A probe for detecting ox-LDL and application thereof

Abstract

This invention discloses a probe for detecting Ox-LDL and its applications, belonging to the field of Ox-LDL detection technology. This invention provides a variety of probe molecules that can specifically and sensitively recognize Ox-LDL and effectively distinguish between high-density lipoprotein (HDL), low-density lipoprotein (LDL), and Ox-LDL. The probe exhibits significantly enhanced fluorescence in the presence of Ox-LDL, while showing almost no fluorescence in pure PBS solution, with low background noise. The probe of this invention is a molecular probe, simple to prepare, stable, easy to store and detect, and can be used as a reagent for detecting Ox-LDL in peripheral blood, cells, and tissues, a lipoprotein identification reagent, an early diagnostic reagent for atherosclerosis, and an early diagnostic reagent for fatty liver.

Description

Technical Field

[0001] This invention relates to the field of Ox-LDL detection technology, and in particular to a probe for detecting Ox-LDL and its application. Background Technology

[0002] Lipoproteins are small particles composed of lipids and proteins, responsible for transporting lipids in the blood and delivering them to tissues. Low-density lipoprotein (LDL) is the main carrier of cholesterol, and its main lipid component is cholesterol esters, while also containing a small amount of free cholesterol. The main apolipoprotein in LDL is ApoB-100. LDL has a spherical structure, approximately 22 nm in diameter, and a density ranging from 1.019 to 1.063 g / cm³. 3 These particles exhibit heterogeneity in size, density, and composition, and 2 to 38 subtypes have been identified depending on the research methods used. The composition and distribution of LDL subtypes vary among individuals and are determined by both genetic and environmental factors. Notably, a high proportion of small, dense LDL subtypes is associated with an increased risk of cardiovascular disease. For example, patients with cardiovascular disease have significantly higher levels of small, dense LDL than healthy controls. Several mechanisms have been proposed to explain how this type of LDL promotes atherosclerosis, with oxidative susceptibility considered to play a key role.

[0003] Under physiological conditions, the human body maintains redox homeostasis. However, when reactive oxygen species (ROS) production increases, components of LDL such as phospholipids, cholesterol esters, and polyunsaturated fatty acids are easily oxidized, forming oxidized low-density lipoprotein (Ox-LDL). LDL oxidation mainly occurs within the blood vessel wall, and its degree is regulated by various factors, including the quantity / size of LDL, its composition, oxidative susceptibility, oxidative stress factors, and endothelial dysfunction. Although the mechanism of action of Ox-LDL in vivo is not yet clear, and the specific spatiotemporal characteristics of LDL oxidation still need to be explored, increasing evidence suggests that Ox-LDL is an important biomarker for cardiovascular disease. Multiple in vivo studies have shown that Ox-LDL levels in patients with cardiovascular disease, diabetes, and hemodialysis are significantly higher than in healthy individuals. Furthermore, by measuring the intima-media thickness of the carotid / femoral arteries, studies have confirmed the correlation between Ox-LDL levels and subclinical atherosclerosis.

[0004] Current methods for detecting Ox-LDL have significant limitations: enzyme-linked immunosorbent assay (ELISA) has limited resolution across different oxidation states (coefficient of variation between subtypes greater than 20%); the thiobarbituric acid reactive substance assay has poor specificity (with 15% to 30% fatty acid cross-reactivity); and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) requires expensive operating resources [see: Reimund, M. et al. Structure of apolipoprotein B100 bound to the low-density lipoprotein receptor. Nature 638 (2025).]. These shortcomings limit the accurate detection of oxidized low-density lipoprotein and its widespread clinical application.

[0005] Furthermore, existing technologies generally cannot simultaneously achieve both molecular-level precision and clinical feasibility. For example, while traditional antibody methods offer high specificity, they suffer from complex synthesis processes, insufficient stability, limited sensitivity, and the potential to induce immunogenicity. Therefore, developing a novel detection tool that can overcome these limitations is particularly urgent.

