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Non-reactive mitochondrial tracking fluorescent probe containing C12 lalkyl chain, and applications thereof

A dodecyl chain and fluorescent probe technology, applied in the field of fluorescent probes, can solve problems affecting the properties of mitochondrial proteins, mitochondrial metabolism, high toxicity of living cells, etc., and achieve intuitive biological detection reagents, low toxicity, The effect of broad application prospects

Inactive Publication Date: 2019-02-01
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, whether it is the existing commercial MitoTracker probe or the reported mitochondrial tracking probe, after staining, it will affect the properties of mitochondrial proteins, thereby affecting the normal metabolic state of mitochondria, and causing high toxicity to living cells.
After searching, non-reactive mitochondrial tracking fluorescent probes containing dodecyl chains and their distribution in labeling or showing mitochondria in normal living cells, living cells with reduced mitochondrial membrane potential, or cells with lost mitochondrial membrane potential The application, or the application in the observation of intracellular mitophagy has not been reported

Method used

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  • Non-reactive mitochondrial tracking fluorescent probe containing C12 lalkyl chain, and applications thereof
  • Non-reactive mitochondrial tracking fluorescent probe containing C12 lalkyl chain, and applications thereof
  • Non-reactive mitochondrial tracking fluorescent probe containing C12 lalkyl chain, and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The synthesis of embodiment 1 ECPI-12

[0031] Take 0.27g 9-(2-ethoxyethyl)-9H-carbazole-3-carbaldehyde (1mmol) and 0.39g 1-dodecyl-4-methylpyridine-1-iodide (1mmol) and add into 5 mL of ethanol and stir. Then add 200 μL tetrahydropyrrole and stir at room temperature for 5 h. After the reaction, the mixture was poured into petroleum ether, and a yellow solid was precipitated. After recrystallization, the final yellow solid product is (E)-1-dodecyl-4-(2-(9-(2-ethoxyethyl)-9H-carbazol-3-enyl) Vinyl)-pyridine-1-iodide salt, about 67% yield.

[0032] 1 H NMR (300MHz, DMSO-d6 ),δ(ppm):8.90(d,J=6.90Hz,2H),8.56(s,1H),8.21(q,J=8.00Hz,4H),7.89(q,J=3.30Hz,1H), 7.75(d,J=8.70Hz,1H),7.69(d,J=8.10Hz,1H),7.49-7.56(m,2H),7.29(t,J=7.35Hz,1H),4.61(t,J =5.25Hz, 2H), 4.47(t, J=7.20Hz, 2H), 3.77(t, J=5.10Hz, 2H), 3.32-3.41(m, 2H), 1.91(s, 2H), 1.26(d ,J=15.60Hz,18H),0.96(t,J=7.05Hz,3H),0.84(t,J=6.60Hz,3H). 13 C NMR(400MHz,DMSO),δ(ppm):153.98,144.39,143.11,142.35,141.37,126.82,126.76...

Embodiment 2

[0037] Embodiment 2 cell culture and dyeing method

[0038] Cell culture:

[0039] HeLa cells were grown in H-DMEM medium containing 10% FBS and 1% penicillin and streptomycin. All cells are in 5% CO 2 cultured in a constant temperature incubator at 37°C.

[0040] Cell staining:

[0041] ECPI-12 was dissolved in DMSO to prepare a stock solution with a concentration of 1 mM.

[0042] HeLa cells were grown on coverslips for 48 hours, then ECPI-12 with a final concentration of 2 μM was added and incubated at 37°C for 30 minutes to complete the staining.

Embodiment 3

[0043] Example 3 Counterstaining experiment of ECPI-12 and MTDR

[0044] MTDR was dissolved in DMSO to prepare a stock solution with a concentration of 0.1 mM.

[0045] ① In living cells: active HeLa cells were first incubated with 0.2 μM MTDR for 30 min, and then incubated with 2 μM ECPI-12 for 30 min; ② In living cells with reduced mitochondrial membrane potential: active HeLa cells were first incubated with 0.2 μM MTDR for 30 min, and then incubated with Incubate with 2μM ECPI-12 for 30min; then treat with 15μM CCCP for 30min; ③In the cells whose mitochondrial membrane potential disappeared: Active HeLa cells were first incubated with 0.2μM MTDR for 30min, then incubated with 2μM ECPI-12 for 30min; POM fixation for 30min. Cells were washed 3 times with PBS before each staining with another probe.

[0046] see results figure 1 .

[0047] figure 1 : Confocal fluorescence pictures of normal active HeLa cells stained by ECPI-12 and MTDR, CCCP-treated HeLa cells and parafor...

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Abstract

The invention discloses a non-reactive mitochondrial tracking fluorescent probe containing a C12 lalkyl chain, and applications of the probe in labeling or displaying of the distribution of mitochondria in cells, and in mitochondrial autophagy observation, wherein the fluorescent probe is a carbazole pyridine salt compound having a structure represented by a formula (I). According to the present invention, the probe can stain mitochondria in normal living cells, and especially can be fixed on mitochondria when the mitochondrial membrane potential is reduced or disappears, such that the resultsshow that the probe can track the dynamic changes of mitochondria; the probe further has good two-photon properties, and can be used for deep tissue imaging; and the probe has low toxicity, can real-timely track the dynamic process of mitochondrial autophagy, and has broad application prospect in the field of fluorescent biomarkers.

Description

technical field [0001] The present invention relates to a fluorescent probe targeting mitochondria and its application, in particular to a non-reactive mitochondrial tracking fluorescent probe containing a dodecyl chain and its use in marking or displaying the distribution of mitochondria in cells, and in Applications in the Observation of Mitophagy. Background technique [0002] Throughout the physiological cycle of cells, tracking the shape and number of mitochondria is of great significance for the study of physiology, pathology and pharmacology. Studies have shown that in the early stage of apoptosis, the shape of mitochondria will change from tubular network structure to point or fragment. There are many tools available to observe the shape and number of mitochondria, such as scanning electron microscopy (SEM), immunofluorescence (IFM) and fluorescence microscopy with fluorescent probes. Among these three methods, the operation steps of SEM and IFM are very complicate...

Claims

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Application Information

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IPC IPC(8): C07D401/06C09K11/06G01N21/64
Inventor 于晓强张若瑶刘志强何秀全
Owner SHANDONG UNIV
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