Lipid droplet targeted photosensitive fluorescent molecule, preparation method and application

Abstract

The invention discloses a lipid droplet targeted photosensitive fluorescent molecule as well as a preparation method and application thereof. The fluorescent molecule has a structure as shown in a formula I, wherein R is selected from at least one of a formula a, a formula b, a formula c, a formula d, a formula e and a formula f; according to the application, the lipid droplet targeting and photosensitivity of fluorescent molecules are utilized to realize proximity labeling of proteins around lipid droplets in cells, and regulatory factors of interaction of the lipid droplets and mitochondria are disclosed through proteome analysis. The probe realizes proximity markers which do not depend on genetic manipulation in the field.

Description

Technical Field

[0001] This application relates to a lipid droplet-targeted photosensitizing fluorescent molecule, its preparation method, and its application, belonging to the field of fluorescent probes. Background Technology

[0002] Lipid droplets interact with other organelles such as the endoplasmic reticulum, mitochondria, and peroxisomes during lipid synthesis, breakdown, and transport. Dysfunction of lipid droplets is frequently associated with a variety of metabolic diseases, including type 2 diabetes, liver disease, and cardiovascular disease. Recent studies have shown that metabolically active lipid droplets promote cancer growth, metastasis, and tumorigenesis by providing energy and resisting cellular stress. Therefore, the morphology, interacting proteome, and lipidome configuration of lipid droplets reveal their important roles in regulating cellular physiology and disease pathology. However, the dynamic properties of lipid droplets, particularly their transient changes in size, number, composition, and suborganelle interactions, present technical challenges for capturing this molecular-level information.

[0003] Significant progress has been made in developing lipid droplet imaging tools, but analytical methods for identifying lipid droplet interaction proteomes and subcellular organelles remain very limited. To decipher the composition of the lipid droplet interaction proteome, conventional density gradient centrifugation is used to separate lipid droplets after cell lysis and enrich related proteins for downstream proteomics analysis. However, this technique may, in principle, co-precipitate subcellular organelles of similar size, such as the endoplasmic reticulum, mitochondria, and endosomes, leading to potential false positives. To capture transient interactions between lipid droplets and other organelles in the cellular environment, engineered ascorbate peroxidase (APEX) proximity labeling technology has been employed, genetically tagging known lipid droplet biomarker proteins, such as perilipid proteins (Plin) family proteins. While APEX technology is highly effective in resolving lipid droplet interaction networks, it requires transfection and genetic fusion of the catalytic enzyme tag to a known protein biomarker.

[0004] Therefore, developing photosensitive fluorescent small molecules can leverage their targeting ability to lipid droplets to achieve in-situ photocatalytic neighbor labeling of proteins surrounding lipid droplets. This technology represents a novel, genetically independent method for protein neighbor labeling, possessing significant scientific and applied value for cell biology and disease research. Summary of the Invention

[0005] This application utilizes the lipid droplet targeting and photosensitivity of fluorescent molecules to achieve proximity labeling of proteins surrounding intracellular lipid droplets, and reveals the regulatory factors of lipid droplet-mitochondrial interactions through proteomic analysis. This probe achieves proximity labeling without relying on genetic manipulation, a first in the field.

[0006] According to one aspect of this application, a lipid droplet-targeted photosensitizing fluorescent molecule...

[0007] The fluorescent molecule has the structure of Formula I:

[0008]

[0009] The R is selected from at least one of formulas a, b, c, d, e, and f;

[0010]

[0011] According to a second aspect of this application, a method for preparing lipid droplet-targeted photosensitizing fluorescent molecules is provided, comprising at least the following steps:

[0012] Step I: Disperse raw material I and 5-bromo-2-furanaldehyde in a mixed solvent, add catalyst and base reagent, and react I to obtain compound I;

[0013] Step II: Disperse compound I and compound II obtained in step I in an organic solvent and react II to obtain the lipid droplet-targeted photosensitizing fluorescent molecule;

[0014] The compound II has at least one structure of formula II;

[0015]

[0016]

[0017] Optionally, the structure of raw material I is as shown in Formula III:

[0018]

[0019] The structure of compound I is shown in Formula IV:

[0020]

[0021] Optionally, the solvent in the mixed solvent is selected from at least one of ethanol, toluene, and dioxane.

