Lipid droplet targeted difunctional ratiometric fluorescent probe and application thereof in preparation of OH detection reagent

Abstract

The invention discloses a lipid droplet targeting difunctional ratiometric fluorescent probe and an application thereof in preparation of an OH detection reagent. The structure of the difunctional ratiometric fluorescent probe is shown as follows: # imgabs0 #. The double-function ratio-type fluorescent probe can respond to OH with different concentrations in a ratio-type fluorescent mode, the detection limit is as low as 1.18 nM, the difference value of two emission peaks is as high as 140 nm, and double-channel crosstalk-free detection can be achieved. A cytotoxicity test shows that the probe is good in biocompatibility, a confocal fluorescence microscopic imaging experiment shows that the probe is good in light stability for HepG2 cells, has the capacity of monitoring endogenous and exogenous OH concentration changes of the cells through two channels, has the capacity of lipid droplet imaging (R2 = 0.85), and is suitable for confocal fluorescence imaging of intracellular lipid droplet tracing.

Description

Technical Field

[0001] The present invention relates to a lipid droplet-targeted bifunctional ratiometric fluorescent probe and its application in the preparation of •OH detection reagents, so as to achieve ratiometric fluorescence imaging of •OH in solutions and cells, and the characteristics of lipid droplet fluorescence tracing, and has the advantages of specific selectivity, efficient detection and good biocompatibility. Background Art

[0002] Lipid droplets are important lipid storage organelles in cells and are widely present in various biological cells. Their structure is mainly composed of a neutral lipid core and a monolayer phospholipid membrane, with a variety of functional proteins on the surface. Lipid droplets are not only involved in energy metabolism, but are also closely related to a variety of cellular functions, such as fatty acid transport and protein degradation. Therefore, in-depth research on the biological characteristics and functions of lipid droplets is of great significance for understanding and treating these diseases.

[0003] Reactive oxygen is a class of highly reactive chemical substances, mainly including hydroxyl radicals (•OH), hydrogen peroxide (H2O2), superoxide anion, hypochlorous acid (HOCl), singlet oxygen ( 1 O2), peroxyl radicals (ROO•), and peroxynitrite (ONOO - ) etc. In biological systems, the redox balance regulated by reactive oxygen species is crucial to maintaining normal cell function. Abnormal content of reactive oxygen species can disrupt the redox balance in cells, causing cell damage and even the occurrence of some diseases, such as cancer, arthritis, atherosclerosis and certain autoimmune diseases. Among them, hydroxyl radical (•OH), as one of the important reactive oxygen species, can be produced through the Fenton reaction. •OH is the most oxidative and harmful type of reactive oxygen species in biological systems. It can react with a variety of biological molecules in the body, causing oxidative damage to substances such as sugars, lipids, proteins and nucleic acids, thereby causing cell mutation or necrosis. •OH is also associated with processes such as aging, inflammation, cancer and cardiovascular disease. •OH has high reactivity. •OH can quickly react with glutathione (GSH) that is abundant in cells. Therefore, unlike other reactive oxygen species with low reactivity and long life, •OH will be quickly consumed where it is produced, resulting in a low concentration in the body. Therefore, the development of methods that can accurately detect •OH in biological systems still faces great challenges.

[0004] So far, the reported methods for determining •OH include nuclear magnetic resonance (NMR), absorption spectroscopy, surface enhanced Raman scattering (SERS), hydrogel, etc. The fluorescence imaging method based on •OH fluorescent probe has attracted much attention because it can eliminate the interference of other signal molecules, has the advantages of specific response to •OH, high sensitivity and real-time performance. The development of a responsive •OH ratio fluorescent probe will provide a powerful tool for real-time imaging of intracellular •OH fluctuations.

[0005] In recent years, fluorescence imaging methods have been widely used to monitor biological processes, but there are still very few specific imaging of •OH, especially ratiometric fluorescent probes. Therefore, the development of a ratiometric fluorescent probe that can respond to •OH will provide a powerful tool for real-time imaging of intracellular •OH fluctuations. Summary of the invention

[0006] In view of the shortcomings of the above-mentioned prior art, the present invention provides a lipid droplet-targeted dual-function ratiometric fluorescent probe and its application in the preparation of •OH detection reagents. The technical problem to be solved is to obtain a molecular structure that responds to •OH and lipid droplet fluorescence tracing in a ratiometric manner through molecular design, requiring that the molecular structure not only has the ability of lipid droplet tracing, but also has a ratiometric fluorescent signal without crosstalk after responding to •OH, and has the advantages of selectivity, high efficiency in •OH detection and good photostability.

