A lipid droplet protein in situ labeled probe, its preparation method and application

Abstract

This invention relates to the field of molecular probe technology for biological systems, and particularly to an in-situ labeling probe for lipid droplet proteins, its preparation method, and its applications. The probe of this invention has the following structure: [structure omitted]. The synthesis method of this probe is simple and easy to implement, and it can specifically locate the lipophilic ends and lipid center regions of the phospholipid monolayer of lipid droplets. It can be used for in-situ, real-time labeling of lipid droplet proteins in living cells and animals. This technology represents the first significant leap from cellular to animal-level in-situ analysis of lipid droplet proteins, providing a powerful tool for systematically elucidating the protein composition and dynamic evolution of lipid droplets, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of molecular probe technology for biological systems, and in particular to a lipid droplet protein in situ labeled probe, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Lipid droplets (LDs) are cellular structures composed of a neutral lipid core, a phospholipid monolayer, and surface proteins. As a highly conserved organelle from prokaryotes to eukaryotes, lipid droplets have attracted considerable attention since the discovery of their signature protein, perilipin, in 1991. Recent studies have shown that lipid droplets function far beyond their traditional role as "lipid reservoirs," exhibiting a highly diverse range of biological functions, primarily dependent on their dynamically changing proteomic networks. Dysfunction of lipid droplets is closely associated with various metabolic diseases, including obesity, diabetes, and cancerous degeneration. Multidimensional analysis of lipid droplet proteins will not only contribute to a deeper understanding of the biological mechanisms of lipid droplets but will also provide new insights into the diagnosis and treatment of related diseases.

[0004] Traditional lipid droplet protein research primarily relies on subcellular organelle separation techniques (Nat. Protoc. 2013, 8, 43-51). This method is cumbersome, prone to protein loss, and fails to reflect true physiological states. The APEX proximity labeling method for lipid droplets (Dev. Cell 2018, 44, 97-112.e7) has achieved a technological breakthrough to some extent in recent years, but its specificity is low, still requiring lipid droplet separation for effective analysis. Recently, we developed a separation-free in-situ analysis platform for live-cell lipid droplet proteins based on a photoaffinity labeling strategy (J. Am. Chem. Soc. 2025, 147, 12, 10724-10736); however, its reliance on UV excitation limits its in vivo application. Therefore, developing next-generation in-situ labeling methods for lipid droplet proteins suitable for live animals, achieving a technological leap from cells to animals, has become an urgent need in the field. This will provide crucial technical support for systematically analyzing the dynamic changes and biological functions of lipid droplet proteomes. Summary of the Invention

[0005] In view of this, the present invention provides an in situ labeling probe for lipid droplets, its preparation method, and its application. The probe provided by the present invention can specifically locate the lipophilic end of the phospholipid monolayer and the lipid center region of lipid droplets, and can be used for in situ and real-time labeling of lipid droplet proteins in living cells and animals.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] In a first aspect, the present invention provides a lipid droplet protein in situ labeling probe, the structural formula of which is shown in formula (1):

[0008]

[0009] Equation (1).

[0010] Lipid droplet proteins are anchored to the phospholipid monolayer of lipid droplets via a hydrophobic structure, and the central region enclosed by this monolayer is a lipophilic region. When a probe is placed in this region, the aqueous environment in the cytoplasm is relatively isolated, effectively avoiding side reactions such as hydrolysis. Simultaneously, the main neighboring proteins of the probe are lip droplet proteins, allowing its covalently reactive warhead to preferentially cross-link with the lip droplet proteins. Based on this mechanism, we designed a class of probes with sulfonyl fluoride warheads that specifically target the lipophilic region of lipid droplets, thus successfully achieving in-situ analysis of the lipid droplet proteome in living cells and animals.

[0011] In a second aspect, the present invention provides a composition comprising the lipid droplet protein in situ labeling probe and excipients described in the first aspect.

[0012] The excipients described in this invention are pharmaceutical excipients, such as solubilizers, emulsifiers, stabilizers, preservatives, etc.

[0013] Thirdly, the present invention provides a detection reagent comprising the lipid droplet protein in situ labeled probe described in the first aspect.

[0014] The reagents include substances that maintain the stability of the in situ labeled probe for lipid droplets and substances that increase solubility, such as dimethyl sulfoxide (DMSO).

