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Lipid droplet probe with high selectivity and large Stokes shift as well as preparation method and application of lipid droplet probe

A highly selective, displacement technology, applied in chemical instruments and methods, fluorescence/phosphorescence, luminescent materials, etc., can solve the problems of strong fluorescence background, poor selectivity, small Stokes displacement, etc., to achieve simple steps, convenient operation, and dyeing speed. quick effect

Active Publication Date: 2019-06-21
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are two commercial lipid droplet fluorescent probes widely used: Nile Red (Nile Red) and BODIPY493 / 503. However, these fluorescent probes still have some important defects: strong fluorescent background, poor selectivity and Small Stokes displacement

Method used

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  • Lipid droplet probe with high selectivity and large Stokes shift as well as preparation method and application of lipid droplet probe
  • Lipid droplet probe with high selectivity and large Stokes shift as well as preparation method and application of lipid droplet probe
  • Lipid droplet probe with high selectivity and large Stokes shift as well as preparation method and application of lipid droplet probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Synthesis of Probe N,N-Dimethyl-3-phenyl-6-(pyridin-4-ylethynyl)naphthalene-1-amine

[0032] Add 0.20mmol 6-bromo-N,N-dimethyl-3-phenylnaphthalene-1-amine, 0.40mmol phenylacetylene, 0.01mmol Pd(PPh) to the reactor 3 Cl 2 , 0.01 mmol CuI, 0.40 mmol diisopropylamine, 1.0 mL THF. Under nitrogen atmosphere, heat to 100°C, keep stirring for 30h, stop the reaction, cool to room temperature, add saturated ammonium chloride solution to wash, then extract with dichloromethane, dry, and distill off the solvent under reduced pressure, the crude product is separated by column chromatography The fluorescent probe was obtained with a yield of 88%. 1 H NMR (400MHz, CDCl 3 ):δ8.62(s,2H),8.22(d,J=8.4Hz,1H),8.11(s,1H),7.71–7.68(m,3H),7.58(d,J=8.1Hz,1H) ,7.50(d,J=6.5Hz,2H),7.43–7.40(m,3H),7.34(s,1H),2.96(s,6H); 13 C NMR (100MHz, CDCl 3 ): δ151.3, 149.8, 141.1, 139.6, 134.5, 132.8, 131.5, 128.9, 127.7, 127.6, 127.4, 125.6, 124.5, 120.6, 119.6, 115.2, 94.4, 86.9, 45.2.

Embodiment 2

[0034] Emission Spectra of Fluorescent Probes in Different Solvents

[0035] Prepare the fluorescent probe N,N-dimethyl-3-phenyl-6-(pyridin-4-ylethynyl)naphthalene-1-amine synthesized in embodiment 1 at a concentration of 1mM in dichloromethane (DCM) Test the mother liquor for use.

[0036] In the test solution, take 3mL solvents of different polarities: dichloromethane (DCM), acetonitrile, ethanol, PBS buffer solution, and then add 30uL of the probe mother solution, so that the concentration of the probe in the test solution is 10uL, and perform fluorescence Scanning (excitation wavelength 405nm, detection wavelength band 400–650nm) to obtain the fluorescence intensity in each system, such as image 3 As shown, with the increase of polar solvent, the spectrum red-shifted and the fluorescence decreased obviously.

Embodiment 3

[0038] Colocalization of Fluorescent Probes with Commercial Ester Droplet Probes

[0039] Dichloromethane (DCM) of N,N-dimethyl-3-phenyl-6-(pyridin-4-ylethynyl)naphthalene-1-amine of the fluorescent probe synthesized in Example 1 with a concentration of 1mM The test mother solution is ready for use; the test mother solution of dichloromethane (DCM) of commercially available Nile Red (special locator for ester drops) with a concentration of 1 mM is prepared for use.

[0040] HepG2 cells that have been cultured, after the cells adhere to the wall, add the polarity-sensitive fluorescent probe N,N-dimethyl-3-phenyl-6-(pyridin-4-ylethynyl) of the present invention respectively Naphthalene-1-amine 10uL and commercially available Nile Red (special locator for ester drops) solution 5uL, discard the medium, wash the cells with PBS buffer 3 times, then perform fluorescence imaging (excitation wavelength of the probe: 405nm , emission band: 450–550nm; excitation wavelength of Nile Red: ...

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Abstract

The invention discloses a lipid droplet probe with high selectivity and large Stokes shift. The lipid droplet probe is characterized in that the chemical name of the probe is N,N-dimethyl-3-phenyl-6-(pyridin-4-yl ethynyl) naphthalen-1-amine which is prepared from 6-bromo-N, N-dimethyl-3-phenylnaphthalen-1-amine and 4-ethynylpyridine by a reaction. The fluorescent probe has large Stokes shift and fluorescence self-quenching inhibition property, has electric neutrality overall, is quite sensitive to polarity and can eliminate background interference. The invention further discloses an application of the probe to specific marking or displaying of form and growth of lipid droplets in living cells or tissue. Experiments verify that the probe is brand-new, has the characteristics of low toxicity, good optical stability, high staining speed, high fluorescence brightness and capability of specifically imaging the lipid droplets in the living cells, thereby having broad application prospects. The invention also provides a synthesis method of the probe. The method adopts simple steps and is convenient to operate.

Description

[0001] 【Technical field】 [0002] The invention relates to an ester droplet probe with high selectivity and large Stokes shift, a preparation method and application thereof, and belongs to the field of organic small molecule fluorescent probes. [0003] 【Background technique】 [0004] Lipid droplets (Lipid droplets, LDs) are composed of a phospholipid monomolecular surface layer and a neutral lipid core. There are many proteins distributed on the surface. They are the main storage places for neutral lipids in cells and are widely found in a variety of animal and plant cells. LDs have long been thought of as inert spheres similar to glycogen that act as energy reservoirs that are used to supply cells with energy when they need it. Therefore, LDs have not received widespread attention for a long time. Recently, research on LDs has developed rapidly. Studies have shown that lipid droplets are not a simple "inert" sphere, but a complex and active multifunctional organelle, and it...

Claims

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Application Information

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IPC IPC(8): C07D213/38C09K11/06G01N21/64
Inventor 周永波苏乐斌潘能董建玉尹双凤
Owner HUNAN UNIV
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