Isophorone derivative fluorescent probe, preparation method and application thereof

Abstract

The application provides a fluorescent probe of isophorone derivative, a preparation method and application thereof, the fluorescent probe is composed of a naphthalene or coumarin part and an isophorone part, the compound can be used as a fluorescent probe, and can distinguish a lipid droplet and an endoplasmic reticulum in a cell in a double emission channel and a double color. The fluorescent probe composed of the isophorone derivative of the application can distinguish organelles between two emission channels without interference, the fluorophore can produce a large emission shift when the polarity changes, and can emit bright fluorescence under high polarity and low polarity conditions, and provides an important molecular tool for promoting the research on the interaction of organelles.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of organic material science and technology, and particularly relates to a fluorescent probe of isophorone derivative, a preparation method and application thereof. BACKGROUND

[0002] Solvatochromic fluorescent probes can exhibit tunable emission colors under different environments, and are extremely important molecular tools in the field of basic biology. So far, such probes have played a crucial role in studying protein aggregation, folding and surface properties, studying lipid raft dynamics, and observing interactions between different organelles. In particular, solvatochromic probes have wide application potential in revealing the elusive complex microenvironment in biology. Intramolecular charge transfer (ICT) mechanism is the main principle guiding the design of solvatochromic fluorophores, and decorating strong electron donors and acceptors is considered to be the only viable strategy for molecular design. However, such fluorophores usually exhibit most of the quenched emission under high polarity conditions, so currently few solvatochromic fluorescent probes have been proposed and applied. Due to the lack of ideal solvatochromic fluorescent probes, it has brought great limitations to design powerful probes for related research.

[0003] Endoplasmic reticulum and lipid droplets are two key organelles in eukaryotic cells. Endoplasmic reticulum plays an indispensable role in protein synthesis and transport, signal transduction, and calcium ion regulation. Endoplasmic reticulum stress can lead to cell death and inflammatory response. Endoplasmic reticulum abnormalities can cause protein folding or folding errors, which are closely related to various diseases including neurodegenerative diseases and diabetes. Lipid droplets are responsible for storing lipids and energy and regulating energy balance. Lipid droplet abnormalities are closely related to fatty liver, neurodegenerative diseases and other diseases. In particular, the important interaction between endoplasmic reticulum and lipid droplets plays a crucial role in the regulation of lipids. It has been reported that lipid droplets can also be synthesized on the endoplasmic reticulum. Therefore, it is necessary to label the endoplasmic reticulum and lipid droplets with fluorescent probes of different fluorescent colors to explore the interaction between the two organelles. SUMMARY

[0004] In order to further study the interaction between the two organelles of endoplasmic reticulum and lipid droplets, for this purpose, we propose an ICT inhibition strategy for designing a solvatochromic probe 2 with strong emission under high polarity conditions, the design of probe 2 contains an electron donor and two acceptors, in particular one of the acceptors (carbonyl) is designed near the electron donor, so that the partial charge transfer is limited in the coumarin moiety.

[0005] Probe 2 shows strong emission ability in all solvents, and has large emission shift when the polarity changes, and is an ideal solvatochromic fluorescent probe for biological imaging applications. Probe 2 is used to observe lipid droplets and endoplasmic reticulum of different emission colors, and probe 1 can illuminate lipid droplets in living cells. Probe 2 is also successfully used to explore lipid droplets and endoplasmic reticulum regulation in living cells and tissues.

[0006] The present application provides a two-color fluorescent probe for distinguishing endoplasmic reticulum and lipid droplets, and the structure of the isophorone derivative is as follows:

[0007]

[0008] The preparation method of the compound 1 is as follows:

[0009]

[0010] In the preparation method, isophorone is mixed with malonitrile in an organic solution, piperidine is added, and the reaction is carried out under reflux conditions. After the reaction is completed, 6-(dimethylamino)-2-naphthaldehyde is mixed with the above reaction product in an organic solution, and piperazine is added dropwise to obtain the product by recrystallization.

[0011] The preparation of compound 2 is as follows:

[0012]

[0013] 3,5,5-trimethylcyclohex-2-enyl malonitrile and 7-(diethylamino) coumarin-3-formaldehyde are mixed in an organic solvent, piperidine is added dropwise, and the reaction is carried out under reflux conditions. After cooling, filtering and recrystallizing in ethanol, compound 2 is obtained.

[0014] Another object of the present application is to provide an indole derivative fluorescent probe which can be used for two-color imaging of lipid droplets and endoplasmic reticulum in cells.

