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CN-122010967-A - Organic fluorescent probe compound and preparation method and application thereof

CN122010967ACN 122010967 ACN122010967 ACN 122010967ACN-122010967-A

Abstract

The invention relates to the technical field of biological detection, in particular to an organic fluorescent probe compound and a preparation method and application thereof. The structural formula of the organic fluorescent probe compound is shown as a formula I, wherein n is selected from 0,2, 4 or 6, and the molecular structure of the organic fluorescent probe compound comprises a spiropyran mother nucleus skeleton and a recognition group, and the spiropyran mother nucleus skeleton and the recognition group are connected through a covalent bond. The organic fluorescent probe compound SP-Cta does not need to destroy the cell structure, avoids the interference of Cu 2+ complexation on CoA steady state, can truly reflect the natural steady state concentration of CoA, and the detection result is more attached to the physiological state of cells.

Inventors

  • ZHAO WENJIE
  • DU YANING
  • Liu Pantong

Assignees

  • 内蒙古大学

Dates

Publication Date
20260512
Application Date
20260202

Claims (10)

  1. 1. An organic fluorescent probe compound is characterized in that the structural formula of the organic fluorescent probe compound is shown as formula I: Wherein n is selected from 0, 2, 4 or 6; The molecular structure of the organic fluorescent probe compound comprises a spiropyran mother nucleus framework and a recognition group, wherein the spiropyran mother nucleus framework is connected with the recognition group through a covalent bond; the structural formula of the recognition group is 。
  2. 2. The organic fluorescent probe compound according to claim 1, wherein the organic fluorescent probe compound is prepared from 1',3',3' -trimethyl-6-nitrospiro [ chromene-2, 2' -indoline ] -5' -amine and The reaction is carried out, wherein, The preparation method comprises the following steps: 3, 6-dibromocarbazole and a compound containing a carboxylic acid group or triphosgene are subjected to substitution reaction in a dark and Ar gas atmosphere under an alkaline condition to obtain an intermediate product; Under the condition of nitrogen protection and no water and oxygen, the mixed solution of triethylamine and acetonitrile is used as a solvent, palladium (I) acetate and tri-o-tolylphosphine are used as catalysts, and under the condition of the existence of the solvent and the catalysts, the intermediate product and 2-vinyl-4, 6-diamino-3, 5-triazine are subjected to Heck coupling reaction to generate a vinyl bridged carbazole-triazine conjugated structure, so that the catalyst is obtained I.e., cta; the structural formula of the compound containing carboxylic acid group is When n in the carboxylic acid group-containing compound is 2, the carboxylic acid group-containing compound is ethyl bromohexanoate; n in the organic fluorescent probe compound is 4 when the carboxylic acid group-containing compound is ethyl 4-bromobutyrate, and n in the organic fluorescent probe compound is 6 when the carboxylic acid group-containing compound is ethyl 6-bromohexanoate; prepared by triphosgene Wherein n is 0.
  3. 3. The organic fluorescent probe compound according to claim 2, wherein the molar ratio of 3, 6-dibromocarbazole to the carboxylic acid group-containing compound is 17.31:28.56.
  4. 4. The organic fluorescent probe compound according to claim 2, wherein the substitution reaction conditions are stirring reaction for 48 to 50 hours at 80 to 85 ℃.
  5. 5. The organic fluorescent probe compound according to claim 2, wherein the molar ratio of the intermediate product to the 2-vinyl-4, 6-diamino-3, 5-triazine is 1.7:12.8.
  6. 6. The organic fluorescent probe compound according to claim 2, wherein the Heck coupling reaction is performed under conditions of condensation reflux reaction at 120 ℃ for 48-50 hours.
  7. 7. A method for preparing the organic fluorescent probe compound according to claim 1, comprising the steps of: Dropwise adding a mixed acid solution of fuming HNO 3 and concentrated H 2 SO 4 into a sulfuric acid solution of 3, 3-dimethyl-1 '-methyl-2-methyleneindole to perform a nitro substitution reaction, and then performing coprecipitation under alkaline conditions to obtain nitro-3, 3-dimethyl-1' -methyl-2-methyleneindole; Mixing nitro-3, 3-dimethyl-1 '-methyl-2-methyleneindole with a hydrochloric acid solution of SnCl 2 , and performing alkalization treatment to obtain amino-3, 3-dimethyl-1' -methyl-2-methyleneindole; The amino-3, 3-dimethyl-1 '-methyl-2-methylene indole and 1, 3-trimethyl-2-methylene indoline are dissolved in an alcohol solution together, and a first condensation reaction is carried out to obtain 1',3',3' -trimethyl-6-nitrospiro [ chromene-2, 2 '-indoline ] -5' -amine, namely SP; in the presence of trifluoromethanesulfonic acid trimethylsilane and pyridine as catalyst, under inert atmosphere, the dichloromethane solution of SP and Mixing, and performing a second condensation reaction to form a covalent bond to obtain an organic fluorescent probe compound; Wherein SP is in contact with The mass ratio of (2) is 12.8:27.6.
  8. 8. The method for preparing an organic fluorescent probe compound according to claim 7, wherein the condition of the second condensation reaction is stirring reaction at room temperature for at least 24 hours.
  9. 9. Use of an organic fluorescent probe compound according to claim 1 for detecting coenzyme a, characterized in that the method of application is: S1, using an organic fluorescent probe compound as a probe, dissolving the probe in an organic solvent, and adjusting the pH value to 7.0-7.6 to obtain a probe working solution; s2, respectively mixing the probe working solution with coenzyme A solutions with different known concentrations to obtain a mixed solution, and incubating the mixed solution for 1-10 min at room temperature; respectively generating different fluorescence signals at 675nm under 400nm excitation light, and obtaining different fluorescence intensity values; establishing a standard curve for detecting the concentration of the coenzyme A by taking coenzyme A solutions with different known concentrations as abscissa; S3, detecting the concentration of the coenzyme A of the actual sample, namely treating the detection target by taking the coenzyme A solution with unknown concentration as the detection target according to the same treatment method in the step S2, then performing fluorescence intensity test to obtain a corresponding fluorescence intensity value, substituting the obtained fluorescence intensity value into a standard curve of the detection of the concentration of the coenzyme A, and calculating to obtain the concentration of the corresponding coenzyme A solution.
  10. 10. The use according to claim 9, wherein the sample to be tested is a living cell in which the coa is located and which is selected from HEK293 cells and HeLa cells when the organic fluorescent probe compound is used for fluorescent imaging of steady state concentrations of coa.

