AIE type near-infrared emission lipid droplet small-molecule fluorescent probe as well as synthesis method and application thereof in cell imaging

Abstract

The invention provides an AIE type near-infrared emission lipid droplet small-molecule fluorescent probe, a synthesis method thereof and application of the AIE type near-infrared emission lipid droplet small-molecule fluorescent probe in cell imaging, and belongs to the technical field of fluorescent probes. The triphenylamine derivative is synthesized to serve as the near-infrared emission lipid droplet fluorescence imaging probe, the fluorescence probe has the AIE effect, red light emission and large Stokes shift, fluorescence quenching caused by probe targeted lipid droplet aggregation can be effectively avoided, and background interference of biomacromolecules is avoided. The probe can target the lipid droplets, the co-localization coefficient of the probe reaches 0.88, and a new tool is provided for lipid droplet related physiological and pathological research.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly relates to an AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe, a synthesis method thereof, and an application thereof in cell imaging. Background Art

[0002] Lipid droplets (LDs) are organelles that store lipids to generate energy and maintain cell homeostasis, mainly containing cholesterol esters and triglycerides. They are ubiquitous and highly dynamic in cells, can bind to most other organelles through the cell membrane, and participate in many important physiological processes (such as cell proliferation, apoptosis, and migration). Research shows that abnormalities in LDs are related to metabolic disorders and diseases such as obesity, diabetes, and cancer. And they have been used as effective cancer markers for cancer diagnosis because cancer cells require more energy (provided by lipid droplets) to accelerate cell proliferation, and there are more lipid droplets in cancer cells than in normal cells. Monitoring the behavior of LDs in vivo is of great significance for the study of LD-related physiological and pathological processes.

[0003] At present, the visualization of cellular LDs can be carried out by label-free imaging techniques such as traditional transmission light microscopy, direct organelle mass spectrometry, Raman microscopy, and coherent anti-Stokes Raman scattering microscopy. These advanced techniques enable us to study the biophysics of cellular LDs, but they require complex sample preparation and data analysis. In addition, the above methods usually require fixing cells or extracting LDs, thus ignoring the possibility of studying the real-time dynamics of LDs in the cellular environment. Fluorescence imaging technology, with its excellent spatio-temporal resolution, good optical properties, and real-time imaging, has become the most commonly used non-invasive cell visualization method at present. Although a considerable number of fluorescent probes have been developed for cytoplasmic LD imaging in recent years, most traditional dyes have inherent aggregation-caused quenching (ACQ) characteristics, that is, the luminescence performance of the luminophore is very poor or completely quenched in the aggregate state. Compared with ACQ fluorophores, AIE fluorophores emit strong fluorescence in the aggregated or solid state. Therefore, constructing novel LD probes based on aggregation-induced emission (AIE)-type fluorophores shows great potential in real-time localization and dynamic monitoring in biomedical applications because they can be well stored in LDs and maintain bright fluorescence intensity. In addition, constructing near-infrared (NIR) fluorescent probes has significant advantages in the field of LD imaging because they have long emission wavelengths that can protect the probes from background fluorescence interference and improve the accuracy of the test. For this reason, the present invention intends to give full play to the advantages of high sensitivity, high spatio-temporal resolution, real-time detection, and non-invasive analysis of the fluorescence probe imaging analysis technology, and develop an AIE-type near-infrared fluorescent probe that can target lipid droplets for real-time in-situ fluorescence imaging monitoring of lipid droplets. Summary of the Invention

[0004] The object of the present invention is to provide a small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission, its synthesis method and application in cell imaging, so as to solve the problems existing in the above-mentioned prior art.

[0005] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission, and its structural formula is:

[0007]

[0008] wherein R is methoxy, hydroxyl or amino.

[0009] Further, the structural formula of the small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission is:

[0010]

[0011] The present invention provides a synthesis method of a small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission, including the following steps:

[0012] 1) React 4-(diphenylamino)phenylboronic acid and 7-bromobenzo[c][1,2,5]thiadiazole-4-carbaldehyde under the action of Pd(PPh 3 ) 4 and K 2 CO 3 by reflux reaction to obtain intermediate 1;

[0013] 2) React intermediate 1 and 1-(6-methoxynaphthalen-2-yl)ethan-1-one under alkaline conditions to obtain a red solid, which is the small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission.

[0014] Further, the molar ratio of 4-(diphenylamino)phenylboronic acid, 7-bromobenzo[c][1,2,5]thiadiazole-4-carbaldehyde, Pd(PPh 3 ) 4 and K 2 CO 3 is 6.5 - 7.0 mmol: 6.0 - 6.8 mmol: 80 - 90 μmol: 10 - 15 mmol.

