare a YJA test solution with a final concentration of 10 μM. Gradually add a gradient concentration of ONOO to this system. - Fluorescence emission spectra were collected. Instrument parameters were measured using a fluorescence spectrophotometer with excitation wavelength λex = 330 nm, emission wavelength λem = 408 nm, and excitation and emission slit widths of 3 nm each. The test temperature was room temperature (25℃). The fluorescence intensity at 408 nm was recorded as a function of ONOO. - The concentration change was analyzed, and a fluorescence titration curve was plotted. The results are shown in Figure 5. The detection limit was calculated using the formula LOD=3σ / k, and the detection limit of this probe YJA against ONOO was determined. - The detection limit was 0.73 nM. Experimental results showed that ONOO... - As the concentration gradually increased, the fluorescence intensity of the system at 408 nm gradually decreased, exhibiting a good concentration-dependent relationship. The linear correlation coefficient R² = 0.9700, indicating that the probe can effectively target ONOO. - Highly sensitive quantitative detection.

[0066] Test probe YJA at different pH values ​​for ONOO - To assess the response, the pH of the test system was adjusted using 0.1 mol / L HCl and 0.1 mol / L NaOH, with pH gradients of 4.0, 5.0, 6.0, 7.0, 7.4, 8.0, 9.0, 10.0, and 11.0. The test system consisted of DMSO / H2O = 6:4 (v / v) and 0.2 mM PBS. 30 μL of YJA probe stock solution and 50 μL of peroxynitrite stock solution were added to 3 mL of blank buffer solution DMSO-H2O (v / v = 6:4). The fluorescence intensity at 408 nm was measured, and a pH-fluorescence intensity curve was plotted with pH on the x-axis and fluorescence intensity on the y-axis. The results are as follows: Figure 6 As shown, the autofluorescence of YJA remains stable within the pH range of 4.0-11, without significant fluctuations; within the physiological pH range of 7.0-11, the probe exhibits stable fluorescence against ONOO. - It has a clear response and stable signal, making it suitable for use in biological fluids and cellular environments.

[0067] Test probe YJA against ONOO -The effect of response time on fluorescence intensity was investigated by adding 30 μL of YJA probe stock solution and 50 μL of peroxynitrite stock solution to 3 mL of blank buffer solution DMSO-H2O (v / v=6:4), and testing the initial fluorescence intensity; ONOO was then rapidly added. - Enable time-scan mode and continuously record the change in fluorescence intensity at 408 nm over time, observing the time required for the signal to stabilize. Add ONOO. - Subsequently, the fluorescence of the system rapidly decreased and stabilized within 1 minute, and the fluorescence signal remained unchanged for the next 30 minutes, indicating that the YJA probe has a rapid response and stable signal, making it suitable for real-time dynamic detection.

[0068] This is a fluorescence imaging experiment of the fluorescent probe YJA in HeLa cells. The cell line was HeLa cells (sourced from Servicebio, Wuhan, China); the working concentration of probe YJA was 10 μM; and exogenous ONOO... - Final concentration 100 μM; cell incubation temperature 37℃. Imaging procedure: HeLa cells were seeded in complete culture medium containing 30 μL YJA and incubated at 37℃ for 30 min. Cells were washed three times with PBS buffer to remove undeclared probes, and fluorescence images were acquired under a laser confocal fluorescence microscope. 50 μL of ONOO was added to the above cells. - The stock solution was incubated at 37°C for another 30 minutes, and fluorescence images were acquired again to compare changes in fluorescence intensity. The experimental results are as follows: Figure 8 As shown in the figure, YJA can efficiently penetrate the cell membrane, exhibiting bright blue fluorescence inside the cell; with the addition of ONOO... - Subsequently, intracellular fluorescence was significantly quenched, indicating that YJA can inhibit ONOO in living cells. - It enables specific fluorescence imaging detection and has good cell membrane permeability and biocompatibility.

[0069] To test the sensitivity of the fluorescent probe YJA on paper, the test strip was immersed in a DMSO / H2O stock solution (6:4 v / v) containing 1 mM of the fluorescent probe for half an hour. The strip was then removed and air-dried to obtain a dried test strip containing the probe. The test strips were then immersed in 0 mM, 0.75 mM, and 1.5 mM peroxynitrite solutions for a few minutes, air-dried, and then irradiated under a 365 nm UV lamp. The results are as follows. Figure 9 As shown, the test strip exhibits bright blue fluorescence when different concentrations of ONOO are added. - After solution, the fluorescence color changes with ONOO - As the concentration increases, the color gradually changes from blue to light blue and then to light yellow; this color change is visible to the naked eye, proving that the fluorescent probe YJA can achieve ONOO. -Rapid on-site visual inspection.

