CN-122010996-A - Preparation method and application of dual-response fluorescent probe for detecting polarity and peroxynitrite

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and property research of a dual-response fluorescent probe for detecting polarity and peroxynitrite. The preparation method of the fluorescent probe is simple, the product yield is high, and the fluorescent probe is suitable for large-scale popularization and use. The probe (Z) -4- (1-cyano-2- (6- (4- (diphenylamino) phenyl) -4-phenyl-4H-dithieno [3,2-b:2',3' -d ] pyrrol-2-yl) vinyl) -1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzyl) pyridin-1-ium bromide (DTBP). The fluorescence intensity is gradually increased with the decrease of the polarity of the solution at 600 nm excitation, and the probe reacts with ONOO − to generate strong fluorescence at 470 nm excitation in an equal proportion of PBS and acetone solvent. As the concentration of ONOO − increases, the fluorescence intensity increases gradually. The probe can specifically detect ONOO − and has good linear relation. The probe also has stronger fluorescence response to the polarity, and the fluorescence emission intensity gradually rises along with the decrease of the polarity.

Inventors

• LI ZHANXIAN
• GAO SIYU
• MA WANLING
• YU MINGMING

Assignees

• 郑州大学

Dates

Publication Date

20260512

Application Date

20260206

Claims (2)

1. 1. A dual response fluorescent probe for detecting polarity and peroxynitrite (Z) -4- (1-cyano-2- (6- (4- (diphenylamino) phenyl) -4-phenyl-4H-dithieno [3,2-b:2',3' -d ] pyrrol-2-yl) vinyl) -1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzyl) pyridin-1-ium bromide DTBP has the structural formula: 。
2. 2. a method for preparing a dual response fluorescent probe for detecting polar and peroxynitrite according to claim 1, which comprises the following steps: Fully dissolving 3,3 '-dibromo-2, 2' -bithiophene, aniline, sodium tert-butoxide, 1 '-binaphthyl-2, 2' -bisdiphenylphosphine and tris (dibenzylideneacetone) dipalladium in toluene Toluene by ultrasonic, refluxing for 12 hours, cooling the reaction liquid to room temperature after the reaction is finished, spin-drying to obtain a crude product, separating and purifying by a column chromatography, and drying to obtain 4-phenyl-4H-dithieno [3,2-b:2',3' -d ] pyrrole A1; Dissolving phosphorus oxychloride in N, N-dimethylformamide DMF cooled by ice water bath under nitrogen atmosphere, stirring for 1 hour, adding a compound A1, reacting for 2 hours at 70 ℃, cooling the mixture to room temperature after the reaction is finished, adjusting the pH value to be neutral by using sodium hydroxide solution, extracting by using dichloromethane, merging organic phases, removing the solvent by rotary evaporation to obtain a crude product, and finally separating and purifying by column chromatography, and drying by a vacuum drying box to obtain 4-phenyl-4H-dithieno [3,2-b:2',3' -d ] pyrrole-2-formaldehyde A2; under the ice water bath condition, N-bromosuccinimide and A2 are dissolved in tetrahydrofuran THF to react for 12 hours at room temperature in a dark environment, and after the reaction is finished, the solvent is directly dried by spin to obtain a product, and the product is dried by a vacuum drying oven to obtain 6-bromo-4-phenyl-4H-dithieno [3,2-b:2',3' -d ] pyrrole-2-formaldehyde A3; fully dissolving 4- (diphenylamino) phenylboric acid and A3 in tetrahydrofuran THF by ultrasonic, adding aqueous solution of potassium carbonate and tetra (triphenylphosphine) palladium, carrying out ultrasonic dissolution, refluxing for 22 hours after nitrogen protection, cooling the reaction solution to room temperature after the reaction is finished, spin-drying to obtain a crude product, separating and purifying by a column chromatography, and drying to obtain 6- (4- (diphenylamino) phenyl) -4-phenyl-4H-dithieno [3,2-b:2',3' -d ] pyrrole-2-formaldehyde A4; Dissolving 4-pyridine acetonitrile and 4-bromomethyl phenylboronic acid pinacol ester in acetonitrile, carrying out ultrasonic treatment to completely dissolve the 4-pyridine acetonitrile and the 4-bromomethyl phenylboronic acid pinacol ester, then heating and refluxing a reaction mixture for 12 hours, cooling to room temperature after the reaction is finished, adding diethyl ether for recrystallization, separating out solid precipitate, carrying out suction filtration, collecting the precipitate, and drying the precipitate by a vacuum drying oven to obtain 4- (cyanomethyl) -1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzyl) pyridine-1-onium bromide B1; Adding A4 and B1 into absolute ethyl alcohol, carrying out ultrasonic treatment to make them fully dissolved, refluxing for 5 hours, cooling reaction liquid to room temperature and spin-drying to obtain crude product, separating and purifying by column chromatography, drying to obtain black solid (Z) -4- (1-cyano-2- (6- (4- (diphenylamino) phenyl) -4-phenyl-4H-dithiophene [3,2-B:2',3' -d ] pyrrol-2-yl) vinyl) -1- (4, 5-tetramethyl-1, 3, 2-dioxaboran-2-yl) benzyl) pyridine-1-onium bromide (DTBP); the synthetic route is as follows: 。

