Preparation method and application of fluorescent probe for distinguishing hydrogen sulfide and glutathione

Abstract

The invention relates to a near-infrared dual-channel fluorescent probe molecule for distinguishing and detecting hydrogen sulfide and glutathione in a living body and application of the near-infrared dual-channel fluorescent probe molecule, and belongs to the technical field of medical chemistry and biological medicine. On the basis of taking a thiophene ester structure functional group as a specific recognition site of biological mercaptan, deep research is carried out, and two near-infrared fluorescent probes YF-O are designed and synthesized. The YF-O uses furyl ester as a recognition unit, the site of the furyl ester initiates Stokes shift of 170 nm, rapid (240 seconds) high-selectivity detection of hydrogen sulfide (600 nm) and glutathione (705 nm) is achieved, and the fluorescence enhancement multiples are 170 times and 161 times respectively. The probe shows excellent biocompatibility and strong signal output, and supports real-time monitoring of an alcoholic liver disease model and a pneumonia model in cells and zebra fish. Research results show that YF-O has potential in evaluation of therapeutic intervention measures, and opens up a new way for probe design and precision medicine.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry and medicine, and in particular to a near-infrared dual-channel fluorescent probe molecule for distinguishing and detecting hydrogen sulfide and glutathione in a living body and applications thereof. Background Art

[0002] Hydrogen sulfide and glutathione are core endogenous molecules in physiological and pathological processes, playing important roles in maintaining redox homeostasis, regulating inflammatory responses, and protecting organs. Hydrogen sulfide exerts anti-inflammatory and antioxidant effects by scavenging reactive oxygen species, inhibiting proinflammatory cytokines, and alleviating inflammation in liver and lung tissue. Glutathione, a key antioxidant in the liver, protects hepatocytes and alveolar cells from oxidative damage. Depletion of both exacerbates oxidative stress and inflammation, reinforcing the pathological interaction between alcoholic liver disease and pneumonia. In-depth understanding of their roles in oxidative stress and inflammation is crucial for elucidating the mechanisms of related diseases and identifying new diagnostic and therapeutic targets. Fluorescence imaging has become a key tool in biomedical research and disease diagnosis due to its non-invasive nature, high sensitivity, and high temporal and spatial resolution. Near-infrared fluorescent probes can visualize and track the distribution of biothiols in biological systems. However, traditional probes present challenges in distinguishing hydrogen sulfide from glutathione due to their similar properties and reaction mechanisms. Developing fluorescent probes that can rapidly, sensitively, and specifically distinguish hydrogen sulfide from glutathione is crucial for advancing the application of imaging technology in research and clinical practice. Summary of the Invention

[0003] Based on the current state of the art, the present invention designed and synthesized a dual-channel near-infrared fluorescent probe, YF-O, that responds to both hydrogen sulfide and glutathione. This probe molecule undergoes selective chemical functional group structural changes in response to the effects of hydrogen sulfide and glutathione in vivo, releasing a fluorescent signal. Further in vitro analysis and imaging verification allow for the differential detection of hydrogen sulfide and glutathione.

[0004] Specifically, the present invention provides a near-infrared fluorescent probe, the structure of which is shown in the following formula (I): The probe molecules involved in the present invention have good specificity, synergistic effects and ultra-low toxic side effects, and can therefore be successfully applied to imaging within biological systems in real-time analysis. It is achieved by releasing two different light signals through a single molecule with high specificity and rapid response to two analytes. The present invention provides a new strategy for the development of multi-purpose fluorescent probes, opens up new ideas for optimizing disease diagnosis schemes, and reveals the potential for in vivo imaging of probe molecules activated by pathological microenvironmental responses. The specific preparation method of the probe molecules shown in the present invention is as follows: Synthesis of Compound YF-O: YF (90 mg, 0.25 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (10 mL), followed by the addition of triethylamine (50 mg, 0.5 mmol, 2.0 equiv). 2-Furanoyl chloride (65 mg, 0.5 mmol, 2.0 equiv) was added dropwise in an ice bath. The reaction mixture was stirred at room temperature for 2 hours and monitored by thin-layer chromatography (TLC). After completion, the reaction was quenched with saturated sodium chloride solution and extracted with dichloromethane (3 × 15 mL). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA, 3:1, v / v) to yield the title compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 This is the fluorescence diagram of the response kinetics of the YF-O probe molecule to hydrogen sulfide.

[0006] Figure 2 This is the fluorescence diagram of the response dynamics of the YF-O probe molecule to glutathione Figure 3 This is the fluorescence graph of the YF-O probe molecule in response to hydrogen sulfide concentration analysis.

[0007] Figure 4 This is the fluorescence graph of the YF-O probe molecule in response to glutathione concentration analysis.

[0008] Figure 5 This is an analysis of the fluorescence response multiples of the YF-O probe molecule to biothiols at different pH values ​​under 600 nm emission.

[0009] Figure 6 This is an analysis of the fluorescence response multiples of the YF-O probe molecule to biothiols at different pH values ​​under 705 nm emission.

[0010] Figure 7 This is the high performance liquid chromatography analysis diagram of hydrogen sulfide and glutathione using YF-O probe molecule.

[0011] Figure 8 This is the imaging analysis diagram of YF-O probe molecules in HepG2 cells.

[0012] Figure 9 This is an imaging analysis diagram of the YF-O probe molecule in zebrafish.

[0013] Experimental method: YF-O (10 µM) and hydrogen sulfide (100 µM) were mixed in PBS (50% DMSO), and the absorption spectrum was scanned every 20 seconds for 240 seconds.