[0022] Optionally, the catalyst is selected from at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, tri(dibenzylideneacetone)palladium, and di(triphenylphosphine)palladium dichloride;

[0023] The alkaline reagent is selected from at least one of potassium carbonate, sodium carbonate, sodium bicarbonate, lithium carbonate, and sodium acetate.

[0024] Optionally, the molar ratio of raw material I, 5-bromo-2-furan carbaldehyde, catalyst, and alkaline reagent is 1:1.1:0.01:2 to 1:5:0.2:5.

[0025] Optionally, the reaction conditions for reaction I are as follows:

[0026] The reaction temperature of reaction I is 20–80°C;

[0027] The reaction time for reaction I is 1 to 12 hours.

[0028] Optionally, the molar ratio of compound I to compound II is 1:1 to 2:1;

[0029] The organic solvent is selected from at least one of 1,4-dioxane, toluene, acetonitrile, and xylene;

[0030] The concentration of compound II is 0.05–1.0 mol / L.

[0031] Optionally, the reaction conditions for reaction II are as follows:

[0032] The reaction temperature for reaction II is 25–120°C;

[0033] The reaction time for reaction II is 1 to 48 hours.

[0034] According to a third aspect of this application, a lipid droplet-targeting photosensitivity fluorescent molecule is provided for protein labeling and enrichment. This fluorescent molecule can both detect lipid droplets in living cells with fluorescence and utilize its photosensitivity to achieve neighboring labeling of proteins surrounding lipid droplets in cells.

[0035] The beneficial effects that this application can produce include:

[0036] 1) The fluorescent probe provided by this invention has excellent lipid droplet targeting and photosensitivity.

[0037] 2) The fluorescent probe provided by this invention can label proteins surrounding lipid droplets in the complex intracellular biological environment.

[0038] 3) The fluorescent probes provided by this invention can reveal extensive interactions between lipid droplets and mitochondria, and further analysis shows that this interaction has an important impact on the regulation of cellular lipid metabolism. Attached Figure Description

[0039] Figure 1 This is a lipid droplet targeting test diagram of the fluorescent molecule prepared in Example 1 of this application;

[0040] Figure 2 This is a photosensitivity test pattern of the fluorescent molecules prepared in Example 1 of this application;

[0041] Figure 3 A map showing the use of fluorescent molecules prepared in Example 1 of this application for neighbor labeling of proteins surrounding lipid droplets;

[0042] Figure 4A diagram of interacting proteins of the fluorescent molecules prepared in Example 1 of this application for enriching lipid droplets in cells;

[0043] Figure 5 The 1H NMR spectrum of compound I prepared in Example 1 of this application;

[0044] Figure 6 The above is the 1H NMR spectrum of the fluorescent molecule prepared in Example 1 of this application;

[0045] Figure 7 The hydrogen NMR spectrum of the fluorescent molecule prepared in Example 2 of this application;

[0046] Figure 8 The hydrogen NMR spectrum of the fluorescent molecule prepared in Example 3 of this application;

[0047] Figure 9 The image shows the hydrogen NMR spectrum of the fluorescent molecule prepared in Example 4 of this application. Detailed Implementation

[0048] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0049] Unless otherwise specified, the raw materials and reagents used in the embodiments of this application were all purchased commercially.

[0050] The NMR data in this embodiment were obtained using a Bruker AVANCE III 700MHz NMR spectrometer.

[0051] The confocal fluorescence microscope used in this embodiment is an Olympic FV1000 FluoView. TM Confocal microscope.

[0052] The mass spectrometer used in this embodiment is an Orbitrap Exploris 480 mass spectrometer.

