A ratiometric fluorescent probe molecule for detecting peroxynitrite and a preparation method and application thereof

Abstract

This invention proposes a ratiometric fluorescent probe molecule for detecting peroxynitrite, its preparation method, and its application, aiming to solve a problem in the technical field of fluorescent sensing materials. The ratiometric fluorescent probe molecules of this invention are HPIP-T-BnB and HPIP-K-BnB. The structural formulas of HPIP-T-BnB and HPIP-K-BnB are: [Insert structural formula here], and the structural formulas of HPIP-K-BnB are: [Insert structural formula here]. The preparation method includes the following steps: 4'-(diphenylamino)-4-(imidazo[1,2-a]pyridin-2-yl)-[1,1'-biphenyl]-3-ol or 4'-(9H-carbazole-9-yl)-4-(imidazo[1,2-a]pyridin-2-yl)-[1,1'-biphenyl]-3-ol undergoes a nucleophilic substitution reaction with 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborhecyclopentane to obtain HPIP-T-BnB and HPIP-K-BnB. The probes prepared in this invention can detect ONOO⁻ with high selectivity, high sensitivity, and quantification, and have been successfully applied to ratiometric fluorescence imaging of exogenous and endogenous ONOO⁻ in live cells. This provides a reliable and crucial tool for mechanism research and drug development in related diseases.

Description

Technical Field

[0001] This invention belongs to the technical field of fluorescent sensing materials, and particularly relates to a fluorescent probe molecule. Background Technology

[0002] peroxynitrite (ONOO) - ) are nitric oxide (NO) and superoxide anion (O2•) in living organisms. - ONOO is a highly reactive oxidizing and nitrating molecule produced through a rapid diffusion-controlled reaction. As an important reactive nitrogen species, it plays a crucial role in physiological processes such as cell signaling and immune defense. However, due to its high reactivity, extremely short lifetime (approximately 10 ms), and extremely low physiological concentration (nanomolar level), ONOO... - Imbalances in ONOO concentration are closely related to various pathological states, such as inflammation, neurodegenerative diseases, cardiovascular diseases, and cancer. Therefore, achieving proper control of ONOO concentration in organisms is crucial. - Real-time, in-situ, and dynamic monitoring is of vital importance for revealing its mechanism of action in physiological and pathological processes, as well as for the early diagnosis of related diseases and the evaluation of drug efficacy.

[0003] Currently, ONOO is being tested. - The methods mainly include electrochemical methods, chromatography, and spectroscopic analysis. Among them, fluorescent probe detection based on small organic molecules is considered the most promising research tool due to its unique advantages of high sensitivity, high spatiotemporal resolution, non-invasiveness, and real-time imaging of live samples. Various ONOO methods based on different recognition groups (such as borate esters, triphenylphosphine, α-keto acids, etc.) have been reported in the existing technology. - Fluorescent probes. For example, Chinese patent CN112079857A discloses an ONOO with a fluoroboron dipyrrole (BODIPY) core structure. - A fluorescent probe, which exhibits weak fluorescence on its own, reacts with ONOO- to generate a strongly fluorescent product, thus enabling the detection of ONOO-. - Highly selective recognition, applicable to fluorescence imaging detection of ONOO- in vitro and in live cells; Chinese patent CN118440101A discloses a near-infrared small molecule fluorescent probe with BOBPY as the fluorescent core and methylboronic acid as the ONOO-. -It possesses specific recognition sites, a maximum emission wavelength of up to 708 nm, a fast response speed (response completed within 1 minute), a detection limit as low as 24 nmol / L, and good biocompatibility, demonstrating potential for applications in in vivo imaging. Chinese patent CN117263967A discloses a benzothiazole-based fluorescent probe using pinacol phenylboronic acid as the recognition unit, which can specifically detect ONOO- through ratiometric fluorescence response, exhibiting good anti-interference capabilities and reducing the influence of environmental factors on the detection results. However, existing ONOO-... - Fluorescent probes still have the following limitations: insufficient selectivity: many probes are not suitable for ONOO. - The response is susceptible to other biological reactive oxygen species (such as H2O2, ClO) - •OH, NO2 - Interference from factors such as [list of factors] leads to false positive signals, making specific detection difficult in complex biological systems. Sensitivity and quantification capabilities are limited: most probes are "on-off" or "enhanced" intensity responses, and their fluorescence signals are easily affected by various non-target factors such as uneven probe concentration distribution, instrument efficiency fluctuations, and microenvironmental changes, making accurate quantitative analysis difficult. Biological application effectiveness is unsatisfactory: some probes have poor water solubility, poor cell membrane penetration, or exhibit biotoxicity, limiting their long-term, dynamic imaging applications at the live cell and in vivo levels. In particular, probes capable of monitoring endogenous ONOO in cells... - The generation of dynamic probes remains very scarce.

