A lipid droplet protein photoaffinity labeling probe and its preparation method and application

Abstract

The present invention proposes a lipid droplet protein photoaffinity labeling probe and its preparation method and application, which belongs to the field of chemical biology technology and the field of organic luminescent materials. The probe provided by the present invention can efficiently detect lipid droplets, can efficiently label lipid droplet proteins in living cells, realize in situ analysis of lipid droplet proteins, and has high spatiotemporal sensitivity and specificity, and has certain application prospects.

Description

Technical Field

[0001] The invention belongs to the field of chemical biology technology and organic luminescent materials, and relates to a biological system molecular probe, and specifically to a lipid droplet protein photoaffinity labeling probe (photoaffinity labeling probe) and a preparation method and application thereof. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Lipid droplets (LDs) are a class of organelles that are conserved from bacteria to mammalian cells. They are composed of a phospholipid monolayer, a lipid center, and LD proteins. As the core organelle of lipid metabolism, the dynamic changes of lipid droplets (size, number, lipid composition, LD protein composition, etc.) are related to a variety of metabolic diseases, such as obesity, diabetes, cancer malignancy, etc.; it is also involved in intracellular signal transduction, gene expression regulation, protein synthesis and degradation, etc.; recent studies have also shown that the dynamic changes of lipid droplets affect the photosensitivity of bird cone cells. In essence, the function of lipid droplets is regulated by LD proteins, and the multi-dimensional analysis of LD proteins in living cells helps to clarify the dynamic properties of lipid droplets and their molecular mechanisms in diseases.

[0004] However, existing lipid droplet proteomics technologies have obvious defects. Among them, the lipid droplet separation method based on gradient density centrifugation (Nat. Protoc. 2013, 8, 43-51) has problems of nonspecific contamination and loss of small lipid droplets, and the proteomic analysis of the separated lipid droplets cannot reflect the actual situation in living cells. The lipid droplet APEX method (Dev. Cell 2018, 44, 97-112.e7) developed in recent years has nonspecific expression outside of lipid droplets, and needs to be combined with lipid droplet separation to effectively carry out omics analysis. At present, there is a lack of effective methods for in situ analysis of lipid droplet proteins in living cells.

[0005] Given the urgency of analyzing the dynamic properties of lipid droplet proteomes in physiological environments, the development of chemical tools that can efficiently and specifically analyze lipid droplet proteins in situ in living cells without lipid droplet isolation is very necessary. Summary of the invention

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a lipid droplet protein photoaffinity labeling probe and its preparation method and application. The probe provided by the present invention can efficiently label lipid droplet proteins in living cells, realize in situ analysis of lipid droplet proteins, and has high spatiotemporal sensitivity and specificity.

[0007] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0008] In a first aspect of the present invention, a lipid droplet protein photoaffinity labeling probe is provided, and its chemical structure is shown in Formula I or Formula II:

[0009]

[0010] The present invention utilizes the property of lipid droplet proteins to locate their lipophilic regions and the property that the active carbene intermediates generated by photoaffinity-labeled probes upon illumination are relatively stable in the lipophilic regions of lipid droplets compared to the external water environment, and combines the lipophilicity / size / rigidity of the molecules to design multiple photoaffinity-labeled probes located in the lipophilic regions of lipid droplets, thereby achieving in situ analysis of lipid droplet proteins without the need for lipid droplet separation or genetic manipulation and with high spatiotemporal sensitivity and specificity.

[0011] The second aspect of the present invention provides a composition, which comprises the above-mentioned photoaffinity labeled probe and auxiliary materials.

[0012] The excipients described in the present invention are pharmaceutical excipients, such as solubilizers, emulsifiers, stabilizers, preservatives, and the like.

[0013] The third aspect of the present invention provides a detection reagent, which includes the above-mentioned photoaffinity labeled probe.

[0014] The reagents include substances for maintaining the stability of the photoaffinity labeled probe and substances for increasing the solubility, such as dimethyl sulfoxide (DMSO).

[0015] The fourth aspect of the present invention provides the use of the above-mentioned photoaffinity labeled probe, composition or detection reagent in lipid droplet detection, in situ detection of lipid droplet protein, 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 use may be for disease or non-disease diagnosis purposes.

[0018] The fifth aspect of the present invention provides the use of the above-mentioned photoaffinity labeled probe, composition or detection reagent 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 may be for the purpose of diagnosing a disease or for the purpose of diagnosing a non-disease.

[0020] In a sixth aspect of the present invention, a method for preparing a lipid droplet protein photoaffinity labeling probe is provided. The method for preparing a lipid droplet photoaffinity labeling probe shown in formula I comprises:

[0021] The process of obtaining the probe of formula I by using compound 5, triethylamine, dimethylamine hydrochloride and ethanol as raw materials according to the following reaction scheme;

[0022]

[0023] In a specific embodiment of the present invention, in the step of generating compound 6, the reaction is carried out at 140-180°C for 2-4 days; preferably, the reaction is carried out at 150°C for 48 hours.

[0024] In a specific embodiment of the present invention, in the step of generating compound 7, the molar ratio of phosphorus oxychloride to compound 6 is 3-5:1 under ice bath and nitrogen protection; phosphorus oxychloride and compound 6 are stirred overnight. The molar ratio of phosphorus oxychloride to compound 6 is preferably 53:11.

[0025] In a specific embodiment of the present invention, in the step of generating compound 3, the added amounts of compound 7, cyanoacetic acid, piperidine and acetonitrile CAN are: 0.5mmol, 2mmol, 0.1mmol, 10ml respectively; the reaction conditions are reflux stirring at 90°C for 5-7h.

[0026] In a specific embodiment of the present invention, in the step of generating compound 1, compound 3, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HATU, and 1-hydroxybenzotriazole hydrate HOBt are first stirred in N,N-dimethylformamide DMF under a nitrogen atmosphere, and then a photo-cross-linker and N-ethyldiisopropylamine DIPEA are added and stirred overnight at room temperature. After extraction and purification, probe compound 1 is obtained.

[0027] In a specific embodiment of the present invention, the first stirring condition is stirring at room temperature for 1-3 hours.

[0028] In a specific embodiment of the present invention, the molar ratio of compound 3 to the photocrosslinking agent in the step is 1:(1-1.5);

[0029] The molar ratio of compound 3 to N-ethyldiisopropylamine in the step is 1:(1.5-3); the molar ratio of compound 3 to HATU in the step is 1:(1.2-2);

[0030] In the steps, the molar ratio of compound 3 to HOBt is 1:(1.2-2).

[0031] In a specific embodiment of the present invention, the first stirring condition is stirring at room temperature for 1-3 hours.

