Preparation and application of ATP fluorescent probe for positioning mitochondria

A fluorescent probe and mitochondrial technology, applied in the field of fluorescent probes, can solve the problems of lack of mitochondrial positioning groups, poor selectivity of biological anions, inconsistency, etc.

Inactive Publication Date: 2017-03-29
XIANGTAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these probes can detect ATP, they have two problems: on the one hand, most of the probes lack the mitochondrial positioning group, which is not conducive to the detection of ATP in the mitochondria; on the other hand, some probes have only one recognition site point, thus poor selectivity to bioanions
However, there are few probes that can be used to detect ATP in mitochondria, and only two probes are reported (document 33: Kurishita, Y.; Kohira, T.; Ojida, A.; Hamachi, I.J.Am.Chem.Soc. 2012, 134, 18779–18789. Literature 34: Srivastava, P.; Razi, S.S.; Ali, R.; Srivastav, S.; Patnaik, S.; 69,179–185.), but these two probes have some limitations, such as: they cannot effectively distinguish ATP from other organophosphate anions, or the detection range is not consistent with the concentration of ATP in mitochondria (1-5mM)
Therefore, it is still challenging to develop a suitable probe to monitor the changes of ATP content in mitochondria

Method used

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  • Preparation and application of ATP fluorescent probe for positioning mitochondria
  • Preparation and application of ATP fluorescent probe for positioning mitochondria
  • Preparation and application of ATP fluorescent probe for positioning mitochondria

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Synthetic route such as figure 1 shown.

[0026] The synthesis of compound 1: add Rhodamine B (2.0g, 4.18mmol) in the 100mL round-bottomed flask that contains 50mL ethanol, after dissolving completely, solution presents purple, and diethylenetriamine (10.0mL, 92.00mmol) is added dropwise under stirring ) into the above reaction system, refluxed for 24h. The solvent was distilled off under reduced pressure. The crude product was separated by column chromatography with an eluent of dichloromethane / ethanol 10:1 (volume ratio) to obtain a yellow solid (compound 1) (0.15 g, yield: 6.40%). 1 H NMR (400MHz, CDCl 3 ):δ7.88(d,J=8.0Hz,1H),7.42(m,2H),7.08(m,1H),6.42(d,J=8.0Hz,2H),6.36(s,2H),6.26 (d, J=8.0Hz, 2H), 3.35-3.18(m, 10H), 2.71(d, J=8.0Hz, 2H), 2.55(t, J=8.0Hz, 2H), 2.20(t, J= 8.0Hz, 2H), 1.16(t, J=8.0Hz, 12H). 13 C NMR (100MHz, CDCl 3 ): δ167.9, 153.6, 153.3, 148.7, 132.2, 131.3, 129.0, 128.9, 128.0, 127.9, 123.7, 122.7, 108.1, 105.7, 97.9, 64.9, 51.7, 51.3, 50.5,...

Embodiment 2

[0029] Solution preparation of fluorescent probe Mito-Rh interacting with ATP

[0030] A certain amount of fluorescent probe was dissolved in water to obtain a probe stock solution with a concentration of 100 μM. Dissolve a certain amount of ATP in water, pour it into a 500mL volumetric flask, add water to dilute to the mark, and obtain ATP with a concentration of 1000mM. The 1000mM ATP aqueous solution was gradually diluted with twice distilled water to obtain a 0.1-100mM ATP aqueous solution. Add 1.0mL probe stock solution and 1.0mL ATP aqueous solution into a 10mL volumetric flask, and dilute to the volume with buffer solution PBS to obtain a fluorescent probe with a concentration of 10μM and an ATP test solution with a concentration of 0.01-100mM.

Embodiment 3

[0032] Determination of Fluorescent Spectrum Properties of Fluorescent Probe Mito-Rh Interaction with ATP

[0033] The PBS buffer solution with a pH value of 7.0 was used as a solvent to measure the fluorescence spectrum of the fluorescent probe interacting with ATP with a Perkin Elmer LS 55 fluorescence spectrophotometer. The results are as follows: figure 2. The concentration of the fluorescent probe is 10μM, the concentration of ATP is 0, 0.1, 0.6, 1.2, 2.0, 3.0, 4.4, 5.6, 6.4, 8.4, 10.0mM, the excitation wavelength is fixed at 520nm, and the emission wavelength range is 530-650nm. The slit width is 5nm / 5nm. Before adding ATP, the fluorescent probe has almost no fluorescence. After adding ATP, the emission peak of rhodamine appears at 583nm. This is because the structure of the probe molecule changes, and the structure changes from the closed ring form of rhodamine to the open ring form. . And with the increase of ATP concentration, the fluorescence intensity of the pro...

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Abstract

The invention discloses preparation and application of an ATP fluorescent probe for positioning mitochondria, wherein the probe has the structural formula defined in the specification and is synthesized by rhodamine B, diethylenetriamine, 5-bromo-valeric acid and triphenylphosphine as raw materials. In the system, ATP induces ring opening of a rhodamine lactam structure, strong fluorescence is produced, and high sensitivity is showed on ATP detection. At the same time, the probe shows good selectivity on the ATP, and is hardly interfered by other biological anions or inorganic anions. When the pH value is 6.0-8.0, ATP determination of the probe is not affected. In addition, the probe can be positioned in the mitochondria, and changes of the content of the ATP in the mitochondria are real-timely monitored.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to the preparation and application of an ATP fluorescent probe for locating mitochondria. Background technique [0002] ATP (adenosine triphosphate) is a biological anion that plays a key role in cellular respiration, enzyme catalysis, energy and signal transmission (Document 1: Knowles, J.R.Annu.Rev.Biochem.1980, 49, 877–919. Document 2: Dennis, P.B.; Jaeschke, A.; Saitoh, M.; Fowler, B.; Kozma, S.C.; , V.; J. Anal. Chem. 2009, 64, 657–673.). Mitochondria are the main place for cells to carry out aerobic respiration, and provide cellular metabolic energy in the form of ATP (Document 4: Knowles, J.R.Annu.Rev. Biochem. 1980, 49, 877–919. Document 5: Higgins, C.F.; Hiles, I.D.; Salmond , G.P.; Gill, D.R.; Downie, J.A.; Evans, I.J.; Holland, I.B.; Gray, L.; , M.F. Nature 2006, 443, 787–795.). Therefore, changes in ATP content will inevitably affect the functi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07F9/6561G01N21/64
CPCC07F9/6561G01N21/6428G01N21/6486
Inventor 李春艳谭凯月
Owner XIANGTAN UNIV
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