[0004] Therefore, it is necessary to develop a method that combines high specificity, high sensitivity, and ratiometric accuracy for quantitative detection, while also possessing good biocompatibility and applicability to both endogenous and exogenous ONOO in living cells. - Novel fluorescent probes for dynamic imaging are a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0005] Addressing the challenge of real-time, in-situ, and dynamic monitoring of peroxynitrite in complex biological systems, this invention proposes a ratiometric fluorescent probe molecule for detecting peroxynitrite, its preparation method, and its applications. This probe can detect ONOO⁻ with high selectivity, high sensitivity, and quantitatively, and has been successfully applied to ratiometric fluorescence imaging of exogenous and endogenous ONOO⁻ in living cells. This provides a reliable and crucial tool for the study of the mechanisms of related diseases and drug development.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A ratiometric fluorescent probe molecule for detecting peroxynitrite, namely HPIP-T-BnB and HPIP-K-BnB, wherein the structural formula of HPIP-T-BnB is:

[0008] The structural formula for HPIP-K-BnB is:

[0009] .

[0010] A method for preparing a ratiometric fluorescent probe molecule for detecting peroxynitrite ions includes the following steps:

[0011] (1) 1-(4-bromo-2-hydroxyphenyl) ethyl ketone, aromatic borate ester, catalyst I and solvent I were mixed and subjected to the Suzuki reaction to obtain compound a;

[0012] (2) Compound a, 2-aminopyridine and catalyst II are mixed and reacted to give compound b by a binucleophilic reaction;

[0013] (3) Compound b, 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane, catalyst III and solvent II are mixed to undergo a nucleophilic reaction to obtain a ratiometric fluorescent probe molecule;

[0014] When the aromatic boronic ester is triphenylamine boronic ester, the ratiometric fluorescent probe molecule is HPIP-T-BnB; when the aromatic boronic ester is 4-(9H-carbazole-9-yl)phenylboronic acid pinacol ester, the ratiometric fluorescent probe molecule is HPIP-K-BnB.

[0015] In step (1), catalyst I includes a palladium catalyst and an auxiliary agent; the palladium catalyst is tetra(triphenylphosphine)palladium or dichlorodi(triphenylphosphine)palladium, and the auxiliary agent is potassium carbonate, sodium carbonate, potassium tert-butoxide or sodium hydroxide or potassium carbonate; the solvent I is toluene, dichloromethane, N,N-dimethylformamide, dioxane or water.

[0016] The molar ratio of 1-(4-bromo-2-hydroxyphenyl)ethyl ketone, triphenylamine borate ester, palladium catalyst, and auxiliaries is 1:0.5:0.2:1-5; the ratio of 1-(4-bromo-2-hydroxyphenyl)ethyl ketone to solvent is 1:5-30 mmol / ml.

[0017] The Suzuki reaction is carried out at a temperature of 80-100°C for a time of 1-24 hours.

[0018] The molar ratio of compound a, 2-aminopyridine, and catalyst II is 1:2-3:1-2; catalyst II is elemental iodine.

[0019] The amphiphilic nucleophilic reaction was carried out at a temperature of 100-110℃ for 12-24 hours.

[0020] The catalyst III is cesium carbonate, potassium carbonate, potassium phosphate, sodium carbonate, or cesium fluoride; the solvent II is acetonitrile, N,N-dimethylformamide, N-methylpyrrolidone, acetone, or dimethyl sulfoxide; the molar ratio of compound b to 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane and catalyst III is 1:1-2:0.5-2; the ratio of compound b to solvent II is 1:15-20 mmol / ml.

[0021] The nucleophilic reaction in step (3) is carried out at a temperature of 60-70℃ for 4-6 hours.