[0032] The purification adopts silica gel column chromatography;

[0033] Furthermore, in the silica gel column chromatography, the ratio of the purification eluent is methanol: dichloromethane = 1: (50-100).

[0034] The seventh aspect of the present invention provides a method for preparing a lipid droplet protein photoaffinity labeling probe represented by formula II, comprising:

[0035] The process of obtaining the probe represented by formula II using compound 8, triethylamine, tert-butyl sarcosine hydrochloride and acetonitrile as raw materials according to the following reaction scheme;

[0036]

[0037] In a specific embodiment of the present invention, in the step of generating compound 9, the reaction conditions are 70-90°C for 60-70h; preferably 80°C for 65h.

[0038] In a specific embodiment of the present invention, in the step of generating compound 10, the molar ratio of phosphorus oxychloride to compound 9 is 3-7:1 under ice bath and nitrogen protection; phosphorus oxychloride and compound 9 are stirred overnight. The molar ratio of phosphorus oxychloride to compound 9 is preferably 5:1.

[0039] In the step of generating compound 4, the reaction conditions are 100° C. and 18-24 h.

[0040] In a specific embodiment of the present invention, in the step of generating compound 2, benzotriazole-1-yl-oxy-tris-pyrrolidine-phosphine hexafluorophosphate PyBOP, 1-hydroxybenzotriazole hydrate HOBt, N-ethyldiisopropylamine DIPEA, and a photo-cross-linker are added to a reaction bottle containing compound 4 and DMF, and the mixture is stirred overnight at room temperature and extracted and purified to obtain the compound 2.

[0041] The molar ratio of the compound 4 to the photocrosslinker is 1:(1-1.5);

[0042] The molar ratio of the compound 4 to N-ethyldiisopropylamine is 1:(1-3);

[0043] The molar ratio of compound 4 to PyBoP is 1:(1.2-2);

[0044] The molar ratio of the compound 4 to HOBt is 1:(1.2-2).

[0045] In a specific embodiment of the present invention, the purification is carried out by silica gel column chromatography;

[0046] Furthermore, in the silica gel column chromatography, the ratio of the purification eluent is methanol: dichloromethane = 1: (50-150).

[0047] One or more of the above technical solutions have the following beneficial effects:

[0048] The photoaffinity labeling probe provided by the present invention has a simple and easy synthesis method. It can be specifically positioned in the lipophilic region of lipid droplets (lipophilic end of lipid droplet phospholipid monolayer and lipid center region) and used for in-situ and real-time labeling of lipid droplet proteins in living cells, realizing for the first time the in-situ analysis of lipid droplet proteins without lipid droplet separation, gene manipulation, high spatiotemporal sensitivity and high specificity, which is of great significance for the mechanism / function analysis of lipid droplet proteins.

[0049] It can perform direct and non-genetic manipulations on various types of cells and has a wide range of applicability. It can analyze lipid droplet proteomes with high temporal and spatial resolution. It is non-invasive and applicable to lipid droplets of different sizes and has broad application prospects.

[0050] 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

[0051] 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.

[0052] Figure 1 This is the H NMR spectrum of the probe shown in Formula I of Example 1 of the present invention.

[0053] Figure 2 This is the NMR carbon spectrum of the probe shown in Formula I of Example 1 of the present invention.

[0054] Figure 3 This is the H NMR spectrum of the probe shown in Formula II of Example 2 of the present invention.

[0055] Figure 4 This is the NMR carbon spectrum of the probe shown in Formula II of Example 2 of the present invention.

[0056] Figure 5 : A is a fluorescence imaging diagram of a co-staining experiment of a lipid droplet photoaffinity labeling probe and a PLIN3 antibody according to the present invention; A: is a fluorescence imaging diagram of a co-staining experiment of a probe shown in Formula I of Example 3 of the present invention and a PLIN3 antibody; B is a fluorescence imaging diagram of a co-staining experiment of a probe shown in Formula II of Example 4 and a PLIN3 antibody.

[0057] Figure 6It is a fluorescence imaging diagram of the co-staining experiment of the photoaffinity labeling probe of the present invention and the commercial dye Lipi-Deep Red for lipid droplets; A is the probe shown in Formula I of Example 5 of the present invention; B is the probe shown in Formula II of Example 6 of the present invention.

[0058] Figure 7 This is a fluorescence imaging diagram of a co-staining experiment with the probe shown in Formula I and other commercial dyes for subcellular organelles.

[0059] Figure 8 This is a fluorescence imaging diagram of a co-staining experiment with the probe shown in Formula II and other commercial subcellular organelle dyes.

[0060] Fig. 9 This is a fluorescence imaging diagram of lipid droplet fusion observed using SIM high-resolution imaging and the probe shown in Formula I.

[0061] Fig.10 This is a fluorescence imaging diagram of a protein gel labeling experiment of the probe shown in Formula I;

[0062] A: The probe shown in Formula I successfully labeled Hela cell proteins; B: The probe shown in Formula I exhibited UV time-dependent labeling; C: The probe shown in Formula I exhibited incubation time-dependent protein labeling; D: Concentration-dependent protein labeling of the probe shown in Formula I; E: The ability of the probe shown in Formula I to label lipid droplet proteins in situ; F: The ability of the probe shown in Formula I to label lipid droplet proteins in situ in living cells.

[0063] Fig.11 This is a fluorescence imaging diagram of a protein gel labeling experiment of the probe represented by Formula II.

[0064] A: The probe shown in Formula II successfully labeled Hela cell proteins; B: The probe shown in Formula II exhibited UV time-dependent labeling; C: The probe shown in Formula II exhibited incubation time-dependent protein labeling; D: The probe shown in Formula II exhibited concentration-dependent protein labeling; E: The ability of the probe shown in Formula II to label lipid droplet proteins in situ; F: The ability of the probe shown in Formula II to label lipid droplet proteins in situ in living cells.

[0065] Fig.12 The schematic diagram is a principle diagram of the in situ detection of lipid droplet proteins using the photoaffinity labeling probe of the present invention. DETAILED DESCRIPTION

[0066] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0067] In the present invention, compound 1 is the photoaffinity labeled probe shown in formula I; compound 2 is the photoaffinity labeled probe shown in formula II.

[0068] In the present invention, the principle schematic diagram of the in situ detection of lipid droplet proteins by the photoaffinity labeled probe is as follows: Fig.12 shown.

[0069] It should be noted that the terms used herein are for describing specific embodiments only and are not intended to be limiting of exemplary embodiments according to the present invention.