[0015] Fourthly, the present invention provides the application of the lipid droplet protein in situ labeled probe described in the first aspect, the composition described in the second aspect, or the detection reagent described in the third aspect in lipid droplet detection, lipid droplet protein in situ detection, fatty liver detection, atherosclerosis detection, diabetic nephropathy detection, neurodegenerative disease detection, fluorescence detection, cell fluorescence imaging, or tissue fluorescence imaging.

[0016] The cells are fixed cells or cells in vivo;

[0017] The application is intended for the diagnosis of diseases or non-diseases.

[0018] Fifthly, the present invention provides the application of the lipid droplet protein in situ labeled probe described in the first aspect, the composition described in the second aspect, or the detection reagent described in the third aspect in the preparation of lipid droplet detection products, lipid droplet protein in situ detection products, fatty liver detection products, atherosclerosis detection products, diabetic nephropathy detection products, neurodegenerative disease detection products, fluorescence detection products, cell fluorescence imaging products, or tissue fluorescence imaging products.

[0019] Specifically, the application can be used for the diagnosis of diseases or for the diagnosis of non-diseases.

[0020] In a sixth aspect, the present invention provides a method for preparing the in-situ labeled probe of the lipid droplet protein described in the first aspect, comprising the following steps:

[0021] (1) Compound 3 was prepared by reacting 4-fluoro-2,1,3-benzoxadiazole with tert-butylsarcosine hydrochloride in the presence of triethylamine;

[0022] (2) In the presence of dimethylformamide (DMF), compound 3 reacts with phosphorus oxychloride to give compound 4;

[0023] (3) Compound 4 reacts with trifluoroacetic acid to give compound 5;

[0024] (4) Compound 5 was reacted with 3-butyn-1-ol in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole hydrate (HOBt) and 4-dimethylaminopyridine (DMAP) to give compound 6.

[0025] (5) In the presence of piperidine, compound 6 reacts with cyanoacetic acid to give compound 2;

[0026] (6) In 1-methylimidazole (NMI) and N,N,N',N' Compound 2 was reacted with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride in the presence of tetramethylchloroformamidine hexafluorophosphate (TCFH) to give compound 1; the synthetic route is as follows:

[0027] .

[0028] Furthermore, in step (1), the solvent is acetonitrile; the reaction temperature is 75-85 ℃, and the reaction time is 60-70 h.

[0029] Further, in step (1), the molar ratio of 4-fluoro-2,1,3-benzoxadiazole to tert-butylsarcosine hydrochloride is 1:4-6; the molar ratio of 4-fluoro-2,1,3-benzoxadiazole to triethylamine is 1:5-7.

[0030] Furthermore, in step (2), the solvent is N,N - Dimethylformamide (DMF); the molar ratio of compound 3 to phosphorus oxychloride is 1:4-6.

[0031] Further, the specific operation of step (2) is as follows: under the protection of ice bath and nitrogen, phosphorus oxychloride is dropped into DMF, stirred for 8-15 min, then DMF containing compound 3 is added, and the reaction is carried out at room temperature for 5-10 h; after the reaction is complete, 10-15% sodium hydroxide solution is added and stirred for 20-40 min, and compound 4 is obtained after post-processing.

[0032] Furthermore, in step (3), the solvent is dichloromethane; the reaction temperature is 20-30 ℃, and the reaction time is 2-4 h.

[0033] Furthermore, in step (3), the ratio of compound 4 to trifluoroacetic acid is 0.1:1-3 mmol / mL.

[0034] Furthermore, in step (4), the solvent is DMF; the reaction temperature is 20-30 ℃, and the reaction time is 10-15 h.

[0035] Furthermore, in step (4), the molar ratio of compound 5 to 3-butyn-1-ol is 1:1-1.2.

[0036] Furthermore, in step (4), the molar ratio of compound 5, EDCI, HOBt and DMAP is 3.5-4:5.5-6:5.5-6:1.

[0037] Furthermore, in step (5), the molar ratio of compound 6 to cyanoacetic acid is 1:1.5-3; the molar ratio of compound 6 to piperidine is 4-6:1.

[0038] Furthermore, in step (5), the solvent is acetonitrile; the reaction time is 5-7 h.

[0039] Further, in step (6), the molar ratio of compound 2 to 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride is 1:1-1.5; the molar ratio of compound 2, NMI and TCFH is 1:2-4:1-1.5.

[0040] Furthermore, in step (6), the solvent is acetonitrile; the reaction temperature is 20-30 ℃, and the reaction time is 10-20 h.

