Lipid peroxidation detection probe based on perylene bisimide-triphenylphosphine structure as well as preparation method and application of lipid peroxidation detection probe

Abstract

The invention provides a lipid peroxidation detection probe based on a perylene bisimide-triphenylphosphine structure and a preparation method and application of the lipid peroxidation detection probe, and belongs to the technical field of lipid peroxidation fluorescent probe design synthesis and visual imaging. The structural formula of the lipid peroxidation detection probe is # imgabs0. The structure of the lipid peroxidation detection probe is optimized on the basis of a perylene bisimide-triphenylphosphine compound Liperfluo, so that the stability of the lipid peroxidation detection probe is greatly improved; the method has good sensitivity and selectivity to ferroptosis marker lipid hydroperoxide, and the pretreatment of a test sample is simple; the method can be used for detecting ferroptosis caused by endogenous and exogenous fibrosarcoma cells; the specific regulation and control mechanism and the biological function of ferroptosis related metabolic diseases are researched; the compound is solid powder, is convenient to store and use, is simple in synthesis method, high in yield and low in cost, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a lipid peroxidation detection probe based on a perylene imide-triphenylphosphine structure, and a preparation method and application thereof. Background Art

[0002] Ferroptosis is a new type of cell death discovered in recent years. It is an oxidative cell death induced by various reasons and is iron-dependent. Its occurrence is caused by the imbalance of the generation and degradation of intracellular lipid reactive oxygen species (ROS), among which the accumulation of lipid peroxides is the most direct cause of cell ferroptosis.

[0003] Phospholipids (PLs) are the main components of biological membranes and lipoproteins and have very important biological functions. Glycerophospholipids are the most abundant phospholipids in the body. According to the different phospholipid heads, they are divided into phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidylserines (PS), phosphatidyl inositols (PI), phosphatidic acid (PA), cardiolipin (CL), etc. Fatty acids are esterified at the sn-1 and sn-2 positions of glycerophospholipids to form their hydrophobic tails, while polyunsaturated fatty acids (PUFAs) rich in diallyl carbon structures are mostly located at the sn-2 position to form unsaturated phospholipids (PUFA-PLs). The abundant bis-allyl carbon units in PUFAs are highly sensitive to reactive oxygen species (ROS), making PUFA-PLs susceptible to enzymatic or non-enzymatic oxidation to form oxidized phospholipids (OxPLs), accompanied by the generation of a variety of other oxidation products. The oxidation of unsaturated phospholipids has a significant impact on the structure and biological activity of phospholipids, affecting the fluidity and permeability of biological membranes and changing the biological functions of lipids and proteins in membranes.

[0004] Phospholipid peroxidation is an important result of oxidative stress. Phospholipid hydroperoxides (PLOOHs), as primary oxidation products, are at the key node of the phospholipid oxidation process. They are closely related to pathological processes such as neurodegenerative diseases, atherosclerosis, and ischemia-reperfusion injury. They are also the key characteristics of cell ferroptosis. For example, many studies have found that the specific accumulation of PSOOH and PIOOH can be observed in AD mouse models and postmortem brain samples of AD patients, suggesting that both phospholipid hydroperoxides are involved in early neurological damage in AD. Compared with the rapid development of ferroptosis mechanism research, the development of ferroptosis-specific biomarkers and related efficient detection technologies is relatively lagging behind, especially the lack of probes that can specifically, highly sensitively, and in real time detect the cellular phospholipid peroxidation process. In recent years, studies have confirmed that phosphatidylethanolamine (PE) is the preferred substrate for phospholipid oxidation in the ferroptosis process, and PEOOH is considered to be the main signal molecule for ferroptosis. Therefore, the precise detection and regulation of various PLOOHs will not only contribute to the biological basis research of oxidative stress and the accurate diagnosis and treatment of related diseases, but also promote the rapid development of precise detection technology of new ferroptosis biomarkers.

