A lipid droplet-targeted dual-functional ratiometric fluorescent probe and its application in the preparation of •OH detection reagents

Abstract

The present invention discloses a lipid droplet-targeted bifunctional ratiometric fluorescent probe and its application in the preparation of a •OH detection reagent, wherein the structure of the bifunctional ratiometric fluorescent probe is as follows: #imgabs0#. The bifunctional ratiometric fluorescent probe of the present invention can respond to different concentrations of •OH with ratiometric fluorescence, with a detection limit as low as 1.18nM and a difference between the two emission peaks as high as 140 nm, enabling dual-channel detection without crosstalk. Cytotoxicity tests have shown that the probe has good biocompatibility, and confocal fluorescence microscopy experiments have shown that the probe has good photostability for HepG2 cells, has the ability to monitor changes in endogenous and exogenous •OH concentrations in cells through dual channels, and has the ability to image lipid droplets (R 2 =0.85), which is suitable for confocal fluorescence imaging of intracellular lipid droplet tracking.

Description

Technical Field

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

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

[0003] Reactive oxygen species are a class of highly reactive chemical substances, including hydroxyl radicals (•OH), hydrogen peroxide (H2O2), superoxide anions, hypochlorous acid (HOCl), singlet oxygen ( 1 O2), peroxyl radicals (ROO•) and peroxynitrite (ONOO - ). In biological systems, the redox balance regulated by reactive oxygen species (ROS) is crucial for maintaining normal cellular function. Abnormal levels of ROS can disrupt cellular redox balance, leading to cell damage and even the development of diseases such as cancer, arthritis, arteriosclerosis, and certain autoimmune diseases. Hydroxyl radicals (•OH), a key ROS species, are produced through the Fenton reaction. •OH is the most potent and harmful ROS species in biological systems. It can interact with a variety of biomolecules in the body, causing oxidative damage to carbohydrates, lipids, proteins, and nucleic acids, leading to cell mutation or necrosis. •OH is also associated with processes such as aging, inflammation, cancer, and cardiovascular disease. •OH is highly reactive and reacts rapidly with glutathione (GSH), which is abundant in cells. Unlike other less reactive and long-lived ROS species, •OH is rapidly consumed at the site of its generation, resulting in low concentrations in the body. Therefore, developing methods for accurately detecting •OH in biological systems remains a significant challenge.

[0004] To date, reported methods for measuring •OH include nuclear magnetic resonance (NMR), absorption spectroscopy, surface-enhanced Raman scattering (SERS), and hydrogels. Fluorescence imaging methods based on •OH fluorescent probes have attracted considerable attention due to their ability to eliminate interference from other signaling molecules, their specific response to •OH, high sensitivity, and real-time performance. The development of a ratiometric fluorescent probe capable of responding to •OH would 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 specific imaging of •OH is still rare, especially ratiometric fluorescent probes. Therefore, the development of ratiometric fluorescent probes that can respond to •OH will provide a powerful tool for real-time imaging of intracellular •OH fluctuations. Summary of the Invention

[0006] To address the shortcomings of the aforementioned prior art, the present invention provides a lipid droplet-targeted dual-function ratiometric fluorescent probe and its application in the preparation of a •OH detection reagent. The technical problem to be solved is to design a molecular structure that exhibits both a ratiometric response to •OH and lipid droplet fluorescence tracing. This molecular structure is required to not only possess lipid droplet tracing capabilities but also produce a crosstalk-free ratiometric fluorescence signal after responding to •OH, demonstrating the advantages of specific selectivity, efficient •OH detection, and excellent photostability.

[0007] This invention designs and synthesizes a ratiometric •OH fluorescent probe, EGS, using the highly-performance quinoline fluorophore. Dihydroquinoline serves as an electron donor and dicyanoisophorone as an electron acceptor to form a D-π-A structure, resulting in an intramolecular charge transfer (ICT) effect and emission at 663 nm. Upon reaction with •OH, the single bond at the carbonyl group is converted to a double bond, disrupting the original D-π-A structure and transforming dihydroquinoline into an electron-withdrawing quinolyl salt. This new D-π-A structure forms with p-methoxyacetophenone, generating a new intramolecular charge transfer (ICT) effect and new emission at 523 nm. EGS exhibits low fluorescence background and large shifts in both emission channels, characteristic of ratiometric probes. EGS's neutral molecular structure also ensures its lipid droplet tracing capabilities. Optical properties and cytotoxicity tests demonstrate that EGS exhibits •OH specificity and good biocompatibility. This study evaluated the feasibility of 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 shown below:

[0009] .

[0010] The preparation method of the dual-function ratiometric fluorescent probe of the present invention comprises the following steps:

[0011] Step 1: Dissolve 5.00 g of isophorone and 2.30 g of malononitrile in 30 mL of anhydrous ethanol, then add 0.5 mL of piperidine and heat under reflux for 8 hours. After the reaction, remove the solvent by vacuum distillation. After extraction and column chromatography (eluent: petroleum ether:ethyl acetate = 30:1), obtain white crystalline intermediate 1.