[0006] Therefore, designing probes for Ox-LDL detection is of great scientific and practical significance. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a probe for detecting Ox-LDL and its applications. This invention synthesizes and screens highly efficient probe molecules capable of detecting Ox-LDL, and verifies their specific recognition ability of lipoproteins with different oxidation levels through quantitative calculations and application to the detection of proteins related to atherosclerosis. For the first time, it achieves fluorescence visualization of Ox-LDL distribution, providing a new method for early disease detection.

[0008] The technical solution of the present invention is as follows:

[0009] The first aspect of this invention protects a probe molecule, the structure of which is shown in general formula (1):

[0010] General formula (1) R represents a conjugated group containing an N-aryl ring.

[0011] Preferably, the probe molecule has any one of the following structures: .

[0012] A second aspect of the present invention protects a composition comprising the probe molecule described in the first aspect, which is formulated as a clinically applicable spray, lyophilized powder injection, or commercial kit by means of the probe molecule as the active ingredient or a combination of the probe molecule and pharmaceutically acceptable excipients.

[0013] A third aspect of the present invention protects a composition comprising the probe molecule described in the first aspect, comprising the probe molecule and a pharmaceutically acceptable carrier.

[0014] The fourth aspect of this invention protects the use of the probe molecule described in the first aspect in the preparation of a product for the detection of oxidized low-density lipoprotein.

[0015] The fifth aspect of this invention protects the use of the probe molecule described in the first aspect in the preparation of products for the identification and detection of oxidized low-density lipoprotein (Ox-LDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL).

[0016] The sixth aspect of this invention protects the use of the probe molecule described in the first aspect in the preparation of a diagnostic reagent for early atherosclerosis.

[0017] The seventh aspect of this invention protects the use of the probe molecule described in the first aspect in the preparation of a diagnostic reagent for early fatty liver.

[0018] The beneficial technical effects of this invention are as follows: This invention obtains Ox-LDL-specific probe molecules through fluorescence screening, achieving effective differentiation between HDL, LDL, and Ox-LDL. Furthermore, the probe exhibits almost no fluorescence in pure PBS solution and can be specifically activated in the presence of Ox-LDL, effectively reducing background noise. Moreover, the probe of this invention is a molecular probe, which offers advantages over traditional antigen-antibody probes, such as simple preparation, easy storage, and simple testing, thus holding promise for widespread application in market screening.

[0019] The probes in this invention exhibit good fluorescence response to different lipoproteins such as HDL, LDL, and Ox-LDL, demonstrating excellent high selectivity. The fluorescence of this type of probe, especially molecule 1j, shows significant changes before and after fluorescence change, and it holds promise for further commercial applications. Attached Figure Description

[0020] Figure 1 The probe 1a of the present invention 1 1H NMR (400 MHz, CDCl3) spectrum.

[0021] Figure 2 This is a high-resolution mass spectrum of probe 1a of the present invention.

[0022] Figure 3 For the probe 1b of the present invention1 1H NMR (400 MHz, CDCl3) spectrum.

[0023] Figure 4 This is a high-resolution mass spectrum of probe 1b of the present invention.

[0024] Figure 5 The probe 1c of the present invention 1 1H NMR (400 MHz, CDCl3) spectrum.

[0025] Figure 6 This is a high-resolution mass spectrum of probe 1c of the present invention.

[0026] Figure 7 The probe 1d of this invention 1 1H NMR (400 MHz, CDCl3) spectrum.

[0027] Figure 8 This is a high-resolution mass spectrum of probe 1d of the present invention.

[0028] Figure 9 The probe 1e of the present invention 1 1H NMR (400 MHz, CDCl3) spectrum.

[0029] Figure 10 This is the high-resolution mass spectrum of probe 1e of the present invention.

[0030] Figure 11 The probe 1f of the present invention 1 1H NMR (400 MHz, CDCl3) spectrum.

[0031] Figure 12 This is a high-resolution mass spectrum of probe 1f of the present invention.