[0005] In recent years, high-sensitivity and high-resolution fluorescence detection technology has been favored by researchers and has been widely used in the detection of various substances. Compared with other monitoring methods, fluorescence imaging technology is sensitive and accurate, can be monitored in real time and has high accuracy. Therefore, in ferroptosis-related scientific research activities, fluorescent probes are also often used to evaluate the process of cell ferroptosis and the level of action of ferroptosis inhibitors. The early developed small molecule probe Liperfluo has been successfully used in the detection of ferroptosis and cell imaging, but the probe has disadvantages such as unstable structure, poor solubility, and weak specificity. Therefore, it is of great significance to improve the stability and solubility of Liperfluo and design and synthesize small molecules that can be used to detect phospholipid hydroperoxides. Summary of the invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a lipid peroxidation detection probe based on a perylene imide-triphenylphosphine structure and a preparation method and application thereof. The compound provided by the present invention has significantly improved stability compared to Liperfluo, has excellent selectivity for phospholipid hydroperoxides, and can be used to prepare detection reagents or drugs for detecting or treating ferroptosis-related diseases; and has obvious fluorescence spectrum changes, has extremely strong selectivity for lipid hydroperoxides, and the phenomenon is obvious and easy to identify.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme to achieve it:

[0008] On the one hand, the present invention provides a lipid peroxidation detection probe molecule compound based on a perylene imide-triphenylphosphine structure, the structural formula of which is shown below:

[0009]

[0010] In the formula, R is at least one of an alkoxy group, an alkyl group, a halogen group, and a halogenated alkyl group.

[0011] Furthermore, the specific R group is selected from at least one of a methoxy group, a methyl group, a fluorine atom, a chlorine atom and a trifluoromethyl group.

[0012] Preferably, the compound is specifically compound I-1 to I-5, and its structural formula is as follows:

[0013]

[0014]

[0015] On the other hand, the present invention also provides the lipid peroxidation detection method based on the perylene imide-triphenylphosphine structure.

[0016] The preparation method of the probe molecule compound comprises the following steps:

[0017]

[0018] (1) Compound 1, p-iodoaniline, CH3COOK, and Pd(OAc)2 were dissolved in DMA, protected by N2, and reacted at 130°C. After the reaction, the mixture was separated and purified to obtain compound 2;

[0019] (2) dissolving the compound 2, N,N-dimethylaniline and trichlorosilane in dry dioxane, protecting with Ar, reacting at 110° C., and separating and purifying after the reaction to obtain compound 3;

[0020] (3) Compound 4 and KOH were dissolved in H2O, and reacted at 90°C. AcOH was added dropwise to the reaction solution, and the reaction was continued. After the reaction was completed, the compound 5 was obtained by separation and filtration.

[0021] (4) The compound 5 and aminotetraglycol monomethyl ether were dissolved in H2O / EtOH (V / V=1:1), reacted at room temperature, and then heated to 90°C for reflux overnight. After completion, the reaction was separated and filtered to obtain compound 6;

[0022] (5) Compound 3, compound 6, Zn(OAc)2 and imidazole were mixed, protected by N2, and reacted at 130°C. After the reaction, the mixture was separated and purified to obtain compound 7, which is a lipid peroxidation detection probe.

[0023] Furthermore, in the step (1), the molar ratio of compound 1: p-iodoaniline: CH3COOK: Pd(OAc)2 is 1-2: 1-3: 1-3: 0.001-0.1.

[0024] Preferably, in step (1), the molar ratio of compound 1: p-iodoaniline: CH3COOK: Pd(OAc)2 is 1:1.2:1.4:0.01.

[0026] Furthermore, in step (2), the molar ratio of compound 2: N,N-dimethylaniline: trichlorosilane is 1-1.1: 8-12: 8-12.

[0028] Preferably, in step (2), the molar ratio of compound 2: N,N-dimethylaniline: trichlorosilane is 1:10:10.

[0029] Furthermore, in step (3), the molar ratio of compound 4:KOH:AcOH is 0.8-1.2:20-30:30-40.

[0031] Preferably, in step (3), the molar ratio of compound 4:KOH:AcOH is 1:28:35.