[0041] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0042] The in-situ labeling probe for lipid droplet proteins provided by this invention is simple and easy to synthesize. This probe can specifically locate the lipophilic ends and lipid centers of the phospholipid monolayer of lipid droplets, and can be used for in-situ, real-time labeling of lipid droplet proteins in living cells and animals. This technology represents a first-ever leap from cellular to animal-level in-situ analysis of lipid droplet proteins, providing a powerful tool for systematically elucidating the protein composition and dynamic evolution of lipid droplets, and has broad application prospects. Attached Figure Description

[0043] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0044] Figure 1 The image shows the hydrogen NMR spectrum of probe 1.

[0045] Figure 2 The image shows the carbon NMR spectrum of probe 1.

[0046] Figure 3 Fluorescence imaging of probe 1 and PLIN3 antibody co-staining experiment;

[0047] Figure 4 Fluorescence imaging of probe 1 co-stained with the commercial lipid droplet dye Lipi-Deep Red;

[0048] Figure 5 The images show fluorescence images of co-staining experiments of probe 1 with commercial dyes for other subcellular organelles; (A) is a fluorescence image of co-staining experiment of probe 1 with the commercial dye MitoTracker Deep Red for mitochondria; (B) is a fluorescence image of co-staining experiment of probe 1 with the commercial dye ER-Tracker Red for endoplasmic reticulum; (C) is a fluorescence image of co-staining experiment of probe 1 with the commercial dye Lyso-Tracker Red for lysosomes; and (D) is a fluorescence image of co-staining experiment of probe 1 with the commercial dye DiD for cell membranes.

[0049] Figure 6 The images show fluorescence images of protein gel labeling experiments for probe 1. Among them, (A) is a fluorescence image of protein gel labeling of probe 1 and DMSO in HeLa cells; (B) is a fluorescence image of protein gel labeling of probe 1 and DMSO in U2OS cells; (C) is a fluorescence image of protein gel labeling of probe 1 incubation time-dependent; (D) is a fluorescence image of protein gel labeling of probe 1 in concentration-dependent; and (E) is a fluorescence image of protein gel labeling of probe 1 after treatment with DMSO, isoproterenol, and rapamycin, respectively.

[0050] Figure 7This is a schematic diagram of probe 1 used for in situ labeling of lipid droplet proteins. Detailed Implementation

[0051] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0053] Example 1

[0054] This embodiment provides a method for synthesizing probe 1, as detailed below:

[0055]

[0056] 21.72 mmol of triethylamine, 3.62 mmol of 4-fluoro-2,1,3-benzoxadiazole, 18.1 mmol of tert-butylsarcosine hydrochloride, and 15 mL of anhydrous acetonitrile were added to a reaction flask, and the mixture was reacted at 80 °C for 65 h. 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 = 1:20, yielding a brown liquid, which was compound 3, in 75% yield.

[0057] Under ice bath and nitrogen protection, 14.6 mmol of phosphorus oxychloride was slowly added dropwise to a reaction flask containing 5 mL of dry DMF. After stirring for 10 min, compound 3 (2.92 mmol) dissolved in 3 mL of dry DMF was slowly added, and the reaction was allowed to proceed at room temperature for 7 h. After the reaction was confirmed to be complete by TLC, 20 mL of 10% NaOH solution was added, and the reaction was stirred for another 30 min. The mixture was extracted three times with 20 mL of dichloromethane 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 = 1:5-20 to obtain a yellow solid, which was compound 4, in 32% yield.

[0058] 0.1 mmol of compound 4 and 2 mL of trifluoroacetic acid were added to a reaction flask containing 2 mL of dichloromethane, and the reaction was carried out at room temperature for 3 h. The solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography with an eluent ratio of methanol:dichloromethane = 1:10-20 to obtain a brown solid, which was compound 5, with a yield of 83%.

[0059] 0.93 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 0.93 mmol of 1-hydroxybenzotriazole hydrate (HOBt), 0.16 mmol of 4-dimethylaminopyridine (DMAP), and 0.62 mmol of compound 5 were added to a reaction flask containing 6 mL of DMF under a nitrogen atmosphere and stirred at 0 °C for 15 min. Then, 0.68 mmol of 3-butyn-1-ol was added to the reaction flask, and the mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography with dichloromethane as the eluent to give a reddish-brown solid, which was compound 6, in 90% yield.