[0053] According to a specific embodiment of this application, a method for preparing a fluorescent molecule includes at least the following steps:

[0054] Compound I was added with compound II, 10% ZnCl2 (1 mol / L THF) and solvent. After reaction, the mixture was quenched, extracted, and separated by column chromatography to obtain fluorescent molecules.

[0055] The structure of compound I is shown in Formula IV:

[0056]

[0057] Compound II is selected from at least one of the compounds represented by formula II:

[0058]

[0059] Optionally, the reaction conditions are: temperature of 20–80°C; time of 1–12 h.

[0060] Optionally, the molar ratio of compound I to compound II is 1:1 to 2:1.

[0061] Optionally, the solvent is selected from dichloromethane, chloroform, carbon tetrachloride, toluene, and acetonitrile.

[0062] Preparation Example 1

[0063] Starting material I (10 mmol), 5-bromo-2-furanaldehyde (11 mmol), tetrakis(triphenylphosphine)palladium (1 mmol), and potassium carbonate (20 mmol) were added to 30 mL of a mixed solvent (toluene, 3 mL ethanol, 3 mL H₂O), and the mixture was purged with gas to ensure the reaction was carried out under argon atmosphere. The reaction mixture was stirred at 100 °C for 18 hours, and then cooled to room temperature. Dichloromethane was added for extraction, the mixture was concentrated, and the organic phase was collected. Finally, the organic phase was purified by column chromatography to give compound I (1.85 g, yield: 54.3%).

[0064] Compound I was characterized structurally and its purity was determined. The results are as follows: 1 H NMR (700MHz, CDCl3): δ9.60 (s, 1H), 7.65 (d, 2H, J = 8.8Hz), 7.28-7.31 (m, 5H), 7.06-7.15 (m, 8H), 6.70 (d, 1H, J = 4.0Hz); 13 C NMR(176MHz, CDCl3): δ176.8,159.9,151.6,149.3,147.0,129.5,126.4,125.2,123.9,122.1,122.0,106.4; HRMS(ESI+)m / z:[M+H] + Calcium 340 1332, found 340 1318; see detailed 1H NMR spectrum. Figure 5 .

[0065] The synthesis route in this embodiment is shown in Figure A:

[0066]

[0067] Example 1

[0068] Compound I (338 mg, 1.0 mmol) and compound II (200 mg, 1.1 mmol) obtained from Preparation Example 1 were dissolved in anhydrous 1,4-dioxane (5 mL), and zinc chloride solution (1 M THF solution, 5 mL) was added. After stirring overnight at room temperature, dichloromethane was added for extraction, the organic phase was collected and concentrated, and finally purified by column chromatography to obtain a fluorescent molecule (454.7 mg, yield: 90.2%).

[0069] The fluorescent molecules were tested using nuclear magnetic resonance spectroscopy, and the results are as follows: 1 H NMR (700MHz, CDCl3): δ8.06 (d, 1H, J = 7.7Hz), 8.01 (m, 1H), 7.87 (m, 3H), 7.78 (t, 1H, J = 7.7H z),7.31(t,4H,J=7.7Hz),7.15(d,4H,J=8.4Hz),7.09-7.13(m,4H),6.86(d,1H,J=3.5Hz); 13 C NMR (176MHz, CDCl3): δ186.7,150.1,147.0,146.7,137.2,135.8,134.2,133.6, 129.5,127.1,125.6,124.3,121.8,121.6,121.3,121.1; HRMS(ESI+)m / z:[M+H] + Calcium 504.1264, found 504.1256. See the detailed 1H NMR spectrum for details. Figure 6 .

[0070] The synthesis route in this embodiment is shown in Figure B:

[0071]

[0072] Example 2

[0073] Compound I (338 mg, 1.0 mmol) and compound II (148 mg, 1.1 mmol) obtained from Preparation Example 1 were dissolved in anhydrous 1,4-dioxane (5 mL), and zinc chloride solution (1 M THF solution, 5 mL) was added. After stirring overnight at room temperature, dichloromethane was added for extraction, the organic phase was collected and concentrated, and finally purified by column chromatography to obtain a fluorescent molecule (308.8 mg, yield: 67.7%).