Description

Preparation method and application of dual-response fluorescent probe for detecting polarity and peroxynitrite Technical Field The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and application of a polar and peroxynitrite dual-response fluorescent probe Background Polarity is a core parameter for regulating and controlling physiological functions of cells and maintaining steady state of microenvironment, and is deeply involved in key physiological processes such as membrane fusion, protein conformational change, enzyme activity regulation, peptide aggregation and the like. Abnormal changes in cell polarity are closely related to occurrence and development of cell dysfunction and diseases, not only are common diseases such as tumors, diabetes mellitus and Alzheimer disease related, but also various pathological states such as cardiovascular diseases, liver cirrhosis and neurodegenerative diseases are related. Because of the complex polarity change and the transient nature, the accurate monitoring of the dynamics is important to analyze the physiological mechanism and the disease pathology, and the fluorescent probe becomes a core tool for the polarity detection by virtue of the advantages of high sensitivity, non-invasiveness, real-time monitoring, high space-time resolution and the like, and the related research has become a focus of attention in the fields of chemistry, biology and medicine. Peroxynitrite (ONOO −) is used as the most reactive and toxic member in the active oxygen family, and is mainly generated by in-situ rapid reaction of nitric oxide and superoxide anions, and coexists in the form of ONOO −/ONOOH acid-base pair at physiological pH, so that the biological detection is very challenging due to the short service life of O 2−, extremely short self-half life and nano-scale steady concentration in cells. ONOO − has the dual functions of physiological regulation and pathological injury, can regulate cell signals through nitrifying amino acid residues, participate in immune defense, and damage biological macromolecules through self and secondary free radicals when excessive, and is closely related to various pathological processes such as cancer, neurodegenerative diseases, inflammation, drug-induced toxicity and the like. Therefore, the development of the probe which has high selectivity, high sensitivity, quick response and good biocompatibility and is suitable for living cells, tissues and living body imaging has important significance for revealing the pathological mechanism of ONO −, discovering the disease biomarker and guiding the early diagnosis and treatment of related diseases. The fluorescent probe has the advantages of flexible synthesis design and various recognition mechanisms, has high selectivity and high sensitivity, can accurately avoid interference of other active species, realizes specific recognition of ONOO-, has high response speed, and provides guarantee for rapid detection of target molecules. The near infrared fluorescent probe has the core advantages of better highlighting in biomedical imaging, has deep tissue penetration capability, low background fluorescence interference, low phototoxicity and photobleaching property, can realize high-resolution dynamic monitoring, and meanwhile, part of probes have subcellular targeting and multi-mode imaging functions, so that the detection accuracy and the application scene are further improved. Polarity and ONOO-are used as important indexes of intracellular microenvironment and are closely related to cell homeostasis and disease progression, and the noninvasive property and good biocompatibility of the fluorescent probe enable the fluorescent probe to track the polarity and ONOO-dynamics in situ in physiological environment, so that an intuitive tool is provided for researching the disease mechanism. In conclusion, the near infrared fluorescent probe with polarity and ONOO-dual response has great necessity, can realize accurate detection and dynamic monitoring of target molecules in organisms, can provide powerful support for early diagnosis of diseases and pathological mechanism analysis, and has wide medical application prospect. Disclosure of Invention The invention aims at a preparation method and property research of a dual-response fluorescent probe for detecting polarity and peroxynitrite. The DTBP has the characteristics of simple synthesis route, good selectivity, high sensitivity, large Stokes displacement and the like, and can effectively detect the ONO − and the polarity under physiological conditions. The double-response near-infrared fluorescent probe (Z) -4- (1-cyano-2- (6- (4- (diphenylamino) phenyl) -4-phenyl-4H-dithiophene [3,2-b:2',3' -d ] pyrrole-2-yl) vinyl) -1- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzyl) pyridine-1-onium bromide (DTBP) has the structural formula: The synthesis process of the fluorescent probe in the invention is as