[0032] Furthermore, in the step (4), the molar ratio of compound 5 to aminotetraglycol monomethyl ether is 0.8-1.2:0.9-1.1.

[0033] Preferably, in step (4), the molar ratio of compound 5 to aminotetraglycol monomethyl ether is 1:1.

[0034] Furthermore, in the step (5), the molar ratio of compound 6: compound 3: Zn(OAc)2 is 0.9-1.1: 1-1.3: 0.4-0.6.

[0036] Preferably, in step (5), the molar ratio of compound 6:compound 3:Zn(OAc)2 is 1:1.2:0.5.

[0037] Preferably, the specific method for separation and purification in step (1) is: after the reaction is completed, the mixed solution is poured into 30 mL of water, the aqueous layer is extracted with dichloromethane, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain a yellow oily compound 2.

[0038] Preferably, the specific method for separation and purification in step (2) is: after the reaction is completed, the mixed solution is poured into 30 mL of water, the aqueous layer is extracted with dichloromethane, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain a white solid compound 3.

[0039] Preferably, the specific method of separation and purification in step (3) is: after the reaction is completed, the mixed reaction is cooled, filtered, washed with water, and dried to obtain a reddish brown solid compound 5.

[0040] Preferably, the specific method of separation and purification in step (4) is: after the reaction is completed, the mixed reaction is cooled, filtered, washed with water, and dried to obtain a reddish-brown solid compound 6.

[0041] Preferably, the specific method for separation and purification in step (5) is: after the reaction is completed, the mixed solution is poured into 30 mL of water, the aqueous layer is extracted with ethyl acetate, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain a red solid compound 7.

[0042] On the other hand, the present invention also provides the use of the lipid peroxidation detection probe in preparing a detection reagent for detecting lipid peroxidation.

[0043] On the other hand, the present invention also provides the use of the lipid peroxidation detection probe in preparing detection reagents and imaging reagents for detecting ferroptosis-related diseases.

[0044] Furthermore, the ferroptosis-related diseases include neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, liver and kidney failure, inflammation, and diabetic complications.

[0045] Furthermore, the drug contains a lipid peroxidation detection probe or a pharmaceutically acceptable salt, excipient or carrier thereof.

[0046] On the other hand, the present invention also provides the use of the lipid peroxidation detection probe in the preparation of antioxidant fluorescent probes or antioxidant indicators.

[0047] Furthermore, the antioxidant fluorescent probe is used for highly sensitive detection of lipid hydroperoxides and has the ability to specifically detect the indicator of lipid peroxidation.

[0048] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0049] 1. The present invention optimizes the structure of the perylene imide-triphenylphosphine compound Liperfluo, and obtains the lipid peroxidation detection probe based on the perylene imide-triphenylphosphine structure through experiments, which greatly improves its stability; it has good sensitivity and selectivity for lipid hydroperoxides, a marker of ferroptosis, and can be used for sensitive detection of ferroptosis and research on ferroptosis-related diseases;

[0050] 2. The preparation method of the lipid peroxidation detection probe based on the perylene imide-triphenylphosphine structure of the present invention has simple steps, simple pretreatment of test samples, high yield, and is suitable for large-scale promotion and application;

[0051] 3. The lipid peroxidation detection probe based on the perylene imide-triphenylphosphine structure described in the present invention can be used as a fluorescent probe, has obvious fluorescence spectrum changes, and can evaluate the monitoring effect in real time through the changes in its own fluorescence spectrum during the detection of ferroptosis.