[0074] The fluorescent molecules were tested using nuclear magnetic resonance spectroscopy, and the results are as follows: ¹H NMR (700MHz, CDCl₃): δ 7.77 (d, 1H, J = 7.0Hz), 7.60 (m, 3H), 7.29 (d, 1H, J = 8.4Hz), 7.27 (t, 4H, J = 7.7Hz), 7.22 (d, 1H, J = 4.2Hz), 7.19 (t, 1H, J = 7.7Hz), 7.11 (d, 4H, J = 7.7Hz), 7.05-7.09 (m, 5H), 6.94 (s, 1H), 6.74 (d, 1H, J = 6.74Hz); 13 C NMR (176MHz, CDCl3): δ183.5,165.4,157.1,148.3,147.9,147.2,144.8,136.2,129.4,125 .4,124.9,124.4,123.6,123.3,122.3,120.3,112.8,107.8,101.8; HRMS(ESI+)m / z:[M+H] + Calcium 456 1594, found 456 1591; see detailed 1H NMR spectrum. Figure 7 .

[0075] The synthesis route in this embodiment is shown in Figure C:

[0076]

[0077] Example 3

[0078] Compound I (338 mg, 1.0 mmol) and compound II (220 mg, 1.1 mmol) obtained from Preparation Example 1 were dissolved in anhydrous 1,4-dioxane (5 mL), and zinc chloride solution (1 M THF solution, 5 mL) was added. After stirring overnight at room temperature, dichloromethane was added for extraction, the organic phase was collected and concentrated, and finally purified by column chromatography to obtain a fluorescent molecule (435.5 mg, yield: 83.4%).

[0079] The fluorescent molecules were tested using nuclear magnetic resonance spectroscopy, and the results are as follows: 1 H NMR (700MHz, CDCl3): δ8.44 (s, 1H), 7.68 (d, 2H, J = 8.4Hz), 7.32 (t, 4H, J = 7.7Hz), 7.16 (d, 3H, J = 7.7 Hz), 7.14 (t, 2H, J = 7.7Hz), 7.07 (d, 2H, J = 8.4Hz), 6.93 (d, 1H, J = 4.2Hz), 4.59 (m, 4H), 1.33 (m, 6H); 13C NMR (176MHz, CDCl3): δ178.6,163.6,161.2,159.1,151.1,150.3,146.5,139.6,133.2,12 9.6,127.1,125.7,124.5,121.3,120.9,110.8,109.3,44.0,43.3; HRMS(ESI+)m / z:[M+H] + Calcium 522.1846, found 522.1861; see detailed 1H NMR spectrum. Figure 8 .

[0080] The synthesis route in this embodiment is shown in Figure D:

[0081]

[0082] Example 4

[0083] Compound I (338 mg, 1.0 mmol) and compound II (172 mg, 1.1 mmol) obtained from Preparation Example 1 were dissolved in anhydrous 1,4-dioxane (5 mL), and zinc chloride solution (1 M THF solution, 5 mL) was added. After stirring overnight at room temperature, dichloromethane was added for extraction, the organic phase was collected and concentrated, and finally purified by column chromatography to obtain a fluorescent molecule (326.1 mg, yield: 68.2%).

[0084] The fluorescent molecules were tested using nuclear magnetic resonance spectroscopy, and the results are as follows: 1 H NMR (700MHz, CDCl3): δ8.44 (s, 1H), 7.70 (d, 2H, J = 8.4Hz), 7.32 (t, 4H, J = 8.4Hz), 7.15 (d, 4H ,J=7.7Hz),7.13(t,2H,J=7.7Hz),7.07(d,2H,J=9.1Hz),6.90(d,1H,J=4.2Hz),3.42(s,6H); 13 C NMR (176MHz, CDCl3): δ162.9,162.8,161.2,151.6,150.5,150.0,146.6,139.1,132.2,12 9.6,126.8,125.6,124.4,121.5,121.2,110.2,108.6,28.9,28.1; HRMS(ESI+)m / z:[M+H] + calcd 478.1761, found 478.1776, see detailed 1H NMR spectrum. Figure 9 .

