A nopinel acridine-based ratiometric fluorescent probe for rapid detection of glutathione, its preparation method and application.

Abstract

This invention discloses a nopinel acridine-based ratiometric fluorescent probe for rapid detection of glutathione, its preparation method, and its applications. The probe is 2-((5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)thiophene-2-yl)methylene)-1H-indene-1,3(2H)-dione (TA-PT-ID). This probe specifically detects glutathione. Under 365 nm ultraviolet light irradiation, the fluorescence color changes from orange to green upon addition of glutathione to the probe solution, with a response time of 1 min, a detection limit of 50 nM, an applicable pH range of 5–9, and a linear detection range of 0–100 μM for glutathione. As a ratiometric fluorescent probe for rapid glutathione detection, this probe exhibits numerous advantages such as good selectivity, high sensitivity, and wide applicability, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and relates to a nopinel acridine ratiometric fluorescent probe capable of rapid detection of glutathione, its preparation method, and its application. Background Technology

[0002] Glutathione (GSH) is a low-molecular-weight tripeptide composed of glutamic acid, cysteine, and glycine. As a key intracellular antioxidant and anti-inflammatory mediator, it is crucial for maintaining homeostasis. Clinical studies have shown that GSH depletion is closely related to the pathological mechanisms of various diseases, including liver fibrosis and diabetes; in particular, the GSH levels in diabetic patients are typically significantly lower than in healthy individuals. Therefore, dynamic monitoring of GSH concentration is of irreplaceable importance for in-depth understanding of the pathological process and clinical diagnosis of diabetes. Constructing ratiometric fluorescent probes that can sensitively respond to changes in GSH levels in the biological microenvironment has become an urgent research need.

[0003] Fluorescent probe technology, with its significant advantages such as high sensitivity, excellent spatiotemporal resolution, and non-invasiveness, has been widely used in bioimaging and environmental analysis. However, existing GSH detection probes generally suffer from technical bottlenecks such as slow response, insufficient sensitivity, narrow linear detection range, and significant cytotoxicity. Therefore, this invention aims to develop a novel ratiometric fluorescent probe. This probe possesses excellent performance characteristics such as rapid response, high sensitivity, and wide detection range, and exhibits good biocompatibility, meeting the needs of live-cell imaging and providing strong technical support for the rapid and accurate detection of glutathione. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is to provide a nopinel acridine-based ratiometric fluorescent probe for rapid detection of glutathione, characterized by rapid response, high sensitivity, and a wide pH adaptability. This probe can be used for qualitative and quantitative detection of glutathione in water, plants, and aquatic animals, and can be applied to glutathione fluorescence imaging in cells. Another technical problem this invention aims to solve is to provide a method for preparing this nopinel acridine-based ratiometric fluorescent probe for rapid detection of glutathione. A further technical problem this invention aims to solve is to provide an application of this nopinel acridine-based ratiometric fluorescent probe for rapid detection of glutathione.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A nopinel acridine-based ratiometric fluorescent probe for rapid detection of glutathione has the following structural formula:

[0007]

[0008] The molecular formula is C 36 H 27 NO2S, chemically named 2-((5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)thiophene-2-yl)methylene)-1H-indene-1,3(2H)-dione, abbreviated as TA-PT-ID.

[0009] The method for preparing a nopinel acridine-based ratiometric fluorescent probe for rapid detection of glutathione specifically includes the following steps:

[0010] (1) 9-(4-bromophenyl)-3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine (TA-PB) was coupled with 5-formyl-2-thiophene boronic acid to prepare compound 5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)thiophene-2-carboxaldehyde (TA-PTC);

[0011] (2) Compound TA-PTC undergoes a condensation reaction with 1,3-indanedione to obtain compound TA-PT-ID.

[0012] The specific preparation steps of TA-PTC in step (1) are as follows:

[0013] 1) Add 1 mmol of compound TA-PB, 1.2–1.5 mmol of 5-formyl-2-thiopheneboronic acid, 0.12–0.15 mmol of [1,1′-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex, 2.0–3.5 mmol of potassium carbonate, and 20–60 mL of methanol / toluene mixed solvent (1:1, v / v) sequentially to a three-necked flask, and reflux under nitrogen protection for 10–30 min until the starting materials have reacted completely;

[0014] 2) After the reaction was completed, the reaction solution was cooled to room temperature, washed three times with distilled water, and the organic phase was dried, filtered, and the solvent was evaporated to obtain the crude product of compound TA-PTC.

[0015] 3) The crude TA-PTC product was further purified by silica gel column chromatography to obtain the compound TA-PTC.

[0016] The specific preparation steps of TA-PT-ID in step (2) are as follows:

[0017] 1) Add 1 mmol TA-PTC, 1-2 mmol of 1,3-indanedion, 0.1-0.2 mL piperidine and 15-50 mL chloroform to a reaction flask, reflux under nitrogen protection for 10-12 h, and monitor with TLC until the reactants have reacted completely.