[0070] Embodiment 1

[0071] This example provides a method for preparing compound 1, and the reaction scheme is as follows:

[0072]

[0073] The specific preparation method of compound 1 is as follows:

[0074] 31mmol triethylamine, 26mmol dimethylamine hydrochloride, 5mmol compound 5, and 30mL ethanol were added to a high pressure reactor and reacted at 150°C for 48h. After the solvent was evaporated under reduced pressure, a 2M hydrochloric acid solution was added, the pH was adjusted to 7, and the product was extracted twice with ethyl acetate, 20mL each time. The obtained organic phase was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography, and the eluent ratio was ethyl acetate: petroleum ether = 1: (20-50) to obtain a yellow solid, which was compound 6, with a yield of 61%.

[0075] In an ice bath and under nitrogen protection, 53mmol of phosphorus oxychloride was slowly dripped into a reaction bottle containing 5mL of dry N,N-dimethylformamide DMF. After stirring for 10min, compound 6 (11mmol) dissolved in 5mL of DMF was slowly added and reacted at room temperature for 2h. After the reaction was complete by spot plate monitoring, ice water was added and stirred overnight until a red solid precipitated. 100mL of 10% NaOH solution was added and the reaction was continued with stirring for 2h. The precipitated red solid was filtered and purified by silica gel column chromatography. The eluent ratio was ethyl acetate: petroleum ether = 1: (5-10) to obtain a yellow solid, which was compound 7 with a yield of 56%.

[0076] 0.5mmol of compound 7, 2mmol of cyanoacetic acid, 0.1mmol of piperidine and 10ml of acetonitrile ACN were added to a reaction bottle and stirred under reflux at 90°C for 6h. The reaction was completed after the plate was monitored, 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: (7-15) to obtain a red solid, namely compound 3, with a yield of 56%.

[0077] 0.12 mmol of compound 3, 0.17 mmol of O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), and 0.17 mmol of 1-hydroxybenzotriazole hydrate (HOBt) were added to a reaction bottle containing 1.5 mL of N,N-dimethylformamide (DMF) under a nitrogen atmosphere and stirred at room temperature for 2 hours. Then, a photo-cross-linker (photo-cross-linker, 0.14 mmol) and N-ethyldiisopropylamine (DIPEA, 0.28 mmol) dissolved in 0.5 mL of DMF were added to the reaction bottle and stirred at room temperature overnight. The reaction was monitored by a plate spotting machine until completion. 2 mL of water was added to quench the reaction, and the mixture was extracted 3 times with 10 mL of ethyl acetate each time. The combined organic phase was 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 methanol: dichloromethane = 1: (100-150) to obtain the target product probe compound 1 with a yield of 9%. The H NMR spectrum and C NMR spectrum of compound 1 are shown as follows: Figure 1 , Figure 2 shown. 1 H-NMR (CDCl3, 400MHz) δ (ppm): 8.79 (s, 1H), 8.47 (d, J = 8.5Hz, 1H), 6.21 (d, J = 8.5Hz, 1H), 3.56 (s, 6H ),3.41-3.34(m,2H),2.05-2.00(m,3H),1.65(t,J=7.3Hz,2H),1.56-1.51(m,2H),1.45-1.40(m,2H). 13 C-NMR(CDCl3,101MHz)δ(ppm):161.38,150.65,145.25,144.69,143.62,136.33,118.92,105.94,10 4.24,97.66,82.85,69.42,42.91,39.87,32.34,30.24,29.85,24.20,13.44.HRMS(ESI)calculated m / zfor[M+H] + 392.1829, found 392.1830.

[0078] Embodiment 2

[0079] This example provides a method for preparing compound 2, and the reaction scheme is as follows:

[0080]

[0081] The specific preparation method of compound 2 is as follows:

[0082] 22mmol triethylamine, 18mmol sarcosine tert-butyl ester hydrochloride, 4mmol compound 8, and 15mL dry acetonitrile were added to a reaction bottle and reacted at 80°C for 65h. The crude product obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography, and the eluent ratio was ethyl acetate: petroleum ether = 1: (20-50) to obtain a brown solid, namely compound 9, with a yield of 75%.

[0083] In an ice bath and under nitrogen protection, 15mmol of phosphorus oxychloride was slowly dripped into a reaction bottle containing 5g of dry DMF. After stirring for 10min, compound 9 (3mmol) dissolved in 3mL of DMF was slowly added and reacted at room temperature for 7h. After the reaction was complete by spot plate monitoring, 10% sodium hydroxide solution was added and stirring was continued for 0.5h. Dichloromethane was added for extraction 3 times, 20mL each time. The combined organic phase was dried with sodium sulfate and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography, and the eluent ratio was ethyl acetate: petroleum ether = 1: (5-20) to obtain a yellow solid, namely compound 10, with a yield of 32%.

[0084] 0.08 mmol of compound 2-methyl-4,6-diphenyl-1,3,5-triazine, 0.08 mmol of compound 10, 0.42 mmol of potassium hydroxide, and 1.5 ml of methanol were added to the reaction bottle and stirred under reflux for 20 hours. The reaction was completed by spot plate monitoring, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of methanol: dichloromethane = 1: (10-20) to obtain a red solid, namely compound 4, with a yield of 21%.

[0085] 0.05mmol benzotriazol-1-yl-oxy-tris-pyrrolidine-phosphine hexafluorophosphate (PyBOP), 0.05mmol 1-hydroxybenzotriazole hydrate (HOBt), 0.04mmol N-ethyldiisopropylamine (DIPEA), and 0.05mmol photo-cross-linker were added to a reaction bottle containing 0.04mmol compound 4 and 1.5mL DMF, stirred at room temperature overnight, and the reaction was monitored by spot plate until complete. 2mL of water was added to quench the reaction, and ethyl acetate was used for extraction 3 times, 10mL each time. After the combined organic phase was 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 methanol: dichloromethane = 1: (100-150) to obtain the target product probe compound 2 with a yield of 30%. The H NMR spectrum and C NMR spectrum of compound 2 are shown as follows: Figure 3 , Figure 4 shown. 1H-NMR (CDCl3, 400MHz) δ (ppm): 8.73 (dd, J = 8.0, 1.8Hz, 4H), 8.45 (d, J = 15.7Hz, 1H), 7.90 (d, J = 15.7Hz, 1H), 7.65-7.49 (m, 7H), 6.31 (d, J=7.9Hz,1H),4.52(s,2H),3.32(s,3H),3.29-3.23(m,2H),2.02-1.93(m,3H),1.63-1.59(m,2H),1.47-1.43(m,2H),1.34-1.30(m,2H). 13 C-NMR (CDCl3, 101MHz) δ (ppm): 172.27, 171.38, 168.82, 149.08, 145.56, 139.78, 136.98, 136.82, 136.36, 132.57, 129.11, 12 8.77,128.58,114.39,106.74,82.81,69.38,58.57,41.25,38.94,32.26,30.08,29.81,24.12,14.27.HRMS(ESI)calculated m / z for[M+H] + 598.2673, found 598.2676.