[0052] The lipid peroxidation detection probe based on the perylene imide-triphenylphosphine structure provided by the present invention can be used to detect endogenous and exogenous ferroptosis in fibrosarcoma cells; and to study the specific regulatory mechanisms and biological functions of ferroptosis-related metabolic diseases; and the lipid peroxidation detection probe is a solid powder, which is easy to store and use, and the synthesis method is simple, the yield is high, the cost is low, and it has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of compound I-1;

[0054] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of compound 1-2;

[0055] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of compound I-3;

[0056] Figure 4 is the hydrogen nuclear magnetic resonance spectrum of compound I-4;

[0057] Figure 5 is the hydrogen nuclear magnetic resonance spectrum of compound I-5;

[0058] Fig. 6A This is a diagram showing the selectivity test results of compound I-1 for detecting lipid hydroperoxides;

[0059] Figure 6B This is a diagram showing the selectivity test results of compound I-2 for detecting lipid hydroperoxides;

[0060] Figure 6C This is a diagram showing the selectivity test results of compound I-3 for detecting lipid hydroperoxides;

[0061] Fig.6D This is a diagram showing the selectivity test results of compound I-4 for detecting lipid hydroperoxides;

[0062] Figure 7 The reaction spectrum and linear relationship between compound I-1 and different concentrations of PEOOH.

[0063] Figure 8 The UV spectrum and fluorescence spectrum of the oxidation state of compound I-1.

[0064] Fig.9A is the stability diagram of compound I-1 itself;

[0065] Fig. 9B is the stability diagram of compound I-2 itself;

[0066] Fig. 9C is the stability diagram of compound I-3 itself;

[0067] Fig.9D is the stability diagram of compound I-4 itself;

[0068] Fig.9E is the stability diagram of compound I-5 itself;

[0069] Fig.9F This is the stability diagram of the compound Liperfluo itself.

[0070] Fig.10 Cell imaging for compound 1-1.

[0071] Fig.11 Cell imaging for compound 1-2.

[0072] Fig.12 Cell imaging of compounds I-1, I-2, Liperfluo, and I-3. DETAILED DESCRIPTION

[0073] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0074] The overall synthesis route of the ferroptosis detection probe based on the perylene imide-triphenylphosphine structure provided by the present invention is as follows:

[0075]

[0076] Among them, (a) DMA, CH3COOK, Pd(OAc) 2, N2, 130°C; (b) dry 1,4-dioxane, N,N-dimethylaniline, trichlorosilane, Ar, 110°C; (c) H2O, KOH, 90°C; AcOH, 90°C; (d) H2O / EtOH (V / V=1:1), stirred at room temperature; 90°C overnight; (e) corresponding triphenylphosphine, imidazole, Zn(OAc)2, N2, 130°C.

[0077] (1) Step a: Compound 1 (1.589 mmol), p-iodoaniline (1.907 mmol), potassium acetate (0.826 mmol) and palladium acetate (0.01589 mmol) were dissolved in DMA (10 mL), replaced with N2 and protected, and reacted at 130°C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, the aqueous phase was extracted with dichloromethane, the organic layer was dried and concentrated. The mixture was separated by column chromatography to obtain a yellow oil, namely compound 2.

[0078] (2) Step b: Compound 2 (0.436 mmol), N,N-dimethylaniline (4.358 mmol), and trichlorosilane (4.358 mmol) were dissolved in dry 1,4-Dioxane (20 mL), and the mixture was reacted at 110° C. for 12 h under Ar protection. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, the aqueous phase was extracted with dichloromethane, the organic layer was dried, and concentrated. The mixture was separated by column chromatography to obtain a white solid, namely compound 3.

[0079] (3) Step c: Compound 4 (5.100 mmol) and KOH (200 mmol) were dissolved in H2O and reacted at 90°C for 5 h. The solution turned green. 10 ml of AcOH (10 mL) was added dropwise to the reaction solution and the reaction was continued. After the reaction was completed, the solution was cooled to room temperature, filtered, washed with water, and dried to obtain a reddish-brown solid, which was Compound 5.

[0080] (4) Step d: The compound 5 (1.040 mmol) was dissolved in H2O / EtOH (V / V=1:1, 20 mL), and aminotetraglycol monomethyl ether (1.040 mmol) was added. After reacting at room temperature for 5 h, the mixture was transferred into a 90°C oil pan overnight. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed with water, and dried to obtain a brick-red solid, which was compound 6.