[0070] Figure 10 The fluorescent probes YJA and ONOO prepared in Example 1 - ONOO's response mechanism - The specificity induces the breakage of ether bonds in the YJA molecule, leading to the decomposition of the probe molecule structure. The highest occupied molecular orbital (HOMO) - lowest unoccupied molecular orbital (LUMO) band gap decreases from 4.0307 eV to 3.7793 eV, weakening the intramolecular charge transfer process and ultimately triggering rapid fluorescence quenching. This reaction is ONOO - This unique reaction, where common substances in other biological systems cannot induce this structural change, is also why YJA reacts to ONOO. - The reason for its high selectivity.

[0071] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A triazine-based fluorescent probe compound YJA, characterized in that, The chemical name of the triazine-based fluorescent probe compound YJA is 4'-(4,6-diphenyl-1,3,5-triazine-2-yl)-[1,1'-biphenyl]-4-yldiphenylphosphine ester. Molecular formula is C 39 H 28 N3O2P, structure is .

2. A process for the preparation of the triazine-based fluorescent probe compound YJA according to claim 1, characterized in that, Includes the following steps: Step 1, Synthesize intermediate 1: 4-(4,6-diphenyl-1,3,5-triazin-2-yl)phenylboronic acid, 4-hydroxybromobenzene, potassium carbonate, and tetra(triphenylphosphine)palladium (0) were dissolved in a tetrahydrofuran / water mixed solvent in a molar ratio of 1:1.2:3:0.

03. The mixture was refluxed at 65-75°C for 12 hours under a nitrogen atmosphere. After the reaction was completed, the solvent was removed by vacuum distillation at 45-55°C. The crude product was purified by silica gel column chromatography to obtain a white solid intermediate 1, namely 4'-(4,6-diphenyl-1,3,5-triazin-2-yl)-[1,1'-biphenyl]-4-ol. Step 2, Synthesis of compound YJA: Intermediate 1 and diphenylphosphonic chloride were dissolved in anhydrous haloalkane solvent at a molar ratio of 1:1.

2. Then an organic base was added, and the reaction was stirred at 20-30℃ for 8 hours. The reaction was monitored by thin-layer chromatography until the end of the reaction. The solvent was removed by vacuum distillation at 45-55℃. The crude solid obtained was purified by silica gel column chromatography to obtain white solid YJA.

3. The method for preparing the triazine-based fluorescent probe compound YJA according to claim 2, characterized in that, In step 1, the volume ratio of the tetrahydrofuran / water mixed solvent is 2-4:1; the reaction temperature is 70℃.

4. The method for preparing the triazine-based fluorescent probe compound YJA according to claim 3, characterized in that, In step 1, the volume ratio of the tetrahydrofuran / water mixed solvent is 3:

1.

5. The preparation method according to claim 2, characterized in that, In step 2, the organic base is triethylamine or N,N-diisopropylethylamine; the anhydrous halocarbon solvent is dichloromethane or chloroform.

6. The preparation method according to claim 5, characterized in that, The organic base mentioned in step 2 is triethylamine, and the anhydrous halocarbon solvent is anhydrous dichloromethane.

7. The application of the triazine-based fluorescent probe compound YJA according to claim 1 in a probe for detecting peroxynitrite ions or endogenous and exogenous peroxynitrite ions in water samples, characterized in that, The water sample was a mixture of dimethyl sulfoxide and water in a volume ratio of 5:5 to 7:3, and adjusted with a phosphate buffer solution with a pH of 7.

4.

8. The application according to claim 7, characterized in that, The detection of peroxynitrite ions by the compound YJA is based on a fluorescence quenching mechanism. In a 0.2 mM PBS buffer system with a dimethyl sulfoxide to water volume ratio of 6:4 and pH 7.4, the detection limit is 0.5 nM-1.0 nM.

9. The application according to claim 8, characterized in that, The detection limit for peroxynitrite ions in the water sample is 0.73 nM.

10. A test strip, characterized in that, It includes a porous substrate material and the triazine-based fluorescent probe compound YJA as described in claim 1, loaded on the porous substrate material.

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