Long-wavelength lipid droplet fluorescent probe with large stokes shift and synthesis and application thereof

Abstract

The application develops a long-wavelength large stokes shift fluorescent dye which can be used for lipid droplet imaging. The amine group introduced in the dye 4th position can regulate the wavelength to reach long wavelength >600nm, wherein the introduction of azetidine can also strongly inhibit intramolecular torsion and improve light stability. Compared with traditional naphthalimide dyes, the fluorescent dye is bridged by a carbon atom substituted with dimethyl at 1,8-position carbonyl, forming a non-coplanar six-membered ring structure. The molar extinction coefficient of the dye in ethanol is 8000M ‑1 cm ‑1 , the emission wavelength reaches 600nm, and the stokes shift in ethanol can reach 225nm. In addition, the dye can quickly label the lipid droplets in cells and be used for lipid droplet fluorescence dynamic imaging.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence imaging technology, specifically relating to a method for synthesizing a class of long-wavelength lipid droplet fluorescent probes with large Stokes shift and their application in the field of fluorescence imaging. Background Technology

[0002] For a long time, lipid droplets were considered glycogen-like particles used solely for energy storage, and thus inert intracellular inclusions. Consequently, they received little attention from researchers for an extended period. However, since the discovery of the first lipid droplet-related protein in 1991, lipidomics and proteomics studies of lipid droplets have rapidly progressed. Research has shown that lipid droplets are not simply energy storage devices but complex, active, and dynamically changing organelles within cells. Unveiling the mechanisms of lipid droplet formation and dynamic changes will accelerate the resolution of human health issues. To meet the requirements for high temporal resolution and in-situ monitoring of lipid droplets, fluorescence imaging technology has gradually gained favor among lipid droplet researchers.

[0003] The development of fluorescence imaging technology has placed high demands on lipid droplet fluorescent dyes. These dyes not only need high stability and brightness, but also large Stokes shifts to facilitate multicolor imaging. However, current commercial lipid droplet dyes are all based on Bodipy and Nile Red dyes, which have small Stokes shifts; for example, Bodipy's Stokes shift is only 15-25 nm. This leads to severe crosstalk between excitation and emission light, resulting in low signal-to-noise ratios and severe fluorescence self-quenching during lipid droplet imaging, limiting their application in dynamic and multicolor imaging of lipid droplets. Therefore, the development of novel lipid droplet fluorescent dyes with large Stokes shifts is particularly urgent. Summary of the Invention

[0004] This invention develops a class of long-wavelength lipid droplet fluorescent probes with large Stokes shifts. Unlike traditional naphthalimide probes where the two carbonyl groups are linked by nitrogen atoms, this class of fluorescent dyes uses dimethyl-substituted carbon atoms for linkage, forming a non-coplanar six-membered ring structure. Furthermore, different amino groups are introduced at position 4 to regulate the dye wavelength to 600 nm and achieve a Stokes shift of 225 nm. This class of dyes can rapidly label intracellular lipid droplets and be used for lipid droplet fluorescence imaging.

[0005] The present invention describes a type of long-wavelength lipid droplet fluorescent probe with large Stokes shift, the structure of which is shown below:

[0006]

[0007] In addition, the present invention also provides a general method for synthesizing the 4-substituted naphthimide dye, the synthesis steps of which are as follows:

[0008]

[0009] The specific synthesis steps are as follows:

[0010] Step 1: Synthesis of intermediate 6-bromo-3-hydroxy-1H-styrene-1-one

[0011] Weigh 2.0-4.0 g of 6-bromonaphthalene anhydride, 3.0-6.0 g of anhydrous zinc chloride, and 10-20 mL of diethyl malonate into a round-bottom flask. Heat the flask to 170-200 °C and react for 10-15 hours. After the reaction is complete, cool to room temperature and add 100-200 mL of 2 mol / L KOH aqueous solution. Filter the solution to obtain a red color. Add 100-200 mL of 2 mol / L acetic acid, and a solid precipitates. Remove the filtrate and dry the solid to obtain the intermediate 6-bromo-3-hydroxy-1H-styrene-1-one.