[0015] Further, in step 1), the reflux reaction is carried out in a solvent, and the solvent is a mixed solvent of ethanol and water. The dosage ratio of 4-(diphenylamino)phenylboronic acid to the solvent is 6.5 - 7.0 mmol: 15 - 25 mL.

[0016] Further, the temperature of the reflux reaction is 70-90 °C, and the time of the reflux reaction is 9-11 h.

[0017] Further, in the step 2), the molar ratio of the intermediate 1 to 1-(6-methoxynaphthalen-2-yl)ethan-1-one is 1:0.9-1.1, the temperature of the reaction is 20-40 °C, and the time of the reaction is 50-80 min.

[0018] Further, the basic condition is sodium hydroxide, and the molar ratio of the sodium hydroxide to the intermediate 1 is 1:1-1.2.

[0019] The present invention also provides an application of an AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe in cell imaging.

[0020] Beneficial effects of the present invention:

[0021] The fluorescent probe of the present invention has an AIE effect, red light emission, and a large Stokes shift, which can effectively avoid fluorescence quenching caused by the aggregation of the probe targeting lipid droplets and avoid background interference of biological macromolecules.

[0022] The probe of the present invention can target lipid droplets, and its co-localization coefficient reaches 0.88, providing a new tool for the physiological and pathological research related to lipid droplets. Description of the drawings

[0023] Figure 1 For the probe TPA-Cou of Example 1 of the present invention in different solvents, (A) ultraviolet-visible absorption spectrum and (B) fluorescence emission spectrum; (C) normalized fluorescence spectrum of TPA-Cou (10 μM) in different polar solvents; (D) linear relationship diagram between the maximum emission wavelength and ET(30).

[0024] Figure 2 For the probe TPA-Cou of Example 1 of the present invention in 0-100% DMSO / H 2 O mixture, (A) fluorescence spectrum and (B) fluorescence intensity diagram under excitation at 480 nm.

[0025] Figure 3 Fluorescence photographs of the probe TPA-Cou of Example 1 of the present invention and BODIPY493 / 503 after staining.

[0026] Figure 4 Fluorescence photographs of A549 cells stained with the probe TPA-Cou of Example 1 of the present invention under different concentrations of oleic acid. Detailed implementation manners

[0027] The present invention provides an AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe, and the structural formula is:

[0028]

[0029] Wherein R is methoxy, hydroxyl or amino group.

[0030] In the present invention, the structural formula of the AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe is preferably:

[0031]

[0032] The present invention provides a synthesis method of an AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe, comprising the following steps:

[0033] 1) React 4-(diphenylamino)phenylboronic acid and 7-bromobenzo[c][1,2,5]thiadiazole-4-carbaldehyde under the action of Pd(PPh 3 ) 4 and K 2 CO 3 by reflux reaction to obtain intermediate 1;

[0034] 2) React intermediate 1 and 1-(6-methoxynaphthalen-2-yl)ethan-1-one under alkaline conditions to obtain a red solid, which is the AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe.

[0035] In the present invention, the synthesis route of the AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe is as follows:

[0036]

[0037] In the present invention, the molar ratio of 4-(diphenylamino)phenylboronic acid, 7-bromobenzo[c][1,2,5]thiadiazole-4-carbaldehyde, Pd(PPh 3 ) 4 and K 2 CO 3 is 6.5-7.0 mmol: 6.0-6.8 mmol: 80-90 μmol: 10-15 mmol, preferably 6.8-7.0 mmol: 6.4-6.6 mmol: 85-88 μmol: 12-14 mmol, and further preferably 6.92 mmol: 6.57 mmol: 86.54 μmol: 13.75 mmol.

[0038] In the present invention, in step 1), the reflux reaction is carried out in a solvent, and the solvent is a mixed solvent of ethanol and water. The dosage ratio of 4-(diphenylamino)phenylboronic acid to the solvent is 6.5-7.0 mmol: 15-25 mL, preferably 6.8-7.0 mmol: 18-22 mL, and further preferably 6.92 mmol: 20 mL.

[0039] In the present invention, the volume ratio of ethanol to water is preferably 5:1.

[0040] In the present invention, the temperature of the reflux reaction is 70-90 °C, preferably 75-85 °C, and more preferably 80 °C; the time of the reflux reaction is 9-11 h, preferably 10 h.

[0041] In the present invention, in step 2), the molar ratio of intermediate 1 to 1-(6-methoxynaphthalen-2-yl)ethan-1-one is 1:0.9-1.1, preferably 1:1.05; the temperature of the reaction is 20-40 °C, preferably 25-35 °C; the time of the reaction is 50-80 min, preferably 60-70 min.

[0042] The basic condition is sodium hydroxide, and the molar ratio of sodium hydroxide to intermediate 1 is 1:1-1.2, preferably 1:1.1.

