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Self-immobilized micromolecular fluorescent probe as well as preparation method and application thereof

A technology of fluorescent probes and small molecules, which is applied in the fields of fluorescence/phosphorescence, chemical instruments and methods, and luminescent materials, etc. It can solve the problems that the fluorescent signal cannot be retained and in-situ detection cannot be realized.

Pending Publication Date: 2022-05-13
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical fluorescent probes have the function of real-time, efficient and high-resolution detection of nitroreductase activity, but the probes reported so far have certain limitations, including the rapid diffusion of probe molecules after reacting with enzymes, and the inability to realize the original bit detection, and the fluorescent signal cannot be retained after the sample is fixed

Method used

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  • Self-immobilized micromolecular fluorescent probe as well as preparation method and application thereof
  • Self-immobilized micromolecular fluorescent probe as well as preparation method and application thereof
  • Self-immobilized micromolecular fluorescent probe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, the synthesis of compound (1-1)

[0032]

[0033] 1. Synthesis of compound (2-3)

[0034] 4-Hydroxyisophthalaldehyde (2-1, 426 mg, 2.8 mmol) and p-nitrobenzyl bromide (2-2, 500 mg, 2.34 mmol) were dissolved in 10 mL of N,N-dimethylformamide (DMF ), followed by adding 415mg of potassium carbonate, stirring and mixing, adding 50mL of water and filtering (the filter membrane adopts ordinary qualitative filter paper), and drying the filter cake at 50°C for 24h to obtain 533mg of the product, namely the compound shown in formula (2-3). Rate 80%.

[0035] H NMR spectrum see figure 1 shown.

[0036] 1 HNMR(600MHz,DMSO)δ10.49(s,1H),9.98(s,1H),8.28-8.30(m,3H),8.19(d,J=8.7Hz,1H),7.84(d,J=8.6 Hz,2H),7.52(d,J=8.7Hz,1H),5.60(s,2H).

[0037] 2. Synthesis of compound (2-4)

[0038]

[0039] At room temperature, the compound represented by formula (2-3) (50 mg, 0.17 mmol) was dissolved in a mixed solvent (dichloromethane / methanol=8:1, v / v, 10 mL), and placed ...

Embodiment 2

[0046] Embodiment 2, the synthesis of compound (I)

[0047]

[0048] 1-Ethyl-2,3,3-trimethylindolium iodide (1-2, 9mg, 0.05mmol) and the compound shown in formula (1-1) prepared in Example 1 (15mg, 0.05mmol ) was dissolved in 4mL of ethanol, then sodium acetate (38mg, 0.46mmol) was added, reacted at room temperature for 4 hours, and the reactant was carried out through a silica gel chromatography column (column height 35cm, diameter 305nm, column height 10cm, silica gel particle size 300 mesh) Separation, using dichloromethane / methanol=80:1 (v / v) as the eluent, the elution rate is 0.3mL / s, eluting 5 column volumes, using dichloromethane / methanol with a volume ratio of 30:1 Thin-layer chromatography monitoring was carried out as a developing agent, and the effluents with Rf of 0.2-0.3 were combined, evaporated under reduced pressure to remove the eluent, and then dried in vacuo at 40°C to obtain 5 mg of the product, which was the small molecule probe shown in formula (I). F...

Embodiment 3

[0051] Example 3, Evaluation of the specific labeling effect of probe FY on Escherichia coli nitroreductase

[0052] 1. Nitroreductase (NTR)

[0053] References (JingBai, Altering the Regioselectivity of a Nitroreductase in the Synthesis of Arylhydroxylamines by Structure-Based Engineering, ChemBioChem, 2015, 16, 1219-1225.), using a nitroreductase-containing Escherichia coli strain to obtain liquid nitroreductase , the protein content is 1.8mg / mL, the specific expression and purification process of nitroreductase is as follows:

[0054] (1) Cultivation of nitroreductase strain

[0055] LB liquid medium formula (take 1L as an example): peptone 10g, yeast extract 5g, sodium chloride 5g, add ultrapure water to 1L.

[0056] LB solid medium formula (taking 1L as an example): peptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, ultrapure water to 1L.

[0057] Streak the plate to obtain a single colony: add Kana (kanamycin) to the sterilized LB solid medium at a vo...

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Abstract

The invention discloses a self-immobilized micromolecular fluorescent probe as well as a preparation method and application thereof. The probe can be used for carrying out in-situ efficient and selective detection and imaging on overexpressed nitroreductase in hypoxic or hypoxic cells. And a chemical tool is provided for exploring the distribution of nitroreductase in cells in an anoxic state and solid tumor hypoxia imaging.

Description

(1) Technical field [0001] The invention relates to a small molecule fluorescent probe and its preparation method and application, in particular to a self-immobilized small molecule fluorescent probe triggered by nitroreductase and its preparation method and application. (2) Background technology [0002] Hypoxia is an important feature of tumors, mainly caused by insufficient oxygen supply and increased oxygen consumption in tumors. In addition to tumor tissue, hypoxia is also closely related to some chronic diseases, such as intracellular infectious diseases, coronary and peripheral artery diseases, alcoholic liver injury, and gastrointestinal inflammation. Therefore, efficient real-time and accurate detection of hypoxia has important clinical significance. Studies have shown that the content of nitroreductase in cells, tissues and tumors under hypoxic conditions is significantly higher than that under normal conditions. Therefore, the expression abundance of nitroreduct...

Claims

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Application Information

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IPC IPC(8): C07D209/12C09K11/06G01N21/64
CPCC07D209/12C09K11/06G01N21/6428G01N21/6458C09K2211/1007C09K2211/1029
Inventor 葛璟燕檀维王书艺郎文杰
Owner ZHEJIANG UNIV OF TECH
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