CN-121974932-A - Dual-channel fluorescent probe for simultaneously detecting nitric oxide and cysteine as well as preparation method and application thereof

CN121974932ACN 121974932 ACN121974932 ACN 121974932ACN-121974932-A

Abstract

The invention discloses a dual-channel fluorescent probe for simultaneously detecting nitric oxide and cysteine, and a preparation method and application thereof, wherein the structural formula of the dual-channel fluorescent probe is as follows: The invention also specifically discloses a preparation method of the double-channel fluorescent probe and application of the double-channel fluorescent probe in dynamic visual detection of NO and Cys in a selective detection system of NO and Cys and a biological cell system and in an iron death and depression cell model. The dual-channel fluorescent probe has the advantages of strong specificity, good selectivity, no spectrum crosstalk and the like.

Inventors

HE GUANGJIE
YANG LINLIN
Bai Shiqiong
XI YANBEI
LIU XIAOBO
LI JING

Assignees

河南医药大学

Dates

Publication Date: 20260505
Application Date: 20251223

Claims (7)

1. A dual-channel fluorescent probe for simultaneously detecting nitric oxide and cysteine is characterized by comprising the following structural formula:
2. a method for preparing a dual-channel fluorescent probe for simultaneously detecting nitric oxide and cysteine according to claim 1, which is characterized by comprising the following specific preparation steps: Step S1, 2-methoxy phenothiazine is dissolved in N, N-dimethylformamide solution and placed in ice bath, sodium hydride is added, stirring and mixing are carried out uniformly, then 3-bromopropyl ammonium bromide is added, stirring and reacting are carried out, and compound 1 is prepared, wherein the corresponding synthetic route is as follows: step S2, carrying out reflux reaction on the compound 1 and 4-bromo-1, 8-naphthalene anhydride in absolute ethyl alcohol to obtain a compound 2, wherein the corresponding synthetic route is as follows: Step S3, uniformly mixing 2- (4- (diethylamino) -2-hydroxybenzoyl) benzoic acid, a compound 2 and methanesulfonic acid, and reacting at 60-80 ℃ to obtain a compound 3, wherein the corresponding synthetic route is as follows: Step S4, dissolving the compound 3, N-hydroxysuccinimide and potassium carbonate in a dimethyl sulfoxide solution, and reacting at 70-90 ℃ to obtain a compound 4, wherein the corresponding synthetic route is as follows: step S5, carrying out reflux reaction on the compound 4 and hydrazine hydrate in a methanol solution at 80-90 ℃ to obtain a compound 5, wherein the corresponding synthetic route is as follows: Step S6, adding the compound 5 into a dichloromethane solution, adding acryloyl chloride and pyridine, and stirring and reacting the mixed system at 30-40 ℃ under the protection of nitrogen to obtain a light yellow solid compound TJ730-NO-Cys, namely a double-channel fluorescent probe, wherein the corresponding synthetic route is as follows:
3. use of the dual channel fluorescent probe of claim 1 for simultaneous selective detection of nitric oxide and cysteine.
4. The use of the dual-channel fluorescent probe according to claim 1 for preparing nitric oxide and cysteine dynamic visualization detection reagents in biological cell systems.
5. Use of the dual channel fluorescent probe of claim 1 for the preparation of nitric oxide and cysteine imaging detection reagents in iron death and depression cell models.
6. The use of the dual-channel fluorescent probe according to claim 1 for preparing imaging detection reagents for dynamic change of nitric oxide and cysteine in the process of death of PC12 cell iron.
7. The use of the dual-channel fluorescent probe according to claim 1 for preparing an imaging detection reagent for dynamic changes of nitric oxide and cysteine in a corticosterone-induced cell depression model.

