Fluorescent probe for sequentially activating and detecting hydrogen sulfide and peroxynitrite as well as preparation method and application of fluorescent probe
By developing a dual-recognition fluorescent probe that can be sequentially activated to detect hydrogen sulfide and peroxynitrite, it solves the problem of difficulty in imaging and monitoring the dynamic changes of these two substances simultaneously with existing technology, and achieves high-sensitivity detection and Bioimaging provides new tools for the study and treatment of ulcerative colitis.
Patent Information
- Application Number
- CN202411453696.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2025-01-21
AI Technical Summary
The prior art is difficult to simultaneously image and monitor the dynamic changes of hydrogen sulfide and peroxynitrite in ulcerative colitis, which affects the study of the pathogenesis, early diagnosis and treatment strategies of the disease.
Develop a sequence to activate the dual -recognition fluorescent probe of hydrogen sulfide and peroxid nitrite. The probe can detect two substances separately and high sensitivity through a specific chemical structure and response mechanism, and Sequential activation is performed.
The high sensitivity detection of micro -hydrogen sulfide and nitrogen nitrite in micromer and Nonorm is realized. It has dual -channel continuous detection capabilities, strong selectivity, and can activate endogenous hydrogen sulfide and peroxid nitrite in order. For the diagnosis and treatment of biological imaging and ulcerative colitis.
Smart Images
Figure CN119330958A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe for sequentially activating and detecting hydrogen sulfide and peroxynitrite, and a preparation method and application thereof. Background Art
[0002] Ulcerative colitis (UC) is the most common inflammatory disease in the colon. It is listed as one of the most difficult diseases to cure by the World Health Organization because of its long course, wide range of lesions, chronicity, and the tendency to develop into cancer. The clinical manifestations of UC include diarrhea, blood in the stool, and mucosal ulcers, often accompanied by symptoms such as weight loss and fever. Therefore, in order to effectively prevent and treat UC, it is particularly important to deeply study its pathogenesis.
[0003] Hydrogen sulfide and peroxynitrite play complex roles in the pathogenesis of UC. Hydrogen sulfide has anti-inflammatory and antioxidant effects, which can reduce intestinal inflammation and help healing by reducing the release of inflammatory factors and inhibiting the overactivation of immune cells. At the same time, hydrogen sulfide can also reduce oxidative stress and protect cells from free radical damage. However, when the concentration of hydrogen sulfide is too high, it may damage the intestinal barrier function, increase intestinal permeability, and thus aggravate the inflammatory response and even cause toxic damage to intestinal epithelial cells. Unlike hydrogen sulfide, peroxynitrite is a potent oxidant that can induce oxidative damage to intracellular proteins, lipids, and DNA. This oxidative damage not only leads to cell dysfunction and apoptosis, but also further worsens the pathological state of UC by activating inflammatory signaling pathways (such as NF-κB) and promoting the release of inflammatory mediators. Therefore, hydrogen sulfide and peroxynitrite are both interdependent and contradictory in the pathogenesis of UC, and it is particularly important to deeply study their dynamic changes and interactions.
[0004] Based on this, it is particularly urgent to develop a dual-channel fluorescent probe that can simultaneously image hydrogen sulfide and peroxynitrite and monitor their dynamic changes during UC. This will provide a new tool for studying the pathogenesis, early diagnosis and treatment strategies of UC. Summary of the invention
[0005] The purpose of the present invention is to overcome the above-mentioned problems existing in the conventional technology and to provide a dual-recognition fluorescent probe for sequentially activating and detecting hydrogen sulfide and peroxynitrite, and a preparation method and application thereof.