[0012] Step 2: Synthesis of intermediate 6-bromo-2,2-dimethyl-1H-styrene-1,3(2H)-dione

[0013] Weigh 0.2-0.4 g of 6-bromo-3-hydroxy-1H-styrene-1-one, 0.5-1.0 g of iodomethane and 0.3-0.6 g of potassium carbonate, dissolve them in 2-5 ml of acetonitrile, react at 60-90 °C for 10-16 hours, cool the reaction solution, remove the solvent under reduced pressure, and then separate and purify by silica gel column chromatography to obtain the intermediate 6-bromo-2,2-dimethyl-1H-styrene-1,3(2H)-dione.

[0014] Step 3: Synthesis of 2,2-dimethyl-1H-styrene-1,3(2H)-dione ester droplet probes with different N-substituted compounds. Weigh 5-20 mg of 6-bromo-2,2-dimethyl-1H-styrene-1,3(2H)-dione and 5-20 mg of secondary amines with different substitutions, add to 3-5 ml of 2-methoxyethanol, heat to 100-140℃ and react for 10-20 hours. After cooling the reaction solution, remove the solvent under reduced pressure and separate and purify by silica gel column chromatography to obtain 2,2-dimethyl-1H-styrene-1,3(2H)-dione ester droplet probes with different N-substituted compounds.

[0015] In step one, 2.0-4.0g of 6-bromonaphthalene anhydride, 3.0-6.0g of anhydrous zinc chloride, 10-20mL of diethyl malonate, 100-200mL of 2mol / L KOH aqueous solution, and 100-200mL of 2mol / L acetic acid are used.

[0016] In step two, add 0.2-0.4g of 6-bromo-3-hydroxy-1H-styrene-1-one, 0.5-1.0g of iodomethane, 0.3-0.6g of potassium carbonate, and 2-5ml of acetonitrile.

[0017] In step three, 10-40 mg of 6-bromo-2,2-dimethyl-1H-styrene-1,3(2H)-dione, 50-100 mg of secondary amines with different substitutions, and 3-5 ml of 2-methoxyethanol are used.

[0018] The aforementioned lipid droplet fluorescent probes can specifically label lipid droplets and achieve fluorescence imaging in living cells and in vivo.

[0019] This invention has the following features:

[0020] These probes have advantages such as low cost of raw materials, simple methods, and easy derivation.

[0021] These probes have a molar extinction coefficient of 8000 M⁻¹ cm⁻¹ in ethanol, an emission wavelength of 600 nm, and a Stokes shift of 225 nm in ethanol.

[0022] Dyes can effectively label lipid droplet structures in cells and have a high signal-to-noise ratio, making them applicable to studies such as lipid droplet growth and fusion.

[0023] This invention introduces an amino group at position 4 of the dye to achieve wavelength modulation to reach long wavelengths >600 nm. The introduction of aziridine further strongly suppresses intramolecular torsion and improves photostability. Compared to traditional naphthalimide dyes, this type of fluorescent dye uses a dimethyl-substituted carbon atom to bridge the 1,8-carbonyl group, forming a non-coplanar six-membered ring structure. This type of dye has a molar extinction coefficient of 8000 M in ethanol. -1 cm -1 The emission wavelength reaches 600 nm, and the Stokes shift in ethanol can reach 225 nm. Furthermore, this type of dye can rapidly label intracellular lipid droplets and be used for dynamic fluorescence imaging of lipid droplets. Attached Figure Description

[0024] Figure 1 : The 1H NMR spectrum of the product in Example 1;

[0025] Figure 2 : The 1H NMR spectrum of the product in Example 2;

[0026] Figure 3 : The 1H NMR spectrum of the product in Example 3;

[0027] Figure 4 : X-ray diffraction crystallography diagram of the product in Example 3;

[0028] Figure 5 : The 1H NMR spectrum of the product in Example 4;

[0029] Figure 6 : The carbon NMR spectrum of the product in Example 4;

[0030] Figure 7 : Normalized UV absorption and fluorescence emission of the product in ethanol in Example 3;

[0031] Figure 8 : Normalized UV absorption and fluorescence emission of the product in ethanol from Example 4

[0032] Figure 9 : This is a confocal image of lipid droplets stained with the product from HeLa cells in Example 3; Detailed Implementation

[0033] This invention provides a method for synthesizing a class of novel long-wavelength fluorescent dyes with large Stokes shifts that can be used for lipid droplet imaging, and their application in the field of fluorescence imaging.