[0086] Embodiment 3

[0087] This example provides a co-staining experiment of compound 1 and PLIN3 antibody

[0088] Compound 1 in the present invention can label lipid droplets in a variety of cells (e.g., human osteosarcoma cells U-2OS or human cervical cancer cells Hela cells) and localize in the lipophilic region of lipid droplets. This embodiment is described using human cervical cancer cells Hela cells as an example:

[0089] The cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Human cervical cancer cell Hela cells were inoculated on coverslips built into a 60mm sterile culture dish and cultured in a 37°C humidified incubator for 12 hours. The coverslip cells were washed 3 times with PBS buffer. 4% paraformaldehyde was added and incubated with the coverslip cells at room temperature for 20 minutes. After the cells were fixed, the cells were washed 3 times with PBS. 10 μg / mL saponin was added and incubated with the coverslip cells for 15 minutes. After the cells were permeabilized, the cells were washed 3 times with PBS. The coverslip cells were incubated with PBST (PBS + 0.1% Tween 20) containing 1% BSA and 1% donkey serum for 30 minutes to block the nonspecific binding of the antibody. The blocking solution was poured out, and the PLIN3 diluted antibody (dissolved in PBST with 1% BSA and 1% donkey serum) was added, and incubated overnight at 4°C in a humidified box. Pour off the solution and wash the cells 3 times with PBS, 5 minutes each time. Add Donkey Anti-Rabbit IgG H&L-Alexa 594 secondary antibody (dissolved in 1% BSA, 1% donkey serum in PBST), incubate the cells for 2 hours at room temperature in the dark. Pour off the solution and wash the cells 3 times with PBS in the dark for 5 minutes each time.

[0090] Add DMEM culture medium containing 10 μM of the probe shown in formula I, and incubate the cells for 1 hour at room temperature for counterstaining. Pour out the solution and wash the cells 3 times with PBS for 5 minutes each time. Finally, seal the coverslip with a drop of mounting medium and seal the coverslip with nail polish to prevent the sample from drying and moving under the microscope. Fluorescence imaging was taken using an ultra-high resolution confocal laser microscope (Leica SP8 STED microscope). The excitation wavelength of the probe shown in formula I is 488 nm, and the fluorescence signal is collected in the 500-630 nm region; PLIN3 is widely expressed in tissues and localized on the surface of newly formed LD lipid droplets. The excitation wavelength is 590 nm, and the fluorescence signal is collected in the 600-680 nm region.

[0091] The results are as follows Figure 5 As shown in A, the green fluorescence signal labeled by the probe shown in Formula I is wrapped by the red fluorescence signal of the lipid droplet structural protein PLIN3, indicating that the probe shown in Formula I can label lipid droplets and locate in the lipophilic region of lipid droplets (lipophilic end of phospholipid monolayer and lipid center).

[0092] Embodiment 4

[0093] This example provides a co-staining experiment of the probe shown in Formula II and the PLIN3 antibody

[0094] Compound 2 in the present invention can label lipid droplets in a variety of cells (e.g., human osteosarcoma cells U-2OS or human cervical cancer cells Hela cells) and localize in the lipophilic region of lipid droplets. This embodiment is described using human cervical cancer cells Hela cells as an example:

[0095] The cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated on coverslips built into a 60mm sterile culture dish and cultured in a humidified incubator at 37°C for 12 hours. The coverslip-loaded cells were washed 3 times with PBS buffer. 4% paraformaldehyde was added and incubated with the coverslip-loaded cells at room temperature for 20 minutes. After the cells were fixed, the cells were washed 3 times with PBS. 10 μg / mL saponin was added and incubated with the coverslip-loaded cells at room temperature for 15 minutes. After the cells were permeabilized, the cells were washed 3 times with PBS. The coverslip-loaded cells were incubated with PBST (PBS+0.1% Tween 20) containing 1% BSA and 1% donkey serum for 30 minutes to block the nonspecific binding of the antibody. The blocking solution was poured out, and the PLIN3 antibody (dissolved in PBST with 1% BSA and 1% donkey serum) diluted 1:200 was added, and incubated overnight at 4°C in a humidified box. Pour off the solution and wash the cells 3 times with PBS, 5 minutes each time. Add Donkey Anti-Rabbit IgG H&L-Alexa 594 secondary antibody (dissolved in 1% BSA, 1% donkey serum in PBST, diluted 1:200), incubate the cells for 2 hours at room temperature in the dark. Pour off the solution and wash the cells 3 times with PBS in the dark for 5 minutes each time.

[0096] Add DMEM culture medium containing 10 μM probe shown in formula II, and incubate the cells for 1 hour at room temperature for counterstaining. Pour out the solution and wash the cells 3 times with PBS for 5 minutes each time. Finally, seal the coverslip with a drop of glycerol and fix the coverslip with nail polish to prevent the sample from drying and moving under the microscope. Fluorescence imaging was taken using an ultra-high resolution confocal laser microscope (Leica SP8 STED microscope). The excitation wavelength of the probe shown in formula II is 480 nm, and the fluorescence signal is collected in the 500-630 nm region; the excitation wavelength of PLIN3 is 590 nm, and the fluorescence signal is collected in the 600-680 nm region.

[0097] The results are as follows Figure 5 As shown in B, the green fluorescence signal labeled by the probe shown in Formula II is wrapped by the red fluorescence signal of the lipid droplet structural protein PLIN3, indicating that the probe shown in Formula II can label lipid droplets and locate in the lipophilic region of lipid droplets (lipophilic end of phospholipid monolayer and lipid center).

[0098] Embodiment 5

[0099] Co-staining experiment of the probe shown in formula I and the commercial lipid droplet dye Lipi-Deep Red

[0100] In the present invention, the probe shown in Formula I can be localized in cells containing lipid droplets, such as human osteosarcoma cells U-2OS or human cervical cancer cells Hela cells. This embodiment is described using Hela cells as an example:

[0101] The cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a sterile confocal culture dish and cultured in a humidified incubator at 37°C for 12 hours. The cells were washed twice with PBS buffer and once with DMEM. DMEM culture medium containing 10 μM of the probe shown in Formula I was added, and the cells were incubated at 37°C for 1 hour. The DMEM solution containing the probe was poured out, and phenol red-free DMEM culture medium containing the commercial lipid droplet dye Lipi-Deep Red (67nM) 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 probe shown in Formula I is 488 nm, and the fluorescence signal is collected in the 500-620 nm region; the excitation wavelength of Lipi-Deep Red is 640 nm, and the fluorescence signal is collected in the 665-700 nm region. The results are shown in Figure 2. Figure 6 As shown in A, the probe represented by Formula I has good co-localization with Lipi-Deep Red (Pearson coefficient 0.93), proving that the probe represented by Formula I is indeed localized in lipid droplets.