[0043] The present invention also provides an application of an AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe in cell imaging.

[0044] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] This example provides an AIE-type near-infrared-emitting lipid droplet small molecule fluorescent probe, and its preparation method includes the following steps:

[0047]

[0048] (1) Preparation of intermediate 1

[0049] 4-(Diphenylamino)phenylboronic acid (6.92 mmol) and 7-bromobenzo[c][1,2,5]thiadiazole-4-carbaldehyde (6.57 mmol) were added to a 100 mL round-bottom flask, and then Pd(PPh 3 ) 4 (86.54 μmol) and K 2 CO 3 (13.75 mmol) were added, and the mixture was refluxed at 80 °C for 10 h in a 20 mL ethanol-water mixed solvent (v / v: EtOH:H 2 O = 5:1). After the reaction was complete, it was purified by column chromatography (the mobile phase was ethyl acetate / petroleum ether with a volume ratio of 8:1), and 1.72 g of a yellow solid (intermediate 1) was obtained, with a yield of 61%; 1 H-NMR (400 MHz, CDCl 3)δ 10.73 (s, 1H), 8.26 (d, J = 4 Hz, 1H), 7.91 (d, J = 8 Hz, 2H), 7.82 (d, J = 8 Hz, 1H), 7.31 (t, J = 8 Hz, 4H), 7.20 (d, J = 8 Hz, 6H), 7.10 (t, J = 8 Hz, 2H).

[0050] (2) Preparation of Fluorescent Probe TPA-Cou

[0051] Intermediate 1 (3.68 mmol) and 1-(6-methoxynaphthalen-2-yl)ethan-1-one (3.65 mmol) were placed in a 100 mL round-bottom flask, and NaOH (3.75 mmol) was added. The reaction was carried out at room temperature for 1 h. After the reaction was completed, it was purified by column chromatography (mobile phase: ethyl acetate / petroleum ether with a volume ratio of 5:1), and 1.66 g of red solid (TPA-Cou) was obtained, with a yield of 76%; 1 H-NMR (400 MHz, CDCl 3 )δ 8.94 (d, J = 16 Hz, 1H), 8.62 (s, 1H), 8.17 (dd, J = 12 Hz, J = 16 Hz, 2H), 7.93 (d, J = 12 Hz, 1H), 7.89 (d, J = 8 Hz, 2H), 7.85 (t, J = 8 Hz, 2H), 7.71 (d, J = 8 Hz, 1H), 7.30 (t, J = 8 Hz, 4H), 7.20 (m, 8H), 7.08 (t, J = 8 Hz, 2H).

[0052] 13 C-NMR (101 MHz, cdcl3) δ 190.03, 159.73, 154.11, 153.89, 148.59, 147.22, 139.70, 137.26, 135.45, 133.62, 131.30, 130.19, 130.14, 130.00, 129.41(6C), 127.94, 127.27, 126.76, 126.57, 126.34, 125.31, 125.12(6C), 123.60, 122.37, 119.65, 105.77, 55.43.

[0053] Example 2

[0054] Spectral Property Determination: Using the fluorescent probe TPA-Cou as the research object, the optical properties of TPA-Cou in different solvents were studied by ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy. The results are as Figure 1As shown in A-1B, the maximum absorption wavelength of TPA-Cou is approximately at 480 nm, and the maximum emission wavelength shows an obvious red shift between 600 nm and 710 nm with the increase of solvent polarity ( Figure 1 C). The Stokes shift is positively correlated with the polarity change. The Stokes shift of TPA-Cou in toluene is 120 nm, while in DMF it is 230 nm, indicating that TPA-Cou has a relatively large Stokes shift. TPA-Cou shows weak emission in DMF and DMSO, and the fluorescence intensity gradually increases with the decrease of solvent polarity. In addition, the maximum emission wavelength of TPA-Cou has a good linear correlation with the solvent polarity parameter E T (30) (R 2 = 0.991) ( Figure 1 D). The above results indicate that the probe TPA-Cou has strong fluorescence emission under low-polarity conditions.

[0055] Figure 1 For Example 1, (A) UV-visible absorption spectra and (B) fluorescence emission spectra of the probe TPA-Cou in different solvents. (C) Normalized fluorescence spectra of TPA-Cou (10 μM) in solvents with different polarities. (D) Linear relationship between the maximum emission wavelength and E T (30).

[0056] In addition, we explored the AIE properties of TPA-Cou. As Figure 2 shown, the fluorescence intensity of TPA-Cou in mixtures of dimethyl sulfoxide (DMSO) and different water fractions (fw) was studied. In pure DMSO solution, TPA-Cou shows weak emission at 650 nm. When the water addition amount increases from 20% to 70%, the fluorescence emission of TPA-Cou gradually increases ( Figure 2 A, 2B), and reaches the maximum value when the water content is 60 - 70%. When the water content is 80 - 100%, the fluorescence intensity of TPA-Cou gradually weakens because the fluorescence intensity of the probe weakens with the increase of polarity. The above results show that the significant increase in the fluorescence intensity of TPA-Cou is activated due to the formation of aggregates.

