A fluorescent dye for lysosome labeling and its synthesis method and application

Abstract

The present invention provides a fluorescent dye for lysosome labeling, a synthesis method and application thereof. The fluorescent dye molecule described in the present invention uses a benzene-substituted naphthylethylene structure as a matrix, and a new type of lysosome-targeted fluorescent dye molecule is designed and synthesized by introducing a dimethylamine group and a cyano group into the molecular structure. The fluorescent dye molecule provided by the present invention has the advantages of simple structure, easy synthesis, long fluorescence emission wavelength, large Stokes shift and good biocompatibility. The emission wavelength of the fluorescent dye molecule in DMSO solution can reach 592nm, the Stokes shift is 191nm, and the fluorescence quantum yield reaches 73.9%, which has excellent fluorescence performance. The fluorescent dye molecule provided by the present invention can target and label lysosomes in living cells, and has broad application prospects.

Description

[Technical field]

[0001] The invention belongs to the field of fluorescent dyes and fluorescent imaging, and in particular relates to a fluorescent dye for lysosome marking and a synthesis method and application thereof. [Background technology]

[0002] Lysosomes are important subcellular structures in eukaryotic cells and are digestive organs in cells. They play an important role in the metabolism of macromolecules, cell membrane circulation, immune recognition, and cell apoptosis. The visual labeling of lysosomes helps to understand the mechanism of action of lysosomes in key physiological processes in cells, which is of great significance for a deeper understanding of the physiological and pathological effects of lysosomes.

[0003] Fluorescence imaging technology has the advantages of high sensitivity, high selectivity, easy operation and little damage to samples. It has been widely used in the fields of fluorescent probes and cell imaging. Laser confocal fluorescence imaging has high temporal and spatial resolution and has become an important method for studying lysosomal visualization markers. The development of fluorescent dye molecules that can be used to target and label lysosomes in living cells has become a research hotspot in the field of fluorescence confocal imaging. However, most lysosome-targeted fluorescent dye molecules still have problems such as complex synthesis routes, poor structural stability, small Stokes shift, high cytotoxicity and long incubation time. Therefore, it is necessary to develop a class of lysosome-targeted fluorescent dye molecules with simple synthesis, good photostability, large Stokes shift, high fluorescence quantum yield and good biocompatibility. The development of fluorescent dye molecules with excellent performance for targeting and labeling lysosomes in living cells will help to further explore the physiological role of lysosomes in cells and has broad application prospects. [Summary of the invention]

[0004] The purpose of the present invention is to provide a fluorescent dye for lysosome labeling and its synthesis method and application in view of the above technical analysis and existing problems. The fluorescent compound described in the present invention has the characteristics of simple structure, easy synthesis, long fluorescence emission wavelength, large Stokes shift and good biocompatibility, and can target and label lysosomes in living cells.

[0005] The present invention discloses the following technical solutions:

[0006] 1. A fluorescent dye for lysosome labeling, characterized in that its structural formula is as shown in Formula I:

[0007]

[0008] 2. Application of a fluorescent dye for lysosome labeling in the preparation of lysosome fluorescence imaging reagent.

[0009] The fluorescent dye provided by the present invention has the advantages of simple preparation method, long fluorescence emission wavelength, large Stokes shift, high fluorescence quantum yield, good photostability and good biocompatibility. The emission wavelength of the dye in DMSO solution can reach 592nm, its Stokes shift is 191nm, and the fluorescence quantum yield reaches 73.9%, showing excellent fluorescence performance, and can be used for specific labeling of lysosomes in living cells and laser confocal fluorescence imaging.

Brief Description of the Drawings

[0010] Figure 1 This is the fluorescence spectrum of the fluorescent dye molecule Lyso-DNBN prepared in Example 1 in DMSO solution, with an excitation wavelength of 401 nm and an emission wavelength of 592 nm.

[0011] Figure 2 Fluorescence confocal images of the fluorescent dye molecule Lyso-DNBN and the commercial lysosome marker dye Lysosomal Blue in SKBR-3 cells prepared in Example 1. The blue channel in the fluorescent image comes from Lysosomal Blue, and the yellow channel comes from the fluorescent dye molecule Lyso-DNBN. [Specific implementation method]

[0012] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be used to limit the scope of protection of the present invention.