[0085] The synthesis route in this embodiment is shown in Figure E:

[0086]

[0087] Test Example 1

[0088] The probe has lipid droplet selectivity

[0089] The fluorescent molecules prepared in Example 1 were used for cell lipid droplet targeting detection. The specific procedure was as follows: Mouse hepatocytes (AML-12) were plated and grown to 80% density. Probes (1-10 μM), commercial lipid droplet dye BODIPY493 / 503 (2 μM), and commercial nuclear staining reagent Hoechst 33342 (1 μg / mL) were added to the cell culture medium and stained for 20 min. After washing the cells with phosphate-buffered saline (PBS), confocal imaging was performed. The results are shown below. Figure 1 As shown in the diagram, the blue signal represents the cell nucleus, the green signal represents lipid droplets, and the red signal represents the stained region of the molecule. When the red and green signals show good co-localization (indicated by yellow), it indicates that the probe has lipid droplet targeting ability.

[0090] Test Example 2

[0091] The probe is photosensitized

[0092] Using the fluorescent molecule prepared in Example 1 as a typical example, the photosensitivity of the detection probe was examined. Several commercially available detection probes were selected, such as dichlorodihydrofluorescein (DCFH), i.e., a reactive oxygen species (ROS) detection probe; and 9,10-anthratridiyl-bis(methylene)dimalonic acid (ABDA), i.e., singlet oxygen (…). 1 O2) detection probe; dihydrorhodamine 123 (DHR123), i.e., superoxide anion (O2) ·- The detection probes and hydroxyphenylfluorescein (HPF), i.e., hydroxyl radical (·OH) detection probes, were used to detect the ROS generation efficiency and the type of ROS generated in compound I, respectively. The results are as follows: Figure 2 As shown, fluorescent molecules, as photosensitizers, have high ROS generation efficiency, and they mainly generate Type-I ROS.

[0093] Test Example 3

[0094] The probe can achieve neighbor labeling of proteins surrounding lipid droplets.

[0095] Using the fluorescent molecule prepared in Example 1 as a typical example, its labeling of proteins surrounding lipid droplets was detected in live cells using immunofluorescence imaging. The specific procedure was as follows: When AML-12 cells reached approximately 80% confluence, they were treated with 200 μL of 100 μmol / L oleic acid (OA) for 12 hours. After treatment, the cells were washed three times with PBS, followed by fixation with 200 μL of 4% formaldehyde fixative for 15 minutes, and then permeation treatment with 200 μL of 0.1% Triton X-100 for 10 minutes. The cells were then incubated with 200 μL of 5 μmol / L fluorescent molecule in the dark for 30 minutes. Afterwards, the cells were washed with PBS, treated with 200 μL of 5 mmol / L NH2-TMT, and illuminated under white light for 5 minutes. After three washes with 1% phosphate-buffered saline (PBST), cells were blocked with 200 μL of 3% bovine serum albumin (BSA) for 15 minutes, followed by overnight incubation with TMT monoclonal antibody at 4°C. Following cell washing, cells were then incubated with goat anti-mouse IgG H&L (Alexa). 488) Incubate at room temperature for 40 minutes. After washing again, stain with 200 μL of 1 μg / mL Hoechst 33342 blue fluorescent dye for 15 minutes. Use an Olympus FV1000 FluoView. TM Imaging was performed using a confocal microscope. The experimental results are as follows: Figure 3 As shown, the probe can effectively label proteins surrounding lipid droplets.

[0096] Test Example 4

[0097] Probes can be used to enrich interacting proteins of lipid droplets in cells.