[0015] Compared with the prior art, the technical scheme of the present application has the following beneficial effects:

[0016] (1) A feasible ICT inhibition strategy is proposed to overcome the emission quenching under high polarity, and a solvatochromic fluorophore is designed.

[0017] (2) An ideal solvatochromic fluorophore is proposed, which can produce a large emission shift when the polarity changes, and can emit bright fluorescence under high and low polarity conditions.

[0018] (3) The fluorophore is used as a fluorescent probe, and the obtained fluorescent probe (compound 1 and compound 2) illuminates lipid droplets and endoplasmic reticulum with different emission colors.

[0019] (4) The fluorescent probe prepared by the application successfully reveals three different lipid accumulation levels induced by lipids (oleic acid > stearic acid > cholesterol).

[0020] (5) The probe is successfully used to observe rapid lipid consumption under high-sugar and fat-free conditions. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The H NMR spectrum of compound 1 is shown in Figure 1. 1 The H NMR spectrum of compound 1 is shown in Figure 1.

[0022] Figure 2 The H NMR spectrum of compound 2 is shown in Figure 2. 1 The H NMR spectrum of compound 2 is shown in Figure 2.

[0023] Figure 3 The cell co-localization imaging of probe 1 and probe 2 is shown in Figure 3.

[0024] Figure 4 The imaging of high-fat treated cells of the probe is shown in Figure 4.

[0025] Figure 5 The imaging of fat-free treated cells of the probe is shown in Figure 5. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme of the application more clear, the following embodiments are used to further illustrate the application, but the protection scope of the application is not limited to these embodiments, and the embodiments are only used to explain the application. Those skilled in the art should understand that any change or equivalent replacement without departing from the concept of the application is included in the protection scope of the application. The application is further described below in combination with embodiments, but the application is not limited to the embodiments.

[0027] The chemicals used in the application are all analytical grade, isophorone, malononitrile and 7-(diethylamino)coumarin-3-carbaldehyde are purchased from Anjie Chemical Technology (Shanghai) Co., Ltd., 6-(dimethylamino)-2-naphthaldehyde is purchased from Zhengzhou Alpha Chemical Co., Ltd., and the solvent used in spectral measurement is chromatographic grade.

[0028] Example 1:

[0029] Preparation of (3,5,5-trimethylcyclohex-2-enylidene)malononitrile:

[0030] Into a flask was added anhydrous ethanol (15 mL), isophorone (3 mL, 20.03 mmol) and malononitrile (1.6 g, 24.22 mmol). 2 drops of piperidine was added to the system, heated to reflux for 8 hours to complete the reaction. Then, the system was cooled to room temperature. The crude product was separated by filtration and washed with ethanol three times. The product was purified by column chromatography with PE / EA (V:V=50:1) as eluent. The pure product was white powder (3.2 g, yield 88%).

[0031] Preparation of compound 1:

[0032] Into a flask was added anhydrous ethanol (15 mL), 6-(dimethylamino)-2-naphthaldehyde (500 mg, 2.51 mmol) and compound (3,5,5-trimethylcyclohex-2-enylidene)malononitrile (560 mg, 3.01 mmol). 2 drops of piperidine was added to the flask to catalyze the reaction. The system was heated to reflux for 8 hours to complete the reaction. The system was cooled to room temperature. The product was separated by filtration and washed with ethanol three times. The crude product was recrystallized with ethanol to obtain a dark red solid (848 mg, yield 92%).

[0033] 1 H NMR (600 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.74 (t, J = 14.5 Hz, 2H), 7.65 (d, J = 8.7 Hz, 1H), 7.40 (s, 2H), 7.25 - 7.19 (m, 1H), 6.94 (s, 1H), 6.87 (s, 1H), 3.05 (s, 6H), 2.62 (s, 2H), 2.58 (s, 2H), 1.03 (s, 6H). (See Figure 1) Figure 1 )

[0034] Preparation of compound 2:

[0035] Into a flask was added anhydrous ethanol (15 mL), compound (3,5,5-trimethylcyclohex-2-enylidene)malononitrile (500 mg, 2.04 mmol) and 7-(diethylamino)coumarin-3-carboxaldehyde (569 mg, 3.05 mmol). 2 drops of piperidine was added dropwise, and the mixture was heated to reflux for 8 hours to complete the reaction. Then, the system was cooled to room temperature. The product was separated by filtration and washed with ethanol three times. The crude product was recrystallized with ethanol to obtain a dark red solid (782 mg, yield 93%).

