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CN-121990985-A - Double-response small molecule fluorescent probe for synchronously detecting NTR and nucleic acid and preparation and application thereof

CN121990985ACN 121990985 ACN121990985 ACN 121990985ACN-121990985-A

Abstract

The invention discloses a double-response small molecular fluorescent probe for synchronously detecting NTR and nucleic acid, and preparation and application thereof, wherein the preparation method comprises the steps of adding 4-fluoro-3-methoxybenzaldehyde, potassium carbonate and methylamine hydrochloride into ethanol, and carrying out reflux reaction to obtain an intermediate NB1; under the protection of nitrogen, NB1, 4-methylpyridine and potassium tert-butoxide are dissolved in N, N-dimethylformamide, heating and stirring are carried out to obtain an intermediate NB2, under the protection of nitrogen, NB2, (4-nitrophenyl) methanesulfonyl chloride and triethylamine are dissolved in dichloromethane to obtain an intermediate NBP after reaction, under the protection of nitrogen, NBP, methyl iodide and triethylamine are dissolved in dichloromethane, and stirring is carried out at room temperature until the reaction is completed to obtain the target product double-response micromolecule fluorescent probe. The invention can synchronously and visually detect NTR activity and nucleic acid state in biological samples, and provides a more reliable technical scheme for tumor accurate detection and mechanism research.

Inventors

  • DU WEI
  • ZHAO TINGTING
  • WANG BAOXIN

Assignees

  • 安徽医科大学

Dates

Publication Date
20260508
Application Date
20260202

Claims (10)

  1. 1. A dual-response small molecule fluorescent probe for synchronously detecting NTR and nucleic acid is characterized by comprising the following structural formula: 。
  2. 2. A method for preparing a dual-response small molecule fluorescent probe for simultaneous detection of NTR and nucleic acid according to claim 1, comprising the steps of: S1, adding 4-fluoro-3-methoxybenzaldehyde, potassium carbonate and methylamine hydrochloride into ethanol, carrying out reflux reaction, and obtaining an intermediate NB1 through extraction, purification and drying; S2, under the protection of nitrogen, dissolving NB1, 4-methylpyridine and potassium tert-butoxide in N, N-dimethylformamide, heating and stirring for reaction, and extracting, purifying and drying to obtain an intermediate NB2; s3, under the protection of nitrogen, dissolving NB2, (4-nitrophenyl) methanesulfonyl chloride and triethylamine in dichloromethane, and after the reaction, purifying and drying to obtain an intermediate NBP; S4, under the protection of nitrogen, dissolving NBP, methyl iodide and triethylamine in dichloromethane, stirring at room temperature until the reaction is completed, purifying and drying to obtain the target product, namely the double-response small-molecule fluorescent probe.
  3. 3. The method for preparing a dual-response small molecular fluorescent probe for synchronously detecting NTR and nucleic acid according to claim 2, wherein in the step S1, the molar ratio of 4-fluoro-3-methoxybenzaldehyde, potassium carbonate and methylamine hydrochloride is (0.3-1): (1.5-3): 1, and/or the reflux reaction temperature is 90-110 ℃ and the reaction time is 6-8 d.
  4. 4. The method of claim 2, wherein in the step S2, the molar ratio of NB1, 4-methylpyridine to potassium tert-butoxide is (0.5-1.5): 5, and/or the reaction temperature is 70-90 ℃ and the reaction time is 1-3 h.
  5. 5. The method for preparing a dual-response small molecule fluorescent probe for simultaneously detecting NTR and nucleic acid according to claim 2, wherein in the step S3, the molar ratio of NB2, (4-nitrophenyl) methanesulfonyl chloride to triethylamine is (0.5-1.5): 1-2): 5, and/or the reaction temperature is 0 ℃ and the reaction time is 3-5 h.
  6. 6. The method for preparing a dual-response small molecule fluorescent probe for simultaneously detecting NTR and nucleic acid according to claim 2, wherein in the step S4, the molar ratio of NBP, methyl iodide and triethylamine is (0.5-1.5): 1-2): 5, and/or the reaction temperature is 20-30 ℃ and the reaction time is 10-15 h.
  7. 7. Use of a dual-response small molecule fluorescent probe according to claim 1 or the method of any one of claims 2-6 for the preparation of a dual-response detection kit for simultaneous detection of NTR activity and nucleic acid status in a biological sample.
  8. 8. The use according to claim 7, wherein the dual-response small molecule fluorescent probe achieves synchronous detection by a dual-channel fluorescent signal, wherein the dual-response small molecule fluorescent probe generates green fluorescence after acting with NTR, has an emission peak of 520-530nm, and generates red fluorescence after binding with nucleic acid, wherein the emission peak of binding with DNA is 605-615nm, and the emission peak of binding with RNA is 603-613nm.
  9. 9. The method of claim 7, wherein the biological sample comprises cells, animal tumor tissue, or human tumor tissue, and the nucleic acid comprises DNA and/or RNA.
  10. 10. A dual response detection kit, comprising the dual response small molecule fluorescent probe of claim 1 or the dual response small molecule fluorescent probe prepared by the preparation method of any one of claims 2-6, wherein the dual response detection kit is used for synchronously detecting the activity of NTR and the state of nucleic acid.

