A lipid droplet fluorescent probe, its preparation method, detection reagent or kit, and its application.

Abstract

The present invention discloses a lipid droplet fluorescent probe and its preparation method, detection reagent or kit and application, belonging to the field of molecular probes for biological systems. Based on the highly lipophilic property of lipid droplets, the present invention utilizes fat-soluble xanthene and quinoline groups and designs a target probe with a donor-acceptor structure through a conjugation expansion strategy. The structure is: The synthesis method of the probe is simple and easy. Based on its lipophilicity, it can be selectively located in lipid droplets, and cells and db / db Lipid droplets in the tissues of type 2 diabetic mice are of great significance for the detection of lipid droplets and the early diagnosis of lipid droplet-related diseases, and have broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of chemical biology technology, more specifically, to the field of biological system molecular probes, and relates to the field of biological system molecular probes, specifically to a lipid droplet fluorescent probe and a preparation method thereof, a detection reagent or a kit and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Lipid droplets (LDs) are highly conserved organelles that store neutral lipids. In addition, they also play an important role in regulating cell metabolism, signal transduction, protein synthesis and degradation, and maintaining cell homeostasis. Lipid droplets are composed of a phospholipid monolayer, lipoproteins, and a lipid center. Except for the hydrophilic area exposed to the cytoplasm, other areas are highly lipophilic. Abnormal deposition of lipids is a common clinical phenomenon in many metabolic-related diseases such as fatty liver and type 2 diabetes, but its role in the occurrence and development of the disease is still unclear. Analyzing the dynamic properties and mechanism of action of lipid droplets is of great significance for the early diagnosis and treatment of lipid droplet-related diseases.

[0004] Fluorescence imaging has the advantages of simple operation, high sensitivity, and fidelity of spatiotemporal in situ information. It has become an important means of visualizing lipid droplets, while super-resolution imaging can more finely observe the structure, biosynthesis, and decomposition of lipid droplets. In recent years, lipid droplet biology has been booming. For example, studies have shown that the dynamic changes of lipid droplets can affect the photosensitivity of bird cones (Dev Cell. 2023, 58(22):2528-2544.e8); recent studies have also found that lipid droplets released into the extracellular space by type 2 innate lymphoid cells can activate and recruit neutrophils, thereby promoting respiratory allergic inflammatory responses (DOI:10.1016 / j.immuni.2024.09.001). These findings further illustrate that the function of lipid droplets is far more extensive than "storing neutral lipids", which also makes it very important to develop fluorescent probes with super-resolution imaging and lipid droplet detection in disease animal model tissues. The probe LD-FG developed by JieChen et al. using a buffering strategy showed excellent super-resolution imaging performance of lipid droplets, but its application at the animal level has not been fully demonstrated (Angew. Chem. Int. Ed. 2021, 60, 25104 – 25113). The development of new chemical probes that can be used for super-resolution imaging and lipid droplet detection in disease animal models has broad prospects for analyzing the mechanism of action of lipid droplets in physiological and pathological environments. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a lipid droplet fluorescent probe and its preparation method, detection reagent or kit and application. Based on the highly lipophilic property of lipid droplets, the present invention utilizes fat-soluble xanthene and quinoline groups and designs a target probe with a donor-acceptor (DA) structure through a conjugation extension strategy. The cLogP value of the probe is 5.87, which realizes the spatiotemporal specific fluorescent labeling and imaging of lipid droplets at the cellular and animal levels.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The first aspect of the present invention provides a lipid droplet fluorescent probe having a structure as shown in formula (I):

[0008]

[0009] Formula (I).

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned lipid droplet fluorescent probe, comprising:

[0011] Compound 2, p-toluenesulfonic acid and 2-methylquinoline react to obtain a lipid droplet fluorescent probe;

[0012] The structure of the compound 2 is as follows:

[0013] .

[0014] In some embodiments of the present invention, the molar ratio of compound 2, p-toluenesulfonic acid and 2-methylquinoline is 1:(1-4):(1-1.5).

[0015] In some embodiments of the present invention, the preparation method comprises: dissolving compound 2, p-toluenesulfonic acid and 2-methylquinoline in an organic solvent, and stirring the mixture at 150-170° C. for reaction.

[0016] In some embodiments of the present invention, the preparation method further comprises the step of separating and purifying to obtain the lipid droplet fluorescent probe after the reaction is completed;

[0017] The separation and purification comprises the steps of adding dichloromethane to the reaction system for liquid-liquid extraction, collecting the organic phase, drying, removing the solvent and purifying by silica gel column chromatography after the reaction is completed.