[0018] 2) After the reaction was completed, the reaction solution was cooled to room temperature, washed three times with distilled water, and the organic phase was dried, filtered, and the solvent was evaporated to obtain the crude product of compound TA-PT-ID.

[0019] 3) The crude product of compound TA-PT-ID was further purified by silica gel column chromatography to obtain the orange solid product TA-PT-ID.

[0020] The application of a nopinel acridine-based ratiometric fluorescent probe in the detection of glutathione. Under 365nm ultraviolet light irradiation, after adding glutathione to the TA-PT-ID fluorescent probe solution, the fluorescence color of the probe solution changes from orange to green, exhibiting a specific response to glutathione.

[0021] Beneficial effects: The compound TA-PT-ID prepared in this invention can specifically recognize glutathione, sensitively detect the content of glutathione in solution, with a response time within 1 min, a detection limit of 50 nM, an applicable pH range of 5–9, and a linear detection range of 0–100 μM for glutathione. As a ratiometric fluorescent probe for detecting glutathione, it has many advantages such as good selectivity, high sensitivity, and wide application range, and has good application prospects. Attached Figure Description

[0022] Figure 1 The fluorescence spectra of the probe TA-PT-ID are shown in the presence of different concentrations of glutathione.

[0023] Figure 2 This is a linear relationship between glutathione concentration and the fluorescence intensity ratio of probe TA-PT-ID at 490 nm and 580 nm;

[0024] Figure 3 This is a bar chart showing the ratio of fluorescence intensity at 490 nm to 580 nm after TA-PT-ID reacts with different analytes such as metal ions, anions, and common amino acids. Detailed Implementation

[0025] The specific embodiments of the present invention will be further described below with reference to specific examples.

[0026] Example 1

[0027] The reaction formula for the synthesis of the fluorescent probe TA-PT-ID is as follows:

[0028]

[0029] 1) 1 mmol of compound TA-PB, 1.5 mmol of 5-formyl-2-thiopheneboronic acid, 0.15 mmol of [1,1′-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex, 2.0 mmol of potassium carbonate, and 40 mL of methanol / toluene mixed solvent (1:1, v / v) were added sequentially to a three-necked flask. The mixture was refluxed under nitrogen protection for 30 min until the reactants were completely reacted. The reaction solution was cooled to room temperature, washed three times with distilled water, and the organic phase was dried, filtered, and the solvent was evaporated to obtain the crude product of compound TA-PTC. The crude product of TA-PTC was further purified by silica gel column chromatography to obtain compound TA-PTC, 310 mg, with a yield of 75%. 1 H NMR (600MHz, DMSO) δ: 9.96 (s, 1H), 8.11 (d, J=3.9Hz, 1H), 8.02 (dd, J=5.4, 3.0Hz, 2H), 7.9 4(d, J=8.3Hz, 1H), 7.88 (d, J=3.9Hz, 1H), 7.66 (t, J=7.6Hz, 1H), 7.53 (d, J=6.3Hz, 1H), 7.4 9-7.45 (m, 1H), 7.42 (t, J=7.0Hz, 1H), 7.35 (d, J=8.4Hz, 1H), 3.13 (t, J=5.6Hz, 1H), 2.82- 2.68 (m, 3H), 2.29 (s, 1H), 1.42 (s, 3H), 1.36 (d, J=9.8Hz, 1H), 0.69 (s, 3H). HRMS (m / z): [M] + Calculated for C 27 H 23 NOS: 409.55, found: 410.16.

[0030] 2) Add 1 mmol TA-PTC, 1.5 mmol 1,3-indanedion, 0.15 mL piperidine and 30 mL chloroform to a reaction flask and reflux for 10 h under nitrogen protection, monitoring with TLC until the reactants are completely reacted. Cool the reaction solution to room temperature, wash three times with distilled water, and dry, filter, and evaporate the solvent to obtain the crude product of compound TA-PT-ID. The crude product of TA-PT-ID is further purified by silica gel column chromatography to obtain an orange solid product TA-PT-ID, 432 mg, with a yield of 80%. 1H NMR (600MHz, DMSO) δ: 7.52 (d, J=4.1Hz, 1H), 7.29 (s, 1H), 7.25 (d, J=7.8Hz, 2H), 7 .13-7.09 (m, 3H), 6.84 (t, J=6.8Hz, 1H), 6.74 (d, J=8.5Hz, 1H), 6.68 (d, J=7.6Hz, 1H), 6.61(s, 1H), 6.55(d, J=8.3Hz, 1H), 2.31(s, 1H), 2.02-1.88(m, 3H), 1.48(s, 1H), 0.60 (s, 3H), 0.55 (d, J=9.7Hz, 1H), 0.35 (s, 3H), 0.13 (s, 3H). HRMS (m / z): [M] + Calculated for C 36 H 27 NO2S: 537.68, found: 538.18.