[0060] 1.12 mmol cyanoacetic acid, 0.11 mmol piperidine, 0.56 mmol compound 6, and 10 mL anhydrous acetonitrile were added to a reaction flask, and the mixture was refluxed and stirred for 6 h. The reaction was monitored for completeness by TLC. The solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography with an eluent ratio of methanol:dichloromethane = 1:10⁻³0, yielding a red solid, which was compound 2, in 71% yield.

[0061] 0.05 mmol of compound 2, 0.07 mmol of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.19 mmol of 1-methylimidazole (NMI), and 0.06 mmol of... N,N,N',N' Tetramethylchloromethanesulfonyl hexafluorophosphate (TCFH) was added to a reaction flask containing 1.5 mL of acetonitrile and stirred at room temperature for 16 h. The reaction was monitored by TLC until complete. The crude product was purified by silica gel column chromatography by evaporation of the solvent under reduced pressure, with an eluent ratio of methanol:dichloromethane = 1:(100-300), yielding the target product, probe compound 1, in 32% yield. The 1H NMR spectrum of probe 1 is shown below. Figure 1 As shown, the carbon NMR spectrum of probe 1 is as follows: Figure 2 As shown. 1 H-NMR (CDCl3, 600 MHz) δ (ppm): δ 8.79 (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 8.00-7.97 (m, 2H), 7.52-7.49 (m, 2H), 6.34 (d, J = 8.3 Hz, 1H), 4.90 (s, 2H), 4.28-4.26 (m, 2H), 3.74-3.70 (m, 2H), 3.40 (s, 3H), 3.08-3.05 (m, 2H), 2.55-2.53(m, 2H), 1.97 (t, J= 2.6 Hz, 1H). 13 C-NMR (CDCl3, 151 MHz) δ(ppm): 168.77,161.14, 150.39, 147.48, 145.23, 144.50, 142.89, 136.29, 130.30, 130.21,129.06, 118.29, 107.81, 105.68, 99.19, 79.60, 70.33, 63.38, 56.65, 41.61, 41.24, 35.94, 19.13.

[0062] Example 2

[0063] Co-staining assay of probe 1 and perilipoplasmin 3 (PLIN3) antibody

[0064] Cells were cultured in Duchenne Spectrophotometry (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. HeLa cells were seeded onto coverslips pre-placed in 60 mm sterile culture dishes and cultured for 12 h. Cells were then washed twice with phosphate-buffered saline (PBS) and once with DMEM. Cells were treated with DMEM medium containing 150 μM oleic acid (OA) for 12 h and washed three times with PBS buffer. Cells were fixed with 4% paraformaldehyde at 37 °C for 20 min and washed three times with PBS. Cells were permeabilized with 10 μg / mL saponin for 15 min and washed three times with PBS buffer. Finally, cells were blocked for 30 min with phosphate-buffered saline (PBST) containing 1% bovine serum albumin (BSA) and 1% donkey serum, along with Tween-20 (0.1%), to prevent non-specific binding. Discard the blocking buffer, add PLIN3 primary antibody prepared with the same dilution, and incubate overnight at 4 °C in a humidified chamber. Discard the primary antibody, wash three times with PBST for 5 min each time. Then add Donkey Anti-Rabbit IgG H&L-Alexa Fluor prepared with the same dilution. ® Incubate with 594 secondary antibody at room temperature in the dark for 2 hours. Finally, discard the secondary antibody and wash three times with PBST for 5 minutes each time, in the dark.

[0065] After completing the aforementioned steps, PBS buffer containing 10 µM probe 1 was added, and cells were incubated at room temperature for 1 h for counterstaining. The staining solution was discarded, and the cells were washed three times with PBS for 5 min each time. Finally, a coverslip was fixed with a drop of mounting medium and the edges were sealed with nail polish to prevent the sample from drying out and moving under the microscope. Fluorescence imaging was performed using a super-resolution confocal laser microscope (Leica SP8 STED). The excitation wavelength of probe 1 was 488 nm, and the fluorescence signal collection range was 500–630 nm; the excitation wavelength of PLIN3 was 590 nm, and the fluorescence signal collection range was 605–690 nm.

[0066] Figure 3 The images show fluorescence imaging results from the co-staining experiment with probe 1 and PLIN3 antibody. The scale bar for each image is 20 μm. Figure 3 As shown, the green fluorescent signal labeled by probe 1 colocalizes with the red fluorescent signal of the lipid droplet structural protein PLIN3, and the green signal is surrounded by the red signal, indicating that probe 1 can specifically label lipid droplets and locate in the lipophilic region between the lipophilic end of the phospholipid monolayer and the internal lipid center of the lipid droplet.