[0018] In some embodiments of the present invention, in the silica gel column chromatography purification step, the eluent ratio is ethyl acetate: petroleum ether = 1: (2-5).

[0019] In some embodiments of the present invention, the preparation method further comprises the following steps:

[0020] In the presence of cesium carbonate, compound 3 and 4-(dimethylamino)-2-hydroxybenzaldehyde are reacted to obtain compound 2; the molar ratio of cesium carbonate, compound 3 and 4-(dimethylamino)-2-hydroxybenzaldehyde is (2.5-3.5):(1.5-2.5):1;

[0021] The structure of compound 3 is as follows:

[0022] .

[0023] In some embodiments of the present invention, the preparation method further comprises the following steps:

[0024] In an ice bath and under nitrogen protection, cyclohexanone was added to an organic solution containing phosphorus tribromide, and the mixture was reacted at room temperature to obtain compound 3; the molar ratio of phosphorus tribromide to cyclohexanone was (2.5-3):1.

[0025] The third aspect of the present invention provides a detection reagent or a kit, which includes the above-mentioned lipid droplet fluorescent probe or the lipid droplet fluorescent probe prepared by the above-mentioned preparation method.

[0026] The fourth aspect of the present invention provides an application of the above-mentioned lipid droplet fluorescent probe or the lipid droplet fluorescent probe prepared by the above-mentioned preparation method or the above-mentioned detection reagent or kit in lipid droplet detection, fatty liver detection, diabetes detection, fluorescence detection, cell fluorescence imaging or tissue fluorescence imaging.

[0027] The fifth aspect of the present invention provides an application of the above-mentioned lipid droplet fluorescent probe or the lipid droplet fluorescent probe prepared by the above-mentioned preparation method or the above-mentioned detection reagent or kit in the preparation of lipid droplet detection, fatty liver detection, diabetes detection, fluorescence detection, cell fluorescence imaging or tissue fluorescence imaging products.

[0028] The beneficial effects of the present invention are:

[0029] In view of the correlation between lipid droplets and metabolic diseases, the present invention is based on the highly lipophilic nature of lipid droplets, utilizes fat-soluble xanthene and quinoline groups, and designs a target probe with a donor-acceptor (DA) structure through a conjugation expansion strategy. The synthesis method of the probe is simple and easy. Its structural formula is shown in formula (1), and its cLogP value is 5.87, which realizes the spatiotemporal specific fluorescence labeling and imaging of lipid droplets at the cellular and animal levels, and provides a new way to analyze the mechanism of action of lipid droplets in physiological and pathological environments at the cellular and animal levels. Under an ultra-high resolution microscope, it can be observed that the lipid droplets labeled by the probe are distinct particles, and there is no unclear boundary. After staining animal tissues with the probe, lipid droplets can be accurately detected when using a confocal microscope for fluorescence imaging. More importantly, the number, intensity and gaps between lipid droplets labeled by the probe are stronger than those of conventional stains.

[0030] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 is the H NMR spectrum of compound 2 in Example 1 of the present invention;

[0033] Figure 2 is the NMR carbon spectrum of compound 2 in Example 1 of the present invention;

[0034] Figure 3 This is the nuclear magnetic hydrogen spectrum of the lipid droplet fluorescent probe obtained in Example 1 of the present invention;

[0035] Figure 4 This is the NMR carbon spectrum of the lipid droplet fluorescent probe obtained in Example 1 of the present invention;

[0036] Figure 5 The UV-visible spectra (A) and fluorescence emission spectra (B) of the lipid droplet fluorescent probe obtained in Example 1 of the present invention in different solvents;

[0037] Figure 6 The fluorescence emission spectra of the lipid droplet fluorescent probe obtained in Example 1 of the present invention in solvent systems with different polarities (A) and the fitting diagram of the maximum fluorescence emission wavelength and solvent polarity (B);

[0038] Figure 7 Fluorescence imaging diagrams of lipid droplet fluorescent probes and other subcellular organelle commercial dyes co-stained with lipid droplets (A), mitochondria (B), endoplasmic reticulum (C), lysosomes (D), and cell membranes (E) obtained in Example 1 of the present invention, and evaluation diagrams of the degree of overlap of fluorescence imaging of commercial dyes and lipid droplet fluorescent probes; wherein, from left to right, the first column is a fluorescence imaging diagram stained with commercial dyes, the second column is a fluorescence imaging diagram stained with lipid droplet fluorescent probes, the third column is an overlap of the fluorescence imaging diagrams of the first column and the second column, and the fourth column is an evaluation of the degree of overlap of fluorescence imaging of commercial dyes and lipid droplet fluorescent probes and the obtained Pearson correlation coefficient (P), the higher the P value, the better the overlap;