[0013] Embodiment 1:

[0014] Synthesis of fluorescent dye molecule Lyso-DNBN having the compound structure of formula I:

[0015]

[0016] 6-Bromo-N,N-dimethylnaphthalene-2-amine (100 mg, 0.40 mmol), palladium acetate (15 mg, 0.07 mmol) and triphenylphosphine (53 mg, 0.20 mmol) were dissolved in a mixed solvent of N,N-dimethylformamide (25 mL) and triethylamine (20 mL). After the reaction solution was heated to 45 °C for 0.5 hours, 4-vinylbenzonitrile (62 μL, 0.54 mmol) was added. The reaction solution was heated to 110 °C for 8 hours. The solvent was removed by vacuum distillation, and the crude product was separated and purified by chromatography. The silica gel used was 300-400 mesh, and the eluent was a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1. The solvent was removed by rotary evaporation to obtain 83 mg of a yellow solid product with a yield of 71%.

[0017] The product structure was characterized by H-NMR, C-NMR and high-resolution mass spectrometry.

[0018] 1 H NMR (500MHz, CDCl3) δ7.74(s,1H),7.71(d,J=9.0Hz,1H),7.63-7.62(m,4H),7.58(d,J=8.4Hz,2H),7.32( d,J=16.3Hz,1H),7.16(dd,J=9.0,2.4Hz,1H),7.10(d,J=16.3Hz,1H),6.90(d,J=2.0Hz,1H),3.08(s,6H). 13 C NMR (126MHz, CDCl3) δ148.1,141.4,134.2,132.0,131.4,128.9,128.1,126.7,125.8 ,125.6,125.5,123.7,122.7,118.2,115.5,108.9,105.2,39.7.HRMS(ESI)calcd.for C 21 H 19 N2[M+H] + :299.1543,found:299.1549.

[0019] Embodiment 2:

[0020] The optical properties of the fluorescent dye molecule Lyso-DNBN prepared in Example 1, such as ultraviolet absorption and fluorescence emission, were studied.

[0021] The fluorescent dye molecule Lyso-DNBN was dissolved in DMSO solution to prepare a concentration of 10 -5 Through UV absorption and fluorescence tests, it was found that the maximum absorption wavelength of the fluorescent dye molecule Lyso-DNBN in DMSO solution was 401nm, the maximum fluorescence emission wavelength was 592nm, and the Stokes shift could reach 191nm. Figure 1 As shown in Figure 2, the fluorescence quantum yield of the fluorescent dye molecule Lyso-DNBN in DMSO solution is 73.9%.

[0022] Embodiment 3:

[0023] Example 1 Co-localization of the fluorescent dye molecule Lyso-DNBN and Lysosomal Blue in SKBR-3 cells

[0024] SKBR-3 cells were incubated with the fluorescent dye molecule Lyso-DNBN (2 μM) prepared in Example 1 and the commercial lysosome marker dye LysoBrite Blue (2 μM), and fluorescence confocal imaging was performed after 10 minutes. The fluorescence staining in the blue channel and the yellow channel was monitored by laser confocal microscopy. Figure 2As shown, the excitation wavelength of the blue channel is 405nm, collecting fluorescence signals from 430 to 450nm, and the excitation wavelength of the yellow channel is 405nm, collecting fluorescence signals from 490 to 530nm. Comparing the fluorescence images of the two channels, it is found that the lysosome blue fluorescence signal in the blue channel and the fluorescent dye molecule Lyso-DNBN fluorescence signal in the yellow channel have a high degree of overlap, and the overlap correlation coefficient can reach 0.93. These results show that the fluorescent dye molecule Lyso-DNBN can target and label lysosomes in living cells.

[0025] The above description is only a preferred embodiment of the present invention, which is merely illustrative and not restrictive of the present invention. It is understood by those skilled in the art that many changes, modifications, and even equivalent changes may be made thereto within the scope defined by the patent claims of the present invention, but all of them will fall within the protection scope of the present invention.

Claims

1. A fluorescent dye for lysosome labeling, characterized in that Its structural formula is shown in Formula I: 。 2. Use of the fluorescent dye according to claim 1 in the preparation of a lysosome fluorescence imaging reagent.

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