[0036] 1H NMR (600 MHz, CDC13-d) δ 7.79 (s, 1H), 7.33 (d, J = 8.8 Hz, 1H), 7.27 - 7.21 (m, 1H), 7.12 (d, J = 16.1 Hz, 1H), 6.83 (s, 1H), 6.67 (d, J = 8.9 Hz, 1H), 6.53 (s, 1H), 3.45 (q, J = 7.1 Hz, 4H), 2.58 (s, 2H), 2.46 (s, 2H), 1.24 (t, J = 7.1 Hz, 6H), 1.06 (s, 6H).(See Figure 1 Figure 2 )

[0037] Example 2: Cell imaging of fluorescent probe 1 (compound 1) and fluorescent probe 2 (compound 2).

[0038] (1) Cell culture:

[0039] HepG2 cells were purchased from Procell Life Science & Technology Co., Ltd. and cultured in H-DMEM (Dulbecco's Modified Eagle's Medium, High Glucose) medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. When performing cell imaging experiments, live HepG2 cells were suspended in culture medium at a concentration of 10000 cells / mL for dilution. 1 mL of the cell suspension was added to a glass-bottom dish and cultured for 24 h to allow the cells to adhere.

[0040] (2) Probe stock solution preparation:

[0041] Compound 1 (1 mM) and compound 2 (1 mM) were dissolved in DMSO to prepare stock solutions. Then, 2 μL of the stock solution was added to 1 mL of medium and mixed uniformly to obtain a working solution (2 μM). The medium in the glass-bottom dish was removed, and 1 mL of the working solution containing 2 μM of the probe was used to incubate the cells for 30 min. The cells were then directly imaged under a Nikon A1MP confocal microscope without further washing procedures. Blue channel: λ ex = 405 nm; Green channel: λ em = 488 nm; Near-infrared channel: λ ex = 561 nm; λ em = 665-735 nm. ex em

[0042] (3) Cell co-localization imaging (See Figure 2 Figure 3 )

[0043] As shown in Figure 2 Figure 3 ​​Live cells were incubated with probes 1 and 2 and imaged in green and near-infrared (NIR) channels, as shown in FIG. 2A. Both probes showed strong fluorescence in both green and near-infrared channels. The green emission of probe 1 and the near-infrared emission overlapped well together and formed a punctate morphology. In addition, the fluorescence signals could also be clearly observed to overlap with the dark dots in the DIC image.

[0044] These results indicated that both green and near-infrared fluorescence of probe 1 were localized in the low-density region in live cells. According to the magnified images of Figure 3

[0045] To confirm the subcellular localization of the green and near-infrared emission of probe 2 in live cells, a colocalization experiment was performed. Probe 2 showed strong emission in green, red and near-infrared channels, while a commercial lipid droplet probe with blue emission failed to do so. Meanwhile, ER-tracker blue (ERB), a commercial blue-emitting fluorescent probe, was also used to colocalize with probe 2. It was clear that the blue fluorescence of ERB had a great overlap with the near-infrared emission of probe 2, with an overlap coefficient of 0.89. These results proved that the near-infrared emission of probe 2 was localized in the endoplasmic reticulum in live cells.

[0046] The polarity of the endoplasmic reticulum membrane was obviously higher than that of the lipid droplet core.

[0047] Probe 2 showed strong emission in both low-polarity and high-polarity environments, and red-shifted emission in high-polarity environment. Therefore, probe 2 successfully illuminated lipid droplets and endoplasmic reticulum with different emission colors.

[0048] FIG. 2C shows the Z-stack images and three-dimensional images of live cells preloaded with probe 2. The three-dimensional images clearly showed that the green emission and the near-infrared emission of probe 2 illuminated lipid droplets and endoplasmic reticulum, respectively. Figure 3

[0049] (4) Cell imaging experiments under high-fat and non-fat conditions:

[0050] Oleic acid, stearic acid and cholesterol were used to mimic high-fat environment. For oleic acid treatment, live HepG2 cells were first adhered in culture dishes and cultured in culture medium containing different amounts of oleic acid (0, 50 μM, 100 μM, 200 μM) for 24 h, and then stained with 2 μM probe 2 for 20 min for probe loading, and imaged directly in green and near-infrared channels under confocal microscope without further washing process.