[0039] Figure 8SIM high-resolution fluorescence imaging images of the lipid droplet fluorescent probe and Nile red obtained in Example 1 of the present invention; wherein A is a SIM high-resolution fluorescence imaging image of 0.1 μM lipid droplet fluorescent probe, B is a SIM high-resolution fluorescence imaging image of 0.1 μM Nile red, C is a SIM high-resolution fluorescence imaging image of 10 μM lipid droplet fluorescent probe, and D is a SIM high-resolution fluorescence imaging image of 10 μM Nile red;

[0040] Fig. 9 The lipid droplet fluorescent probe and Nile red obtained in Example 1 of the present invention are db / db Fluorescence imaging of lipid droplets in mouse kidney. DETAILED DESCRIPTION

[0041] The present invention discloses a lipid droplet fluorescent probe and a preparation method, a detection reagent or a kit and an application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] The present invention discloses a probe based on a xanthene-quinoline structure, the structure of which is shown in formula (I), and is used to selectively identify lipid droplets in cells or animal tissues. Based on the highly lipophilic property of lipid droplets, the present invention utilizes fat-soluble xanthene and quinoline groups and designs a target probe with a donor-acceptor (DA) structure through a conjugation extension strategy, with a cLogP value of 5.87, to achieve spatiotemporal specific fluorescence labeling and imaging of lipid droplets at the cellular and animal levels.

[0043] The purpose of the present invention is to provide a probe that can be used for super-resolution imaging and detection of lipid droplets in disease animal models, and to analyze the mechanism of action of lipid droplets in physiological and pathological environments at the cellular and animal levels. This probe generates a fluorescent signal after excitation, and the fluorescent signal is collected. Under an ultra-high resolution microscope, it can be observed that the lipid droplet particles marked by the probe are distinct, and there is no unclear boundary, indicating that the probe has the ability of ultra-high resolution fluorescence imaging of lipid droplets. After staining animal tissues with the probe, lipid droplets can be accurately detected when fluorescence imaging is performed using a confocal microscope. More importantly, the number, intensity and gaps between lipid droplets marked by the probe are stronger than those of conventional stains, indicating that the probe has excellent ability to image lipid droplets in animal tissues.

[0044] The first typical embodiment of the present invention provides a lipid droplet fluorescent probe, which is excited at 488 nm, has fluorescence emission in the 530-630 nm region, and can collect fluorescence signals, and has a structure as shown in formula (I):

[0045]

[0046] Formula (I).

[0047] A second typical embodiment of the present invention provides a method for preparing the above-mentioned lipid droplet fluorescent probe, comprising:

[0048] Compound 2, p-toluenesulfonic acid and 2-methylquinoline react to obtain a lipid droplet fluorescent probe;

[0049] The structure of the compound 2 is as follows:

[0050] .

[0051] The synthetic route is:

[0052]

[0053] In some examples of this embodiment, the molar ratio of Compound 2, p-toluenesulfonic acid and 2-methylquinoline is 1:(1-4):(1-1.5).

[0054] In some examples of this embodiment, the preparation method comprises: dissolving compound 2, p-toluenesulfonic acid and 2-methylquinoline in an organic solvent, and stirring the mixture at 150-170° C. for reaction.

[0055] Wherein, the organic solvent includes but is not limited to N,N-dimethylformamide (DMF) or dichloromethane.

[0056] The stirring reaction is carried out at 160° C. Preferably, the stirring reaction is carried out at 160° C. for 4 h.

[0057] In some examples of this embodiment, the preparation method further comprises the step of separating and purifying to obtain the lipid droplet fluorescent probe after the reaction is completed;

[0058] The separation and purification comprises the steps of adding dichloromethane to the reaction system for liquid-liquid extraction, collecting the organic phase, drying, removing the solvent and purifying by silica gel column chromatography after the reaction is completed.

[0059] In some examples of this embodiment, in the silica gel column chromatography purification step, the eluent ratio is ethyl acetate:petroleum ether=1:(2-5).

[0060] It should be noted that when purification is performed by silica gel column chromatography, gradient elution is performed, and the content of ethyl acetate in the eluent gradually increases, that is, the polarity of the eluent gradually increases, until the target product is obtained.