[0034] Example 1

[0035] The synthetic route and product structure of intermediate compound 1 are as follows:

[0036]

[0037] Weigh 2.77 g (10 mmol) of 6-bromonaphthalene anhydride, 4.08 g (30 mmol) of anhydrous zinc chloride, and 15 mL of diethyl malonate into a round-bottom flask. Heat to 180 °C and react for 14 hours. After the reaction is complete, cool to room temperature, add 200 mL of 2 mol / L KOH aqueous solution, and filter to collect the red solution. Add 200 mL of 2 mol / L acetic acid, precipitate a solid, remove the filtrate, and dry the solid to give compound 1 (508.6 mg, 1.86 mmol, 18%). 1 H NMR (700MHz, DMSO) δ8.39(d,J=8.4Hz,1H),8.34(d,J=7.2Hz,1H),8.11(d,J=7.8Hz,1H),8.08(d,J=7.8Hz,1H),7.86(t,J=7.8Hz,1H),5.91(s,1H).

[0038] Example 2

[0039] The synthetic route and product structure of intermediate compound 2 are as follows:

[0040]

[0041] Compound 1 (274 mg, 1 mmol), iodomethane (622 μL, 10 mmol), and potassium carbonate (414.6 mg, 3 mmol) were weighed into a sealed tube, dissolved in 2 mL of acetonitrile, and stirred at 80 °C for 12 h. After cooling the reaction solution to room temperature, the solvent was removed under reduced pressure, and the solution was purified by silica gel column chromatography (volume ratio: petroleum ether: ethyl acetate = 10:1) to obtain compound 2 (7.6 mg, 0.025 mmol, 2.5%). 1 H NMR (700MHz, CDCl3) δ8.45(d,J=8.5Hz,1H),8.33(d,J=7.2Hz,1H),8.09(d,J=7.8Hz,1H),7.90(d,J=7.8Hz,1H),7.71(t,J=7.8Hz,1H),1.40(s,6H).

[0042] Example 3

[0043] The synthetic route and product structure of the molecular C-AN are as follows:

[0044]

[0045] Weigh 6.6 mg (0.12 mmol) of aziridine and 7 mg (0.023 mmol) of compound 2 into 2 mL of 2-methoxyethanol solution. Heat the mixture to 120 °C and reflux for 10 hours. After the reaction is complete, cool the reaction solution to room temperature, remove the solvent under reduced pressure, and then separate and purify by silica gel column chromatography (volume ratio, petroleum ether:ethyl acetate = 10:1) to obtain product C-AN (5.2 mg, 0.018 mmol, 81%). 1 H NMR (700MHz, CDCl3) δ8.33(t,J=7.3Hz,2H),8.25(d,J=8.4Hz,1H),7.50(t,J=7.8Hz ,1H),6.46(d,J=8.4Hz,1H),4.49(t,J=7.5Hz,4H),2.59–2.53(m,2H),1.48(s,6H).

[0046] Example 4

[0047] The synthetic route and product structure of the C-DMAN molecule are as follows:

[0048]

[0049] Weigh dimethylamine hydrochloride (9.4 mg, 0.12 mmol), compound 2 (7 mg, 0.023 mmol), and 2-5 drops of triethylamine into a solution of 2-methoxyethanol. Heat the mixture to 120 °C and reflux for 10 hours. After the reaction is complete, cool the reaction solution, remove the solvent under reduced pressure, and then separate and purify the product C-DMAN (4.6 mg, 0.017 mmol, 76%) by silica gel column chromatography (volume ratio: petroleum ether: ethyl acetate = 10:1) to obtain the product. 1 H NMR (700MHz, CDCl3) δ8.42(d,J=8.4Hz,1H),8.35(d,J=7.1Hz,1H),8.33(d,J=8. 1Hz,1H),7.62(t,J=7.8Hz,1H),7.12(d,J=8.1Hz,1H),3.05(s,6H),1.48(s,6H). 13 C NMR(176MHz, CDCl3)δ200.14(s),198.19(s),156.86(s),133.43(s),131.25(s),130.37(s),128. 82(s),127.43(s),126.40(s),124.82(s),120.12(s),113.33(s),58.12(s),44.69(s),22.99(s).

[0050] The dyes to be tested were dissolved in dimethyl sulfoxide solution to prepare 2mM stock solutions of different dyes. Test solutions of different concentrations were prepared as needed to detect changes in fluorescence spectra and intracellular nuclear fluorescence imaging.