[0022] Application of a ratiometric fluorescent probe molecule in the preparation of a fluorescent probe for detecting peroxynitrite ions.

[0023] The beneficial effects of this invention are:

[0024] This invention provides two novel methods for detecting peroxynitrite (ONOO). - A ratiometric fluorescent probe molecule was discovered, and its borate ester group was found to act as a recognition unit, capable of interacting with ONOO. - It undergoes a specific oxidative shearing reaction, while remaining unresponsive to other common reactive oxygen species (ROS), thus effectively avoiding cross-interference. When the recognition group is ONOO... - After cleavage, the chemical structure and electronic distribution of the probe change significantly, resulting in a marked shift in its fluorescence emission spectrum (ratiometric signal change), thus enabling the detection of ONOO. - It offers highly sensitive and accurate quantitative detection, with detection limits down to the nanomolar level. The probe molecules exhibit good lipid solubility and cell membrane permeability, and low toxicity to live cells. Successfully applied in live-cell imaging experiments, it can clearly visualize exogenously added ONOO. - It can more sensitively detect endogenous ONOO stimuli induced by lipopolysaccharide (LPS) and other stimuli. - The dynamics of ONOO generation provide a basis for studying ONOO under physiological and pathological conditions. - This provides a powerful tool for understanding biological functions. The probe has a simple synthetic route, readily available starting materials, mild reaction conditions, convenient post-processing, and is easy to purify to obtain high-purity products. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The proton NMR spectrum of the stimulus-responsive molecule HPIP-T-BnB.

[0027] Figure 2 The carbon NMR spectrum of the stimulus-responsive molecule HPIP-T-BnB.

[0028] Figure 3 The proton NMR spectrum of the stimulus-responsive molecule HPIP-K-BnB.

[0029] Figure 4 The carbon NMR spectrum of the stimulus-responsive molecule HPIP-K-BnB.

[0030] Figure 5 Fluorescence emission spectra of the stimulus-responsive molecule HPIP-K-BnB at different ratios of tetrahydrofuran to water.

[0031] Figure 6 The fluorescence emission spectra of the stimulus-responsive molecule HPIP-T-BnB at different ratios of tetrahydrofuran to water were obtained.

[0032] Figure 7 To stimulate the response molecule HPIP-K-BnB (10 μM) at different equivalents of peroxynitrite (ONOO) - The fluorescence emission spectrum under ( ).

[0033] Figure 8 To stimulate the response molecule HPIP-T-BnB (10 μM) at different equivalents of peroxynitrite (ONOO) - The fluorescence emission spectrum under ( ).

[0034] Figure 9 To stimulate the response molecule HPIP-K-BnB (10 μM) under different interfering factors and peroxynitrite (ONOO) - The fluorescence emission spectrum under ( ).

[0035] Figure 10 Photographs before and after irradiation with a 365nm lamp after adding ONOO- and other interfering substances to the HPIP-K-BnB solution to stimulate the response molecule. (a) Before irradiation; (b) After irradiation.

[0036] Figure 11To stimulate the response molecule HPIP-K-BnB (10 μM) under different interfering factors and peroxynitrite (ONOO) - Intracellular fluorescence imaging.

[0037] Figure 12 Intracellular fluorescence imaging of the stimulus-responsive molecule HPIP-K-BnB (10 μM) under different drug concentrations.

[0038] Figure 13 Intracellular fluorescence imaging of the stimulus-responsive molecule HPIP-K-BnB (10 μM) under different drug induction. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] Used for detecting peroxynitrite (ONOO) - The method for preparing ratiometric fluorescent probe molecules, the structural formula of compound HPIP-T-BnB is as follows:

[0042]

[0043] The general reaction formula is:

[0044]