[0031] Example 2

[0032] The compound TA-PT-ID was prepared into 1×10 -5 Glutathione was dissolved in PBS buffer solution (pH = 7.4, 1‰ Tween 80) to prepare solutions with concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μmol / L. Fluorescence emission spectra of the probe TA-PT-ID in the presence of different concentrations of glutathione were measured using a fluorescence spectrophotometer by fluorescence titration. The results are shown below. Figure 1 As shown in the figure. The results indicate that as the glutathione concentration in the solution gradually increases from 0 mol / L to 100 μmol / L, the fluorescence emission intensity of this compound at 490 nm gradually increases, while the fluorescence intensity at 580 nm gradually decreases. The fluorescence intensity ratio of the probe (I 490 / I 580 The correlation between GSH concentration and GSH concentration was robustly linear in the range of 0-100 μM (R0). 2 =0.9919)( Figure 3 This demonstrates that the compound can be used as a ratiometric fluorescent probe for the sensitive detection of glutathione. The linear detection range for glutathione is 0-100 μM, the response time is within 1 min, the detection limit is 50 μM, and the applicable pH range is 5-9.

[0033] Example 3

[0034] TA-PT-ID is formulated into 1×10 -5A 100 μM solution was prepared by dissolving various analytes, including metal ions, anions, and common amino acids, in PBS buffer solution (pH = 7.4, 1‰ Tween 80). Fluorescence emission spectra of the TA-PT-ID assay in the presence of these analytes were measured using a fluorescence spectrophotometer via fluorescence titration. The results are shown below. Figure 3 As shown. After the compound TA-PT-ID reacted with glutathione, the fluorescence intensity ratio at 490 nm and 580 nm increased significantly, while the addition of Fe... 3+ Cu 2+ Mg 2+ Ca 2+ Zn 2+ Ni 2+ Metal ions, F - I - ,Br - HS - NO 2- Comparative observations were conducted with different analytes, including anions and amino acids such as Tyr, Gln, and Cys, and it was found that these analytes had no significant effect on the fluorescence spectrum of TA-PT-ID. This indicates that the compound can be used as a ratiometric fluorescent probe for the selective detection of glutathione.

Claims

1. A nopinene-acridine ratiometric fluorescent probe for rapid detection of glutathione, its preparation method, and its application, characterized in that, The fluorescent probe is 2-((5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)thiophene-2-yl)methylene)-1H-inden-1,3(2H)-dione, abbreviated as TA-PT-ID, and its structural formula is:

2. The method for preparing the norpinyl acridine-based ratiometric fluorescent probe TA-PT-ID for rapid detection of glutathione as described in claim 1, characterized in that, Includes the following steps: (1) 9-(4-bromophenyl)-3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine (TA-PB) was coupled with 5-formyl-2-thiophene boronic acid to prepare compound 5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)thiophene-2-carboxaldehyde (TA-PTC); (2) Compound TA-PTC undergoes a condensation reaction with 1,3-indanedione to obtain compound TA-PT-ID.

3. The method for preparing a ratiometric fluorescent probe for rapid detection of glutathione nopinel acridine according to claim 2, characterized in that, The specific preparation steps of TA-PTC in step (1) are as follows: 1) Add 1 mmol of compound TA-PB, 1.2–1.5 mmol of 5-formyl-2-thiopheneboronic acid, 0.12–0.15 mmol of [1,1′-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex, 2.0–3.5 mmol of potassium carbonate, and 20–60 mL of methanol / toluene mixed solvent (1:1, v / v) sequentially to a three-necked flask, and reflux under nitrogen protection for 10–30 min until the starting materials have reacted completely; 2) After the reaction was completed, the reaction solution was cooled to room temperature, washed three times with distilled water, and the organic phase was dried, filtered, and the solvent was evaporated to obtain the crude product of compound TA-PTC. 3) The crude TA-PTC product was further purified by silica gel column chromatography to obtain the compound TA-PTC.

4. The method for preparing a ratiometric fluorescent probe for rapid detection of glutathione nopinel acridine according to claim 2, characterized in that, The specific preparation steps of TA-PTC in step (2) are as follows: 1) Add 1 mmol TA-PTC, 1-2 mmol of 1,3-indanedion, 0.1-0.2 mL piperidine and 15-50 mL chloroform to a reaction flask, reflux under nitrogen protection for 10-12 h, and monitor with TLC until the reactants have reacted completely. 2) After the reaction was completed, the reaction solution was cooled to room temperature, washed three times with distilled water, and the organic phase was dried, filtered, and the solvent was evaporated to obtain the crude product of compound TA-PT-ID. 3) The crude product of compound TA-PT-ID was further purified by silica gel column chromatography to obtain the orange solid product TA-PT-ID.

5. The application of the norpinel acridine ratiometric fluorescent probe of claim 1 in the detection of glutathione.

6. The application according to claim 5, characterized in that, The fluorescent probe TA-PT-ID can specifically react with glutathione. Under 365nm UV light irradiation, after adding glutathione to the probe solution, its fluorescence color changes from orange to green, with a response time of less than 1 minute, a detection limit of 50 nM, an applicable pH range of 5–9, and a linear detection range of 0–100 μM for glutathione.

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