[0102] Embodiment 6

[0103] Co-staining experiment of the probe shown in formula II and the commercial lipid droplet dye Lipi-Deep Red

[0104] In the present invention, the probe shown in Formula II can be localized in cells containing lipid droplets, such as human osteosarcoma cells U-2OS or human cervical cancer cells Hela cells. This embodiment is described using Hela cells as an example:

[0105] The cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a confocal culture dish and cultured in a humidified incubator at 37°C for 12 hours. The cells were washed twice with PBS buffer and once with DMEM. DMEM culture medium containing 10 μM of the probe shown in Formula II was added, and the cells were incubated at 37°C for 1 hour. The DMEM solution containing the probe was poured out, and DMEM culture medium containing the commercial lipid droplet dye Lipi-Deep Red (67nM) 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 SP8STED microscope). The excitation wavelength of the probe shown in Formula II is 480nm, and the fluorescence signal is collected in the 520-630nm region; the excitation wavelength of Lipi-Deep Red is 640nm, and the fluorescence signal is collected in the 665-700nm region. The results are shown in Figure 2. Figure 6 As shown in Figure B, the probe represented by Formula II has good co-localization with Lipi-Deep Red (Pearson coefficient 0.92), proving that the probe represented by Formula II is indeed localized in lipid droplets.

[0106] Embodiment 7

[0107] Co-staining experiments with the probe shown in formula I and other subcellular organelles

[0108] In the present invention, the probe shown in Formula I has strong specificity, can specifically target lipid droplets, and can achieve this result in a variety of cells. This example is introduced using Hela cells as an example:

[0109] The cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a confocal culture dish and cultured in a humidified incubator at 37°C for 12 hours, washed twice with PBS buffer, and washed once with DMEM. DMEM culture medium containing 10 μM of the probe shown in Formula I was added, and the cells were incubated at 37°C for 1 hour. The DMEM solution containing the probe was poured out, and DMEM culture medium containing the mitochondrial commercial dye MitoTracker Deep Red (67nM), the endoplasmic reticulum commercial dye ER-Tracker Red (100nM), the lysosomal commercial dye Lyso-Tracker Red (50nM), or the 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, respectively. Fluorescence imaging was taken using an ultra-high resolution confocal laser microscope (Leica SP8STED microscope). The excitation wavelength of the probe shown in Formula I is 488nm, and the fluorescence signal is collected in the 500-620nm region; the excitation wavelength of MitoTrackerDeep Red is 635nm, and the fluorescence signal is collected in the 650-700nm region; the excitation wavelength of ER-Tracker Red is 587nm, and the fluorescence signal is collected in the 600-650nm region; the excitation wavelength of Lyso-Tracker Red is 600nm, and the fluorescence signal is collected in the 620-700nm region; the excitation wavelength of DID is 635nm, and the fluorescence signal is collected in the 650-700nm region. The results are shown in Figure 2. Figure 7 As shown, the probe shown in Formula I does not co-localize with organelles such as mitochondria, endoplasmic reticulum, lysosomes, and cell membranes, demonstrating the excellent ability of the probe shown in Formula I to selectively target lipid droplets, indicating that it has strong specificity.

[0110] Embodiment 8

[0111] Co-staining experiments with the probe shown in formula II and other subcellular organelles

[0112] In the present invention, the probe shown in Formula II has strong specificity, can specifically target lipid droplets, and can achieve this result in a variety of cells. This example is introduced using Hela cells as an example:

[0113] The cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a confocal culture dish and cultured in a humidified incubator at 37°C for 12 hours, washed twice with PBS buffer, and washed once with DMEM. DMEM culture medium containing 10 μM of the probe shown in Formula II was added, and the cells were incubated at 37°C for 1 hour. The DMEM solution containing the probe was poured out, and DMEM culture medium containing the mitochondrial commercial dye MitoTracker Deep Red (67nM), the endoplasmic reticulum commercial dye ER-Tracker Red (100nM), the lysosomal commercial dye Lyso-Tracker Red (50nM), or the 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, respectively. Fluorescence imaging was taken using an ultra-high resolution confocal laser microscope (Leica SP8STED microscope). The excitation wavelength of the probe shown in Formula II is 480nm, and the fluorescence signal is collected in the region of 520-630nm; the excitation wavelength of MitoTrackerDeep Red is 635nm, and the fluorescence signal is collected in the region of 650-700nm; the excitation wavelength of ER-Tracker Red is 587nm, and the fluorescence signal is collected in the region of 600-650nm; the excitation wavelength of Lyso-Tracker Red is 600nm, and the fluorescence signal is collected in the region of 620-700nm; the excitation wavelength of DID is 635nm, and the fluorescence signal is collected in the region of 650-700nm. The results are shown in Figure 2. Figure 8 As shown, the probe shown in Formula II does not co-localize with organelles such as mitochondria, endoplasmic reticulum, lysosomes, and cell membranes, demonstrating the excellent ability of the probe shown in Formula II to selectively target lipid droplets, indicating that the probe shown in Formula II has strong specificity.

[0114] Embodiment 9

[0115] Lipid droplet SIM ultra-high resolution fluorescence imaging of the probe shown in formula I

[0116] In the present invention, the probe shown in Formula I has ultra-high resolution fluorescence imaging function in a variety of cells such as human osteosarcoma cells U-2OS or human cervical cancer cells Hela cells. This embodiment is described using Hela cells as an example:

[0117] The cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a confocal culture dish and cultured in a humidified incubator at 37°C for 12 hours. Oleic acid (200 μM) was added, and the cells were incubated at 37°C for 4 hours to induce lipid droplet formation. The DMEM solution containing oleic acid was poured out, and DMEM culture medium containing the probe shown in Formula I (10 μM) was added, and the cells were incubated at 37°C for 1 hour. Time-dependent dynamic fluorescence imaging was captured using a DeltaVision OMX Flex ultra-high resolution microscope. The excitation wavelength of the probe shown in Formula I is 488 nm, and the fluorescence signal is collected in the 500-620 nm region. The results are shown in Figure 2. Fig. 9 As shown, five fusion events were captured within 20 seconds using the probe shown in Formula I and SIM super-resolution imaging, reflecting the superior ability of the probe shown in Formula I to label the dynamics of lipid droplets.