[0045] The preparation of compound a was as follows: 1-(4-bromo-2-hydroxyphenyl)ethyl ketone (2.15 g, 10 mmol), 4-(diphenylamino)phenylboronic acid (3.47 g, 12 mmol), tetra(triphenylphosphine)palladium (1.15 g, 1 mmol), and potassium carbonate (4.24 g, 40 mmol) were added to a 100 mL Schlenk flask, and 1,4-dioxane (50 mL) and water (15 mL) were injected under argon protection. The mixture was refluxed overnight at 90 °C under argon atmosphere. After cooling to room temperature, the mixture was extracted three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (dichloromethane / petroleum ether) to obtain the target product a (3.03 g, 80%). NMR was as follows. Figure 1-2 As shown, 1H NMR (600 MHz, CDCl3) δ12.37 (s, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.28 (t, J= 7.8 Hz, 4H), 7.17 (s, 1H), 7.13 (dd, J = 15.6, 8.3 Hz, 8H), 7.06 (t, J =7.3 Hz, 2H), 2.64 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 203.77 (s), 162.82 (s), 148.60 (s), 147.34 (s), 132.41 (s), 131.16 (s), 129.41 (s), 127.92 (s), 124.95 (s), 123.52 (s), 122.92 (s), 118.19 (s), 117.24 (s), 115.45 (s), 77.23(s), 77.02 (s), 76.81 (s), 26.53(s).

[0046]

[0047] The preparation method of compound HPIP-T is as follows: Compound a (1.14 g, 3 mmol), 2-aminopyridine (650 mg, 6.9 mmol), and elemental iodine (914.4 mg, 3.6 mmol) were added to a 75 mL high-pressure reaction tube. The mixture was reacted at 110 °C for four hours. After the temperature dropped to 70 °C, excess sodium hydroxide solution was added, and the reaction was continued at 100 °C for one hour. After cooling to room temperature, the pH of the mixture was adjusted to 7 with dilute hydrochloric acid, followed by extraction three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain the target product HPIP-T (404.9 mg, 29.8%). NMR was as follows. Figure 5-6 As shown, 1H NMR (600 MHz, CDCl3) δ 12.73 (s, 1H), 8.17 (d, J = 6.7 Hz, 1H), 7.89 (s, 1H), 7.62 (dd, J =16.5, 8.5 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 7.8 Hz, 4H), 7.23 (d, J = 7.9 Hz, 1H), 7.16 – 7.11 (m, 7H), 7.04 (t, J = 7.4 Hz, 2H), 6.87 (t,J = 6.7 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 157.64 (s), 147.70 (s), 143.56(s), 134.48 (s), 129.28 (s), 127.57 (s), 126.08 (s), 125.34 (s), 125.12 (s),124.50 (s), 123.76 (s), 122.95 (s), 117.41 (s), 116.80 (s), 115.41 (s), 113.13 (s), 106.61 (s).

[0048] Preparation of compound HPIP-T-BnB: HPIP-T (200 mg, 0.44 mmol) was dissolved in acetonitrile (20 mL) in a 50 mL round-bottom flask. Cesium carbonate (143.4 mg, 0.44 mmol) and 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (156.8 mg, 0.53 mmol) were added in portions. The reaction mixture was heated at 70°C for 4 hours, and then concentrated under reduced pressure. The residue was diluted with water and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The crude product was carefully washed with cold ethanol and lyophilized to give a yellow solid HPIP-T-Bn (28% yield). 1HNMR (400 MHz, CDCl3) δ 8.49 (d, J = 8.1 Hz, 1H), 8.15 (s, 1H), 8.02 (d, J =6.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 9.0 Hz, 1H), 7.55 (d, J =7.8 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.2 Hz, 1H), 7.28 (d, J =8.0 Hz, 3H), 7.24 (s, 1H), 7.14 (d, J = 8.1 Hz, 7H), 7.04 (t, J = 7.3 Hz,2H), 6.72 (t, J = 6.7 Hz, 1H), 1.37 (s, 13H). 13 C NMR (151 MHz, CDCl3) δ135.19 (s), 129.32 (d, J = 9.5 Hz), 127.73 (s), 127.06 (s), 124.48 (s),123.82 (s), 123.00 (s), 77.22 (s), 77.01 (s), 76.80 (s), 24.91 (s).