[0051] ​​Lipid-free conditions were simulated using Duchenne Modified Eagle Medium (DMEM) with a serum content of less than 10%. Live HepG2 cells were first adhered to glass-bottomed culture dishes and then cultured for 24 h in DMEM containing different serum proportions (0%, 1%, 2%, and 5%). Afterward, the cells were stained with 2 μM Probe 2 for 20 min to load the probe and were directly imaged in the green and near-infrared channels under a confocal microscope without further washing.

[0052] To assess the consumption of lipids accumulated under a high-fat environment, cells were treated sequentially under high-fat and lipid-free conditions. Viable HepG2 cells were cultured for 24 hours in media containing the same concentration (50 μM) of oleic acid, stearic acid, and cholesterol. Then, the cells were stained with 2 μM probe 2 for 20 minutes to load the probe, and imaging was performed directly in the green and near-infrared channels under a confocal microscope without additional steps.

[0053] Changes in the endoplasmic reticulum, particularly lipid droplets, were investigated using probe 2 under both high-fat and lipid-free conditions. (See attached image) Figure 4 As shown in Figure A, in the control group cells, probe 2 illuminated lipid droplets and endoplasmic reticulum with different emission colors, and the morphology of lipid droplets and endoplasmic reticulum could be clearly observed in the magnified image. Conversely, after culturing cells in a medium containing 50 μM oleic acid for 24 hours, the number of lipid droplets increased dramatically, while the number of endoplasmic reticulum remained almost unchanged. Furthermore, further treatment of cells with 100 μM and 200 μM oleic acid resulted in a further increase in the number of lipid droplets with increasing oleic acid concentration, while the amount of endoplasmic reticulum remained unchanged. Therefore, oleic acid incubation can lead to a significant increase in lipid droplets in live cells, but does not have a significant effect on the number of endoplasmic reticulum. Free oleic acid is harmful to live cells; it migrates in the form of triglycerides and encapsulates within lipid droplets. This is the reason for the significant increase in lipid droplets.

[0054] The lipids in the culture medium are mainly derived from serum (typically 10%), therefore, live cells were cultured in DMEM (high glucose) with low serum content (0%, 1%, 2%, and 5%) to simulate lipid-free conditions. As shown in Figure B, compared to the control group containing 10% serum, the number of lipid droplets in cells cultured for 24 hours in medium containing 5% serum was significantly reduced. Furthermore, the reduction in serum content also led to a further reduction in lipid droplet count; in serum-free DMEM, lipid droplets disappeared. These results confirm that even under high glucose conditions, lipids in live cells can be rapidly consumed under lipid-free conditions. Additionally, as shown in the attached... Figure 4 As shown in Figure B, the residual lipid droplets after lipid-free treatment exhibit a yellow pseudo-color, indicating an overlap between the green and near-infrared signals. These results suggest that in a lipid-free environment, the close contact between the endoplasmic reticulum and lipid droplets increases, which is beneficial for lipid consumption.

[0055] Saturated phospholipids, unsaturated phospholipids, and cholesterol are three important classes of lipids that regulate the physical properties of liposomes. Subsequently, cells were cultured for 24 hours with 50 μM stearic acid, oleic acid, and cholesterol, respectively, as shown in the attached figure. Figure 5 As shown in Figure A, compared with the control group cultured in the culture medium, the number of lipid droplets in cells cultured with all three lipids was significantly increased. Notably, the degree of lipid droplet increase induced by the three lipids differed (oleic acid > stearic acid > cholesterol). These results indicate that ingestion of foods containing these three lipids leads to lipid accumulation in living cells. Among them, oleic acid most readily causes lipid accumulation in cells.

[0056] Since lipid-free treatment can lead to a reduction in lipid droplet volume and lipid consumption, cells pre-incubated with three different lipids were cultured in serum-free DMEM, as shown in the attached figure. Figure 5 As shown in Figure B. These results indicate that, after 24 hours of treatment under lipid-free conditions, the lipids accumulated during high-fat treatment can be reversibly and rapidly consumed by lipid-free treatment. Furthermore, as shown in the attached figure... Figure 5 As shown in Figure B, cells cultured in DMEM after treatment with stearic acid exhibited more low-density lipoprotein than cells treated with oleic acid and cholesterol. These results suggest that stearic acid lipids are more difficult to consume compared to oleic acid and cholesterol lipids.

Claims

1. Use of an isophorone derivative for the preparation of a dual-color imaging marker of intracellular lipid droplets and endoplasmic reticulum fluorescent probes, characterized in that, The molecular structure of the isophorone derivative is as follows: 。

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