[0012] Step 2: Under nitrogen, 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. The mixture was heated at 60°C for 7 hours. After cooling to room temperature, a large amount of yellow precipitate formed. This was filtered, rinsed with cold ethanol, and air-dried to obtain Intermediate 2 as a yellow powder.

[0013] Step 3: Under nitrogen, 0.25 g of intermediate 2 and 400 μL of methyl trifluoromethanesulfonate were dissolved in dichloromethane. Stirring at room temperature for 24 h produced a large amount of yellow precipitate. The yellow precipitate was filtered and rinsed with ether to obtain intermediate 3 as a light yellow powder.

[0014] Step 4: Dissolve 0.08 g of intermediate 3 and 0.044 g of p-methoxyacetophenone in 10 mL of a 1:1 ethanol / water mixture. Add 0.0118 g of NaOH and react at 55°C for 5 h. After the reaction, extract the product with dichloromethane, remove the solvent, and separate by column chromatography (eluent: petroleum ether:ethyl acetate = 20:1) to obtain EGS, a purple-black solid powder.

[0015] The synthetic route is as follows:

[0016]

[0017] Application of the dual-function ratiometric fluorescent probe of the present invention in the preparation of a •OH detection reagent.

[0018] The detection reagent is in I 523nm / I 663 nm The fluorescence ratio at 40 nm is linearly related to the concentration of •OH.

[0019] 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 quinolyl salt structure with electron-withdrawing ability, disrupting the original D-Π-A structure. The newly formed quinolyl salt structure forms a new D-Π-A structure with the electron-donating p-methoxyacetophenone, forming a new emission channel at 523nm, thereby forming two different channels of fluorescence, with the ability to ratiometrically detect •OH. 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:

[0020]

[0021] The detection method is as follows:

[0022] The EGS of the present invention was dissolved in DMSO to prepare a 2 mM mother solution, and 15 μL of the mother solution was taken in 3 mL of a mixed solvent of PBS and DMSO containing different concentrations of •OH (PBS:DMSO=6 / 4), and the fluorescence spectrum and UV spectrum of 10 μM EGS in different test solutions were obtained. With the increase of •OH, the absorbance of EGS increased at 550 nm. 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 shift before and after the reaction of EGS 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 shift difference was 140 nm, with almost no crosstalk, and it had the ability of ratiometric luminescence detection. Its fluorescence ratio had a good linear relationship with the concentration of •OH (R 2 =0.98), EGS responded normally to •OH in the physiological pH range and responded well to •OH in cells, with good imaging of intracellular lipid droplets (Pr=0.85).

[0023] The present invention is a dual-function ratiometric fluorescent probe for detecting •OH and imaging lipid droplets in cells. It can respond to different concentrations of •OH with a dual-channel ratiometric fluorescence, with an effective minimum detection limit as low as 1.18nM. Cytotoxicity tests have shown that EGS has good biocompatibility. Confocal fluorescence microscopy experiments have shown that EGS has good photostability in HepG2 cells and has the ability to dual-channel monitor changes in endogenous and exogenous •OH concentrations in cells. EGS also has the ability to image lipid droplets (R 2 =0.85), which is suitable for confocal fluorescence imaging of intracellular lipid droplet tracking. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 3D fluorescence images showing the fluorescence emission peak changes during the reaction between EGS and •OH at different reaction times. (a) EGS (10 μM) and •OH (200 μM) reaction for 0 min; (b) EGS (10 μM) and •OH (200 μM) reaction for 10 min; (c) EGS (10 μM) and •OH (200 μM) reaction for 35 min; (d) EGS (10 μM) and •OH (200 μM) reaction for 50 min.

[0025] 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).

[0026] Figure 3 Figure 3. UV fluorescence titration and linear relationship after the reaction of EGS with •OH, as well as detection limit test. (a) Changes in the 523 nm emission peak after the reaction of EGS (10 μM) and •OH (0-200 μM); (b) Changes in the 663 nm emission peak after the reaction of EGS (10 μM) and •OH (0-200 μM); (c) Changes in the 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) shows a good linear relationship with the concentration of •OH (R 2 =0.98), with the lowest detection limit of 1.18 nM.

[0027] Figure 4 This is a pH stability test of EGS. This test investigates the pH stability of EGS (10 μM) after reacting with •OH (200 μM) for 10 minutes to determine whether EGS is affected by physiological pH during the reaction with •OH.

[0028] Figure 5 This is a selectivity test for 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.

[0029] 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.

[0030] 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 the reaction lasted for 30 minutes before confocal imaging was performed to explore the ability of EGS to detect •OH from outside the cells.