Description

Double-response small molecule fluorescent probe for synchronously detecting NTR and nucleic acid and preparation and application thereof Technical Field The invention relates to a double-response small molecular fluorescent probe for synchronously detecting NTR and nucleic acid, and preparation and application thereof, belonging to the field of biological detection technology and fluorescent probes. Background Cancer is a malignant disease seriously threatening the life health of human beings, hypoxia is one of the core characteristics of the microenvironment of solid tumors, the environment can obviously induce the abnormally high expression of Nitroreductase (NTR), and NTR is used as a key functional enzyme for playing an anticancer role of Hypoxia Activated Prodrug (HAP), and the key action mechanism of a hypoxia activated prodrug treatment system is formed by the prodrug activation process mediated by the NTR and the DNA damage effect caused by cytotoxic metabolites after activation. Therefore, the dynamic association of the two key processes is synchronously monitored, and the dynamic association is a core premise for realizing accurate tumor treatment and assessing the curative effect of the prodrug. However, the existing detection technology has a plurality of difficult-to-break limitations that firstly, the traditional detection means such as High Performance Liquid Chromatography (HPLC), immunoblotting (Western blot) and the like can only realize the quantitative analysis of the end point of a target molecule, the dynamic association process of NTR activation and DNA damage cannot be traced in real time, secondly, the DNA damage detection technologies such as gamma-H2 AX immunofluorescence staining, comet experiments and the like are mutually independent of an NTR-mediated prodrug activation detection system, the two technologies lack of matching in space-time dimension, and direct causal association is difficult to establish, thirdly, the currently reported fluorescent probe detection tools are single target response type, only can specifically identify NTR activity or only target bound nucleic acid, and double monitoring of NTR activity and nucleic acid state cannot be synchronously realized in the same detection system. The prior patent relates to a near infrared two-photon dual-mode linkage response NTR & Cys fluorescent probe, a preparation method and application thereof (CN 116675636B), wherein NTR+Cys is used as a dual-response target point, the dual-response is dependent on chemical conversion reaction among small molecules, NTR catalyzes nitro reduction to amino, cys and the probe to generate nucleophilic addition/cyclization reaction, the detection mode driven by the dual-chemical reaction is realized, cys is used as a pan metabolite, and cross interference of thiol substances such as homocysteine, glutathione and the like in a living body is easy to occur. Disclosure of Invention Aiming at the problems in the prior art, the invention provides a dual-response small molecular fluorescent probe for synchronously detecting NTR and nucleic acid, and preparation and application thereof, has the characteristics of high sensitivity, high specificity and high spatial resolution, can synchronously and visually detect NTR activity and nucleic acid state in biological samples, and is suitable for tumor diagnosis and prodrug curative effect evaluation. In order to achieve the above purpose, the dual-response small molecule fluorescent probe for synchronously detecting NTR and nucleic acid has the following structural formula: 。 The invention also provides a preparation method of the dual-response small molecule fluorescent probe for synchronously detecting NTR and nucleic acid, which comprises the following steps: S1, adding 4-fluoro-3-methoxybenzaldehyde, potassium carbonate and methylamine hydrochloride into ethanol, carrying out reflux reaction, and obtaining an intermediate NB1 through extraction, purification and drying; S2, under the protection of nitrogen, dissolving NB1, 4-methylpyridine and potassium tert-butoxide in N, N-dimethylformamide, heating and stirring for reaction, and extracting, purifying and drying to obtain an intermediate NB2; s3, under the protection of nitrogen, dissolving NB2, (4-nitrophenyl) methanesulfonyl chloride and triethylamine in dichloromethane, and after the reaction, purifying and drying to obtain an intermediate NBP; S4, under the protection of nitrogen, dissolving NBP, methyl iodide and triethylamine in dichloromethane, stirring at room temperature until the reaction is completed, purifying and drying to obtain the target product, namely the double-response small-molecule fluorescent probe. As a modification, in the step S1, the molar ratio of 4-fluoro-3-methoxybenzaldehyde to potassium carbonate to methylamine hydrochloride is (0.3-1): (1.5-3): 1, and/or the reflux reaction temperature is 90-110 ℃ and the reaction time is 6-8 d. As a modification, in the step