[0061] In some examples of this embodiment, the preparation method comprises:

[0062] 0.2 mmol of compound 2, 0.66 mmol of p-toluenesulfonic acid and 0.24 mmol of 2-methylquinoline were added to a reaction bottle containing 5 mL of DMF, stirred at 160°C for 4 hours, and the reaction was monitored by spot plate until completion. After cooling to room temperature, 10 mL of water was added to quench the reaction, and the mixture was extracted with dichloromethane three times, 30 mL each time. The combined organic phases were dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of ethyl acetate: petroleum ether = 1:(2-5) to obtain the target probe.

[0063] In some examples of this embodiment, the preparation method further comprises the following steps:

[0064] In the presence of cesium carbonate, compound 3 and 4-(dimethylamino)-2-hydroxybenzaldehyde are reacted to obtain compound 2; the molar ratio of cesium carbonate, compound 3 and 4-(dimethylamino)-2-hydroxybenzaldehyde is (2.5-3.5):(1.5-2.5):1;

[0065] The structure of compound 3 is as follows:

[0066] .

[0067] The preparation method comprises: adding compound 3, cesium carbonate and 4-(dimethylamino)-2-hydroxybenzaldehyde into an organic solvent, and stirring the reaction at room temperature.

[0068] Wherein, the organic solvent includes but is not limited to N,N-dimethylformamide (DMF) or dichloromethane.

[0069] The preparation method further comprises the steps of removing insoluble matter after the reaction is completed, washing the filter residue with chloroform, removing the solvent from the filtrate and purifying it by silica gel column chromatography.

[0070] Wherein, in the silica gel column chromatography purification step, the eluent ratio is ethyl acetate:petroleum ether=1:(3-10).

[0071] The preparation method comprises: adding 12 mmol of compound 3, 18 mmol of cesium carbonate and 6 mmol of 4-(dimethylamino)-2-hydroxybenzaldehyde to a reaction bottle containing 20 mL of dry DMF, stirring at room temperature for 48 h; monitoring the reaction to be complete by spot plate, filtering to remove insoluble matter, washing the filter residue with dichloromethane three times, 30 mL each time, evaporating the solvent of the filtrate under reduced pressure, purifying the obtained crude product by silica gel column chromatography, and the eluent ratio is ethyl acetate: petroleum ether = 1: (3-10), to obtain compound 2.

[0072] It should be noted that when the crude product was purified by silica gel column chromatography, gradient elution was still performed, and the content of ethyl acetate in the eluent gradually increased.

[0073] In some examples of this embodiment, the preparation method further comprises the following steps:

[0074] In an ice bath and under nitrogen protection, cyclohexanone was added to an organic solution containing phosphorus tribromide, and the mixture was reacted at room temperature to obtain compound 3; the molar ratio of phosphorus tribromide to cyclohexanone was (2.5-3):1.

[0075] The preparation method comprises: slowly dropping phosphorus tribromide into a mixed solution of N,N-dimethylformamide and chloroform in an ice bath and under nitrogen protection, stirring for reaction, slowly adding cyclohexanone, reacting at room temperature for 10-14 h, and adding ice water to quench the reaction.

[0076] Wherein, the volume ratio of N,N-dimethylformamide to chloroform is 1-1.5:5, preferably 1.1:5.

[0077] The preparation method further comprises the steps of adjusting the reaction system to neutrality after the reaction is completed, adding dichloromethane to perform liquid-liquid extraction on the aqueous phase, collecting the organic phase, drying, and removing the solvent.

[0078] The preparation method comprises: slowly dripping 131 mmol of phosphorus tribromide into a reaction bottle containing 11 mL of dry DMF and 50 mL of chloroform in an ice bath and under nitrogen protection, stirring and reacting for 30 min, slowly adding 49 mmol of cyclohexanone, and reacting at room temperature for 12 h; after adding ice water to quench the reaction, adjusting the pH of the solution to 7 with sodium bicarbonate; after stratification, extracting the aqueous phase with dichloromethane three times, 100 mL each time; drying the combined organic phase with sodium sulfate, and evaporating the solvent under reduced pressure to obtain compound 3.

[0079] A third typical embodiment of the present invention provides a detection reagent or a kit, which includes the above-mentioned lipid droplet fluorescent probe or the lipid droplet fluorescent probe prepared by the above-mentioned preparation method.

[0080] A fourth typical embodiment of the present invention provides an application of the above-mentioned lipid droplet fluorescent probe or the lipid droplet fluorescent probe prepared by the above-mentioned preparation method or the above-mentioned detection reagent or kit in lipid droplet detection, fatty liver detection, diabetes detection, fluorescence detection, cell fluorescence imaging or tissue fluorescence imaging.