[0031] Figure 8 This is a test using EGS 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

[0032] The present invention will be further described below by way of examples.

[0033] Example 1: Synthesis of EGS

[0034] 0.08 g of intermediate 3 and 0.044 g of p-methoxyacetophenone were dissolved in 10 mL of a 1:1 ethanol / water mixture. 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. EGS was obtained as a purple-black solid powder by column chromatography (40% yield).

[0035] 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.

[0036] Example 2: Fluorescence changes of EGS in response to •OH in solvent

[0037] In order to explore the changes in fluorescence of EGS in response to •OH (200 μM) in solvent, 3D fluorescence was used to monitor the reaction process. Figure 1 As can be clearly seen in the figure, after EGS reacts with •OH, the intensity of the characteristic peak at 663nm gradually decreases and eventually disappears, while the intensity of the characteristic peak at 523nm gradually increases. This indicates that as EGS reacts with •OH, the signal in one fluorescence channel gradually turns off while the signal in the other gradually turns on, suggesting potential application as a ratiometric probe.

[0038] Example 3: Channel displacement difference and reaction time of EGS in response to •OH in solvent

[0039] 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 As can be seen in (a), 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. This shows that EGS has a very low crosstalk fluorescence emission characteristic, which is very beneficial to the ratiometric signal of the probe. Thanks to this advantage, EGS can also perform dual-channel imaging to detect •OH in cells. Figure 2As 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.

[0040] Example 4: Optical titration and linear relationship of EGS in response to •OH in solvent

[0041] 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 the reaction between EGS and •OH can emit a good ratiometric fluorescence signal and accurately monitor •OH in the solvent.

[0042] Example 5: pH stability of EGS in response to •OH in solvent

[0043] In order to investigate the pH stability of EGS in response to •OH (200 μM) in solvents. Figure 4 , comparing the changes in the fluorescence ratio of EGS and •OH in different pH solvents after ten minutes of reaction, this shows that EGS can respond normally to •OH within the pH of the physiological environment.

[0044] Example 6: Selective response experiment of EGS in response to •OH in solvent

[0045] 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, and has a good response ability only to •OH, and the fluorescence ratio after response is much higher than that of other detectors. This will have the potential to be used for real-time monitoring of •OH concentration in cells.

[0046] Example 7: Lipid droplet imaging of EGS

[0047] To investigate 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 = 550 nm) and the green channel of BODIPY (λ em = 510±20 nm; λ ex = 488 nm) overlapped well, and the Pearson colocalization coefficient between EGS and BODIPY was calculated to be 0.85. These results indicate that EGS has the ability to track lipid droplets.

[0048] Example 8: Exogenous OH test of EGS in cells

[0049] 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, while the fluorescence intensity of the 663 nm channel decreased with the increase of •OH concentration (Channel I: λ em = 523±20 nm. λ 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 errors in a single channel and causing erroneous signal feedback.

[0050] Example 9: Endogenous OH Test of EGS in Cells

[0051] 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 to the control group, after LPS was added to cells to induce oxidative stress, the green channel increased and the red channel decreased with increasing LPS induction time. Cells produce more •OH after LPS-induced oxidative stress. These results demonstrate that both channels of the EGS well capture changes in •OH during this process, which holds promise for monitoring changes in •OH in vivo.

Claims

1. A lipid droplet-targeting dual-function ratiometric fluorescent probe, referred to as EGS, characterized in that Its structure is as follows: 。 2. The method for preparing the dual-function ratiometric fluorescent probe according to claim 1, characterized in that The steps include: Step 1: Dissolve isophorone and malononitrile in anhydrous ethanol, add piperidine, and heat under reflux for reaction; after the reaction, remove the solvent by distillation under reduced pressure, and obtain white crystalline intermediate 1 after extraction and column chromatography; Step 2: Under nitrogen protection, intermediate 1, 3-quinolinecarboxaldehyde, and a catalytic amount of piperidine were dissolved in acetonitrile, and the mixture was heated to 60°C for reaction. After the reaction, it was cooled to room temperature to produce a large amount of yellow precipitate, which was filtered, rinsed with ethanol, and air-dried to obtain intermediate 2 as a yellow powder. Step 3: Under nitrogen protection, intermediate 2 and methyl trifluoromethanesulfonate were dissolved in dichloromethane and stirred at room temperature for 24 h to produce a large amount of yellow precipitate. The yellow precipitate was filtered and rinsed with ether to obtain light yellow powder intermediate 3; Step 4: Dissolve 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 colorless solid powder EGS. The synthetic route is as follows: 。 3. Use of the dual-function ratiometric fluorescent probe according to claim 1 in the preparation of a ·OH detection reagent.

4. The use according to claim 3, characterized in that: The detection reagent is in I 523nm / I 663 nm The fluorescence ratio at 100 nm 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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