[0007] The present invention designs and synthesizes a ratiometric •OH fluorescent probe EGS using quinoline with excellent performance as a fluorophore. A D-Π-A structure is formed with dihydroquinoline as an electron donor and dicyanoisophorone as an electron acceptor, thereby generating an intramolecular charge transfer (ICT) effect and causing emission at 663nm. After reacting with •OH, the single bond at the carbonyl group is converted into a double bond, disrupting the original D-Π-A structure, making dihydroquinoline an electron-withdrawing quinoline salt structure, and forming a new D-Π-A structure with p-methoxyacetophenone, generating a new intramolecular charge transfer (ICT) effect and causing new emission at 523nm. The two emission channels of EGS have low fluorescence background and large displacement, which are characteristics of ratiometric probes. At the same time, the molecular structure of EGS is neutral, which also ensures its ability to trace lipid droplets. Tests of optical properties and cytotoxicity show that the EGS has •OH specificity and good biocompatibility. This study evaluated the feasibility of using EGS for living cell •OH-specific fluorescence imaging and lipid droplet imaging through analysis of cell confocal fluorescence imaging, aiming to provide a convenient chemical tool for the visualization detection of •OH and the study of related diseases.

[0008] The lipid droplet-targeting dual-function ratiometric fluorescent probe of the present invention is abbreviated as EGS, and its structure is as follows: .

[0009] The preparation method of the dual-function ratiometric fluorescent probe of the present invention comprises the following steps: Step 1: Dissolve 5.00 g of isophorone and 2.30 g of malononitrile in 30 mL of anhydrous ethanol, add 0.5 mL of piperidine and heat under reflux for 8 hours; after the reaction, remove the solvent by distillation under reduced pressure, and obtain white crystalline intermediate 1 after extraction and column chromatography (eluent: petroleum ether: ethyl acetate = 30:1).

[0010] Step 2: Under nitrogen protection, 0.20 g of intermediate 1, 0.20 g of 3-quinolinecarboxaldehyde and a catalytic amount of piperidine were dissolved in 5 mL of acetonitrile, and the mixture was heated at 60°C for 7 hours. After cooling to room temperature, a large amount of yellow precipitate was produced, which was filtered and rinsed with cold ethanol and air-dried to obtain intermediate 2 as a yellow powder.

[0011] Step 3: Under nitrogen protection, 0.25 g of intermediate 2 and 400 μL of methyl trifluoromethanesulfonate were dissolved in dichloromethane. After stirring at room temperature for 24 h, a large amount of yellow precipitate was produced. The yellow precipitate was filtered and rinsed with ether to obtain light yellow powder intermediate 3.

[0012] Step 4: 0.08 g of intermediate 3 and 0.044 g of p-methoxyacetophenone were dissolved in a mixed solution of ethanol and water (10 mL V / V = 1:1), and then 0.0118 g of NaOH was added and the mixture was reacted at 55 °C for 5 h. After the reaction, the product was extracted with dichloromethane, and then the solvent was removed and separated by column chromatography (eluent: petroleum ether: ethyl acetate = 20:1) to obtain purple-black solid powder EGS.

[0013] The synthetic route is as follows:

[0014] Application of the dual-functional ratiometric fluorescent probe of the present invention in the preparation of •OH detection reagents.

[0015] The detection reagent is in I 523nm / I 663 nm The fluorescence ratio at is linearly related to the concentration of •OH.

[0016] The bifunctional ratiometric fluorescent probe of the present invention uses dihydroquinoline as an electron donor and dicyanoisophorone as an electron acceptor to form a D-Π-A structure, thereby emitting fluorescence at 663nm. After reacting with •OH, the molecular structure changes to EGS-1. The entire reaction process transforms the carbonyl group of EGS into a single bond, and dihydroquinoline is transformed into a quinolate structure with electron-withdrawing ability, disrupting the original D-Π-A structure. The newly formed quinolate structure and p-methoxyacetophenone with electron-donating ability form a new D-Π-A structure to form a new emission channel at 523nm, thereby forming fluorescence in two different channels, which has the ability to detect •OH in a ratiometric manner. EGS has good cell compatibility and has the prospect of being used in cell confocal imaging. Since the EGS molecule is a neutral molecule, EGS itself has the ability to enter lipid droplets. Its response mechanism to •OH is shown as follows:

[0017] The detection method is as follows: The EGS of the present invention was dissolved in DMSO to prepare a 2 mM mother solution, and 15 μL of each mother solution was taken into 3 mL of a mixed solvent of PBS and DMSO (PBS:DMSO=6 / 4) containing different concentrations of •OH to obtain the fluorescence spectrum and UV spectrum of 10 μM EGS in different test solutions. With the increase of •OH, the absorbance of EGS increased at 550nm. The fluorescence intensity at 663 nm decreased, and new fluorescence emission appeared at 523 nm and the fluorescence intensity gradually increased. In addition, the dual-channel displacement of EGS before and after the reaction with •OH was large. The emission before the reaction was at 663 nm, and the emission after the reaction was at 523 nm. The emission displacement difference was 140nm, with almost no crosstalk, and it has the ability of ratiometric luminescence detection. Its fluorescence ratio has a good linear relationship with the concentration of •OH (R 2 =0.98), EGS can respond normally to •OH in the physiological pH range, can respond well to •OH in cells, and the imaging of intracellular lipid droplets is good (Pr=0.85).

[0018] The dual-function ratiometric fluorescent probe for detecting •OH and lipid droplet imaging in cells of the present invention can respond to different concentrations of •OH with dual-channel ratiometric fluorescence, and the effective minimum detection limit is as low as 1.18nM. Cytotoxicity tests show that EGS has good biocompatibility, and confocal fluorescence microscopy experiments show that EGS has good photostability for HepG2 cells, has the ability to dual-channel monitor changes in endogenous and exogenous •OH concentrations in cells, and has the ability to image lipid droplets (R 2 =0.85), which is suitable for confocal fluorescence imaging of intracellular lipid droplet tracing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The 3D fluorescence graphs show the changes in the fluorescence emission peaks of the EGS and •OH reactions at different reaction times. (a) EGS (10 μM) and •OH (200 μM) react for 0 min; (b) EGS (10 μM) and •OH (200 μM) react for 10 min; (c) EGS (10 μM) and •OH (200 μM) react for 35 min; (d) EGS (10 μM) and •OH (200 μM) react for 50 min.

[0020] Figure 2 (a) shows the difference in fluorescence emission peaks before and after the reaction of EGS and •OH; (b) shows the change in the ratio of the dual-channel emission peak intensities over time when EGS (10 μM) reacts with •OH (200 μM) and •OH (200 μM).

[0021] Figure 3 The UV fluorescence titration and linear relationship and detection limit test after the reaction of EGS and •OH. (a) The change of 523 nm emission peak after the reaction of EGS (10 μM) and •OH (0-200 μM); (b) The change of 663 nm emission peak after the reaction of EGS (10 μM) and •OH (0-200 μM); (c) The change of UV absorption spectrum after the reaction of EGS (10 μM) and •OH (0-200 μM); (d) The fluorescence ratio after the reaction of EGS (10 μM) and •OH (0-25 μM) showed a good linear relationship with the concentration of •OH (R 2 =0.98), and the minimum detection limit was 1.18 nM.

[0022] Figure 4 This is a pH stability test of EGS. This test was conducted to investigate the pH stability of EGS (10 μM) after reacting with •OH (200 μM) for 10 min, and to investigate whether EGS was affected by the pH in the physiological range during the reaction with •OH.

[0023] Figure 5 It is a selective test of EGS. The fluorescence intensity changes after EGS (10 μM) reacted with common metal ions (100 μM), reactive oxygen species (100 μM), enzymes (100 μM) and •OH (200 μM) were investigated.

[0024] Figure 6 This is a co-localization study of lipid droplet imaging of EGS. EGS and 1 μg / mL lipid droplet dye (BODIPY) were co-incubated in HepG2 cells and then confocal imaging was performed to explore the lipid droplet imaging ability of EGS.

[0025] Figure 7This is a test in which EGS is used to detect exogenous •OH. After incubating cells with EGS (10 μM), 30 μM and 60 μM •OH were added respectively and reacted for 30 min. Confocal imaging was then performed to explore the ability of EGS to detect •OH from outside the cells.

[0026] Figure 8 This is a test in which EGS is used to detect endogenous •OH. After inducing oxidative stress in cells with 1 μM lipopolysaccharide (LPS) for 30 minutes and 60 minutes respectively, EGS (10 μM) was added and incubated for 30 minutes before confocal imaging was performed to explore the ability of EGS to monitor •OH produced in cells. DETAILED DESCRIPTION

[0027] The present invention will be further described below by way of examples.

