CN-122010890-A - Organic fluorescent probe for detecting hypoxia, preparation method and application

Abstract

The invention relates to the technical field of organic micromolecular fluorescent probes, in particular to an organic fluorescent probe for detecting hypoxia, a preparation method and application thereof, wherein the organic fluorescent probe is a compound DCM-Azo-pNO 2 , and the structure of the organic fluorescent probe is shown as a formula (I). The invention is characterized in that the electrophilic property and bond dissociation energy of azo bonds are obviously enhanced by introducing a strong electron-withdrawing substituent-NO 2 on azo aromatic hydrocarbon, so that the azo aromatic hydrocarbon can be quickly and efficiently reduced and broken by azo reductase. Under the anaerobic environment, the azo bond of the organic fluorescent probe is specifically reduced, the intramolecular charge transfer effect is recovered, and the obvious change of fluorescence from off to on is realized. Compared with the traditional probe, the fluorescence enhancement multiple of the organic fluorescence probe is up to 690 times, the fluorescence enhancement multiple of the organic fluorescence probe is improved by 3-6 times, the quenching efficiency is greatly improved, the detection limit of the simulated enzyme SDT is reduced to 0.12 mu M, and the detection sensitivity is greatly improved.

Inventors

• ZHAO WENJIE
• Bai Linghe
• ZHAO XIN
• JIN HUIYING
• ZHANG ZHENQI

Assignees

• 内蒙古大学

Dates

Publication Date

20260512

Application Date

20260202

Claims (8)

1. 1. The organic fluorescent probe for detecting hypoxia is characterized in that the organic fluorescent probe is a compound DCM-Azo-pNO 2 , and the structure of the organic fluorescent probe is shown as a formula (I): Formula (I).
2. 2. The method for preparing an organic fluorescent probe according to claim 1, comprising the steps of: (1) Taking tetrahydrofuran as a solvent, and carrying out condensation reaction on 2' -hydroxyacetophenone and ethyl acetate under the action of sodium hydride to obtain a compound 1; (2) Taking acetic acid as a solvent, and carrying out catalytic ring-closure reaction on the compound 1 and sulfuric acid to obtain a compound 2; (3) Taking acetic anhydride as a solvent, and carrying out Knoevenagel condensation reaction on the compound 2 and malononitrile to obtain a compound 3; (4) In a nitrogen atmosphere, toluene is used as a solvent, and the compound 3 and acetamidobenzaldehyde are subjected to condensation reaction under the catalysis of piperidine and acetic acid to obtain a fluorophore DCM-NH 2 ; (5) Performing oxidation reaction on 4-nitroaniline and potassium peroxymonosulfate in a nitrogen atmosphere by taking dichloromethane and water as solvents to obtain a compound 4, wherein the mass ratio of the 4-nitroaniline to the potassium peroxymonosulfate is 0.2:1.74; (6) Diazotizing and coupling the compound 4 with a fluorophore DCM-NH 2 by taking acetic acid as a solvent to obtain DCM-Azo-pNO 2 , wherein the mass ratio of the compound 4 to the fluorophore DCM-NH 2 is 60:63.8; 。
3. 3. The method of claim 2, wherein in step (6), the diazotisation coupling reaction is carried out at a temperature of 40 ℃ for a period of 12 hours.
4. 4. Use of the organic fluorescent probe according to claim 1 for preparing a preparation for detecting hypoxia.
5. 5. The use according to claim 4, wherein the formulation is for detecting a hypoxic state at the cellular level.
6. 6. The use according to claim 5, wherein the cells are HeLa cells.
7. 7. The use of claim 4, wherein the formulation is used to detect the hypoxic state of tissue levels.
8. 8. The use of claim 7, wherein the tissue is tumor tissue.