[0006] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:
[0007] The present invention provides a fluorescent probe for sequentially activating and detecting hydrogen sulfide and peroxynitrite, wherein the fluorescent probe has the following structural formula:
[0008]
[0009] The present invention also provides a method for preparing a fluorescent probe for sequentially activating and detecting hydrogen sulfide, wherein (E)-2-(benzo[d]thiazol-2-yl)-3-(7-hydroxycoumarin-3-yl)acrylonitrile is added to N,N-diisopropylethylamine and stirred, and then 2,4-dinitrobenzenesulfonyl chloride is added and stirred to obtain the target probe BCH. The synthetic route is:
[0010]
[0011] Furthermore, the molar ratio of the (E)-2-(benzo[d]thiazol-2-yl)-3-(7-hydroxycoumarin-3-yl)acrylonitrile to N,N-diisopropylethylamine is 1:2.
[0012] Furthermore, the molar ratio of the (E)-2-(benzo[d]thiazol-2-yl)-3-(7-hydroxycoumarin-3-yl)acrylonitrile to 2,4-dinitrobenzenesulfonyl chloride is 1:4.
[0013] Furthermore, when the target probe is generated by reaction, the reaction conditions are: first stirring the reaction at 0°C for 0.2 to 1 hour, and then reacting at room temperature for 2 to 10 minutes.
[0014] Furthermore, after the target probe is generated by the reaction, the product is separated and purified by vacuum filtration.
[0015] The invention also provides an application of a fluorescent probe for sequentially activating and detecting hydrogen sulfide and peroxynitrite in cells and organisms.
[0016] The beneficial effects of the present invention are:
[0017] 1. The fluorescent probe of the present invention can detect hydrogen sulfide and peroxynitrite at micromole and nanomole levels, respectively, and therefore has high sensitivity.
[0018] 2. The fluorescent probe of the present invention has the ability of dual-channel continuous detection of analytes.
[0019] 3. The fluorescent probe of the present invention has good selectivity and specifically recognizes hydrogen sulfide and peroxynitrite, but has no response to other active oxygen, active nitrogen, active sulfur and other substances.
[0020] 4. The fluorescent probe of the present invention can sequentially activate and detect endogenous hydrogen sulfide and peroxynitrite in cells, and can be used for biological imaging.
[0021] 5. The fluorescent probe of the present invention can be used to simultaneously detect changes in the concentrations of hydrogen sulfide and peroxynitrite during the onset of ulcerative colitis.
[0022] 6. The synthesis method is simple, the raw materials are easily available and the yield is high.
[0023] Of course, any product implementing the present invention does not necessarily need to achieve all of the above advantages at the same time. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.
[0025] Figure 1 The absorption and fluorescence intensity response diagram and linear diagram of the fluorescent probe BCH to different concentrations of hydrogen sulfide and peroxynitrous acid;
[0026] The concentration of the probe is 10 μM, the concentration of hydrogen sulfide is 0-500 μM, and the concentration of peroxynitrite is 0-100 μM; the detection system is a mixed solution of DMF and PBS buffer (volume ratio is 3:7), and the excitation wavelengths are 530 nm and 425 nm, respectively.
[0027] Figure 2 To test the selectivity and anti-interference of the fluorescent probe BCH;
[0028] The concentration of the probe is 10 μM, the concentration of hydrogen sulfide is 0-500 μM, and the concentration of peroxynitrite is 0-100 μM; the detection system is a mixed solution of DMF and PBS buffer (volume ratio is 3:7), and the excitation wavelengths are 530 nm and 425 nm, respectively.
[0029] Figure 3 The time response and pH stability response diagram of probe BCH;
[0030] Among them, the concentration of the probe is 10μM, the concentration of hydrogen sulfide is 500μM, and the concentration of peroxynitrite is 100μM; the detection system is a mixed solution of DMF and PBS buffer (volume ratio is 3:7), and the excitation wavelengths are 530nm and 425nm, respectively.
[0031] Figure 4 To use the fluorescent probe BCH to perform fluorescence imaging of endogenous hydrogen sulfide and peroxynitrite in HCT116 cells;
[0032] Among them, the concentration of the probe is 10μM, red channel: excitation wavelength is 530nm, emission wavelength range is 550 to 605nm; blue channel: excitation wavelength is 375nm, emission wavelength collection range is 410 to 460nm; *★*P<0.001, *★★*P<0.0001; scale bar: 200μm.