[0081] A fifth typical embodiment of the present invention provides an application of the above-mentioned lipid droplet fluorescent probe or the lipid droplet fluorescent probe prepared by the above-mentioned preparation method or the above-mentioned detection reagent or kit in the preparation of lipid droplet detection, fatty liver detection, diabetes detection, fluorescence detection, cell fluorescence imaging or tissue fluorescence imaging products.

[0082] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments. Unless otherwise specified, the raw materials, reagents, consumables and instruments used in the present invention are all common commercial products and can be purchased from the market.

[0083] Example 1

[0084] The synthesis of lipid droplet fluorescent probe, the synthesis route is as follows:

[0085]

[0086] The synthesis method comprises the following steps:

[0087] In an ice bath and under nitrogen protection, 131 mmol of phosphorus tribromide was slowly dripped into a reaction bottle containing 11 mL of dry DMF and 50 mL of chloroform. After stirring for 30 min, 49 mmol of cyclohexanone was slowly added and reacted at room temperature for 12 h. After adding ice water to quench the reaction, the pH of the solution was adjusted to 7 with sodium bicarbonate. After stratification, the aqueous phase was extracted three times with dichloromethane, 100 mL each time, and the organic phases were combined. After the combined organic phases were dried with sodium sulfate, the solvent was evaporated under reduced pressure to obtain a yellow oil, namely compound 3, with a yield of 60%.

[0088] 12 mmol of compound 3, 18 mmol of cesium carbonate and 6 mmol of 4-(dimethylamino)-2-hydroxybenzaldehyde were added to a reaction bottle containing 20 ml of dry DMF and stirred at room temperature for 48 h. The reaction was monitored by spot plate, the insoluble matter was removed by filtration, and the filter residue was washed three times with dichloromethane, 30 mL each time. The solvent of the filtrate was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography, the eluent ratio was ethyl acetate: petroleum ether = 1: (3-10), and an orange-yellow solid was obtained, which was compound 2, with a yield of 87%.

[0089] like Figure 1 and Figure 2 As shown, 1H NMR (400 MHz, CDCl3) δ 10.29 (s, 1H), 7.01 (d, J = 8.5Hz, 1H), 6.62 (s, 1H), 6.44 (dd, J = 8.5, 2.5 Hz, 1H), 6.39 (d, J = 2.5 Hz, 1H), 3.02 (s, 6H), 2.54 (t, J = 6.1 Hz, 2H), 2.44 (t, J = 6.1 Hz, 2H), 1.70 (p, J = 6.1Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 187.36, 161.89, 153.94, 152.09, 128.02,127.48, 123.94, 111.74, 111.00, 108.26, 98.05, 40.42, 30.02, 21.75, 20.77.

[0090] 0.2 mmol of compound 2, 0.66 mmol of p-toluenesulfonic acid and 0.24 mmol of 2-methylquinoline were added to a reaction bottle containing 5 mL of dry DMF and stirred at 160°C for 4 h. The reaction was monitored by spot plate. After cooling to room temperature, 10 mL of water was added to quench the reaction, and the mixture was extracted three times with 30 mL of dichloromethane each time, and the organic phases were combined. After the combined organic phases were dried over sodium sulfate, the solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography with an eluent ratio of ethyl acetate: petroleum ether = 1: (2-5) to obtain the target product, i.e., lipid droplet fluorescent probe, with a yield of 39%.

[0091] like Figure 3 and Figure 4 As shown, 1 H NMR (400 MHz, CDC l3 ) δ 8.15 (d, J = 16.2 Hz, 1H), 8.06- 8.01 (m, 2H), 7.77 - 7.72 (m, 2H), 7.67 - 7.63 (m, 1H), 7.44 - 7.41 (m,1H), 6.88 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 16.2 Hz, 1H), 6.43 (d, J= 2.5 Hz, 1H),6.33 (dd, J = 8.4, 2.5 Hz, 1H), 6.24 (s, 1H), 3.01 (s, 6H), 2.58 (t, J = 6.2 Hz,2H), 2.49 (t, J = 6.2 Hz, 2H), 1.81 (p, J = 6.2 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ157.95, 154.59, 151.45, 149.99, 148.66, 135.83, 129.98, 129.58, 128.97,127.54, 127.01, 126.46, 126.00, 125.45, 124.45, 121.99, 119.10, 111.94,109.92, 107.05, 98.71, 40.63, 30.14, 25.11, 21.43. ESI-MS calculated for [M+H] + 381.2; Found 381.5.

