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Fluorescent probe with double response to viscosity and nitroreductase as well as preparation method and application of fluorescent probe

A technology of fluorescent probes and reductases, applied in chemical instruments and methods, biochemical equipment and methods, luminescent materials, etc., to achieve the effect of simple synthesis and easy application

Pending Publication Date: 2022-05-06
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Studies have shown that small molecule fluorescent probes have a wide range of applications in the detection of the intracellular microenvironment due to their high sensitivity, real-time analysis, and non-invasive detection; Probes have hardly been reported. Therefore, the development of new fluorescent probes capable of dual response viscosity and nitroreductase has good application value.

Method used

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  • Fluorescent probe with double response to viscosity and nitroreductase as well as preparation method and application of fluorescent probe
  • Fluorescent probe with double response to viscosity and nitroreductase as well as preparation method and application of fluorescent probe
  • Fluorescent probe with double response to viscosity and nitroreductase as well as preparation method and application of fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the synthetic preparation of probe M440

[0028]

[0029] Synthesis of compound 1: add anhydrous DMF (10.0mL, 130.4mmol) and potassium tert-butoxide (2.4g, 21.3mmol) in a 50mL round bottom flask, under nitrogen protection, reflux and stir at 60°C for 7 hours, then cool to At room temperature, the reaction solution was poured into ice water, a yellow solid precipitated out, filtered, then washed three times with methanol / water (V:V=1:1), and dried to obtain compound 1 as a yellow solid. 1 HNMR (400MHz, DMSO-d6) δ8.50(d, J=4.9Hz, 2H), 7.44(d, J=8.7Hz, 2H), 7.34(d, J=5.9Hz, 2H), 7.26(d, J=16.3Hz, 1H), 6.80(d, J=16.2Hz, 1H), 6.71(d, J=8.8Hz, 2H), 3.03(s, 6H). 13 C NMR (100MHz, DMSO-d6) δ145.51, 143.77, 140.95, 128.66, 124.69, 123.08, 118.70, 115.26, 106.79, 34.91;

[0030] Synthetic preparation of probe M440: Add 20.0mL of anhydrous acetonitrile, compound 1 (1.122g, 5.0mmol) and p-nitrobenzyl bromide (1.082g, 5.0mmol) in a 50mL two-necked flask, and then ...

Embodiment 2

[0031] Example 2: Response of Probe M440 to Viscosity in Solution

[0032] The probe M440 in Example 1 was dissolved in DMSO to prepare a stock solution with a concentration of 1.0 mM. Take 10 μL of the above mother liquor and add 1 mL of different volume ratios (methanol:glycerol=90:10; 80:20; 70:30; 60:40; 50:50; 40:60; 30:70; 20:80; 10:90 ,0:100) in the methanol / glycerol system, and then perform fluorescence detection, the results are as follows image 3 As shown, with the increase of glycerol volume specific gravity, the fluorescence intensity of the probe gradually increases, indicating that the probe M440 can have a good response relationship to the viscosity in the glycerol system.

Embodiment 3

[0033] Example 3: Response of probe M440 to nitroreductase in solution

[0034] Prepare the probe M440 in Example 1 with a final concentration of 10 μM. In the presence of 500 μM NADPH, add different concentrations (0-5.0 μg / mL) of nitroreductase and react fully at 37°C for 60 minutes before performing fluorescence detection. ,Such as Figure 4 As shown, it can be observed that the fluorescence intensity of the emission peak near 520nm is continuously enhanced, indicating that the probe M440 can have a good response relationship to nitroreductase in the solution system.

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Abstract

The invention belongs to the technical field of organic small molecule fluorescent probes, and particularly relates to a fluorescent probe with double responses to viscosity and nitroreductase as well as a preparation method and application of the fluorescent probe. The preparation method comprises the following steps: adding anhydrous DMF (Dimethyl Formamide) and potassium tert-butoxide, refluxing in nitrogen, cooling to room temperature, pouring reaction liquid into ice water, filtering, washing and drying to obtain a compound 1; and adding anhydrous acetonitrile, a compound 1 and p-nitrobenzyl bromide, refluxing under the protection of N2, cooling to room temperature, filtering, and purifying by column chromatography to obtain the probe. The probe is remarkably sensitive to the viscosity change in a solution and the detection of nitroreductase, and the fluorescence intensity of the probe at 610nm is remarkably enhanced along with the increase of the viscosity of the solution; under the participation of NADPH, along with the increase of the concentration of nitroreductase, the probe M440 is reduced into a compound 1 by nitroreductase, and the fluorescence of the compound 1 at 520 nm is obviously enhanced; cell experiments show that the probe can be well positioned in mitochondria, and the change of mitochondrial viscosity in cells and the generation of nitroreductase under an anoxic condition are detected.

Description

technical field [0001] The invention belongs to the technical field of small organic molecule fluorescent probes, and in particular relates to a fluorescent probe double-responsive to viscosity and nitroreductase, a preparation method and application thereof. Background technique [0002] The prior art discloses that the viscosity of mitochondria in cells is an important influencing factor for material exchange, signal transmission and interaction between intracellular biomolecules, and abnormal viscosity is used to judge diseases such as cell malignancies, Alzheimer's disease, diabetes and atherosclerosis. reference index. Studies have disclosed that when the oxygen concentration in cells is insufficient, it will lead to cellular hypoxia, and hypoxia is one of the characteristics of tumor tissue. Studies have shown that cells or tissues will overexpress nitroreductase under hypoxic conditions, and nitroreductase It can be used as a common marker of hypoxia in tumor tissue....

Claims

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Application Information

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IPC IPC(8): C07D213/38C09K11/06C12Q1/02C12Q1/26
CPCC07D213/38C09K11/06G01N33/5005C12Q1/26C09K2211/1007C09K2211/1029
Inventor 张炜佳黄晖唐美麟
Owner FUDAN UNIV
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