[0033] Figure 5 To use the fluorescent probe BCH to dynamically image the concentration of hydrogen sulfide in mice with ulcerative colitis;
[0034] The excitation wavelength is 530 nm, and the emission wavelength collection range is 550 to 605 nm. DETAILED DESCRIPTION
[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0036] The fluorescent probe provided by the present invention can be used for the detection of hydrogen sulfide and peroxynitrite. The probe has the advantages of dual-channel emission, good ion selectivity, low biological toxicity, etc. It can be used for the detection of exogenous and endogenous hydrogen sulfide and peroxynitrite in cells and organisms, and has high commercial application value. The relevant specific embodiments of the present invention are as follows:
[0037] Example 1 Synthesis of fluorescent probe BCH
[0038] Compound (E)-2-(Benzo[d]thiazol-2-yl)-3-(7-hydroxycoumarin-3-yl)acrylonitrile (86.5 mg, 0.25 mmol) was dissolved in 5 mL of dichloromethane, and N,N-diisopropylethylamine (90 μL, 0.5 mmol) was slowly added dropwise at 0°C. Then 2,4-dinitrobenzenesulfonyl chloride (252.6 mg, 0.95 mmol) was added to the reaction mixture, and stirring was continued at room temperature for 5 min. After the reaction was completed, bright yellow solid BCH was obtained by vacuum filtration with a yield of 91%.
[0039] 1 H NMR (600 MHz, DMSO-d 6)δ9.12(d,J=2.3Hz,1H),8.88(s,1H),8.63(dd,J=8.7,2.2Hz,1H),8.35(d,J=8.6Hz,1H),8.20(d,J=9.3Hz,2H),8.13(d,J=8.2 Hz,1H),8.03(d,J=8.6Hz,1H),7.61(t,J=7.7Hz,1H),7.55(t,J=7.6Hz,1H),7.49(d,J=2.3Hz,1H),7.29(dd,J=8.6,2.2Hz,1H). 13 C NMR (101 MHz, DMSO-d 6 )δ162.4,158.9,154.7,153.4,152.1,151.7,148.6,142.6,140.2,134.9,134.2,132.4,131.0, 128.2,127.9,127.3,124.0,123.2,121.8,121.8,119.7,118.8,115.4,111.2,109.8.HRMS:m / z calculated for BCH(C 25 H 13 N 4 O 9 S 2 + ,[M+H] + ),577.0118;found,577.0134.
[0040] Example 2 UV absorption and fluorescence spectra of BCH under different concentrations of hydrogen sulfide and peroxynitrite
[0041] Different volumes of hydrogen sulfide and peroxynitrite solutions were added to the probe solution (10 μM) respectively. After the reaction was complete, fluorescence detection was performed (excitation wavelengths were 530 nm and 425 nm, respectively) to detect the relative fluorescence intensity of each system. The hydrogen sulfide concentration-relative fluorescence intensity and peroxynitrite concentration-relative fluorescence intensity curves were drawn, such as Figure 1 As shown in A, BCH exhibits an absorption peak at 420 nm. After the addition of hydrogen sulfide, the peak red-shifts to 515 nm, indicating the formation of a specific reaction product BC. When peroxynitrite is introduced, the probe exhibits an absorption peak at 420 nm ( Figure 1 D), which indicates that it can react with peroxynitrite and hydrogen sulfide to form new substances. Figure 1As shown in Figure 2, the probe exhibited weak fluorescence in DMF-PBS buffer (3 / 7, v / v). However, after the addition of hydrogen sulfide (0-500 μM), the fluorescence signal at 585 nm was significantly enhanced, which was attributed to the cleavage and release of the fluorescent benzenesulfonic acid group. Interestingly, when peroxynitrite (0-100 μM) was added in the presence of hydrogen sulfide (1.0 mM), a clear emission peak appeared at 465 nm ( Figure 1 E), indicating that BCH can monitor hydrogen sulfide and peroxynitrite simultaneously through dual channels. In addition, the fluorescence emission intensity shows good linear relationships with the concentrations of hydrogen sulfide (0-500 μM) and peroxynitrite (0-100 μM), respectively ( Figure 1 C and 1F). The detection limits of probe BCH for hydrogen sulfide and peroxynitrite were 1.6 μM and 0.05 μM, respectively. These findings indicate that BCH is capable of simultaneously detecting hydrogen sulfide and peroxynitrite, highlighting its potential as an efficient bifunctional fluorescent probe in chemical analysis.