[0032] Figure 13 The probe of this invention is 1g 1 1H NMR (400 MHz, CDCl3) spectrum.

[0033] Figure 14 This is a high-resolution mass spectrum of the probe 1g of this invention.

[0034] Figure 15 For the probe of the present invention, 1h 1 1H NMR (400 MHz, CDCl3) spectrum.

[0035] Figure 16 This is a high-resolution mass spectrum of the probe of this invention after 1 hour.

[0036] Figure 17 The probe 1i of the present invention 11H NMR (400 MHz, CDCl3) spectrum.

[0037] Figure 18 This is the high-resolution mass spectrum of probe 1i of the present invention.

[0038] Figure 19 The probe 1j of this invention 1 1H NMR (400 MHz, CDCl3) spectrum.

[0039] Figure 20 The probe 1j of this invention 13 C NMR (400 MHz, CDCl3) spectrum.

[0040] Figure 21 This is the high-resolution mass spectrum of probe 1j of the present invention.

[0041] Figure 22 This is the UV absorption spectrum of probe 1a-1j of the present invention in PBS.

[0042] Figure 23 This is the fluorescence fold response of probe 1a-1j of the present invention to Ox-LDL before and after the reaction.

[0043] Figure 24 The fluorescence titration response of the probe 1j invented to Ox-LDL.

[0044] Figure 25 The fluorescence spectra of the specific response of the probe 1j to different lipoproteins are shown.

[0045] Figure 26 The normalized fluorescence spectra of the probe 1j invented in response to different polar environments.

[0046] Figure 27 The fluorescence spectra of the probe 1j invented in response to different polar environments.

[0047] Figure 28 The fluorescence spectrum of the probe 1j invented was used to simulate environments with different viscosity.

[0048] Figure 29 The invention probe 1j is used in intracellular fluorescence confocal imaging.

[0049] Figure 30 The probe 1j invented is used for imaging fluorescence lifetime in cells. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0051] The method for preparing the probe molecule for detecting Ox-LDL in this invention includes the following steps:

[0052] Specifically, glycine tert-butyl hydrochloride was dispersed in tert-butanol, and sodium hydroxide and the corresponding aldehyde were added to continue the reaction. Then, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester was prepared and added. After the reaction was complete, the mixture was extracted with water and CH2Cl2. The organic layer was dried with anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography to obtain a solid, which was the probe molecule.

[0053] The corresponding aldehyde is any one of 4-(dibutylamino)benzaldehyde, 8-hydroxyjulonidine-9-carboxaldehyde, julonidine-9-carboxaldehyde, 4-(dipropylamino)benzaldehyde, 4-(dimethylamino)benzaldehyde, 4-(dimethylamino)-2-methoxybenzaldehyde, 4-(diethylamino)benzaldehyde, 4-(diethylamino)-2-methylbenzaldehyde, 4-(dimethylamino)-2-hydroxybenzaldehyde, and 4-(diethylamino)-2-hydroxybenzaldehyde.

[0054] The intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester needs to be prepared and used immediately. Its preparation method is as follows:

[0055] Specifically, glycine tert-butyl hydrochloride, ethyl acetylimine hydrochloride, and sodium carbonate are dissolved in a water / ether mixture, and then stirred at high speed. After the reaction is complete, the ether phase is collected, dried with anhydrous sodium sulfate, and the solvent is removed to obtain a white oily liquid, which is the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester.

[0056] The probe molecules of this invention employ an "off-on" fluorescence response strategy based on the twisted intramolecular charge transfer (TICT) mechanism. In the free state (e.g., PBS solution), the single bonds between the donor and acceptor fragments in the probe molecule are free to rotate. Upon photoexcitation, they rapidly undergo intramolecular torsion, prompting the system to return to the ground state via an efficient TICT nonradiative relaxation pathway, resulting in extremely weak fluorescence and a very low background signal. When the probe specifically recognizes and embeds into the hydrophobic cavity of the target protein Ox-LDL, its molecular conformation and rotational degrees of freedom are strongly restricted by the protein microenvironment, and the TICT process is significantly suppressed. At this point, the excited-state energy is mainly released through radiative transitions, thereby generating a strong fluorescence signal.