[0118] Embodiment 10

[0119] Protein gel labeling experiment (in-gel labeling) of the probe shown in formula I

[0120] In the present invention, the probe shown in Formula I can achieve in situ labeling of lipid droplet proteins in living cells in a variety of cells such as human osteosarcoma cells U-2OS or human cervical cancer cells Hela cells. This embodiment is described using Hela cells as an example:

[0121] Probe and DMSO control experiment: The cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. The DMEM solution was poured out, and DMEM culture medium containing the probe shown in Formula I (20 μM) or DMSO was added, and the cells were incubated at 37°C in the dark for 45 minutes. The DMEM culture medium containing the probe or DMSO was poured out, washed 3 times with PBS, and DMEM culture medium was added. The cells were illuminated with ultraviolet light (UV, 365nm) for 20 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, and cell lysis solution (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40 μM) was added. The reaction was allowed to react for 2 hours at room temperature in the dark. Frozen acetone and methanol were added to precipitate the protein. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey DLx imager (Ex = 680 nm). The results are shown in Figure 2. Fig.10As shown in A, the probe shown in Formula I successfully labeled Hela cell proteins, while DMSO labeling was weak.

[0122] Probe illumination time dependence experiment: Cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. The DMEM solution was poured out, and DMEM culture medium containing the probe shown in Formula I (20 μM) was added, and the cells were incubated at 37°C in the dark for 45 minutes. The DMEM culture medium containing the probe was poured out, washed 3 times with PBS, and DMEM culture medium was added. Ultraviolet light (UV, 365nm) was used for illumination for 20 minutes, 10 minutes, 5 minutes, and 0 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, and cell lysis solution (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added to ultrasonically disrupt the cells, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40 μM) was added. The reaction was allowed to react for 2 hours at room temperature in the dark. Frozen acetone and methanol were added to precipitate the protein. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey DLx imager (Ex = 680 nm). The results are shown in Figure 2. Fig.10 As shown in B, the probe shown in Formula I exhibits UV time-dependent labeling, and obvious protein labeling can be obtained after 5 minutes of illumination.

[0123] Probe incubation time dependence experiment: Cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. The DMEM solution was poured out, and DMEM culture medium containing the probe shown in Formula I (20 μM) was added. The cells were incubated at 37°C in the dark for 120 minutes, 60 minutes, 30 minutes, 15 minutes, and 0 minutes. The DMEM culture medium containing the probe was poured out, washed 3 times with PBS, and DMEM culture medium was added. Ultraviolet light (UV, 365nm) was used for 20 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, and cell lysis solution (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added to ultrasonically disrupt the cells, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40 μM) was added. The reaction was allowed to react for 2 hours at room temperature in the dark. Frozen acetone and methanol were added to precipitate the protein. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey DLx imager (Ex = 680 nm). The results are shown in Figure 2. Fig.10 As shown in C, the probe shown in Formula I exhibits incubation time-dependent protein labeling, and obvious protein labeling can be obtained after incubation for 15 minutes.

[0124] Probe concentration dependence experiment: Cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. The DMEM solution was poured out, and DMEM culture medium containing the probe shown in Formula I (40μM, 20μM, 10μM, 5μM, 2.5μM, 0μM) was added, and the cells were incubated at 37°C in the dark for 45 minutes. The DMEM culture medium containing the probe was poured out, washed 3 times with PBS, and DMEM culture medium was added. The cells were illuminated with ultraviolet light (UV, 365nm) for 20 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, and cell lysis solution (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added. The cells were ultrasonically disrupted and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40 μM) was added. The reaction was allowed to react for 2 hours at room temperature in the dark. Frozen acetone and methanol were added to precipitate the protein. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey DLx imager (Ex = 680 nm). The results are shown in Figure 2. Fig.10 As shown in D, the probe of formula I exhibits protein labeling that is dependent on the probe concentration. Clear imaging is possible at 10 μM.

[0125] Lipolysis and lipophage induction experiment: cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. Oleic acid (150 μM) was added and the cells were incubated at 37°C for 12 hours to induce lipid droplet formation. The DMEM solution containing oleic acid was poured out and washed with DMEM 3 times. DMEM (2% FBS) culture medium containing isoproterenol (Isoproterenol, 10 μM), rapamycin (Rapamycin, 5 μM), or DMSO was added respectively and the cells were incubated for 12 hours. DMEM was washed 3 times, and DMEM culture medium containing the probe shown in Formula I (20 μM) was added, and the cells were incubated at 37°C in the dark for 45 minutes. Pour out the DMEM culture medium containing the probe, wash three times with PBS, add DMEM culture medium, and irradiate with ultraviolet light (UV, 365nm) for 20 minutes under ice bath conditions. After irradiation, collect the cells by centrifugation, add cell lysis buffer (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4), ultrasonically disrupt the cells, and collect the supernatant by centrifugation. Adjust the protein concentration to 2mg / ml, add click chemistry reagent (Cyanine5.5-N3, 40μM), and react at room temperature in the dark for 2 hours. Add frozen acetone and methanol to precipitate the protein respectively. The obtained protein particles are separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging is captured using Odyssey DLx imager (Ex=680nm). The results are as shown in the figure. Fig.10 As shown in E, the protein labeled with the probe shown in Formula I is degraded in lipid droplets after lipolysis or lipophagy activation, and the protein labeling signal is significantly reduced, indicating that the designed photoaffinity labeling probe has the ability to label lipid droplet proteins in situ.

[0126] Diacylglycerol acyltransferase inhibitor experiment: Cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a humidified incubator at 37°C for 12 hours. Oleic acid (150 μM) or diacylglycerol acyltransferase inhibitors (DGATi, A-922500, 20 μM; PF-06424439, 20 μM) were added, and the cells were incubated at 37°C for 18 / 48 hours. The DMEM solution containing oleic acid or inhibitors was poured out and washed with DMEM three times. DMEM culture medium containing the probe shown in Formula I (20 μM) was added, and the cells were incubated at 37°C in the dark for 45 minutes. The DMEM culture medium containing the probe was poured out, washed with PBS three times, and DMEM culture medium was added. The cells were illuminated with ultraviolet light (UV, 365nm) for 20 minutes under ice bath conditions. After irradiation, the cells were collected by centrifugation, cell lysis buffer (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40μM) was added. The reaction was carried out at room temperature in the dark for 2 hours. Frozen acetone and methanol were added to precipitate the protein respectively. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was taken using Odyssey DLx imager (Ex=680nm). The results are shown in Fig.10 As shown in F, after the addition of DGAT inhibitor, the synthesis of neutral lipids was inhibited, the formation of lipid droplets was stopped accordingly, and most of the proteins labeled by the probe shown in Formula I disappeared. These results fully prove that the probe shown in the photoreactive affinity formula I has the ability to in situ label lipid droplet proteins in living cells.