[0049] Example 2

[0050] Used for detecting peroxynitrite (ONOO) - The method for preparing ratiometric fluorescent probe molecules, the structural formula of compound HPIP-K-BnB is as follows:

[0051]

[0052] The general reaction formula is:

[0053]

[0054] Compound b was prepared as follows: 1-(4-bromo-2-hydroxyphenyl)ethyl ketone (2.15 g, 10 mmol), 9-[4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)phenyl]-9H-carbazole (4.43 g, 12 mmol), and tetrakis(triphenylphosphine)palladium (0.578 g, 0.5 mmol) were added to a 100 mL Schlenk flask. Toluene (40 mL) and 2 mol / L sodium carbonate solution (15 mL) were then added under argon protection. The mixture was refluxed overnight at 90 °C under argon atmosphere. After cooling to room temperature, the mixture was extracted three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (dichloromethane / petroleum ether) to obtain the target product b (2.12 g, 56.2%). NMR was performed as follows. Figure 3-4 As shown, 1 H NMR (600 MHz, CDCl3) δ 12.40 (s, 1H), 8.16 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 8.2 Hz, 3H), 7.68 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.31 (t, J = 7.4 Hz, 3H), 7.24 (d, J = 8.2 Hz, 1H), 2.69 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 204.04 (s), 162.83 (s), 148.10 (s), 140.71 (s), 138.28 (d, J = 9.8 Hz), 131.37 (s), 128.69 (s), 127.39 (s), 126.06 (s), 123.59 (s), 120.39 (s), 120.20 (s), 118.86 (s), 117.81 (s),116.51 (s), 109.80 (s), 77.23 (s), 77.02 (s), 76.80 (s), 26.66 (s).

[0055]

[0056] The preparation method of compound HPIP-K was as follows: Compound b (1.13 g, 3 mmol), 2-aminopyridine (650 mg, 6.9 mmol), and elemental iodine (914.4 mg, 3.6 mmol) were added to a 75 mL high-pressure reaction tube. The mixture was reacted at 110 °C for four hours. After the temperature dropped to 70 °C, excess sodium hydroxide solution was added, and the reaction was continued at 100 °C for one hour. After cooling to room temperature, the pH of the mixture was adjusted to 7 with dilute hydrochloric acid, followed by extraction three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain the target product HPIP-T (449.7 mg, 33.24%). NMR was as follows. Figure 7-8 As shown, 1 H NMR (600 MHz, CDCl3) δ 12.84 (s, 1H), 8.20 (d, J = 6.7 Hz, 1H), 8.16 (d, J = 7.8 Hz, 2H), 7.94 (s, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.72 (d, J 7.24 (d, J = 1.7 Hz, 1H), 6.89 (t, J = 6.7 Hz,1H). 13C NMR (151 MHz, CDCl3) δ 157.80 (s), 143.62 (s), 140.91 (s), 139.76(s), 128.30 (s), 127.30 (s), 126.29 (s), 125.97 (s), 125.34 (d, J = 18.9 Hz), 123.46 (s), 120.29 (s), 119.96 (s), 116.88 (s), 116.09 (s), 115.67 (s), 113.25 (s), 109.91 (s).

[0057] The preparation method of compound HPIP-K-BnB is as follows: HPIP-K (200 mg, 0.44 mmol) was dissolved in acetonitrile (20 mL) in a 50 mL round-bottom flask. Cesium carbonate (143.4 mg, 0.44 mmol) and 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (156.8 mg, 0.53 mmol) were added in portions. After heating at 70°C for 4 hours, the reaction mixture was concentrated under reduced pressure. The residue was diluted with water and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The crude product was carefully washed with cold ethanol and lyophilized to give a yellow solid HPIP-K-BnB in 42% yield. 1 H NMR (600 MHz, CDCl3) δ 8.59 (d, J = 8.0 Hz, 1H), 8.20 (s, 1H), 8.16 (d,J = 7.8 Hz, 2H), 8.04 (d, J = 6.7 Hz, 1H), 7.91 (d, J = 7.9 Hz, 2H), 7.85 (d,J = 8.3 Hz, 2H), 7.67 – 7.62 (m, 3H), 7.59 (d, J = 7.9 Hz, 2H), 7.48 (t, J =9.1 Hz, 3H), 7.43 (t, J = 7.6 Hz, 2H), 7.36 (s, 1H), 7.30 (t, J = 7.4 Hz, 2H), 7.18 – 7.14 (m, 1H), 6.73 (t, J = 6.7 Hz, 1H), 1.37 (s, 12H). 13C NMR(151 MHz, CDCl3) δ 156.31 (s), 144.50 (s), 140.84 (d, J = 16.8 Hz), 140.58(s), 139.93 (d, J = 17.3 Hz), 137.02 (s), 135.23 (s), 129.50 (s), 128.46 (s),127.36 (s), 127.06 (s), 125.99 (s), 125.80 (s), 124.61 (s), 123.47 (s),122.22 (s), 120.29 (d, J = 12.2 Hz), 120.00 (s), 117.25 (s), 112.93 (s),111.99 (s), 110.95 (s), 109.88 (s), 83.99 (s), 77.24 (s), 77.03 (s), 76.82(s), 70.76 (s), 24.92 (s).