[0057] This probe exhibits sensitivity to both environmental polarity and viscosity, but its high specificity in recognizing and signal activation of Ox-LDL is primarily attributed to the dramatic increase in local microenvironment viscosity following binding to the target protein, rather than a simple change in solvent polarity. This TICT inhibition mechanism, triggered by specific biorecognition events and dominated by viscosity changes, endows the probe with an extremely high signal-to-noise ratio and excellent anti-interference capabilities, making it particularly suitable for highly selective, high-contrast fluorescence imaging of Ox-LDL in complex biological systems.

[0058] Example 1 A method for preparing a probe 1a capable of targeting Ox-LDL, a characteristic biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 4-(dibutylamino)benzaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0059] The intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester was prepared by the following method: Glycine tert-butyl hydrochloride (1.0 eq.), ethyl acetylimine hydrochloride (1.1 eq.), and sodium carbonate (1.0 eq.) were dissolved in a water / ether (1:1, v / v) mixture. The mixture was then stirred at high speed at room temperature. After the reaction was complete, the ether phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed to obtain a white oily liquid, which was the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester.

[0060] probe 1a 1 H NMR spectrum ( Figure 1 ): 1 H NMR (400 MHz, CDCl3): δ 7.95 (d, J = 8.8 Hz,2H), 6.98 (s, 1H), 6.54 (d, J = 9.0 Hz, 2H), 4.17 (s, 2H), 3.25 – 3.17 (m, 4H), 2.18 (d, J= 2.2 Hz, 3H), 1.49 (s, 4H), 1.38 (s, 9H), 1.26 (q, J = 7.4 Hz, 4H), 0.87 (t, J = 7.3 Hz, 6H). High-resolution mass spectrometry ( Figure 2 ): Calculate m / z [M+H] + The value was 428.2908, and the measured value was 428.2881.

[0061] Example 2 A method for preparing probe 1b capable of targeting Ox-LDL, a characteristic biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 8-hydroxyjulonidine-9-carboxaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0062] Among them, probe 1b 1 H NMR spectrum ( Figure 3 ): 1 H NMR (400 MHz, CDCl3) δ 14.17 (s, 1H), 7.07 (s, 1H), 6.71 (s, 1H), 4.29 (s, 2H), 3.22 (q, J = 6.3 Hz, 4H), 2.72 (t, J =6.5 Hz, 2H), 2.63 (t, J = 6.3 Hz, 2H), 2.27 (s, 3H), 1.95 – 1.88 (m, 4H), 1.45 (s, 9H). High-resolution mass spectrometry ( Figure 4 ): Calculate m / z [M+H] + The value is 412.2236, and the measured value is 412.2217.

[0063] Example 3 A method for preparing probe 1c capable of targeting Ox-LDL, a biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, julonidine-9-carboxaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0064] Among them, probe 1c 1 H NMR spectrum ( Figure 5 ): 1 H NMR (400 MHz, CDCl3)δ 7.53 (s, 2H), 6.92 (s, 1H), 4.19 (s, 2H), 3.16 (dd, J = 6.6, 4.9 Hz, 4H), 2.68 (t, J = 6.3 Hz, 4H), 2.21 (s, 3H), 1.89 – 1.83 (m, 4H), 1.38 (s, 9H). High-resolution mass spectrometry ( Figure 6 ): Calculate m / z[M+H] + The value is 396.2287, and the measured value is 396.2268.