[0067] Example 3

[0068] Co-staining experiment of probe 1 and the commercial lipid-drop dye Lipi-Deep Red

[0069] Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. HeLa cells were seeded into sterile confocal culture dishes and cultured for 12 h, followed by washing twice with PBS and once with DMEM. Cells were then treated with DMEM medium containing 150 μM oleic acid (OA) for 12 h, followed by washing twice with PBS and once with DMEM. Subsequently, DMEM medium containing 10 µM probe 1 was added, and the cells were incubated at 37 °C for 1 h. The probe 1 medium was removed, and phenol red-free DMEM containing 67 nM Lipi-Deep Red (a commercial lipid-droplet dye) was added, followed by incubation at 37 °C for 15 min. Fluorescence imaging was performed using a Leica SP8 STED super-resolution confocal laser microscope. The excitation wavelength of probe 1 is 488 nm, and the fluorescence signal collection range is 500-630 nm; the excitation wavelength of Lipi-Deep Red is 640 nm, and the signal collection range is 665-700 nm.

[0070] Figure 4 The images show fluorescence images of probe 1 co-stained with the commercial lipid dye Lipi-Deep Red, with scale bars of 20 μm. Figure 4As shown, the fluorescence signal of probe 1 highly overlaps with that of the lipid droplet dye Lipi-Deep Red, and its Pearson colocalization coefficient reaches 0.93, proving that probe 1 is indeed localized to the lipid droplet.

[0071] Example 4

[0072] Co-staining experiment of probe 1 and other subcellular organelles

[0073] Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37 ℃ in a 5% CO2 incubator. HeLa cells were seeded in confocal culture dishes and cultured for 12 h. After 12 h, the cells were washed twice with PBS and once with DMEM. The cells were then treated with DMEM medium containing 150 μM oleic acid (OA) for 12 h, followed by washing twice with PBS and once with DMEM. Subsequently, DMEM medium containing 10 µM probe 1 was added and incubated at 37 ℃ for 1 h. After removing the probe-containing medium, DMEM medium containing different organelle dyes for mitochondria, endoplasmic reticulum, lysosomes, and cell membranes was added: MitoTracker Deep Red (67 nM) for 20 min, ER-Tracker Red (100 nM) for 20 min, Lyso-Tracker Red (50 nM) for 20 min, or DiD (1:400 dilution) for 5 min, all at 37 ℃. Fluorescence imaging was performed using a super-resolution confocal laser microscope (Leica SP8 STED). The excitation / collection wavelengths for probe 1 were 488 nm / 500-630 nm; for MitoTracker Deep Red, 635 nm / 650-700 nm; for ER-Tracker Red, 587 nm / 600-650 nm; for Lyso-Tracker Red, 600 nm / 620-700 nm; and for DiD, 635 nm / 650-700 nm.

[0074] Figure 5 The images show fluorescence images of probe 1 co-stained with commercial dyes for other subcellular organelles; (A) shows a fluorescence image of probe 1 co-stained with the commercial dye MitoTracker Deep Red for mitochondria; (B) shows a fluorescence image of probe 1 co-stained with the commercial dye ER-Tracker Red for endoplasmic reticulum; (C) shows a fluorescence image of probe 1 co-stained with the commercial dye Lyso-Tracker Red for lysosomes; and (D) shows a fluorescence image of probe 1 co-stained with the commercial dye DiD for cell membranes. The scale bar is 20 μm. Figure 5As shown in (A), the fluorescence signal of probe 1 does not significantly overlap with the signal of the mitochondrial dye; as Figure 5 As shown in (B), the fluorescence signal of probe 1 does not significantly overlap with the signal of the endoplasmic reticulum dye; as Figure 5 As shown in (C), the fluorescence signal of probe 1 does not significantly overlap with the signal of the lysosomal (C) dye; as Figure 5 As shown in (D), the fluorescence signal of probe 1 does not significantly overlap with the signal of the cell membrane (D) dye, indicating that probe 1 can specifically target lipid droplets and has good selectivity.