[0098] AML-12 cells were washed with Hank's balanced salt solution (HBSS) after OA treatment, followed by treatment with 5 mL of the fluorescent molecule obtained in Example 1 at a concentration of 1 μmol / L for 15 min. After three HBSS washes, cells were treated with 5 mL of 5 mmol / L propargylamine under white light at room temperature for 5 min. After washing with PBS, cells were collected, sonicated, and the proteome was obtained. This proteome was gradually added to pre-chilled acetone and incubated overnight at -20°C. After centrifugation, the precipitate was dissolved in 4% sodium dodecyl sulfate (SDS), clicked with 0.3 mmol / L biotin-PEG4-N3, and rotated at the bottom for 1 hour at room temperature. After the click reaction, it was gently mixed with cold acetone and incubated overnight at -20°C. After centrifugation, the supernatant was discarded, and the precipitate was washed twice in pre-chilled acetone and centrifuged at 14,000 g for 5 min at 4°C. The resulting precipitate was redissolved in 6 mL of 1 mol / L urea and 50 mmol / L ammonium bicarbonate solution. Approximately 50 μL of pretreated Mag Sepharose magnetic beads were washed twice each with 8 mol / L urea and 2 mol / L sodium chloride, mixed with a proteomic solution, and rotated at room temperature for 4 hours. The beads were then washed with 50 mmol / L ammonium bicarbonate solution, treated with 10 mmol / L dithiothreitol (DTT) for 1 hour, and then treated with 20 mmol / L iodoacetamide (IAA) in the dark for 25 minutes. After a second wash, the beads were digested with trypsin at a 1:30 trypsin / protein ratio (w / w) at 37 °C for 12 hours. The digested samples were separated by high-pH reversed-phase chromatography and analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS / MS). The results are as follows: Figure 4 As shown, the probe can be used to enrich lipid droplet proteins.

[0099] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A lipid droplet-targeted photosensitizing fluorescent molecule, characterized in that, The fluorescent molecule has the structure of Formula I: The R is selected from at least one of formulas a, b, c, d, e, and f; 2. A method for preparing a lipid droplet-targeted photosensitizing fluorescent molecule, characterized in that, At least the following steps are included: Step I: Disperse raw material I and 5-bromo-2-furanaldehyde in a mixed solvent, add catalyst and base reagent, and react I to obtain compound I; Step II: Disperse compound I and compound II obtained in step I in an organic solvent and react II to obtain the lipid droplet-targeted photosensitizing fluorescent molecule; The compound II has at least one structure of formula II; The structure of raw material I is shown in Formula III: The structure of compound I is shown in Formula IV:

3. The preparation method according to claim 2, characterized in that, The solvent in the mixed solvent is selected from at least one of ethanol, toluene, and dioxane.

4. The preparation method according to claim 2, characterized in that, The catalyst is selected from at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, tri(dibenzylideneacetone)palladium, and di(triphenylphosphine)palladium dichloride.

5. The preparation method according to claim 2, characterized in that, The alkaline reagent is selected from at least one of potassium carbonate, sodium carbonate, sodium bicarbonate, lithium carbonate, and sodium acetate.

6. The preparation method according to claim 2, characterized in that, The molar ratio of raw material I, 5-bromo-2-furan carbaldehyde, catalyst, and alkaline reagent is 1:1.1:0.01:2 to 1:5:0.2:

5.

7. The preparation method according to claim 2, characterized in that, The reaction conditions for reaction I are as follows: The reaction temperature of reaction I is 20–120°C; The reaction time for reaction I is 1 to 24 hours.

8. The preparation method according to claim 2, characterized in that, The molar ratio of compound I to compound II is 1:1 to 2:1; The organic solvent is selected from at least one of 1,4-dioxane, toluene, acetonitrile, and xylene; The concentration of compound II is 0.05–1.0 mol / L.

9. The preparation method according to claim 2, characterized in that, The reaction conditions for reaction II are as follows: The reaction temperature for reaction II is 25–120°C; The reaction time for reaction II is 1 to 48 hours.

10. The application of a lipid droplet-targeting photosensitizing fluorescent molecule in protein labeling and enrichment, characterized in that, The fluorescent molecule is selected from claim 1 or prepared by any one of the preparation methods in claims 2 to 9.

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