[0065] Example 4 A method for preparing a probe 1d capable of targeting Ox-LDL, a characteristic biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 4-(dipropylamino)benzaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0066] Among them, probe 1d 1 H NMR spectrum ( Figure 7 ): 1 H NMR (400 MHz, CDCl3). δ 8.02 (d,J = 9.0Hz, 2H), 7.08 (s, 1H), 6.63 (d, J = 9.2 Hz, 2H), 4.27 (s, 2H), 3.36 – 3.22 (m,4H), 2.28 (s, 3H), 1.63 (q, J = 7.6 Hz, 4H), 1.46 (s, 9H), 0.93 (t, J = 7.4 Hz, 6H). High-resolution mass spectrometry ( Figure 8 ): Calculate m / z [M+H] + The value was 400.2600, and the measured value was 400.2574.

[0067] Example 5 A method for preparing a probe 1e capable of targeting Ox-LDL, a characteristic biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 4-(dimethylamino)benzaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0068] Among them, probe 1e 1 H NMR spectrum ( Figure 9 ): 1 ¹H NMR (400 MHz, CDCl₃) δ 8.10 – 7.99 (m, 2H), 7.09 (s, 1H), 6.71 – 6.66 (m, 2H), 4.26 (s, 2H), 3.02 (s, 6H), 2.29 (s, 3H), 1.46 (s, 9H). High-resolution mass spectrometry (H NMR) Figure 10 ): Calculate m / z [M+H] + The value is 344.1974, and the measured value is 344.1957.

[0069] Example 6 A method for preparing a probe 1f capable of targeting Ox-LDL, a biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 4-(dimethylamino)-2-methoxybenzaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0070] Among them, probe 1f 1 H NMR spectrum ( Figure 11 ): 1 H NMR (400 MHz, CDCl3) δ 8.73 (d, J = 9.0Hz, 1H), 7.68 (s, 1H), 6.37 (dd, J = 9.0, 2.5 Hz, 1H), 6.08 (d, J = 2.4 Hz, 1H), 4.27 (s, 2H), 3.86 (s, 3H), 3.05 (s, 6H), 2.28 (s, 3H), 1.46 (s, 9H). High-resolution mass spectrometry ( Figure 12 ): Calculate m / z [M+H] + The value was 374.2080, but the measured value was 374.2069.

[0071] Example 7 A method for preparing 1g of a probe capable of targeting Ox-LDL, a characteristic biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 4-(diethylamino)benzaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0072] Of which 1g of probe 1 H NMR spectrum ( Figure 13 ): 1H NMR (400 MHz, CDCl3) δ 8.07 – 7.98 (m,2H), 7.09 (s, 1H), 6.70 – 6.64 (m, 2H), 4.28 (s, 2H), 3.42 (q, J = 7.1 Hz, 4H), 2.30 (s, 3H), 1.47 (s, 9H), 1.20 (t, J = 7.1 Hz, 6H). High-resolution mass spectrometry ( Figure 14 ): Calculate m / z[M+H] + The value is 372.2287, and the measured value is 372.2275.

[0073] Example 8 A method for preparing a probe capable of targeting Ox-LDL, a biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 4-(diethylamino)-2-methylbenzaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0074] Among them, probe 1h 1 H NMR spectrum ( Figure 15 ): 1 H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 9.1Hz, 1H), 7.31 (s, 1H), 6.52 (dd, J = 9.1, 2.8 Hz, 1H), 6.36 (d, J = 2.7 Hz, 1H), 4.20 (s, 2H), 3.32 (q, J = 7.1 Hz, 5H), 2.38 (s, 3H), 2.22 (s, 3H), 1.39 (s,9H), 1.11 (t, J = 7.0 Hz, 6H). High-resolution mass spectrometry ( Figure 16 ): Calculate m / z [M+H] +The value is 386.2444, and the measured value is 386.2426.

[0075] Example 9 A method for preparing a probe 1i capable of targeting Ox-LDL, a characteristic biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 4-(dimethylamino)-2-hydroxybenzaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0076] Among them, probe 1i 1 H NMR spectrum ( Figure 17 ): 1 H NMR (400 MHz, CDCl3) δ 14.15 (s, 1H), 7.18 – 7.09 (m, 2H), 6.25 (dd, J = 8.8, 2.6 Hz, 1H), 6.18 (d, J = 2.6 Hz, 1H), 4.31 (s, 2H), 3.04 (s, 6H), 2.29 (s, 3H), 1.47 (s, 9H). High-resolution mass spectrometry ( Figure 18 ): Calculate m / z [M+H] + The value was 360.1923, but the measured value was 360.1903.