[0058] The ratiometric fluorescent probe molecules HPIP-T-BnB and HPIP-K-BnB prepared in Examples 1 and 2 for the detection of peroxynitrite (ONOO⁻) were systematically characterized.

[0059] Test method:

[0060] Compounds HPIP-T-BnB and HPIP-K-BnB were prepared into 1 mM stock solutions using DCM and stored for later use. 100 μL of each stock solution was pipetted into a 10 mL volumetric flask. After the solvent was allowed to evaporate completely at room temperature, different test solvents were added to bring the volume to the required mark. The flasks were then shaken well to obtain 10 μM test solutions. 100 μL of each stock solution was pipetted into a 10 mL volumetric flask. After the solvent had completely evaporated, a THF / H₂O mixed solvent was added to bring the volume to the required level (where the water volume was 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%). The solutions were allowed to stand for several hours to promote molecular aggregation. This solution was used to test the aggregation behavior of compounds HPIP-T-BnB and HPIP-K-BnB in THF / H₂O mixed solvents with different water contents. The fluorescence emission spectra of both compounds in different ratios of tetrahydrofuran / water mixed solvents are shown below. Figure 5-6 As shown.

[0061] To further evaluate the effects of HPIP-K-BnB and HPIP-T-BnB on ONOO - Based on its quantitative response capability, a systematic fluorescence titration experiment was conducted. For example... Figure 7 and 8 As shown, the results indicate that when ONOO - As the concentration was gradually increased from 0 to 150 μM (equivalent to 0-15 times the probe concentration), the fluorescence intensity of HPIP-K-BnB at 395 nm gradually decreased; simultaneously, a new emission peak appeared at approximately 540 nm, and the fluorescence intensity continued to increase. This emission peak position highly coincides with the emission peak of HPIP-K, indicating that the probe exhibits high fluorescence intensity at 0-150 nm. - A structural transformation occurred under the influence of the ONOO force. This phenomenon can be attributed to ONOO. - The specific oxidative cleavage of the BnB-terminated group releases the hydroxyl group, thereby restoring the zwitterion ESIPT channel under photoexcitation conditions and realizing the transition from short-wavelength emission to long-wavelength emission.

[0062] Further investigation was conducted to examine the response of HPIP-K-BnB to various potential interfering substances, such as... Figure 9 and 10 As shown, only when ONOO is added - At that time, the emission peak at 543 nm was significantly enhanced, while the fluorescence changes caused by other analytes were negligible. Even in complex environments where multiple reactive oxygen species and other interfering substances coexist, HPIP-K-BnB showed good fluorescence performance against ONOO. - It still exhibits excellent recognition specificity.

[0063] Furthermore, HPIP-K-BnB molecules also exhibit excellent imaging capabilities in the cellular environment: intracellular fluorescence imaging results under different conditions of coexistence of interfering substances and ONOO⁻ are shown in [Figure number missing]. Figure 11 Intracellular fluorescence imaging under different drug concentrations is shown in the figure. Figure 12 ; and intracellular fluorescence imaging results induced by different types of drugs are shown in Figure 13 .

[0064] Example 3

[0065] Used for detecting peroxynitrite (ONOO) - The method for preparing ratiometric fluorescent probe molecules, the structural formula of compound HPIP-T-BnB is as follows:

[0066]

[0067] The preparation method of compound a is as follows: 1-(4-bromo-2-hydroxyphenyl)ethyl ketone (10 mmol), 4-(diphenylamino)phenylboronic acid (5 mmol), tetrakis(triphenylphosphine)palladium (0.1 mmol), and potassium carbonate (10 mmol) were added to a 100 mL Schlenk flask, and 1,4-dioxane (35 mL) and water (15 mL) were injected under argon protection. The mixture was refluxed overnight at 80 °C under argon atmosphere. After cooling to room temperature, the mixture was extracted three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (dichloromethane / petroleum ether) to obtain the target product a.