[0081] (5) Step e: Compound 6 (0.5111 mmol), compound 3 (0.562 mmol), Zn(OAc)2 (0.266 mmol) and imidazole were mixed, replaced and protected by N2, and reacted at 130°C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, the aqueous phase was extracted with ethyl acetate, the organic layer was dried and concentrated. The mixture was separated by column chromatography to obtain a red solid, which was Compound 7.

[0082] Example 1: Preparation of Compound I-1

[0083]

[0084] In this example, the compound I-1 was prepared by the experimental method described above. Figure 1 As shown, 1HNMR(400MHz,Chloroform-d)δ8.69(d,J=7.9Hz,2H),8.63–8.51(m,6H),7.47–7.40(m,2H),7.39–7.31(m,6H),6.93(d,J=8.9Hz,4H),4. 46(t,J=6.0Hz,2H),3.87(d,J=5.1Hz,2H),3.83(s,6H),3.74–3.70(m,2H),3.65–3.57(m,8H),3.50(dd,J=5.8,3.4Hz,2H),3.34(s,3H).

[0085] Example 2: Preparation of Compound I-2

[0086]

[0087] In this example, the compound I-2 was prepared by the experimental method described above. Figure 2 As shown, 1 HNMR(400MHz,Chloroform-d)δ8.67(d,J=7.9Hz,2H),8.58(d,J=7.9Hz,2H),8.53 (d,J=8.1Hz,2H),8.48(d,J=8.1Hz,2H),7.47(dd,J=8.4,6.8Hz,2H),7.36–7.29(m ,6H),7.22–7.16(m,4H),4.46(t,J=6.0Hz,2H),3.86(t,J=5.9Hz,2H),3.72(dd,J =5.9,3.6Hz,2H),3.65–3.57(m,8H),3.53–3.47(m,2H),3.34(s,3H),2.37(s,6H).

[0088] Example 3: Preparation of Compound I-3

[0089]

[0090] In this example, the compound I-3 was prepared by the experimental method described above. Figure 3 As shown, 1HNMR(400MHz,Chloroform-d)δ8.65(d,J=8.0Hz,2H),8.55(d,J=8.0Hz,2H),8.49(d,J=8.1Hz,2H),8.43(d,J=8.1Hz,2H),7.47–7.42(m,2H),7.41–7. 35(m,6H),7.10(t,J=8.8Hz,4H),4.45(t,J=5.9Hz,2H),3.86(t,J=5.9Hz, 2H),3.74–3.70(m,2H),3.65–3.57(m,8H),3.52–3.49(m,2H),3.34(s,3H).

[0091] Example 4: Preparation of Compound I-4

[0092]

[0093] In this example, the compound I-4 was prepared by the experimental method described above. Figure 4 As shown, 1 HNMR(400MHz,Chloroform-d)δ8.68(d,J=8.0Hz,2H),8.60–8.47(m,6H),7.41–7.35(m,7H),7.35–7.27(m,5H),4.46(t, J=6.0Hz,2H),3.86(t,J=6.0Hz,2H),3.72(dd,J=5.9,3.6Hz,2H),3.66–3.56(m,8H),3.50(d,J=9.2Hz,2H),3.34(s,3H).

[0094] Example 5: Preparation of Compound I-5

[0095]

[0096] In this example, the compound I-5 was prepared by the experimental method described above. Figure 5 As shown, 1HNMR(400MHz,Chloroform-d)δ8.66(d,J=8.0Hz,2H),8.56(d,J=8.0Hz,2H),8. 51(d,J=8.1Hz,2H),8.45(d,J=8.2Hz,2H),7.65(d,J=7.9Hz,4H),7.55–7.48(m, 6H),7.45(dd,J=8.5,1.3Hz,2H),4.45(t,J=6.0Hz,2H),3.86(t,J=6.0Hz,2H), 3.72(dd,J=5.9,3.6Hz,2H),3.65–3.57(m,8H),3.52–3.48(m,2H),3.34(s,3H).