[0077] Example 10 A method for preparing a probe 1j capable of targeting Ox-LDL, a characteristic biomarker of atherosclerosis, includes the following steps: Glycine tert-butyl hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butanol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then, 4-(diethylamino)-2-hydroxybenzaldehyde (1.0 eq.) was added and stirred overnight at room temperature. Next, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added. The mixture was stirred overnight at room temperature, and then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by column chromatography using petroleum ether:ethyl acetate (3:1) as eluent to give a yellow solid.

[0078] Among them, probe 1j 1 H NMR spectrum ( Figure 19 ): 1 H NMR (400 MHz, CDCl3) δ 14.16 (s, 1H), 7.11 (s, 1H), 7.09 (d, J = 8.9 Hz, 1H), 6.20 (dd, J = 8.8, 2.5 Hz, 1H), 6.14 (d, J = 2.6 Hz, 1H), 4.28 (s, 2H), 3.36 (q, J = 7.1 Hz, 4H), 2.26 (s, 3H), 1.44 (s,9H), 1.17 (t, J = 7.1 Hz, 6H).

[0079] 13 C NMR spectrum ( Figure 20 ): 13 C10 NMR (101 MHz, CDCl3) δ 167.19, 166.64, 161.04, 153.14, 151.85, 138.82, 132.07, 127.12, 109.46, 104.66, 99.28, 83.09, 44.62, 42.17, 27.97, 14.79, 12.80. High-resolution mass spectrometry (C101 MHz, CDCl3) Figure 21 ): Calculate m / z [M+H] + The value is 388.2236, and the measured value is 388.2223.

[0080] Example 11 A spray containing probe 1j is prepared as follows: Take 5 mg of probe 1j prepared in Example 10, dissolve it in 10 mL of sterile physiological saline, add 0.1% (w / v) of cosolvent (polysorbate 80), filter it through a 0.22 μm microporous membrane for sterilization, and dispense it into a brown glass bottle equipped with a spray pump to obtain the probe molecule spray.

[0081] Example 12 A lyophilized powder injection containing probe 1i is prepared as follows: 10 mg of probe 1i prepared in Example 9 and 20 mg of mannitol are dissolved in 1 mL of phosphate-buffered saline (PBS, pH 7.4). After sterilization by filtration through a 0.22 μm microporous membrane, the solution is dispensed into vials and freeze-dried to obtain a white, loose, lumpy or powdery lyophilized powder injection. The lyophilized powder injection is reconstituted with water for injection before use.

[0082] Example 13 A commercial kit containing 1g of probe comprises: Component A: a bottle of DMSO stock solution (1 mM concentration) containing 1 mg of the probe prepared in Example 7; Component B: 10 mL of 10× concentrated PBS buffer (pH 7.4); Component C: a series of Ox-LDL standards at different concentrations (for preparing a standard curve); and an instruction manual describing the detection steps in detail.

[0083] Example 14 Probes 1a-1j prepared in Examples 1-10 were dissolved in DMSO to prepare stock solutions of each probe with a concentration of 2 mM. The UV absorption spectroscopy test was performed using the 1j test system as an example, and the method was as follows: PBS buffer (25...) was used. o The test solvent was C (pH=7.4), and the system contained 10 μmol of probe 1j and 50 μg / mL Ox-LDL. After obtaining the mixed solution, it was shaken well at room temperature and allowed to stand for 15 min before recording the UV absorption spectrum of probe 1j. The wavelength value of the maximum absorption of each compound was recorded for excitation fluorescence spectrum. The same method was used to perform the above tests on 1a-1i, and the UV absorption spectra of the probe molecules are shown below. Figure 22 As shown.