[0075] Example 5

[0076] Protein gel labeling experiment with probe 1

[0077] Probe and DMSO control experiments: Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37 ℃ in a 5% CO2 incubator. U2OS / HeLa cells were seeded into 6-well plates and cultured for 12 h. After culturing, the original medium was discarded, and DMEM medium containing 20 µM probe 1 or an equal volume of DMSO was added. The cells were incubated at 37 ℃ for 90 min. After incubation, the culture medium was removed, cells were collected and centrifuged, washed once with PBS, and cell lysis buffer (20 mM Tris-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added. Cells were sonicated and centrifuged to collect the supernatant. The protein concentration was adjusted to 2 mg / mL, and Cyanine 5.5-N3 (final concentration 40 µM) was added. The reaction was carried out at room temperature in the dark for 2 h. Subsequently, proteins were precipitated with frozen acetone and methanol, respectively. The resulting protein precipitates were separated by SDS-PAGE gel electrophoresis. Finally, fluorescence imaging was performed using an Odyssey DLx imager (excitation wavelength 700 nm).

[0078] Figure 6 The images show fluorescence images of protein gel labeling experiments using probe 1; (A) is a fluorescence image of protein gel labeling using probe 1 and DMSO in HeLa cells; (B) is a fluorescence image of protein gel labeling using probe 1 and DMSO in U2OS cells; (C) is a fluorescence image of protein gel labeling using probe 1 incubation time-dependently; (D) is a fluorescence image of protein gel labeling using probe 1 in concentration-dependently; and (E) is a fluorescence image of protein gel labeling using probe 1 after treatment with DMSO, isoproterenol, and rapamycin, respectively. Figure 6 As shown in (A) and (B), probe 1 can effectively label proteins in U2OS / HeLa cells, while the DMSO control group only shows a weak background signal.

[0079] Probe incubation time-dependent experiment: Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin and incubated at 37 ℃ in a 5% CO2 incubator. U2OS cells were seeded into 6-well plates and cultured for 12 h. After the original medium was discarded, DMEM medium containing 20 µM probe 1 was added, and the cells were incubated at 37 ℃ in the dark for 120, 60, 30, 15, or 0 min. After incubation, the culture medium was removed, cells were collected by centrifugation, and washed once with PBS. Cell lysis buffer (20 mM Tris-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was then added, and the cells were sonicated and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / mL, and Cyanine 5.5-N3 (final concentration 40 µM) was added. The reaction was carried out at room temperature in the dark for 2 h. After the reaction, the proteins were precipitated with frozen acetone and methanol, respectively, and the obtained proteins were separated by SDS-PAGE gel electrophoresis. Finally, fluorescence imaging analysis was performed using an Odyssey DLx imager (excitation wavelength 700 nm).

[0080] The results are as follows Figure 6 As shown in (C), the labeling effect of probe 1 on the protein is time-dependent, and a significant labeling signal can be observed after 15 min of incubation.

[0081] Probe concentration-dependent assay: Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. U2OS cells were seeded into 6-well plates and cultured for 12 h. After the original medium was discarded, DMEM medium containing different concentrations of probe 1 (0, 2.5, 5, 10, 20, 40 µM) was added, and the cells were incubated at 37°C for 90 min. After incubation, the culture medium was removed, cells were collected by centrifugation, washed once with PBS, and cell lysis buffer (20 mM Tris-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added. Cells were sonicated and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / mL, and Cyanine 5.5-N3 (final concentration 40 µM) was added. The reaction was carried out at room temperature in the dark for 2 h. Subsequently, the protein was precipitated with frozen acetone and methanol, and the obtained proteins were separated by SDS-PAGE gel electrophoresis. Finally, fluorescence imaging was performed using an Odyssey DLx imager (excitation wavelength 700 nm).

[0082] The results are as follows Figure 6 As shown in (D), the labeling of the protein by probe 1 is concentration-dependent.

[0083] Lipolysis and lipophage induction assay: Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. U2OS cells were seeded into 6-well plates and cultured for 12 h. Then, 150 µM oleic acid was added, and incubation continued for another 12 h to induce lipid droplet formation. The oleic acid-containing medium was then discarded, and the cells were washed three times with DMEM. Cells were then treated for 12 h with DMEM containing 10 µM isoproterenol, 5 µM rapamycin, or an equal volume of DMSO (solvent control) (containing 2% FBS). After treatment, the cells were washed three times with DMEM, and then incubated in DMEM medium containing 20 µM probe 1 at 37°C in the dark for 90 min. After incubation, the probe-containing medium was removed, and the cells were collected by centrifugation and washed once with PBS. Cells were lysed by sonication with cell lysis buffer (20 mM Tris-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4), and the supernatant was collected after centrifugation. The protein concentration was adjusted to 2 mg / mL, and Cyanine 5.5-N3 (final concentration 40 µM) was added. The reaction was carried out at room temperature in the dark for 2 h. After the reaction, the proteins were precipitated sequentially with frozen acetone and methanol. The resulting proteins were separated by SDS-PAGE gel electrophoresis, and finally, fluorescence imaging was performed using an Odyssey DLx imager (excitation wavelength 700 nm).