[0097] Example 6

[0098] Based on the perylene imide-triphenylphosphine structure-based ferroptosis detection probe molecule provided by the present invention, the probe solution is prepared as follows: weigh an appropriate amount of red solid powder probe compounds I-1 to I-5, dissolve them in a certain amount of dimethyl sulfoxide (DMSO) to obtain a stock solution with a concentration of 1 mM. Dilute the stock solution with methanol, measure the final concentration of the solution used to be 1.0 μM, and record the ultraviolet and fluorescence spectra at 37°C.

[0099] Example 7

[0100] Preparation of analytes for selective experiments: H2O2, HClO, t-BuOOH, CumOOH, ·O2 - ,ONOO - , ·OH, PCOOH, PEOOH, PSOOH and other reactive oxygen species (ROS) were prepared as follows. H2O2 solution was diluted from 30% H2O2 solution and the concentration was measured by light absorbance at 240nm (ε=43.6M-1cm-1). Commercial sodium hypochlorite solution was used as the source of hypochlorite and the molar absorbance of 350M-1cm-1 was used to measure the hypochlorite (ClO - ) concentration. t-BuOOH, CHP and ONOO- were purchased directly. ·O2 - , produced by KO2. Hydroxyl radicals (·OH) are produced by mixing FeSO4 with 10 equivalents of H2O2. 2+ The concentration of ·OH was calculated by the concentration. PCOOH, PEOOH and PSOOH were produced by the corresponding PC, PE and PS respectively through the induction of azobisisoheptanenitrile.

[0101] The experimental results are as follows Figures 6A-6DAs shown, the horizontal axis is the probe molecules (I-1~4) and the types of added ROS, including H2O2, HClO, t-BuOOH, CumOOH, ·O2 - ,ONOO - , ·OH, PCOOH, PEOOH, PSOOH, probe selectivity test conditions Ex = 488nm, Em = 535nm; from Figures 6A-6D It can be seen that these probe molecules have good selectivity for phospholipid hydroperoxides compared with various reactive oxygen species, confirming their specific response to ferroptosis biomarkers. At the same time, the probe molecules have a good linear relationship with the concentration of phospholipid hydroxides ( Figure 7 ). Therefore, these molecules can be further studied as ferroptosis biomarker probes.

[0102] Example 8

[0103] Based on the perylene imide-triphenylphosphine structure-based ferroptosis detection probe molecule of the present invention, the stability evaluation condition is room temperature, not protected from light, and the fluorescence intensity of the probe molecule is tested at the same time interval. The stability of the probe molecule is evaluated by comparing the increase in fluorescence at 535nm after oxidation with Liperfluo. The stock solution is diluted with methanol, and the final concentration of the solution used is measured to be 1.0μM, Ex=488nm, Em=535nm.

[0104] After oxidation of Liperfluo and its derivatives, the peak shape and peak position remain unchanged, the excitation wavelength is 488nm, and the maximum emission peak appears at 535nm ( Figure 8 ). After a long period of observation, the experimental results are as follows Figures 9A-9F As shown in the figure, it can be seen from the line graph that after the introduction of the electron-withdrawing group -CF3, the fluorescence intensity is significantly weakened and almost unstable, with 67.6% of the oxidation state in 1 hour. The introduction of electron-donating groups such as -OCH3 increases the fluorescence intensity. In addition, the introduction of -OCH3 / -CH3 can significantly increase the stability of Liperfluo. At 48h, the oxidation state accounts for 30% and 36%, both lower than Liperfluo's 48.8%. At the same time, -OCH3 improves the solubility of the probe (Table 1).

[0105] Table 1 Solubility of different probes

[0106] Chloroform Ethanol DMSO water I-1 >3 mg / mL <0.5mg / mL <1.8mg / mL Insoluble I-2 >3 mg / mL <0.5mg / mL <1.2mg / mL Insoluble I-3 >3 mg / mL <0.5mg / mL <3mg / mL Insoluble I-4 >3 mg / mL <0.5mg / mL <1.8mg / mL Insoluble I-5 >3 mg / mL <0.5mg / mL <10mg / mL Insoluble Liperfluo >3 mg / mL <0.5mg / mL <1.2mg / mL Insoluble