[0092] Performance Verification:

[0093] 1. UV-visible spectra test of lipid droplet fluorescent probe prepared in Example 1 in different solvents

[0094] The lipid droplet fluorescent probe prepared in Example 1 was dissolved in DMSO to prepare a 10 mM probe stock solution. 1 μL was taken out from the probe stock solution and added to a cuvette containing 499 μL toluene (Toluene), ethyl acetate (EA), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetonitrile (ACN), isopropanol (IPA), ethanol (EtOH) and water (H2O), respectively. The working concentration of the probe was 20 μM, and the absorption spectra of the lipid droplet fluorescent probe in different solvents were measured using a UV-visible spectrophotometer.

[0095] The results are as follows Figure 5 As shown in (A), the maximum absorption peak of the lipid droplet fluorescent probe in toluene solvent is 445 nm. Compared with lipophilic solvents such as toluene, the absorption of lipid droplet fluorescent probe in polar solvents such as ethanol, isopropanol, and water is reduced.

[0096] 2. Fluorescence emission spectrum test of lipid droplet fluorescent probe prepared in Example 1 in different solvents

[0097] The lipid droplet fluorescent probe prepared in Example 1 was dissolved in DMSO to prepare a 10 mM probe stock solution. 1 μL was taken out from the probe stock solution and added to a cuvette containing 999 μL toluene (Toluene), ethyl acetate (EA), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetonitrile (ACN), isopropanol (IPA), ethanol (EtOH) and water (H2O), respectively. The working concentration of the probe was 10 μM. The fluorescence emission spectra of the lipid droplet fluorescent probe in different solvents were measured using a fluorescence spectrophotometer, with an excitation and emission bandwidth of 5 nm, a data interval of 1 nm, and a scanning speed of 200 nm / min.

[0098] The results are as follows Figure 5 As shown in (B), the maximum emission wavelength of the lipid droplet fluorescent probe is between 600-650 nm, reaching the near-infrared emission range; the fluorescence emission intensity is significantly affected by the solvent, and the fluorescence intensity in low-polarity toluene, dichloromethane and ethyl acetate solvents is significantly stronger than that in high-polarity isopropanol, ethanol and water.

[0099] To further illustrate the polarity sensitivity of the lipid droplet fluorescent probe, the fluorescence emission spectra of the lipid droplet fluorescent probe in mixed solvents of 1,4-dioxadioxane / water at different ratios (0 to 99% 1,4-dioxadioxane) were also measured, and the maximum fluorescence emission wavelength and the percentage of 1,4-dioxadioxane reflecting polarity were fitted. The results are shown in Figure 2. Figure 6 (A) Figure 6 As shown in (B), the two showed an excellent linear correlation, indicating that the lipid droplet fluorescent probe exhibited typical polarity sensitivity.

[0100] 3. Co-staining experiment of lipid droplet fluorescent probe prepared in Example 1 and lipid droplet commercial dye Lipi-Deep Red

[0101] HeLa cells were cultured in a humidified incubator at 37°C in DMEM medium containing 1% antibiotics (streptomycin and penicillin) and 10% FBS. The cells were seeded in a sterile confocal culture dish and cultured in the incubator for 12 hours, then washed twice with PBS buffer and once with DMEM. DMEM culture medium containing 2.5 µM lipid droplet fluorescent probe was added, and the cells were incubated at 37°C for 1 hour. The DMEM solution containing lipid droplet fluorescent probe was poured out, and phenol red-free DMEM culture medium containing lipid droplet commercial dye Lipi-Deep Red (LDtracker, 67 nM) was added, and the cells were incubated at 37°C for 15 minutes. Fluorescence imaging was taken using an ultra-high resolution confocal laser microscope (Leica SP8 STED microscope). The excitation wavelength of the lipid droplet fluorescent probe was 488 nm, and the fluorescence signal was collected in the 530-630 nm region; the excitation wavelength of Lipi-Deep Red was 635 nm, and the fluorescence signal was collected in the 650-700 nm region.

[0102] The results are as follows Figure 7 As shown in (A), the lipid droplet fluorescent probe has good colocalization with Lipi-Deep Red (Pearson coefficient 0.83), proving that the probe is indeed localized in lipid droplets.