Description

Dual-channel fluorescent probe for simultaneously detecting nitric oxide and cysteine as well as preparation method and application thereof Technical Field The invention belongs to the technical field of dual-channel fluorescent probes and nitric oxide and cysteine fluorescent detection, and particularly relates to a dual-channel fluorescent probe for simultaneously detecting nitric oxide and cysteine, and a preparation method and application thereof. Background Depression is a high incidence of psychotic disorder that still constitutes a significant public health challenge due to its complex pathogenesis and limited efficacy of traditional therapies. Although current antidepressants (e.g., selective 5-hydroxytryptamine reuptake inhibitors) are widely used in clinical practice, about 30% of patients respond poorly to treatment, indicating that their underlying mechanisms have not yet been fully elucidated. Iron death is an emerging form of programmed cell death characterized by excessive accumulation of iron-dependent lipid peroxides, which is closely related to the pathogenesis of a variety of neurodegenerative diseases. Potential regulatory mechanisms for iron death, including lipid peroxidation, iron ion accumulation, and amino acid metabolism, have also attracted considerable attention. There is increasing evidence underscores the contribution of iron death to depression and its underlying mechanisms. Therefore, the physiological process and the regulation mechanism of iron death are researched, and the iron death is used as potential diagnosis and treatment means of depression, so that the iron death has great significance for future drug development. Nitric Oxide (NO) is a signaling molecule that catalyzes the production of L-arginine by nitric oxide synthase in living cells. Previous studies have found that elevated intracellular Nitric Oxide Synthase (NOS) activity in depressed patients results in upregulation of nitric oxide, further confirming that NO is a critical mediator in the pathological process of depression. Nitric oxide can interact with Reactive Oxygen Species (ROS) and produce peroxynitrite (ONOO -), which not only exacerbates oxidative damage and triggers LPO to initiate effective iron death, but also increases intracellular GSH metabolites. Although cysteine (Cys) is an active form of the active sulfur species (RSS), a core metabolic node of iron death, its uptake and metabolism directly affects GSH-GPX4 axis function and regulates the susceptibility to iron death. Nitric oxide and cysteine have important and interrelated roles in the occurrence of iron death (ferroptosis) and redox regulation. By revealing the dynamic association of NO and Cys in the channel, the method effectively regulates the iron death process and advances the detection and treatment of depression to have important significance. Therefore, it is important to develop detection tools for NO and Cys related detection and research in biological systems. Fluorescence imaging is a powerful detection technique, known as high sensitivity, high selectivity and high spatial-temporal resolution, capable of visualizing and quantitatively analyzing biomolecules in living systems. Various fluorescent probes have been developed for detecting nitric oxide or cysteine with high sensitivity and good selectivity, wherein o-phenylenediamine-based NO probes are activated by formation of a benzotriazole ring, which structure is sensitive to thiol-containing substances. Although cysteine-specific probes generally rely on nucleophilic substitution reactions of sulfhydryl groups, the reactions are susceptible to interference by GSH and the like. The single channel fluorescent probes reported so far are mostly limited to detection of a single molecule, and cannot reveal the interaction mechanism between nitric oxide and cysteine. Although synchronous imaging of these biomarkers can be achieved theoretically by only mixing two single probes, the penetration capacity and metabolic differences can significantly affect performance and detection accuracy in biological imaging applications. Therefore, the design of the dual-response fluorescent probe provides a new idea for solving the problem through the synergistic effect of spectral separation and specific recognition groups. However, no fluorescent probe is currently available that can meet the need for simultaneous monitoring of nitric oxide and cysteine during iron death. Disclosure of Invention Aiming at the technical problems and the research situation faced by the fluorescent probe for detecting the NO/Cys value at present, the invention provides a dual-channel fluorescent probe for simultaneously detecting nitric oxide and cysteine and a preparation method thereof, when the NO content of the dual-channel fluorescent probe is increased, 1,3, 4-oxadiazole heterocycle is formed to cause the ring-opening reaction of a phenothiazine rhodamine unit, so that the fluorescent intensity of an N