[0107] Note: Measured at room temperature

[0108] Example 9

[0109] HT1080 cells were pretreated with RLS3 (2.5 μM) for 30 min to induce ferroptosis. Then, the nuclear dye Hoechst and probe molecules (such as I-1 and I-2) were incubated with the cells for 30 min, and the cell imaging of the probe molecules was observed by confocal microscopy. The experimental results are shown in Figure 2. Fig.10 and Fig.11 As shown in the figure, the probe molecules have good ferroptosis imaging ability. The cells were incubated with Erastin (10 μM) for 3 h, and then I-1, I-2, Liperfluo and I-3 probe molecules were added respectively. After incubation for 30 min, the cells were imaged by confocal microscopy ( Fig.12 ), compared with the commercial probe Liperfluo, probes I-1, I-2, and I-3 all have good cell imaging capabilities. Therefore, I-1, I-2, and I-3 can be used for the imaging of ferroptosis in cells.

[0110] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A lipid peroxidation detection probe based on perylene imide-triphenylphosphine structure, characterized in that: The structural formula of the lipid peroxidation detection probe is as follows: In the formula, R is at least one of an alkoxy group, an alkyl group, a halogen group, and a halogenated alkyl group.

2. The lipid peroxidation detection probe according to claim 1, characterized in that The R is selected from at least one of a methoxy group, a methyl group, a fluorine atom, a chlorine atom and a trifluoromethyl group.

3. The lipid peroxidation detection probe according to claim 1, characterized in that The lipid peroxidation detection probe is specifically compounds I-1 to I-5, and their structural formulas are as follows:

4. The method for preparing a lipid peroxidation detection probe based on a perylene imide-triphenylphosphine structure according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) Compound 1, p-iodoaniline, CH3COOK, and Pd(OAc)2 were dissolved in DMA, protected by N2, and reacted at high temperature. After the reaction, the oily compound, i.e., Compound 2, was obtained by separation and purification; (2) dissolving the compound 2, N,N-dimethylaniline and trichlorosilane in dry dioxane, protecting with Ar, reacting at high temperature, and separating and purifying after the reaction to obtain compound 3; (3) Compound 4 was dissolved in H2O, KOH was added, and the reaction was carried out at high temperature. AcOH was dropped into the reaction solution, and the reaction was continued at high temperature. After the reaction was completed, the compound 5 was obtained by separation and filtration; (4) Dissolve the compound 5 and aminotetraglycol monomethyl ether in H2O / EtOH and react at room temperature. After reaction, separate and filter to obtain a brick-red solid, which is compound 6; (5) Compound 6, compound 3, Zn(OAc)2 and imidazole are mixed, protected by N2, and reacted at high temperature. After the reaction is completed, a red solid compound is obtained by separation and purification, which is compound 7, which is a lipid peroxidation detection probe.

5. The preparation method according to claim 4, characterized in that: In the step (1), the molar ratio of compound 1: p-iodoaniline: CH3COOK: Pd(OAc)2 is 1-2: 1-3: 1-3: 0.001-0.

1.

6. The preparation method according to claim 4, characterized in that: In the step (2), the molar ratio of compound 2: N,N-dimethylaniline: trichlorosilane is 1-1.1: 8-12: 8-12.

7. The preparation method according to claim 4, characterized in that: In the step (3), the molar ratio of compound 4: KOH: AcOH is 0.8-1.2: 20-30: 30-40.

8. The preparation method according to claim 4, characterized in that: In the step (4), the molar ratio of compound 5: aminotetraglycol monomethyl ether is 0.8-1.2: 0.9-1.1, and the volume ratio of H2O / EtOH V / V is 1:1; in the step (5), the molar ratio of compound 6: compound 3: Zn(OAc)2 is 0.9-1.1: 1-1.3: 0.4-0.

6.

9. Use of the lipid peroxidation detection probe according to any one of claims 1 to 3 in the preparation of a detection reagent for detecting lipid peroxidation and ferroptosis-related diseases.

10. The use according to claim 9, characterized in that: The ferroptosis-related diseases include neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, liver and kidney failure, inflammation, and diabetic complications.

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