Description

Organic fluorescent probe for detecting hypoxia, preparation method and application Technical Field The invention relates to the technical field of organic small molecule fluorescent probes, in particular to an organic fluorescent probe for detecting hypoxia, a preparation method and application thereof. Background The anoxic microenvironment of the solid tumor is a key index for evaluating the malignancy of the solid tumor, predicting the chemoradiotherapy resistance and guiding the personalized treatment. Therefore, development of a technology capable of accurately and rapidly detecting hypoxia has important clinical significance. Currently, there are various technical means for hypoxia detection. Optical imaging is a research hotspot because of its non-radiation, low cost, and direct coupling with commercial hand-held/endoscopic devices. Previous studies have utilized azo reductase (AzoR) to cleave-n=n-bonds to construct fluorescent probes. However, the probes adopt symmetrical or weak electron-donating/electron-withdrawing substituted azo frameworks, so that the problem of low quenching efficiency exists, and the fluorescence enhancement times before and after hypoxia are only 3-6 times. Therefore, there is a need to develop an organic fluorescent probe for detecting hypoxia with high quenching efficiency. Disclosure of Invention In order to solve the problems, the invention provides an organic fluorescent probe for detecting hypoxia, a preparation method and application thereof. The invention is realized by the following technical scheme: An organic fluorescent probe for detecting hypoxia, which is a compound DCM-Azo-pNO 2, and has a structure shown in a formula (I): Formula (I). The preparation method of the organic fluorescent probe comprises the following steps: (1) Tetrahydrofuran is used as a solvent, and 2' -hydroxyacetophenone and ethyl acetate are subjected to condensation reaction under the action of sodium hydride to obtain a compound 1. (2) And (3) taking acetic acid as a solvent, and carrying out catalytic ring-closure reaction on the compound 1 and sulfuric acid to obtain a compound 2. (3) And (3) carrying out Knoevenagel condensation reaction on the compound 2 and malononitrile by taking acetic anhydride as a solvent to obtain a compound 3. (4) And (3) in a nitrogen atmosphere, using toluene as a solvent, and carrying out condensation reaction on the compound 3 and acetamidobenzaldehyde under the catalysis of piperidine and acetic acid to obtain a fluorophore DCM-NH 2. (5) And (3) oxidizing 4-nitroaniline and potassium peroxymonosulfate in a nitrogen atmosphere by taking methylene dichloride and water as solvents to obtain a compound 4, wherein the mass ratio of the 4-nitroaniline to the potassium peroxymonosulfate is 0.2:1.74. (6) And (3) diazotizing and coupling the compound 4 with a fluorophore DCM-NH 2 by taking acetic acid as a solvent to obtain DCM-Azo-pNO 2, wherein the mass ratio of the compound 4 to the fluorophore DCM-NH 2 is 60:63.8. 。 Preferably, in step (6), the diazotisation coupling reaction is carried out at a temperature of 40 ℃ for a period of 12 hours. The application of the organic fluorescent probe in preparation of preparations for detecting hypoxia. Preferably, the formulation is used to detect the hypoxic state at the cellular level. Preferably, the cells are HeLa cells. Preferably, the formulation is used to detect the hypoxic state of tissue levels. Preferably, the tissue is a tumor tissue. Compared with the prior art, the invention has the following beneficial effects: The invention provides an organic fluorescent probe for detecting hypoxia, which is a compound DCM-Azo-pNO 2, and has a structure shown in a formula (I). The invention is characterized in that the electrophilic property and bond dissociation energy of azo bonds are obviously enhanced by introducing a strong electron-withdrawing substituent-NO 2 on azo aromatic hydrocarbon, so that the azo aromatic hydrocarbon can be quickly and efficiently reduced and broken by azo reductase. Under the anaerobic environment, the azo bond of the organic fluorescent probe is specifically reduced, the intramolecular charge transfer effect is recovered, and the obvious change of fluorescence from off to on is realized. Experiments prove that the fluorescence enhancement multiple of the organic fluorescent probe is up to 690 times, compared with the traditional probe, the fluorescence enhancement multiple of the organic fluorescent probe is only 3-6 times, the quenching efficiency is greatly improved, the detection limit of the simulated enzyme SDT is as low as 0.12 mu M, and the detection sensitivity is greatly improved. In addition, the kit shows high specific response to the azo reductase, but hardly responds to other common ions, reducing substances and other interferents in organisms, so that the accuracy and reliability of the detection result are ensured. The invention is proved in a cell model and a mouse ische