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Application of merocyanine-coumarin fluorophore to detection of hydrogen sulfide in mitochondria

A technology of coumarin and hydrogen sulfide, applied in fluorescence/phosphorescence, organic chemistry, luminescent materials, etc., can solve the problems of inability to meet actual needs, long response time, etc., and achieve the effect of rapid response, high sensitivity and fast response

Active Publication Date: 2020-02-18
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the probe has high sensitivity (detection limit 40nM) and good selectivity, its response time is too long (7min), which cannot meet the actual needs

Method used

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  • Application of merocyanine-coumarin fluorophore to detection of hydrogen sulfide in mitochondria
  • Application of merocyanine-coumarin fluorophore to detection of hydrogen sulfide in mitochondria
  • Application of merocyanine-coumarin fluorophore to detection of hydrogen sulfide in mitochondria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the preparation of coumarin aldehyde derivative

[0029] Preparation of Compound 2: Dissolve 3.88g of 4-N,N diethyl salicylaldehyde, 6.0mL of diethyl malonate and 2mL of hexahydropyridine in 65mL of absolute ethanol, and heat to reflux for 6h. Spin to dry the solvent, add 40 mL of concentrated hydrochloric acid and 40 mL of glacial acetic acid into the eggplant-shaped bottle, and reflux for six hours. After cooling to room temperature, the reaction solution was poured into 200 mL of ice water, and 40% NaOH was added dropwise with stirring in an ice bath until pH ≈ 5, and a large amount of yellow precipitate was obtained. Suction filtration, washing the filter cake with a large amount of ice water. Drying in vacuo yielded 3.66 g of product, yield 83.9%.

[0030] Synthesis of Compound Co: N at 30°C 2 Under protection, slowly drop 6mL of anhydrous DMF into 6mL of POCl 3 After stirring for 30 min, a reddish-brown solution was obtained. 4.56g of compound 2...

Embodiment 2

[0031] Embodiment 2: Preparation of CMC series probes

[0032] Indole salt synthesized according to literature (Pardal A.C., Ramos S.S., Santos P.F., Reis L.V., Almeida P., Synthesis and Spectroscopic Characterization of N-Alkyl Quaternary AmmoniumSalts Typical Precursors of Cyanines, Molecules, 2002, 3:320-330) Derivative (MR) 1.0mmol and coumarin aldehyde derivative 1.0mmol were dissolved in 20mL ethanol solution. Heated to reflux for 12h, and the solvent was spin-dried under reduced pressure after the reaction was completed. The resulting residue was separated by column chromatography (CH 2 Cl 2 :CH 3 OH, 20:1 to 10:1), to obtain the corresponding CMC series products.

[0033] CM-NC 1 , Yield: 56%. 1 H NMR (300MHz, Chloroform-d) δ10.05(s, 1H), 8.59(d, J=15.9 Hz, 1H), 8.11(d, J=9.1Hz, 1H), 8.01(d, J=15.9Hz ,1H),7.69–7.37(m,4H),6.71(dd,J=9.1, 2.4Hz,1H),6.47(d,J=2.2Hz,1H),4.31(s,3H),3.52(q, J=7.1Hz,4H),1.84(s,7H),1.29(t,J=7.1Hz,6H). 13 C NMR(75MHz,Chloroform-d)δ181.17...

Embodiment 3

[0035] Example 3: Probe CM-NC 6 Fluorescence Spectrum of Response Speed ​​to Hydrogen Sulfide

[0036] The test concentration of the fluorescence spectrum used in the present invention is 10 μM, the solvent is 20 mM PBS solution with pH=7.4 mixed with 2% DMSO, and when the emission spectrum is measured, the excitation wavelength is 475 nm. NaHS was used as the hydrogen sulfide donor. Add the fluorescent probe to the PBS solution, add 20 equivalents of NaHS and mix well, then perform the corresponding fluorescence spectrum test at regular intervals. Such as figure 1 As shown, to 10μM CM-NC 6 When 200 μM NaHS was added to the PBS solution, the emission peak of the merocyanine / coumarin hybrid fluorophore at 650 nm decreased significantly with time, and nearly disappeared after 2 min. Through the fluorescence change rate curve (F 0 -F t ) / F 0 It can be seen that the reaction is close to equilibrium when the reaction reaches 40s, and the reaction degree reaches more than 95...

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Abstract

The invention discloses application of a merocyanine-coumarin fluorophore serving as a hydrogen sulfide fluorescent probe to detection of hydrogen sulfide in mitochondria. The structure of the merocyanine-coumarin fluorophore is shown in a formula defined in the description, wherein C6H13 refers to linear or branched hexyl. The merocyanine-coumarin fluorophore is not prone to being influenced by adetection environment, such as a probe concentration and other conditions, is not disturbed by biological mercaptan, can selectively react with hydrogen sulfide, has the advantages of fluorescence ratio response effects, strong selectivity, high sensitivity, rapid response and the like and realizes intracellular exogenous and endogenous hydrogen sulfide ratio imaging; and besides, the probe can perform mitochondrial locating, is expected to play a role in bioscience and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of biological analysis and detection, and specifically relates to the application of a merocyanine-coumarin fluorophore as a hydrogen sulfide fluorescent probe in the detection of hydrogen sulfide in organelles Background technique [0002] Hydrogen sulfide is the third gas signal molecule discovered after NO and CO in living organisms. It is used as a cell protection agent and gas transmitter in various tissues, and has the functions of controlling vasodilation and neuromodulation. Abnormal levels of hydrogen sulfide have also been linked to diseases such as Alzheimer's, Down syndrome, diabetes and cirrhosis of the liver. The research on the physiological and pathological functions of hydrogen sulfide has become a hot topic widely concerned by chemists and biologists, and the development of accurate and fast hydrogen sulfide detection methods is the technical basis of these related studies. Fluorescent probes and im...

Claims

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Application Information

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IPC IPC(8): C07D405/06C09K11/06G01N21/64
CPCC07D405/06C09K11/06G01N21/6486G01N21/6447C09K2211/1029C09K2211/1088
Inventor 何卫江郭子建方红宝
Owner NANJING UNIV
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