[0051] Example 5

[0052] Spectroscopic testing of C-AN in ethanol. Take 7.5 μL of C-AN stock solution and add it to 3 mL of ethanol to prepare a 5 μM fluorescent probe test solution, and perform UV-Vis absorption spectroscopy and fluorescence emission spectroscopy (excitation light 400 nm) testing.

[0053] Figure 7 The concentration of the fluorescent probe was 5 μM. The absorption and fluorescence spectra of C-AN in ethanol are shown. The molar extinction coefficient of C-AN in ethanol reaches 8800 M. -1 cm -1 It has a maximum emission wavelength of 600 nm, a quantum yield of 0.014, and a Stokes shift of 225 nm.

[0054] Example 6

[0055] Spectroscopic testing of C-DMAN in ethanol. Take 7.5 μL of C-DMAN stock solution and add it to 3 mL of ethanol to prepare a 5 μM fluorescent probe test solution, and perform UV-Vis absorption spectroscopy and fluorescence emission spectroscopy (excitation light 400 nm).

[0056] Figure 8 The concentration of the fluorescent probe was 5 μM. The absorption and fluorescence spectra of C-DMAN in ethanol are shown. The molar extinction coefficient of C-DMAN in ethanol reaches 8500 M. -1 cm -1 It has a maximum emission wavelength of 613 nm, a quantum yield of 0.019, and a Stokes shift of 222 nm.

[0057] Example 7

[0058] Fluorescence imaging of HeLa after C-AN staining. 2.5 μL of C-AN stock solution was dissolved in 1 mL of HeLa culture medium and incubated at room temperature for 30 minutes before fluorescence confocal imaging (excitation light 405 nm).

[0059] Figure 9 In this study, C-AN can accurately locate lipid droplets in HeLa cells and has an extremely high signal-to-noise ratio, making it suitable for fluorescence imaging studies of lipid droplet dynamics.

Claims

1. A long-wavelength lipid droplet fluorescent probe with a large Stokes shift, characterized in that: Its structural formula is shown below for one or two of the compounds. 。 2. A method for synthesizing the fluorescent probe according to claim 1, characterized in that: The specific method for this synthesis is as follows: (1) Synthesis of intermediate 6-bromo-3-hydroxy-1H-finaren-1-one Weigh 2.0-4.0 g of 6-bromonaphthalene anhydride, 3.0-6.0 g of anhydrous zinc chloride, and 10-20 mL of diethyl malonate, add them to a container, and heat to 170-200 ℃ for 10-15 hours. After the reaction is complete, cool to room temperature, add 100-200 mL of 1-3 mol / L KOH aqueous solution, filter and collect the solution; add 100-200 mL of 1-3 mol / L acetic acid, and a solid precipitates; remove the filtrate, dry the solid to obtain the intermediate 6-bromo-3-hydroxy-1H-finaden-1-one. (2) Synthesis of intermediate 6-bromo-2,2-dimethyl-1H-finaren-1,3(2H)-dione Weigh 0.2-0.4 g of 6-bromo-3-hydroxy-1H-finaren-1-one, 0.5-1.0 g of iodomethane and 0.3-0.6 g of potassium carbonate, dissolve in 2-5 ml of acetonitrile, react at 60-90 °C for 10-16 hours, remove the solvent by vacuum distillation, and then separate and purify to obtain the intermediate 6-bromo-2,2-dimethyl-1H-finaren-1,3(2H)-dione; (3) Different N Synthesis of a substituted 2,2-dimethyl-1H-finaene-1,3(2H)-dione drop probe Weigh out 10-40 mg of 6-bromo-2,2-dimethyl-1H-finasterene-1,3(2) H )-Diketones and 50-100 mg of amines with different substitutions were added to 3-5 ml of 2-methoxyethanol, and the mixture was heated to 100-140 °C and reacted for 10-20 hours. After removing the solvent under reduced pressure, the mixture was separated and purified to obtain different amines. N -Substituted 2,2-dimethyl-1H-finaene-1,3(2H)-dione drop probe.

3. The synthesis method as described in claim 2, characterized in that: In step (3), the different substituted amines are aziridine and / or dimethylamine hydrochloride.

4. The use of the fluorescent probe of claim 1 in fluorescence imaging or as a molecular probe, wherein the use is not for disease diagnosis or treatment purposes.

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