[0103] 4. Co-staining experiment of lipid droplet fluorescent probe prepared in Example 1 and other subcellular organelles

[0104] HeLa cells were cultured in DMEM medium containing 1% antibiotics (streptomycin and penicillin) and 10% FBS in a humidified incubator at 37°C. After incubation for 12 hours in a confocal culture dish, the cells were washed twice with PBS buffer and once with DMEM. DMEM culture medium containing 2.5 µM lipid droplet fluorescent probe was added, and the cells were incubated at 37°C for 1 hour. The DMEM solution containing lipid droplet fluorescent probe was poured out, and DMEM culture medium containing mitochondrial commercial dye Mitotracker Deep Red (67nM), endoplasmic reticulum commercial dye ERtracker Red (100 nM), lysosomal commercial dye Lysotracker Red (50 nM) or cell membrane commercial dye DID (1:400 dilution) was added, and the cells were incubated at 37°C for 20 minutes, 20 minutes, 20 minutes, and 5 minutes. Fluorescence imaging was taken using an ultra-high resolution confocal laser microscope (Leica SP8 STED microscope). The excitation wavelength of the lipid droplet fluorescent probe is 488 nm, and the fluorescence signal is collected in the 530-630 nm region; the excitation wavelength of Mitotracker DeepRed is 635 nm, and the fluorescence signal is collected in the 650-700 nm region; the excitation wavelength of ERtracker Red is 590 nm, and the fluorescence signal is collected in the 600-650 nm region; the excitation wavelength of Lysotracker Red is 600 nm, and the fluorescence signal is collected in the 620-670 nm region; the excitation wavelength of DID is 635 nm, and the fluorescence signal is collected in the 650-750 nm region.

[0105] The results are as follows Figure 7 (B)- Figure 7 As shown in (E), the lipid droplet fluorescent probe does not co-localize with organelles such as mitochondria, endoplasmic reticulum, lysosomes, and cell membranes, demonstrating that the lipid droplet fluorescent probe has excellent ability to selectively target lipid droplets.

[0106] 5. Lipid droplet SIM ultra-high resolution fluorescence imaging of the lipid droplet fluorescent probe prepared in Example 1

[0107] HeLa cells were cultured in a humidified incubator at 37°C in DMEM medium containing 1% antibiotics (streptomycin and penicillin) and 10% FBS. After the cells were seeded in a confocal culture dish and cultured in a humidified incubator at 37°C for 12 hours, oleic acid (150 µM) was added and the cells were incubated at 37°C for 3 hours to induce lipid droplet formation. The DMEM solution containing oleic acid was poured out, and DMEM culture medium containing lipid droplet fluorescent probes (0.1µM, 10 µM) and Nile red (0.1µM, 10 µM) were added, and the cells were incubated at 37°C for 1 hour. After washing the cells twice with PBS buffer, they were placed under a DeltaVision OMX Flex ultra-high resolution microscope for fluorescence imaging. The excitation wavelength of the lipid droplet fluorescent probe was 488 nm, and the fluorescence signal was collected in the 530-630 nm region.

[0108] The results are as follows Figure 8 As shown, the lipid droplet particles marked by the lipid droplet fluorescent probe are distinct, while the boundaries of some lipid droplets marked by Nile red are unclear, reflecting that the lipid droplet fluorescent probe has the ability to perform ultra-high resolution fluorescence imaging of lipid droplets.

[0109] 6. The lipid droplet fluorescent probe prepared in Example 1 db / db Fluorescence imaging of lipid droplets in mouse kidneys

[0110] Animals: Heterozygous BKS db mice ( db / m Mice (BKS.Cg-Dock7m+ / +Lepr db / J, Stock No. 000642) were purchased from Jackson Laboratory, USA. db / m Homozygous mice can be obtained by self-fertilization db / db Mice. The selected mice were all around 30 weeks old and had similar appearance and body size. db / db Mice and control mice db / m The kidney tissue of the mice was perfused with normal saline placed at 4°C in advance. After that, the kidney tissue was fixed in 4% paraformaldehyde for 24 hours. The kidney tissue was embedded in OCT embedding medium and cut into 10 µm slices using a freezing microtome and stored in a -20°C refrigerator.

[0111] Tissue imaging: The frozen sections were placed at room temperature, rewarmed for 30 minutes, and washed three times with PBS for 5 minutes each. Nile red (10 µM) was stained for 10 minutes at room temperature, lipid droplet fluorescent probe (10 µM) was stained for 1 hour, and washed three times with PBS for 5 minutes each. Finally, the sections were mounted with glycerol, fixed with nail polish, and fluorescent imaging was performed using a Leica SP8 confocal microscope.