[0127] Embodiment 11

[0128] Protein gel labeling experiment of the probe shown in formula II (in-gel labeling)

[0129] In the present invention, the probe shown in Formula II can achieve in situ labeling of lipid droplet proteins in living cells in a variety of cells such as human osteosarcoma cells U-2OS or human cervical cancer cells Hela cells. This embodiment is described using Hela cells as an example:

[0130] Probe and DMSO control experiment: Cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. The DMEM solution was poured out, and DMEM culture medium containing the probe shown in Formula II (20 μM) or DMSO was added, and the cells were incubated at 37°C in the dark for 45 minutes. The DMEM culture medium containing the probe or DMSO was poured out, washed 3 times with PBS, and DMEM culture medium was added. The cells were illuminated with ultraviolet light (UV, 365nm) for 20 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, cell lysis solution (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagents (Cyanine5.5-N 3, 40μM) and reacted at room temperature in the dark for 2 hours. Frozen acetone and methanol were added to precipitate the protein. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was taken using an Odyssey DLx imager (Ex = 680nm). The results are shown in Fig.11 As shown in A, the probe shown in Formula II successfully labeled Hela cell proteins, while DMSO labeling was weak.

[0131] Probe illumination time dependence experiment: Cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. The DMEM solution was poured out, and DMEM culture medium containing the probe shown in Formula II (20 μM) was added, and the cells were incubated at 37°C in the dark for 45 minutes. The DMEM culture medium containing the probe was poured out, washed 3 times with PBS, and DMEM culture medium was added. Ultraviolet light (UV, 365nm) was used for illumination for 20 minutes, 10 minutes, 5 minutes, and 0 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, and cell lysis solution (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added to ultrasonically disrupt the cells, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40 μM) was added. The reaction was allowed to react for 2 hours at room temperature in the dark. Frozen acetone and methanol were added to precipitate the protein. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey DLx imager (Ex = 680 nm). The results are shown in Figure 2. Fig.11 As shown in B, the probe represented by Formula II exhibits UV time-dependent labeling, and obvious protein labeling can be obtained after 5 minutes of illumination.

[0132] Probe incubation time dependence experiment: Cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. The DMEM solution was poured out, and DMEM culture medium containing the probe shown in Formula II (20 μM) was added. The cells were incubated at 37°C in the dark for 120 minutes, 60 minutes, 30 minutes, 15 minutes, and 0 minutes. The DMEM culture medium containing the probe was poured out, washed 3 times with PBS, and DMEM culture medium was added. Ultraviolet light (UV, 365nm) was used for 20 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, and cell lysis solution (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added. The cells were ultrasonically disrupted and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40 μM) was added. The reaction was allowed to react for 2 hours at room temperature in the dark. Frozen acetone and methanol were added to precipitate the protein. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey DLx imager (Ex = 680 nm). The results are shown in Figure 2. Fig.11 As shown in C, the probe represented by Formula II exhibits protein labeling that is dependent on the incubation time, and obvious protein labeling can be obtained after incubation for 1 hour.

[0133] Probe concentration dependence experiment: Cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. The DMEM solution was poured out, and DMEM culture medium containing the probe shown in Formula II (40μM, 20μM, 10μM, 5μM, 0μM) was added, and the cells were incubated at 37°C in the dark for 45 minutes. The DMEM culture medium containing the probe was poured out, washed 3 times with PBS, and DMEM culture medium was added. The cells were illuminated with ultraviolet light (UV, 365nm) for 20 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, and cell lysis solution (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added. The cells were ultrasonically disrupted and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40 μM) was added. The reaction was allowed to react for 2 hours at room temperature in the dark. Frozen acetone and methanol were added to precipitate the protein. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey DLx imager (Ex = 680 nm). The results are shown in Figure 2. Fig.11 As shown in D, the probe shown in Formula II exhibits protein labeling that is dependent on probe concentration.

[0134] Lipolysis and lipophage induction experiment: The cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a humidified incubator at 37°C for 12 hours. DMEM (2% FBS) culture medium containing isoproterenol (Isoproterenol, 20 μM), rapamycin (Rapamycin, 10 μM), or DMSO was added, and the cells were incubated for 24 hours. Wash with DMEM three times, add DMEM culture medium containing the probe shown in Formula II (20 μM), and incubate the cells at 37°C in the dark for 45 minutes. Pour out the DMEM culture medium containing the probe, wash with PBS three times, add DMEM culture medium, and irradiate with ultraviolet light (UV, 365nm) for 20 minutes under ice bath conditions. After irradiation, the cells were collected by centrifugation, cell lysis buffer (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was added, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2mg / ml, and click chemistry reagent (Cyanine5.5-N3, 40μM) was added. The reaction was carried out at room temperature in the dark for 2 hours. Frozen acetone and methanol were added to precipitate the protein respectively. The obtained protein particles were separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was taken using Odyssey DLx imager (Ex=680nm). The results are shown in Fig.11 As shown in E, the protein labeled with the probe shown in Formula II, after lipolysis or lipophagy activation, lipid droplets are degraded and the protein labeling signal is significantly reduced, indicating that the designed photoaffinity labeling probe has the ability to label lipid droplet proteins in situ.

[0135] Lipolysis and lipophagy inhibition experiment: The cells were cultured in a 37°C humidified incubator in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). Hela cells were inoculated in a 6-well cell culture plate and cultured in a 37°C humidified incubator for 12 hours. DMEM (2% FBS) culture medium containing triglyceride lipase inhibitor (Atglistatin, 40 μM), lysosomal inhibitor chloroquine (Chloroquine, 40 μM), or DMSO was added, and the cells were incubated for 24 hours. DMEM was washed 3 times, and DMEM culture medium containing the probe shown in Formula II (20 μM) was added, and the cells were incubated at 37°C in the dark for 45 minutes. Pour out the DMEM culture medium containing the probe, wash 3 times with PBS, add DMEM culture medium, and irradiate with ultraviolet light (UV, 365nm) for 20 minutes under ice bath conditions. After illumination, collect the cells by centrifugation, add cell lysis buffer (20mM Tris-HEPES, 100mM KCl, 2mM MgCl2, pH7.4), disrupt the cells by ultrasound, and collect the supernatant by centrifugation. Adjust the protein concentration to 2mg / ml, add click chemistry reagent (Cyanine5.5-N3, 40μM), and react at room temperature in the dark for 2 hours. Add frozen acetone and methanol to precipitate the protein respectively. The protein particles obtained by centrifugation are separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging is captured using Odyssey DLx imager (Ex=680nm). The results are shown in Fig.11 As shown in F, after lipolysis or lipophagy is inhibited, the protein labeled with the probe shown in Formula II has its lipid droplet degradation inhibited and the protein labeling signal is significantly increased, indicating that the designed photoaffinity labeling probe has the ability to label lipid droplet proteins in situ.