[0014] Experimental results: YF-O showed a rapid response to hydrogen sulfide, and the reaction was completed within 240 seconds.

[0015] Experimental method: YF-O (10 µM) and glutathione (1 mM) were mixed in PBS (50% DMSO), and the absorption spectrum was scanned every 20 seconds for 240 seconds.

[0016] Experimental results: YF-O showed a rapid response to glutathione, and the reaction was completed within 240 seconds.

[0017] Experimental method: YF-O (10 µM) was mixed with different concentrations of hydrogen sulfide (0-1 mM) in PBS (containing 50% DMSO) and incubated until the reaction equilibrium, and then the absorption spectra were collected.

[0018] Experimental results: The response of YF-O to hydrogen sulfide reached its optimal level within its physiological concentration range, reflecting its good biological adaptability.

[0019] Experimental method: YF-O (10 µM) and glutathione (0-2 mM) were mixed in PBS (50% DMSO) and the absorption spectrum was scanned after incubation to the final state.

[0020] Experimental results: The response of YF-O to glutathione reached its optimal level within its physiological concentration range, reflecting its good biological adaptability.

[0021] Experimental Methods: YF-O (10 µM) and its mixtures with hydrogen sulfide / cysteine / homocysteine ​​(100 µM) or glutathione (1 mM) were incubated in phosphate-buffered saline (PBS, pH 6.0–8.0, 50% DMSO) and sodium carbonate buffer (pH 9.0–11.0, 50% DMSO) at 37°C. Fluorescence emission at 600 nm was recorded under 535 nm excitation. All experiments were repeated three times to ensure reproducibility.

[0022] Experimental results: Through tests at different pH values, it was found that YF-O is suitable for the pH value of the physiological environment and can release fluorescence in a more alkaline environment.

[0023] Experimental Methods: YF-O (10 μM) and its mixtures with hydrogen sulfide / cysteine / homocysteine ​​(100 μM) or glutathione (1 mM) were incubated in phosphate-buffered saline (PBS, pH 6.0–8.0, 50% DMSO) and sodium carbonate buffer (pH 9.0–11.0, 50% DMSO) at 37°C. Fluorescence emission was recorded at 705 nm under 535 nm excitation. All experiments were repeated three times to ensure reproducibility.

[0024] Experimental results: Through tests at different pH values, it was found that YF-O is suitable for the pH value of the physiological environment and can release fluorescence in a more alkaline environment.

[0025] Experimental Methods: 1 mL of YF-O (10 μM) was incubated with either hydrogen sulfide (100 μM) or glutathione (1 mM) in phosphate-buffered saline (PBS) containing 50% dimethyl sulfoxide (DMSO) at 37°C. The reaction mixture was analyzed by HPLC using a Shimadzu LCMS-2020 system equipped with a Wondasil C18 Superb reversed-phase column (5 μm, 4.6 × 150 mm). The mobile phase consisted of methanol and water at a flow rate of 0.4 mL / min. The detection wavelength was set at 254 nm.

[0026] Experimental results: New peaks with significantly different retention times were observed when YF-O reacted with hydrogen sulfide and glutathione compared with the original probe through high-performance liquid chromatography, further verifying the reaction mechanism of YF-O with hydrogen sulfide and glutathione.

[0027] Experimental Methods: HepG2 cells were pretreated with N-acetyl-L-cysteine ​​(NEM, 100 μM) for 30 minutes, followed by treatment with glutathione (GSH, 1 mM), cysteine ​​(Cys, 100 μM), homocysteine ​​(Hcy, 100 μM), and hydrogen sulfide (H2S, 100 μM), and finally incubated with YF-O (10 μM) for 30 minutes. The cells were washed three times with PBS and then imaged under a laser confocal microscope (Olympus FV-3000).

[0028] Experimental results: The results showed that HepG2 cells in the control group exhibited strong fluorescence, but the addition of NEM to shield intracellular thiols significantly reduced the fluorescence. Parallel experimental groups introducing different thiols also exhibited significantly stronger fluorescence. Dual-channel intracellular imaging of hydrogen sulfide and glutathione was achieved in the yellow and red channels, respectively.

[0029] Three-day-old zebrafish were placed in imaging culture dishes containing culture medium and treated with various concentrations of ethanol for 30 minutes to establish an alcohol stimulation model. The group treated with the highest ethanol concentration was divided into two parallel experiments, one of which was further treated with NAC (final concentration 100 μM) for 4 hours. The fluorescent probe YF-O (final concentration 10 μM) was then added and incubated for an additional 30 minutes. After treatment, the zebrafish were gently washed three times with PBS buffer to remove residual probe. Finally, the zebrafish were imaged using a laser confocal microscope (Olympus FV-3000) to observe changes in the probe signal.

[0030] Experimental results: Similar to the intracellular imaging results, YF-O is achieving dual-channel imaging of hydrogen sulfide and glutathione in zebrafish.

Claims

1. The present invention discloses a method for preparing and using a near-infrared dual-channel fluorescent probe for distinguishing hydrogen sulfide from glutathione. Its structure is shown in the following formula (I). The fluorescent probe molecule of the present invention can be used for dual-channel detection of hydrogen sulfide and glutathione, emitting characteristic fluorescent signals in different wavelengths, enabling real-time monitoring of pathological hydrogen sulfide and glutathione abnormalities: （Ⅰ）。 2. A near-infrared dual-channel fluorescent probe for distinguishing hydrogen sulfide from glutathione, characterized in that The molecular design of the dual-response recognition site of the furanyl ester structure is shown in the following formula (I): （Ⅰ）。

Images