Description

Organic fluorescent probe compound and preparation method and application thereof Technical Field The invention relates to the technical field of biological detection, in particular to an organic fluorescent probe compound and a preparation method and application thereof. Background Coenzyme a (Coenzyme A, coA) is a widely occurring cofactor that plays an important role in many enzymatic reactions and cellular physiological processes, such as the processes of the citrate cycle, sterol biosynthesis, amino acid metabolism, ketone body production and fatty acid metabolism. The change in CoA concentration not only regulates cellular metabolism, but also acts as a substrate allosteric modulator. Post-translational modification of histones and other proteins can control the fate of a variety of other proteins through acetyl-CoA (acyl-CoA). Thus, deregulation of CoA concentration levels can lead to various pathological changes in the body. Since Lipmann and its colleagues in the 50 th century of 19 discovered CoA, various methods of assaying CoA have been established. Traditional methods include thin layer chromatography, enzyme activity analysis, capillary electrophoresis, high Performance Liquid Chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS), and electrochemical methods. However, these methods all require lysis of the cells and consume large amounts of sample. While HPLC and LC-MS/MS enable simultaneous detection of multiple CoA species, they rely on expensive instrumentation and suffer from significant lack of sensitivity and selectivity without derivatization or radiolabeling. In recent years, nanomaterials such as gold nanoparticles (AuNPs), cadmium telluride quantum dots (CdTe QDs), carbon quantum dots (CDs), metal organic framework Materials (MOFs), and the like have been introduced into the CoA analysis field, pushing the establishment of new methods. However, such methods generally rely on the complexation of Cu 2+ with CoA during the assay, which can interfere with the steady state concentration of CoA in the system, and thus make it difficult to accurately determine its true steady state level. In addition, organic fluorescent probes based on SNAP-TAG are currently the only probe method for detecting CoA using fluorescent signals. However, the construction of this probe relies on the expression and purification of the protein, which not only results in high costs, but also limits its application in vivo imaging. Disclosure of Invention Aiming at the defects existing in the prior art, the invention provides an organic fluorescent probe compound, a preparation method and application thereof, and the invention constructs the organic fluorescent probe compound for CoA detection without Cu 2+ assistance, solves the problems of protein dependence, high cost and inapplicability to in-vivo imaging of SNAP-TAG probes based on protein design in the existing coenzyme A detection fluorescent probe, and overcomes the Cu 2+ dependence of the existing CoA detection nano fluorescent probe. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: A first object of the present invention is to provide an organic fluorescent probe compound having a structural formula: Wherein n is selected from 0, 2, 4 or 6, n is the number of the repeated units of-CH 2 -, and the larger the number of the repeated units of-CH 2 -, the larger the folding and deformation amplitude of the organic fluorescent probe compound molecule is, the better the response to CoA can be achieved. The molecular structure of the organic fluorescent probe compound comprises a spiropyran mother nucleus framework and a recognition group, wherein the spiropyran mother nucleus framework is connected with the recognition group through a covalent bond, and the recognition group has the structural formula of。 Preferably, the organic fluorescent probe compound is prepared from 1',3',3' -trimethyl-6-nitrospiro [ chromene-2, 2' -indolidin ] -5' -amine andThe reaction is carried out, wherein,The synthetic route of (2) is as follows: The preparation method comprises the following steps: and in the dark and Ar gas atmosphere, carrying out substitution reaction on the 3, 6-dibromocarbazole and a compound containing a carboxylic acid group or triphosgene under an alkaline condition to obtain an intermediate product. Under the condition of nitrogen protection and no water and oxygen, the mixed solution of triethylamine and acetonitrile is used as a solvent, palladium (I) acetate and tri-o-tolylphosphine are used as catalysts, and under the condition of the existence of the solvent and the catalysts, the intermediate product and 2-vinyl-4, 6-diamino-3, 5-triazine are subjected to Heck coupling reaction to generate a vinyl bridged carbazole-triazine conjugated structure, so that the catalyst is obtainedI.e. Cta. Wherein the structural formula of the compound containing carboxylic acid group isWherein