[0084] Example 15 PBS buffer (25) oIn a system (C, pH=7.4), probes 1a-1j were added to a concentration of 10 μmol. The fluorescence spectrum and absolute fluorescence quantum yield at 509 nm were measured. Then, 50 μg / mL Ox-LDL was added to each probe in a PBS buffer system, and the fluorescence spectrum and absolute fluorescence quantum yield at 509 nm were measured again. The change in absolute fluorescence quantum yield before and after the addition of Ox-LDL was calculated. The results are as follows: Figure 23 As shown. From Figure 23 The results show that after adding probe 1a-1j to the PBS buffer system, the fluorescence intensity of all systems remained almost unchanged. However, after adding Ox-LDL, the fold change in the reaction of probes 1a-1i gradually increased, with the absolute fluorescence quantum yield of 1i changing by more than 40-fold. Probe 1j showed the largest change in absolute fluorescence quantum yield before and after the reaction with Ox-LDL, exceeding 90-fold. This indicates that probe 1j can be considered the ideal probe selected and has the best recognition effect on Ox-LDL.

[0085] Example 16 In the PBS buffer system (25) o Add probe 1j to a solution (C, pH=7.4) to a probe concentration of 10 μmol, then add different concentrations (0, 1, 2, 3, 5, 10, 25, 50 μg / mL) of Ox-LDL. Incubate at room temperature for 15 minutes, then test the fluorescence spectrum (excitation / emission wavelengths are set according to probe characteristics). The results are as follows: Figure 24 As shown. From Figure 24 As can be seen, after adding probe 1j to the PBS buffer system, the fluorescence intensity of the system is almost non-existent. However, with the increase of the added Ox-LDL concentration, the fluorescence intensity of probe 1j at 509 nm increases significantly. At different Ox-LDL concentrations, the fluorescence intensity shows a good linear or positive correlation with the Ox-LDL concentration, indicating that the probe of this invention can be used for the quantitative detection of Ox-LDL within a certain concentration range.

[0086] Example 17 In a PBS buffer system (25℃, pH=7.4), 1j of probe was added to a concentration of 10 μmol. Then, 50 μg / mL of HDL, LDL, and 0x-LDL were added respectively, and the fluorescence spectra were measured. The results are as follows: Figure 25 As shown. From Figure 25 It can be seen that as the degree of lipoprotein oxidation (degree of oxidation: Ox-LDL>LDL>HDL) increases, the fluorescence emission of probe 1j gradually red-shifts and the intensity gradually increases, indicating that the probe can effectively distinguish between HDL, LDL and Ox-LDL.

[0087] Example 18 The fluorescence spectra of probe 1j under different polarities were tested in various test solvent systems. The concentration of probe 1j in each solvent was 10 μmol. The solvents were: xylene, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, methanol, N,N-dimethylformamide, dimethylsulfoxide, and formamide. The results are as follows: Figure 26 As shown, from Figure 26 The results show that as the solvent polarity increases, the emission wavelength of the probe gradually red-shifts, indicating that probe 1j is more sensitive to polarity response. This can explain the changes in fluorescence emission shift of 1j in the three different polarity lipoprotein environments of HDL, LDL and Ox-LDL.

[0088] Example 19 The fluorescence spectra of probe 1j under different polarity environments were tested in different test solvent systems and solutions of 50 μg / mL Ox-LDL. The concentration of probe 1j was 10 μmol in all cases. The solvents were: xylene, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, methanol, N,N-dimethylformamide, dimethylsulfoxide, and formamide. The results are as follows: Figure 27 As shown. From Figure 27 The results show that the fluorescence intensity of probe 1j reaches its maximum in the presence of Ox-LDL, while the fluorescence caused by polar solvents is not obvious. This indicates that although probe 1j is more sensitive to polarity, it is difficult to achieve specific targeting of proteins by polarity alone. However, it can provide a reasonable explanation for the changes in fluorescence emission shift of 1j in different polar lipoprotein environments.