[0057] Figure 2 For Example 1, (A) fluorescence spectra and (B) fluorescence intensities of the probe TPA-Cou in 0 - 100% DMSO / H 2 O mixtures under 480 nm excitation.

[0058] Example 3

[0059] Application of the fluorescent probe TPA-Cou in cell imaging

[0060] To further evaluate the sub - colocalization ability of the probe in cells, a commercial lipid droplet targeting reagent (BODIPY493 / 503) and TPA - Cou were used for imaging after co - incubation. As Figure 3 shown, the red and green channels overlapped well, and the merged fluorescence showed a large area of yellow. The Pearson coefficient was as high as 0.88, indicating that TPA - Cou has good lipid droplet targeting ability. Figure 3 Fluorescence photograph after staining with the probe TPA - Cou (100 nM) and BODIPY493 / 503 (100 nM) in Example 1. Red channel: λ ex = 488 nm; λ em = 600 - 750 nm. Green channel: λ ex = 488 nm; λ em = 503 - 580 nm.

[0061] Subsequently, the accumulation of LDs in living cells was monitored using TPA - Cou. As is well known, cells can produce new LDs to store lipids, so when cells are incubated with oleic acid - containing medium, the number of LDs increases. As Figure 4 shown, A549 cells were exposed to different oleic acid concentrations for 4 h and stained with TPA - Cou. In the absence of oleic acid, only weak red fluorescence (B2) was observed. When A549 cells were treated with higher oleic acid concentrations, the fluorescence signal was significantly enhanced, indicating that TPA - Cou can monitor more LDs (C2 and D2).

[0062] From the above examples, it can be seen that the present invention provides an AIE - type near - infrared - emitting lipid droplet small - molecule fluorescent probe, its synthesis method, and its application in cell imaging. The fluorescent probe of the present invention has an AIE effect, red light emission, and a large Stokes shift, which can effectively avoid fluorescence quenching caused by the aggregation of the probe targeting lipid droplets and avoid background interference of biological macromolecules. The probe of the present invention can target lipid droplets, and its co - localization coefficient reaches 0.88, providing a new tool for lipid droplet - related physiological and pathological research.

[0063] The above - mentioned is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission, characterized in that, the structural formula is: wherein R is methoxy, hydroxy or amino.

2. The small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission according to claim 1, characterized in that, the structural formula is:

3. A synthesis method of the small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission according to claim 1 or 2, characterized in that, comprises the following steps: 1) 4-(Diphenylamino)phenylboronic acid and 7-bromobenzo[c][1,2,5]thiadiazole-4-carbaldehyde are subjected to a reflux reaction under the action of Pd(PPh 3 ) 4 and K 2 CO 3 to obtain intermediate 1; 2) React intermediate 1 and 1-(6-methoxynaphthalen-2-yl)ethan-1-one under alkaline conditions to obtain a red solid, which is the small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission.

4. The synthesis method according to claim 3, characterized in that, The molar ratio of the 4-(diphenylamino)phenylboronic acid, 7-bromobenzo[c][1,2,5]thiadiazole-4-carbaldehyde, Pd(PPh 3 ) 4 and K 2 CO 3 is 6.5 - 7.0 mmol: 6.0 - 6.8 mmol: 80 - 90 μmol: 10 - 15 mmol.

5. The synthesis method according to claim 3 or 4, characterized in that, in step 1), the reflux reaction is carried out in a solvent, the solvent is a mixed solvent of ethanol and water, and the dosage ratio of 4-(diphenylamino)phenylboronic acid to the solvent is 6.5 - 7.0 mmol: 15 - 25 mL.

6. The synthesis method according to claim 5, characterized in that, the temperature of the reflux reaction is 70 - 90 °C, and the time of the reflux reaction is 9 - 11 h.

7. The synthesis method according to claim 3 or 6, characterized in that, in step 2), the molar ratio of intermediate 1 to 1-(6-methoxynaphthalen-2-yl)ethan-1-one is 1: 0.9 - 1.1, the temperature of the reaction is 20 - 40 °C, and the time of the reaction is 50 - 80 min.

8. The synthesis method according to claim 7, characterized in that, the alkaline condition is sodium hydroxide, and the molar ratio of sodium hydroxide to intermediate 1 is 1: 1 - 1.

2.

9. Application of the small molecule fluorescent probe for lipid droplets with AIE-type near-infrared emission according to claim 1 or 2 in cell imaging.

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