[0028] Example 1: Synthesis of EGS 0.08 g of intermediate 3 and 0.044 g of p-methoxyacetophenone were dissolved in a mixed solution of ethanol and water (10 mL v / v=1:1), and then 0.0118 g of NaOH was added and the mixture was reacted at 55 °C for 5 h. After the reaction, the product was extracted with dichloromethane. The solvent was then removed. Purple-black solid powder EGS was obtained by column chromatography (yield 40%).

[0029] 1 H NMR (600 MHz, CHLOROFORM- D ) δ 7.78 (d, J = 8.9 Hz, 2H), 7.28 (dd, J = 7.7, 1.5 Hz, 1H), 7.20-7.15 (m, 1H), 6.98 (td, J = 7.4, 1.1 Hz, 1H),6.88-6.85 (m, 2H), 6.84 (d, J = 8.1 Hz, 1H), 6.78 (d, J = 15.5 Hz, 1H), 6.60(d, J = 37.6 Hz, 2H), 6.23 (d, J = 15.4 Hz, 1H), 4.69 (dd, J = 7.9, 4.1 Hz,1H), 3.85 (s, 3H), 3.33 (s, 3H), 3.23 (dd, J = 15.4, 7.9 Hz, 1H), 2.95 (dd,J = 15.4, 4.2 Hz, 1H), 2.51 (s, 2H), 2.37-2.28 (m, 2H), 1.02 (d, J = 2.5 Hz,6H). 13 C NMR (101 MHz) 120.70, 119.61,115.05, 114.08, 113.81, 112.76, 112.43, 77.48, 77.16, 76.84, 73.50, 55.66,46.77, 43.05, 39.80, 38.99, 33.77, 31.99, 28.24, 28.13.

[0030] Example 2: Fluorescence changes of EGS in response to •OH in solvent In order to explore the fluorescence changes of EGS in response to •OH (200 μM) in solvent, 3D fluorescence was used to monitor the reaction process. Figure 1 It can be clearly seen that after EGS reacts with •OH, the characteristic peak intensity of EGS at 663nm gradually weakens until it disappears completely, while the characteristic peak intensity of EGS at 523nm gradually increases after the reaction with •OH. This indicates that after EGS reacts with •OH, the signal of one fluorescence channel gradually turns off, and the signal of another fluorescence channel gradually turns on, which has the application prospect of ratiometric probe.

[0031] Example 3: Channel displacement difference and reaction time of EGS in response to •OH in solvent In order to explore the channel shift of EGS in response to •OH (200 μM) in solvent, the emission channels of EGS before and after the response to •OH were compared. Figure 2 (a) It can be seen that the emission channel of EGS and •OH before the response is located at 663nm, and the emission channel after the response is located at 553nm. The difference between the two channels is 140nm, which shows that EGS has the characteristics of extremely low crosstalk fluorescence emission, which is extremely beneficial to the ratio signal of the probe. Thanks to this advantage, EGS can also perform dual-channel imaging detection of •OH in cells. Figure 2 As shown in (b), the reaction time of EGS in response to •OH (200 μM) in the solvent was monitored, and it can be seen that the reaction between EGS and •OH was basically completed at 30 min.

[0032] Example 4: Optical titration and linear relationship of EGS in response to •OH in solvent To investigate the optical titration of EGS in response to •OH in solvent, fluorescence titration was performed using •OH (200 μM). Figure 3 (a) and Figure 3 (b) It can be seen that as the •OH concentration of EGS increases, the fluorescence intensity at 663 nm gradually decreases from left to right, and the fluorescence intensity at 523 nm gradually increases. Figure 3 (c) It can be seen that with the addition of •OH, the absorption of EGS at 550 nm gradually decreases. Figure 3 (d) It can be seen that the fluorescence ratio signal of EGS shows a good linear relationship in the detection of •OH (0-25μM), and the linear correlation coefficient R 2 =0.98, and the minimum detection limit was 1.18 nM. This indicates that EGS and •OH can emit a good ratiometric fluorescence signal after reaction, and •OH can be accurately monitored in the solvent.

[0033] Example 5: pH stability of EGS in response to •OH in solvent In order to investigate the pH stability of EGS in response to •OH (200 μM) in solvents. Figure 4 , comparing the changes in fluorescence ratio of EGS and •OH in different pH solvents after ten minutes of reaction, this indicates that EGS can respond normally to •OH within the pH of the physiological environment.