[0112] The results are as follows Fig. 9 As shown, db / mNo lipid droplets were detected in the mouse kidneys. db / db In the kidneys of mice, both Nile red and lipid droplet fluorescent probes detected abnormal lipid deposition (dot-shaped fluorescent signals). More importantly, the number and intensity of lipid droplets marked by the lipid droplet fluorescent probe of the present invention as well as the gaps between lipid droplets were stronger, indicating that the lipid droplet fluorescent probe has excellent ability to image lipid droplets in animal tissues.

[0113] Example 2

[0114] The synthesis of the lipid droplet fluorescent probe is different from that of Example 1 in that 0.2 mmol of compound 2, 0.2 mmol of p-toluenesulfonic acid and 0.2 mmol of 2-methylquinoline are added to a reaction bottle containing 5 mL of dry DMF, and the remaining steps are consistent with those of Example 1. The target product, i.e., the lipid droplet fluorescent probe, is obtained with a yield of 33%.

[0115] The H-NMR spectrum and C-NMR spectrum of the lipid droplet fluorescent probe obtained in Example 2 are consistent with those of the lipid droplet fluorescent probe obtained in Example 1.

[0116] Example 3

[0117] The synthesis of the lipid droplet fluorescent probe is different from that of Example 1 in that 0.2 mmol of compound 2, 0.8 mmol of p-toluenesulfonic acid and 0.3 mmol of 2-methylquinoline are added to a reaction bottle containing 5 mL of dry DMF, and the remaining steps are consistent with those of Example 1. The target product, i.e., the lipid droplet fluorescent probe, is obtained with a yield of 37%.

[0118] The H-NMR spectrum and C-NMR spectrum of the lipid droplet fluorescent probe obtained in Example 3 are consistent with those of the lipid droplet fluorescent probe obtained in Example 1.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A lipid droplet fluorescent probe, characterized in that It has a structure as shown in formula (I): Formula (I).

2. A method for preparing a lipid droplet fluorescent probe according to claim 1, characterized in that: include: Compound 2, p-toluenesulfonic acid and 2-methylquinoline react to obtain a lipid droplet fluorescent probe; The structure of the compound 2 is as follows: 。 3. The preparation method according to claim 2, characterized in that: The molar ratio of compound 2, p-toluenesulfonic acid and 2-methylquinoline is 1:(1-4):(1-1.5).

4. The preparation method according to claim 2, characterized in that: The preparation method comprises: dissolving compound 2, p-toluenesulfonic acid and 2-methylquinoline in an organic solvent, and stirring and reacting at 150-170° C.; The preparation method further comprises the step of separating and purifying after the reaction to obtain the lipid droplet fluorescent probe; The separation and purification comprises the steps of adding dichloromethane to the reaction system for liquid-liquid extraction, collecting the organic phase, drying, removing the solvent and purifying by silica gel column chromatography after the reaction is completed.

5. The preparation method according to claim 4, characterized in that: In the silica gel column chromatography purification step, the eluent ratio is ethyl acetate: petroleum ether = 1: (2-5).

6. The preparation method according to claim 2, characterized in that: The preparation method further comprises the following steps: In the presence of cesium carbonate, compound 3 and 4-(dimethylamino)-2-hydroxybenzaldehyde are reacted to obtain compound 2; the molar ratio of cesium carbonate, compound 3 and 4-(dimethylamino)-2-hydroxybenzaldehyde is (2.5-3.5):(1.5-2.5):1; The structure of compound 3 is as follows: 。 7. The preparation method according to claim 6, characterized in that: The preparation method further comprises the following steps: In an ice bath and under nitrogen protection, cyclohexanone was added to an organic solution containing phosphorus tribromide, and the mixture was reacted at room temperature to obtain compound 3; the molar ratio of phosphorus tribromide to cyclohexanone was (2.5-3):

1.

8. A detection reagent or kit, characterized in that: The invention comprises the lipid droplet fluorescent probe according to claim 1 or the lipid droplet fluorescent probe prepared by any preparation method according to claims 2-7.

9. Use of the lipid droplet fluorescent probe according to claim 1, or the lipid droplet fluorescent probe prepared by any preparation method according to claims 2 to 7, or the detection reagent or kit according to claim 8 in lipid droplet detection; The lipid droplet detection is not intended for the diagnosis of disease.

10. Use of the lipid droplet fluorescent probe according to claim 1, or the lipid droplet fluorescent probe prepared by the preparation method according to any one of claims 2 to 7, or the detection reagent or kit according to claim 8 in preparing a lipid droplet detection product.

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