[0136] Embodiment 12

[0137] Pull-down experiment of lipid droplet proteins in living cells using probe 1 / 2 of Formula I or Formula II

[0138] In the present invention, the probe shown in Formula I or the probe shown in Formula II can capture lipid droplet proteins in living cells. This example is introduced by taking the lipid droplet protein pull-down experiment of the probe shown in Formula I in U-2OS cells as an example:

[0139] The cells were cultured in a humidified incubator at 37°C in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). U-2OS cells were inoculated in a 10 cm cell 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 18 hours to induce lipid droplet formation. The DMEM solution containing oleic acid was poured out and washed with DMEM three times. DMEM culture medium containing probe 1 (20 μM) shown in Formula I was added and the cells were incubated at 37°C in the dark for 45 minutes. The DMEM culture medium containing the probe was poured out, washed with PBS three times, and DMEM culture medium was added. The cells were illuminated with ultraviolet light (UV, 365 nm) for 20 minutes under ice bath conditions. After illumination, the cells were collected by centrifugation, cell lysis solution (20 mM Tris-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Bio-N3, 40 μM) was added. The reaction was carried out at room temperature in the dark for 2 hours. Frozen acetone, chloroform, and methanol were added to precipitate / wash the protein, and the protein particles were collected by centrifugation. PBS containing 0.2% SDS was added to re-dissolve the precipitated protein particles. Streptavidin magnetic beads were added to enrich the protein captured by the cross-linking probe 1 shown in Formula I, and incubated at room temperature for 4 hours. The magnetic beads were collected by magnet and washed in the following order: 1% SDS / PBS (PBS solution containing 1% SDS; SDS: sodium dodecyl sulfate; PBS: sodium chloride 8g; potassium chloride 0.2g; disodium hydrogen phosphate 1.44g; potassium dihydrogen phosphate 0.24g, replenished to 1L) 1 time, 5% SDS / PBS 2 times, 1% SDS / PBS 1 time, PBS 1 time. The magnetic beads were collected by magnet, SDS-PAGE protein loading buffer was added, heated at 90°C for 20 minutes, and the upper buffer containing the pulled-down protein was collected by centrifugation. After quantitative LC-MS / MS analysis, the results are shown in Table 1. Without the need for lipid droplet separation, the probe 1 shown in Formula I successfully captured lipid droplet proteins from living cells; the lipid droplet proteins captured from living cells by the probe shown in Formula II are also shown in Table 1.

[0140] Table 1 High-confidence lipid droplet proteins identified by probe 1 shown in Formula I or probe 2 shown in Formula II combined with quantitative LC-MS / MS.

[0141]

[0142]

[0143] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A lipid droplet protein photoaffinity labeling probe, characterized in that: The chemical structure is shown in Formula 1 or Formula 2:

2. A composition, characterized in that The composition comprises the photoaffinity labeled probe according to claim 1 and auxiliary materials.

3. A detection reagent, characterized in that: The reagent comprises the photoaffinity labeled probe according to claim 1.

4. Use of the lipid droplet protein photoaffinity labeling probe according to claim 1, the composition according to claim 2 or the detection reagent according to claim 3 in lipid droplet detection or in situ detection of lipid droplet proteins; The application is for non-disease diagnosis purposes.

5. Use of the lipid droplet protein photoaffinity labeling probe according to claim 1, the composition according to claim 2 or the detection reagent according to claim 3 in the preparation of a lipid droplet detection product or a lipid droplet protein in situ detection product.

6. A method for preparing a lipid droplet protein photoaffinity labeling probe, characterized in that: The method for preparing the lipid droplet protein photoaffinity labeling probe shown in Formula 1 comprises: The process of obtaining probe 1 using compound 5, triethylamine, dimethylamine hydrochloride and ethanol as raw materials according to the following reaction route; 7. The method for preparing a lipid droplet protein photoaffinity labeling probe according to claim 6, characterized in that: In the step of generating compound 1, compound 3, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HATU, and 1-hydroxybenzotriazole hydrate HOBt are first stirred in N,N-dimethylformamide DMF under a nitrogen atmosphere, and then a photo-cross-linker and N-ethyldiisopropylamine DIPEA are added and stirred overnight at room temperature. After extraction and purification, probe compound 1 is obtained.

8. The method for preparing a lipid droplet protein photoaffinity labeling probe according to claim 7, characterized in that: In the step, the molar ratio of compound 3 to the photocrosslinking agent is 1:(1-1.5); or, In the steps, the molar ratio of compound 3 to N-ethyldiisopropylamine is 1:(1.5-3); or, In the steps, the molar ratio of compound 3 to HATU is 1:(1.2-2); or, In the steps, the molar ratio of compound 3 to HOBt is 1:(1.2-2); The purification was carried out by silica gel column chromatography.

9. The method for preparing a lipid droplet protein photoaffinity labeling probe according to claim 8, characterized in that: In the silica gel column chromatography, the ratio of the purification eluent is methanol: dichloromethane = 1: (50-100).

10. A method for preparing a lipid droplet protein photoaffinity labeling probe, characterized in that: The method for preparing the lipid droplet protein photoaffinity labeling probe shown in Formula 2 comprises: The process of obtaining probe 2 using compound 8, triethylamine, sarcosine tert-butyl ester hydrochloride and acetonitrile as raw materials according to the following reaction scheme; 11. The method for preparing a lipid droplet protein photoaffinity labeling probe according to claim 10, characterized in that: In the step of generating compound 2, benzotriazole-1-yl-oxy-tris-pyrrolidine-phosphine hexafluorophosphate PyBOP, 1-hydroxybenzotriazole hydrate HOBt, N-ethyldiisopropylamine DIPEA, and photo-cross-linker are added to a reaction bottle containing compound 4 and DMF, stirred overnight at room temperature, and extracted and purified to obtain the compound.

12. The method for preparing a lipid droplet protein photoaffinity labeling probe according to claim 11, characterized in that: The molar ratio of the compound 4 to the photocrosslinking agent is 1:(1-1.5); or, The molar ratio of the compound 4 to N-ethyldiisopropylamine is 1:(1-3); or, The molar ratio of compound 4 to PyBoP is 1:(1.2-2); or, The molar ratio of the compound 4 to HOBt is 1:(1.2-2); or, The purification was carried out by silica gel column chromatography.

13. The method for preparing a lipid droplet protein photoaffinity labeling probe according to claim 12, characterized in that: In the silica gel column chromatography, the ratio of the purification eluent is methanol: dichloromethane = 1: (50-150).

Images