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Preparation and application of peroxide nitroso (ONOO<->) fluorescent probe based on hemicyanine dye

A technology of peroxynitroso and fluorescent probes, applied in the field of fluorescent probes, can solve the problems of high reactivity, low selectivity of reactive oxygen species, limited applications, difficulties, etc.

Active Publication Date: 2018-06-29
XIANGTAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are some problems in these probes: (1) the emission wavelength of the fluorophores of the probes (such as fluorescein, naphthalimide, fluoropyrrole, etc.) is relatively short, which limits the application of these probes in vivo; (2) The selectivity of these previously reported probes for highly reactive reactive oxygen species is low because ONOO - with ClO - and H 2 o 2 All belong to highly reactive active oxygen, effectively distinguishing ONOO - with ClO - 、H 2 o 2 It is very difficult; (3) Most of these fluorescent probes use a single fluorescence emission peak intensity change as a response signal, which is easily affected by some environmental factors (such as equipment efficiency, temperature, probe concentration, etc.)
However, no cyanine dye-based probes have been used to detect ONOO - , so a cyanine-based probe was designed and synthesized to detect ONOO - is very necessary

Method used

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  • Preparation and application of peroxide nitroso (ONOO&lt;-&gt;) fluorescent probe based on hemicyanine dye
  • Preparation and application of peroxide nitroso (ONOO&lt;-&gt;) fluorescent probe based on hemicyanine dye
  • Preparation and application of peroxide nitroso (ONOO&lt;-&gt;) fluorescent probe based on hemicyanine dye

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Synthesis of fluorescent probes

[0027] Synthetic route such as figure 1 . In a 100 mL round bottom flask, 3-nitrophenol (174 mg, 1.25 mmol) and sodium hydride (30.8 mg, 1.25 mmol) were dissolved in 6 mL of anhydrous DMF and stirred at room temperature under nitrogen for 10 minutes. Then, IR-780 (334 mg, 0.5 mmol) was added via syringe and stirring was continued at room temperature for 12 hours. Concentrated under reduced pressure, the residue was dissolved in CH 2 Cl 2 , washed three times with deionized water, anhydrous Na 2 SO 4 Dried, filtered and concentrated, the resulting material was dissolved in 30 mL of methanol. Add SnCl 2 (2 g, 10 mmol) and concentrated HCl (2 mL), the reaction solution was heated to 70 °C and stirred overnight. Then, with saturated Na 2 CO 3 Neutralize, remove the precipitate by filtration, and wash with CH 2 Cl 2 washing. The collected filtrate and washings were treated three times with water and washed with anhydrous Na 2 S...

Embodiment 2

[0029] Fluorescent Probes and ONOO - Solution preparation

[0030] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in DMF to make 1×10 -3 M probe solution. ONOO - Solution preparation: 0.70M H 2 o 2 solution, 0.60M HCl solution, 0.60M NaNO 2 The solution was mixed, and 1.5M NaOH solution was added rapidly, the excess H 2 o 2 Remove with manganese dioxide and store in a freezer at -20°C. Thaw before use, ONOO - The determination of concentration needs to measure the absorbance A of the solution at 302nm, and the calculation formula is: C ONOO- =A / 1.67 (mM).

Embodiment 3

[0032] Fluorescent Probes and ONOO - Determination of the fluorescence spectrum of the action

[0033] figure 2 for fluorescent probes with ONOO - The fluorescence spectrum of the action, the concentration of the fluorescent probe is 5 μM, ONOO - The concentrations are 0, 5, 10, 15, 20, 25, 30, 35, 40, 50 μM in turn. The first excitation wavelength is 680nm, and the emission wavelength range is 690-750nm; the second excitation wavelength is 360nm, and the emission wavelength range is 420-520nm. The slit width is 5.0 nm / 5.0 nm, and the fluorescence measurement instrument used is a Hitachi F4600 fluorescence spectrophotometer. From figure 2 It can be seen that joining ONOO - Before, the fluorescent probe had a near-infrared (708nm) fluorescence emission peak; join ONOO - Afterwards, a blue emission peak appeared in the visible region (460nm). This is because the probe molecules are ONOO - Oxidation, resulting in cleavage, the smaller the conjugated structure, resultin...

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Abstract

The invention relates to preparation and application of a peroxide nitroso (ONOO<->) fluorescent probe based on a hemicyanine dye. A structural formula of the fluorescent probe is shown in the attached figure. The invention provides a preparation method of the fluorescent probe which is synthesized by adopting IR-780, 3-nitrophenol, stannous chloride and the like as raw materials. The fluorescentprobe is characterized in that the fluorescent probe is a ratio peroxide nitroso fluorescent probe with near-infrared effect and high selectivity; firstly, the fluorescent probe has higher sensitivityon ONOO<->, and the specific value of fluorescence intensity (F460 / F708) is enhanced by 153 times; then, the fluorescent probe has higher selectivity on ONOO<->, and is free from the interference byother active oxygen, active nitrogen, active sulfur and biological mercaptan; the fluorescent probe can quickly act with ONOO<->, and the response time is controlled within 80s; the fluorescent probehas been successfully applied into the study of cell imaging, and can be used for detecting the change of content of ONOO<-> in the cells.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, in particular to the preparation and application of peroxynitroso fluorescent probes based on semicyanine dyes. Background technique [0002] Peroxynitroso (ONOO - ) is an important reactive oxygen species, which is composed of nitric oxide (NO) and superoxide anion (O 2 . - ) reaction product (R. Radi, J. Biol. Chem., 2013, 288, 26464-26472). As a strong oxidizing agent and strong nucleophile, ONOO - It can react with a variety of biomolecules, such as proteins, lipids, nucleic acids, etc., and eventually lead to cell death (L. Liaudet, G. Vassalli, P. Pacher, Front. Biosci., 2009, 14, 4809-4814). And, ONOO - It can also participate in the signal transduction process as a signal molecule (B.C.Dickinson, C.J.Chang, Nat.Chem.Biol., 2011, 7, 504-511). In addition, abnormal levels of peroxynitroso are associated with some common diseases, such as cardiovascular disease, neurodegener...

Claims

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Application Information

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IPC IPC(8): C09B23/10C09K11/06G01N21/64
CPCC09B23/105C09K11/06C09K2211/1029C09K2211/1088G01N21/6486
Inventor 李春艳周东叶
Owner XIANGTAN UNIV
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