[0084] The results are as follows Figure 6 As shown in (E), after treatment with isoproterenol (a lipolysis inducer) or rapamycin (a lipophage inducer), lipid droplets were degraded, and the protein labeling signal corresponding to probe 1 was significantly reduced. This result indicates that the designed labeling probe has the ability to label lipid droplet-related proteins in situ.

[0085] Example 6

[0086] Live cell lipid droplet protein pull-down assay with probe 1

[0087] Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. U2OS cells were seeded in 10 cm culture dishes and cultured for 12 h. Then, 150 µM oleic acid was added, and the cells were incubated for another 18 h to induce lipid droplet formation. The oleic acid-containing medium was discarded, and the cells were washed three times with DMEM. Subsequently, DMEM medium containing 20 µM probe 1 was added, and the cells were incubated at 37 °C for 90 min. After incubation, the culture medium was removed, and the cells were washed once with PBS. Cells were collected by centrifugation, and cell lysis buffer (20 mM Tris-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added. Cells were sonicated and the supernatant was collected by centrifugation. The protein concentration in the supernatant was adjusted to 2 mg / mL, and Biotin-N3 (final concentration 40 µM) was added. The reaction was carried out at room temperature in the dark for 2 h. After the reaction, the protein was precipitated and washed with frozen acetone and methanol, and the protein precipitate was collected by centrifugation. The precipitate was resuspended in PBS containing 0.2% sodium dodecyl sulfate (SDS), and then streptavidin magnetic beads were added. The mixture was incubated at room temperature for 4 h to enrich the protein captured by probe 1 crosslinking. After incubation, the magnetic beads were collected under a magnetic field and washed sequentially with the following solutions: 1% SDS / PBS (once), 5% SDS / PBS (twice), 1% SDS / PBS (once), and PBS (once). After washing, SDS-PAGE protein loading buffer was added to the magnetic beads, and the mixture was heated at 90 °C for 20 min. The supernatant was collected by centrifugation to obtain the pull-down protein sample.

[0088] The results of quantitative LC-MS / MS analysis are shown in Table 1. This method eliminates the need for traditional lipid droplet separation steps; probe 1 successfully captured lipid droplet proteins in situ at the live cell level.

[0089] Table 1. Lipodroplet proteins identified by probe 1 binding quantitative LC-MS / MS

[0090]

[0091] Example 7

[0092] Animals: Specific Pathogen Free (SPF) level db / db Mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All selected mice were approximately 8-10 weeks old, with similar appearance, body size, weight, and blood glucose levels.

[0093] Five days before the experiment, mice were moved to the laboratory to acclimatize. The laboratory temperature was maintained at 20-25 ℃, with a light / dark cycle of 12 h / 12 ​​h, and mice had free access to food and water. On the day of the experiment, probe 1 (15 mg / kg) was dissolved in physiological saline and administered to mice (n = 3) via intraperitoneal injection. Twelve h after administration, mice were euthanized by CO2 asphyxiation, and their hearts were perfused with pre-cooled physiological saline. After perfusion, tissues such as the heart, kidneys, and liver were separated. After all mice were collected, lysis buffer (20 mM Tris-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added to each tissue for grinding and lysis, and the supernatant was collected by centrifugation. The protein concentration in the supernatant was uniformly adjusted to 2.5 mg / mL, and Biotin-N3 (final concentration 40 µM) was added. The reaction was carried out at 4 ℃ in the dark for 12 h. After the reaction, the protein was precipitated and washed with frozen acetone and methanol sequentially, and the protein precipitate was collected by centrifugation. The precipitate was resuspended in PBS containing 0.2% SDS, and then streptavidin magnetic beads were added. The mixture was incubated at 4 °C for 12 h to enrich the protein captured by probe 1 crosslinking. After incubation, the magnetic beads were collected under a magnetic field and washed sequentially with the following solutions: 1% SDS / PBS (once), 5% SDS / PBS (twice), 1% SDS / PBS (once), and PBS (once). After washing, SDS-PAGE protein loading buffer was added to the magnetic beads, and the mixture was heated at 90 °C for 20 min. The supernatant was collected by centrifugation to obtain the pull-down protein sample. A schematic diagram of probe 1 for in situ labeling of lipid droplet proteins is shown below. Figure 7 As shown.