[0068] The preparation method of compound HPIP-T is as follows: Compound a (1 mmol), 2-aminopyridine (3 mmol), and elemental iodine (1 mmol) were added to a 75 mL high-pressure reaction tube. The mixture was reacted at 100 °C for 5 hours. After the temperature dropped to 70 °C, excess sodium hydroxide solution was added, and the reaction was continued at 100 °C for 2 hours. After cooling to room temperature, the pH of the mixture was adjusted to 7 with dilute hydrochloric acid, followed by extraction three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain the target product HPIP-T.

[0069] Preparation of compound HPIP-T-BnB: In a 50 mL round-bottom flask, HPIP-T (1 mmol) was dissolved in acetonitrile (15 mL). Cesium carbonate (0.5 mmol) and 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (1 mmol) were added in portions. The reaction mixture was heated at 60°C for 6 hours, and then concentrated under reduced pressure. The residue was diluted with water and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The crude product was carefully washed with cold ethanol and lyophilized to obtain yellow solid HPIP-T-Bn.

[0070] Example 4

[0071] Used for detecting peroxynitrite (ONOO) - The method for preparing ratiometric fluorescent probe molecules, the structural formula of compound HPIP-T-BnB is as follows:

[0072]

[0073] Compound a was prepared as follows: 1-(4-bromo-2-hydroxyphenyl)ethyl ketone (10 mmol), 4-(diphenylamino)phenylboronic acid (20 mmol), tetrakis(triphenylphosphine)palladium (0.1 mmol), and potassium carbonate (10 mmol) were added to a 500 mL Schlenk flask, and 100 mL of dichloromethane was injected under argon protection. The mixture was refluxed overnight at 100 °C under argon atmosphere. After cooling to room temperature, the mixture was extracted three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (dichloromethane / petroleum ether) to obtain the target product a.

[0074] The preparation method of compound HPIP-T is as follows: Compound a (1 mmol), 2-aminopyridine (2 mmol), and elemental iodine (2 mmol) were added to a 75 mL high-pressure reaction tube. The mixture was reacted at 110 °C for 10 hours. After the temperature dropped to 70 °C, excess sodium hydroxide solution was added, and the reaction was continued at 100 °C for 2 hours. After cooling to room temperature, the pH of the mixture was adjusted to 7 with dilute hydrochloric acid, followed by extraction three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain the target product HPIP-T.

[0075] Preparation of compound HPIP-T-BnB: In a 50 mL round-bottom flask, HPIP-T (1 mmol) was dissolved in acetonitrile (15 mL). Cesium carbonate (2 mmol) and 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (2 mmol) were added in portions. The reaction mixture was heated at 70°C for 6 hours, and then concentrated under reduced pressure. The residue was diluted with water and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The crude product was carefully washed with cold ethanol and lyophilized to obtain yellow solid HPIP-T-Bn.

[0076] Example 4

[0077] Used for detecting peroxynitrite (ONOO) - The method for preparing ratiometric fluorescent probe molecules, the structural formula of compound HPIP-K-BnB is as follows:

[0078]

[0079] Compound b was prepared as follows: 1-(4-bromo-2-hydroxyphenyl)ethyl ketone (10 mmol), 9-[4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)phenyl]-9H-carbazole (20 mmol), and tetra(triphenylphosphine)palladium (2 mmol) were added to a 100 mL Schlenk flask. Toluene (50 mL) and 2 mol / L sodium carbonate solution (15 mL) were added under argon protection. The mixture was refluxed overnight at 90 °C under argon atmosphere. After cooling to room temperature, the mixture was extracted three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (dichloromethane / petroleum ether) to obtain the target product b (2.12 g, 56.2%).

[0080] The preparation method of compound HPIP-K is as follows: Compound b (5 mmol), 2-aminopyridine (10 mmol), and elemental iodine (5 mmol) were added to a 75 mL high-pressure reaction tube. The mixture was reacted at 110 °C for four hours. After the temperature dropped to 70 °C, excess sodium hydroxide solution was added, and the reaction was continued at 100 °C for one hour. After cooling to room temperature, the pH of the mixture was adjusted to 7 with dilute hydrochloric acid, followed by extraction three times with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. After solvent removal, the organic residues were purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain the target product HPIP-T.