[0089] Example 20 Mixed solutions of different viscosities (i.e., different cP values) were prepared using a water / glycerol system, with the water content increasing in a gradient from 0% to 100%, corresponding to different cP values. The fluorescence spectrum changes of probe 1j in different viscosity environments were then tested, and the results are as follows: Figure 28 As shown. From Figure 28The results show that the emission intensity of the probe gradually increases with increasing viscosity in the system, indicating that probe 1j is highly sensitive to viscosity. This suggests that the binding of the probe to the protein is likely due to molecular rotational restriction. This could explain the enhanced fluorescence emission wavelength of 1j in different polar lipoprotein environments.

[0090] Example 21 The photophysical properties of probes 1a-1j prepared in Examples 1-10 were detected using ultraviolet and fluorescence spectroscopy, and the results are shown in Table 1.

[0091] Table 1. Photophysical properties of probes 1a-1j

[0092] As shown in Table 1, the absolute fluorescence quantum yield of 1j was the highest after the addition of Ox-LDL, reaching 14.7%.

[0093] Example 22 Fluorescence imaging was used to qualitatively assess the ability of probe 1j to distinguish different apolipoproteins in live cells. Figure 29 Fluorescence imaging revealed no fluorescence in untreated cells when probe 1j was used alone. Cells treated with low-density lipoprotein (LDL) exhibited significantly higher fluorescence granularity and intensity than those treated with high-density lipoprotein (HDL). LDL treatment induced a diffuse granular fluorescence pattern, while HDL produced a very weak signal. However, upon co-incubation with Ox-LDL, the probe selectively labeled large, dot-like aggregates exhibiting strong green fluorescence, confirming the specificity of the response. The experiments demonstrated a correlation between apolipoprotein oxidation levels and cellular oxidative stress.

[0094] Example 23 Using lifetime imaging technology, the ability of probe 1j to distinguish different apolipoproteins in living cells was quantitatively evaluated. Figure 30 To confirm these results, we performed co-staining experiments using the nuclear probe Hoechst 33342 for fluorescence lifetime imaging microscopy. After incubation with HDL or LDL, the fluorescence lifetime of probe 1j in diffuse distribution mode was approximately 1.41 ± 0.6 ns. Notably, Ox-LDL treatment elicited a unique response, characterized by an extended fluorescence lifetime (1.65 ± 0.5 ns) in the perinuclear endoplasmic reticulum region.

[0095] In summary, these imaging studies demonstrate that probe 1j can achieve precise and highly specific localization of oxidized apolipoproteins within living cells. Crucially, the observed change in fluorescence lifetime—which gradually increases with increasing oxidation—provides a quantitative indicator for distinguishing protein aggregation states. This phenomenon is consistent with the probe's activation mechanism, namely its sensitivity to the local polarity and viscosity of the microenvironment.

[0096] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A probe molecule, characterized in that, The structure of the probe molecule is shown in general formula (1): General formula (1) R represents a conjugated group containing an N-aryl ring.

2. The probe molecule according to claim 1, characterized in that, The probe molecule has a specific structure that is any one of the following: 。 3. A composition comprising the probe molecule of claim 1 or 2, characterized in that, Clinically applicable sprays, lyophilized powder injections, or commercial kits are prepared from the active ingredient being the probe molecule or a combination of the probe molecule and pharmaceutically acceptable excipients.

4. A composition comprising the probe molecule of claim 1 or 2, characterized in that, It includes the probe molecule and a pharmaceutically acceptable carrier.

5. The use of the probe molecule of claim 1 or 2 in the preparation of a product for the detection of oxidized low-density lipoprotein.

6. The use of the probe molecule of claim 1 or 2 in the preparation of a product for the identification and detection of oxidized low-density lipoprotein, low-density lipoprotein and high-density lipoprotein.

7. The use of the probe molecule of claim 1 or 2 in the preparation of a diagnostic reagent for early atherosclerosis.

8. The use of the probe molecule of claim 1 or 2 in the preparation of a diagnostic reagent for early fatty liver.

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