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Fluorescent probe for detecting biological mercaptan in lysosome and preparation method and application of fluorescent probe

A technology of fluorescent probes and biothiols, which is applied in the field of analytical chemistry to achieve the effects of low preparation cost, fast response and easy promotion

Inactive Publication Date: 2020-04-28
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the current lack of fluorescent probes for detecting endogenous biothiols at the organelle level, the present invention provides a fluorescent probe for detecting biothiols in lysosomes, which has fast response speed and strong anti-interference ability

Method used

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  • Fluorescent probe for detecting biological mercaptan in lysosome and preparation method and application of fluorescent probe
  • Fluorescent probe for detecting biological mercaptan in lysosome and preparation method and application of fluorescent probe
  • Fluorescent probe for detecting biological mercaptan in lysosome and preparation method and application of fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Synthesis of Fluorescent Probe CM-NBD

[0034] (1) Compound a1 (1 mmol), a2 (1 mmol), 1-hydroxybenzotriazole (0.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide ( 3 mmol) into a 25 mL single-necked flask, dissolved with N,N-dimethylformamide (5 mL), stirred at room temperature for 10 min, and the catalyst N,N-diisopropylethylamine (0.1 mL) was slowly Add dropwise into the flask and stir at room temperature for 5h. After the reaction, use water-dichloromethane to extract, and the compound (I) obtained after the lower organic phase is spin-dried:

[0035] ;

[0036] (2) Compound (I) (0.36 mmol) was stirred with a3 (0.36 mmol) and potassium carbonate (0.3 mmol) in N,N-dimethylformamide (3 mL) at room temperature for 5 h. After the reaction was complete, filter , the filtrate was spin-dried by distillation under reduced pressure, and purified by column chromatography with dichloromethane:methanol (v / v)=25:1 as the chromatographic solution to obtain a ye...

Embodiment 2

[0039] Example 2 Fluorescence spectra of fluorescent probe CM-NBD ​​under different biothiols

[0040] Prepare 1 mM DMSO mother solution of the fluorescent probe CM-NBD ​​obtained in Example 1 for future use. Prepare 5 parts of 2 mL PBS buffer solution (containing 20% ​​DMSO, pH=5) in advance, add 20 μL of probe mother solution, and add 20 μL of 10 mM biothiol (Cys, Hcy, GSH, Na 2 S). Fluorescence detection is then performed (λ ex =405 nm and 470 nm), calculate the fluorescence intensity in each system, the results are as follows figure 2 shown. Depend on figure 2 It can be seen that in the presence of Cys and Hcy, the probe produces two different fluorescence emissions at 465 nm and 565 nm under the excitation of two independent wavelengths. However, adding Na to the probe 2 S and GSH will only fluoresce blue at 465 nm. It indicated that the fluorescent probe CM-NBD ​​could be used to detect biothiols.

Embodiment 3

[0041] Example 3 Selectivity of Fluorescent Probe CM-NBD

[0042] Prepare 1 mM DMSO mother solution of the fluorescent probe CM-NBD ​​obtained in Example 1 for future use. Prepare 28 copies of 2 mL fluorescent probe CM-NBD ​​buffer solution (containing 20% ​​DMSO, pH=5) obtained in Example 1 in advance, add 20 μL of the probe mother solution to each, and then add 20 μL of the concentration of 10 mM AlCl 3 , BaCl 2 , CaCl 2 , CuCl 2 、CuSO 4 , MgCl 2 , FeCl 3, SnCl 2 , FeCl 2 , ZnCl 2 , KCl 2 、KNO 3 , NaSCN, H 2 o 2 , HClO, Na 2 SO 4 , NaBr, NaCl 2 , NaClO, NaF, NaI, NaNO 2 、NaNO 3 , Cys, Hcy, GSH, Na 2 S in PBS solution. Fluorescence detection is then performed (λ ex =405 nm and 470 nm); calculate the fluorescence intensity in each system, as shown in 3. Depend on image 3 It can be seen that the selectivity of the fluorescent probe to biothiol is much higher than that of other substances, indicating that the fluorescent probe has the characteristic of sp...

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Abstract

The invention provides a fluorescent probe for detecting biological mercaptan in lysosome. The chemical structural formula of the fluorescent probe is shown in the specification. According to the probe, in the presence of Cys and Hcy, the probe generates two different fluorescence emissions of blue and green at 480nm and 550nm respectively under the excitation of two independent wavelengths. However, when GSH and Na2S are added into the probe, blue fluorescence can only be generated at 480 nm. The difference can be reasonably attributed to the fact that the NBD-GSH / SH intermediate is differentfrom the NBD Cys / Hcy, and an intramolecular cyclization rearrangement reaction cannot occur. Meanwhile, due to the fact that morpholine has lysosome targeting, lysosome can be distinguished from other organelles. The fluorescent probe has the advantages of good lysosome targeting, strong specificity, fast response and the like and can be used for real-time visual determination of biological mercaptan in living cell lysosome, and can be obtained through chemical synthesis and has the advantages of simple and feasible synthesis process, cheap and easily available raw materials, low preparationcost and easiness in popularization.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a fluorescent probe for detecting biothiols in cell lysosomes and an application thereof. Background technique [0002] Reactive sulfur species (RSS), including small molecular biothiols and hydrogen sulfide, are a class of bioactive molecules that play important physiological roles in the human body. Cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH) are the three most common small molecule biothiols. They are closely related and have essential differences and pharmacological effects in many biological processes. Hydrogen sulfide is one of the simplest biological thiols, a toxic gas with an unpleasant smell, and has been discovered together with carbon monoxide and nitric oxide as an important small molecule for signal transduction. Cys is the precursor of glutathione, which can form iron sulfide complexes with iron ions. However, abnormal l...

Claims

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Application Information

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IPC IPC(8): C07D413/12C09K11/06G01N21/64
CPCC07D413/12C09K11/06G01N21/6486C09K2211/1048C09K2211/1033C09K2211/1088
Inventor 林伟英景新颖于法祺
Owner UNIV OF JINAN
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