[0034] Example 6: Experiment on the selective response of EGS to •OH in solvent In order to explore the specificity of EGS in response to •OH (200 μM) in solvent, EGS was used to detect common enzymes, metal ions, reactive oxygen species, etc. in solvent. Figure 5 This indicates that EGS is not affected by other metal ions, enzymes, and other reactive oxygen species, but has a good response to •OH, and the fluorescence ratio after response is much higher than that of other detectors. This will hopefully be used for real-time monitoring of •OH concentration in cells.

[0035] Example 7: Lipid droplet imaging of EGS In order to explore the ability of EGS to image lipid droplets in cells, we used lipid droplet dye (BODIPY) and EGS to perform co-localization studies in HepG2 cells. Figure 6 As shown, the red channel of EGS (λ em = 663 ± 20 nm; λ ex = 550nm) and the green channel of BODIPY (λ em = 510±20 nm; λ ex= 488 nm) overlapped well, and the Pearson colocalization coefficient of EGS and BODIPY was calculated to be 0.85. These results indicate that EGS has the ability to track lipid droplets.

[0036] Example 8: Exogenous OH test of EGS in cells Whether EGS can be used to detect •OH in cells is very important, because it reflects whether EGS can be used to monitor the concentration changes of •OH in cells. Figure 7 In the monitoring process of exogenous •OH in HepG2 cells, compared with the control group, after incubation with •OH in other groups, the fluorescence intensity of the 523 nm channel increased with the increase of •OH concentration, and the fluorescence intensity of the 663 nm channel decreased with the increase of •OH concentration (ChannelⅠ:λ em = 523±20 nm. ex =440 nm. Channel II: λ em = 663 ± 20 nm; λ ex = 550 nm), which shows that both channels of EGS can monitor the concentration changes of exogenous •OH in cells. Dual-channel fluorescence verification is more stable than traditional single-channel fluorescence, avoiding erroneous signal feedback caused by errors in a single channel.

[0037] Example 9: Endogenous OH test of EGS in cells In order to explore the ability of EGS to monitor the changes of •OH in cells, HepG2 cells were induced with LPS for different time periods of oxidative stress ( Figure 8 ). Compared with the control group, after LPS was added to cells to induce oxidative stress, the green channel increased with the increase of LPS induction time, while the red channel decreased with the increase of LPS induction time. Cells will produce more •OH after LPS-induced oxidative stress. The results show that the two channels of EGS can well capture the changes of •OH in this process, which will hopefully be used to monitor the changes of •OH in organisms.

Claims

1. A lipid droplet-targeted dual-function ratiometric fluorescent probe, referred to as EGS, characterized in that Its structure is as follows: 。 2. The method for preparing the dual-functional ratiometric fluorescent probe according to claim 1, characterized in that The steps include: Step 1: dissolving isophorone and malononitrile in anhydrous ethanol, adding piperidine and heating under reflux for reaction; after the reaction, removing the solvent by distillation under reduced pressure, and obtaining a white crystalline intermediate 1 after extraction and column chromatography; Step 2: Under nitrogen protection, the intermediate 1, 3-quinolinecarboxaldehyde and a catalytic amount of piperidine are dissolved in acetonitrile, and the mixture is heated at 60°C for reaction; after the reaction is completed, it is cooled to room temperature to produce a large amount of yellow precipitate, which is filtered and rinsed with ethanol and air-dried to obtain yellow powder intermediate 2; Step 3: Under nitrogen protection, intermediate 2 and methyl trifluoromethanesulfonate were dissolved in dichloromethane. After stirring at room temperature for 24 h, a large amount of yellow precipitate was produced. The yellow precipitate was filtered and washed with ether to obtain light yellow powder intermediate 3. Step 4: Dissolve the intermediate 3 and p-methoxyacetophenone in a mixed solution of ethanol and water, then add NaOH and react the mixture at 55°C for 5 h; after the reaction, extract the product with dichloromethane, remove the solvent, and separate by column chromatography to obtain a color solid powder EGS; The synthetic route is as follows: 。 3. Use of the dual-functional ratiometric fluorescent probe according to claim 1 in the preparation of •OH detection reagents.

4. The use according to claim 3, characterized in that: The detection reagent is in I 523nm / I 663 nm The fluorescence ratio at is linearly related to the concentration of •OH.

5. The use according to claim 3, characterized in that: The detection reagent targets lipid droplets.

6. The use according to claim 3, characterized in that: The detection limit of the detection reagent for •OH is 1.18 nM.

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