[0081] The preparation method of compound HPIP-K-BnB is as follows: In a 100 mL round-bottom flask, HPIP-K (1 mmol) was dissolved in N-methylpyrrolidone (15 mL). Sodium carbonate (1 mmol) and 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (2 mmol) were added in portions. The reaction mixture was heated at 70°C for 4 hours, and then concentrated under reduced pressure. The residue was diluted with water and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The crude product was carefully washed with cold ethanol and lyophilized to obtain yellow solid HPIP-K-BnB.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ratiometric fluorescent probe molecule for detecting peroxynitrite, characterized in that, The ratiometric fluorescent probe molecules are HPIP-T-BnB and HPIP-K-BnB. The structural formula of HPIP-T-BnB is: The structural formula for HPIP-K-BnB is: 。 2. The method for preparing the ratiometric fluorescent probe molecule for detecting peroxynitrite ions as described in claim 1, characterized in that, Includes the following steps: (1) 1-(4-bromo-2-hydroxyphenyl) ethyl ketone, aromatic borate ester, catalyst I and solvent I were mixed and subjected to the Suzuki reaction to obtain compound a; (2) Compound a, 2-aminopyridine and catalyst II are mixed and reacted to give compound b by a binucleophilic reaction; (3) Compound b, 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane, catalyst III and solvent II are mixed to undergo a nucleophilic reaction to obtain a ratiometric fluorescent probe molecule; When the aromatic boronic ester is triphenylamine boronic ester, the ratiometric fluorescent probe molecule is HPIP-T-BnB; when the aromatic boronic ester is 4-(9H-carbazole-9-yl)phenylboronic acid pinacol ester, the ratiometric fluorescent probe molecule is HPIP-K-BnB.

3. The method for preparing a ratiometric fluorescent probe molecule for detecting peroxynitrite ions according to claim 2, characterized in that, In step (1), catalyst I includes a palladium catalyst and an auxiliary agent; the palladium catalyst is tetra(triphenylphosphine)palladium or dichlorodi(triphenylphosphine)palladium, and the auxiliary agent is potassium carbonate, sodium carbonate, potassium tert-butoxide or sodium hydroxide or potassium carbonate; the solvent I is toluene, dichloromethane, N,N-dimethylformamide, dioxane or water.

4. The method for preparing a ratiometric fluorescent probe molecule for detecting peroxynitrite according to claim 3, characterized in that, The molar ratio of 1-(4-bromo-2-hydroxyphenyl)ethyl ketone, triphenylamine borate ester, palladium catalyst and auxiliaries is 1:0.5:0.2:1-5; the ratio of 1-(4-bromo-2-hydroxyphenyl)ethyl ketone to solvent is 1:5-30 mmol / ml.

5. The method for preparing a ratiometric fluorescent probe molecule for detecting peroxynitrite according to claim 2, characterized in that, The Suzuki reaction was carried out at a temperature of 80-100°C for 12-24 hours.

6. The method for preparing a ratiometric fluorescent probe molecule for detecting peroxynitrite ions according to claim 2, characterized in that, The molar ratio of compound a, 2-aminopyridine, and catalyst II is 1:2-3:1-2; catalyst II is elemental iodine.

7. The method for preparing a ratiometric fluorescent probe molecule for detecting peroxynitrite ions according to claim 2, characterized in that, The amphiphilic reaction is carried out at a temperature of 100-110℃ for 1-24 hours.

8. The method for preparing a ratiometric fluorescent probe molecule for detecting peroxynitrite according to any one of claims 2-7, characterized in that, The catalyst III is cesium carbonate, potassium carbonate, potassium phosphate, sodium carbonate, or cesium fluoride; the solvent II is acetonitrile, N,N-dimethylformamide, N-methylpyrrolidone, acetone, or dimethyl sulfoxide; the molar ratio of compound b to 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane and catalyst III is 1:1-2:0.5-2; the ratio of compound b to solvent II is 1:15-20 mmol / ml.

9. The method for preparing a ratiometric fluorescent probe molecule for detecting peroxynitrite according to claim 8, characterized in that, The nucleophilic reaction in step (3) is carried out at a temperature of 60-70℃ for 4-6 hours.

10. The use of the ratiometric fluorescent probe molecule of claim 1 in the preparation of a fluorescent probe for detecting peroxynitrite ions.

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