[0094] The results of quantitative LC-MS / MS analysis are shown in Table 2. This method eliminates the need for traditional lipid droplet separation steps; probe 1 successfully captured lipid droplet proteins in situ at the live animal level.

[0095] Table 2. Lipodroplet proteins identified by probe 1 binding to quantitative LC-MS / MS

[0096]

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lipid droplet protein in situ labeling probe, characterized in that, Its structural formula is shown in equation (1): Equation (1).

2. A composition, characterized in that, The composition comprises the lipid droplet protein in situ labeling probe and excipients as described in claim 1.

3. A detection reagent, characterized in that, The reagent includes the lipid droplet protein in situ labeling probe as described in claim 1.

4. The application of the lipid droplet protein in situ labeled probe of claim 1, the composition of claim 2, or the detection reagent of claim 3 in lipid droplet detection, lipid droplet protein in situ detection, fluorescence detection, cell fluorescence imaging, or tissue fluorescence imaging; the application is for non-disease diagnosis purposes.

5. The use of the lipid droplet protein in situ labeled probe of claim 1, the composition of claim 2, or the detection reagent of claim 3 in the preparation of lipid droplet detection products, lipid droplet protein in situ detection products, fluorescence detection products, cell fluorescence imaging products, or tissue fluorescence imaging products.

6. The method for preparing the in-situ labeled lipid droplet protein probe as described in claim 1, characterized in that, Includes the following steps: (1) Compound 3 was prepared by reacting 4-fluoro-2,1,3-benzoxadiazole with tert-butylsarcosine hydrochloride in the presence of triethylamine; (2) In the presence of DMF, compound 3 reacts with phosphorus oxychloride to give compound 4; (3) Compound 4 reacts with trifluoroacetic acid to give compound 5; (4) In the presence of EDCI, HOBt and DMAP, compound 5 reacts with 3-butyn-1-ol to give compound 6; (5) In the presence of piperidine, compound 6 reacts with cyanoacetic acid to give compound 2; (6) Compound 2 was reacted with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride in the presence of NMI and TCFH to give compound 1; the synthetic route is as follows: 。 7. The preparation method according to claim 6, characterized in that, In step (1), the solvent is acetonitrile; the reaction temperature is 75-85 ℃, and the reaction time is 60-70 h; And / or, in step (1), the molar ratio of 4-fluoro-2,1,3-benzoxadiazole to tert-butylsarcosine hydrochloride is 1:4-6; the molar ratio of 4-fluoro-2,1,3-benzoxadiazole to triethylamine is 1:5-7; And / or, in step (2), the solvent is DMF; the molar ratio of compound 3 to phosphorus oxychloride is 1:4-6; And / or, the specific operation of step (2) is as follows: under the protection of ice bath and nitrogen, phosphorus oxychloride is dropped into DMF, stirred for 8-15 min, then DMF containing compound 3 is added, and the reaction is carried out at room temperature for 5-10 h; after the reaction is complete, 10-15% sodium hydroxide solution is added and stirred for 20-40 min, and compound 4 is obtained after post-processing.

8. The preparation method according to claim 6, characterized in that, In step (3), the solvent is dichloromethane; the reaction temperature is 20-30 ℃, and the reaction time is 2-4 h; And / or, in step (3), the ratio of compound 4 to trifluoroacetic acid is 0.1:1-3 mmol / mL; And / or, in step (4), the solvent is DMF; the reaction temperature is 20-30 ℃, and the reaction time is 10-15 h; And / or, in step (4), the molar ratio of compound 5 to 3-butyn-1-ol is 1:1-1.2; And / or, in step (4), the molar ratio of compound 5, EDCI, HOBt and DMAP is 3.5-4:5.5-6:5.5-6:

1.

9. The preparation method according to claim 6, characterized in that, In step (5), the molar ratio of compound 6 to cyanoacetic acid is 1:1.5-3; the molar ratio of compound 6 to piperidine is 4-6:

1. And / or, in step (5), the solvent is acetonitrile; the reaction time is 5-7 h.

10. The preparation method according to claim 6, characterized in that, In step (6), the molar ratio of compound 2 to 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride is 1:1-1.5; the molar ratio of compound 2, NMI and TCFH is 1:2-4:1-1.

5. And / or, in step (6), the solvent is acetonitrile; the reaction temperature is 20-30 ℃, and the reaction time is 10-20 h.

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