[0042] Example 3 Selectivity and anti-interference of fluorescent probe BCH
[0043] In the study of small molecule fluorescent probes, selectivity is a key feature and is essential for the accurate detection of target substances in complex biological systems. To evaluate the selectivity of BCH for hydrogen sulfide and peroxynitrite, we screened a variety of reactive oxygen species (ROS), reactive nitrogen species (RNS), metal ions, and amino acids as potential interferents. In the presence of hydrogen sulfide, the fluorescence intensity of BCH in the red channel was significantly enhanced, while under the influence of other analytes, its fluorescence signal remained almost unchanged ( Figure 2 A). Further studies showed that in the presence of hydrogen sulfide, only peroxynitrite could cause a significant fluorescence enhancement, while the other tested substances did not show significant fluorescence fluctuations ( Figure 2 B). These results indicate that BCH has excellent selectivity and can effectively distinguish hydrogen sulfide from peroxynitrite. In addition, the fluorescence intensity of BCH in the coexistence of hydrogen sulfide, peroxynitrite and other substances was evaluated by competition experiments ( Figure 2 C, D), the results show that the fluorescence response of BCH to hydrogen sulfide and peroxynitrite remains unchanged in the presence of other analytes, highlighting its high selectivity and anti-interference properties.
[0044] Example 4 Effects of time and pH on the response of fluorescent probe BCH to hydrogen sulfide and peroxynitrite
[0045] The reaction characteristics of the probe BCH in the presence of hydrogen sulfide and peroxynitrite were investigated by time-dependent fluorescence spectroscopy. Figure 3As shown in A, when hydrogen sulfide is added to the probe BCH reaction system, the fluorescence intensity of the reaction system at 585 nm increases significantly with the extension of the reaction time and reaches the maximum value after 6 minutes of treatment. Figure 3 As shown in Figure B, after reacting with 1mM hydrogen sulfide for 30 minutes, 100μM peroxynitrite was added, and the fluorescence intensity of the reaction system at 465nm increased significantly and reached the maximum value after 5 minutes of treatment. Further studies have shown that the fluorescence characteristics of the probe BCH for hydrogen sulfide and peroxynitrite remain consistent within the pH range of 3 to 11, especially under the physiological pH conditions of the biological system ( Figure 3 C, D), the fluorescence characteristics of the probe remain stable. These results indicate that the probe BCH has potential application value in detecting hydrogen sulfide and peroxynitrite in biological systems.
[0046] Example 5 Fluorescence imaging of endogenous hydrogen sulfide and peroxynitrite in HCT116 cells using fluorescent probe BCH
[0047] HCT-116 cells were divided into five groups for imaging endogenous hydrogen sulfide and peroxynitrite. The control group was imaged without any additional treatment. The probe group was imaged 1 h after the addition of BCH (10 μM). For the hypoxia-reperfusion (OGD / R) group, cells were initially cultured in sugar-free DMEM medium, 0.5 mmol / L sodium sulfite was added for 30 min, and then transferred to high glucose DMEM medium for incubation for 30 min. BCH was then added and imaging was performed after 1 h, 2 h, and 3 h of incubation to evaluate the dynamic changes of hydrogen sulfide and peroxynitrite and their biological effects. Due to the overexpression of hydrogen sulfide in HCT116 cells, HCT116 cells with the addition of the probe (a) showed obvious fluorescence in the red channel compared with the control group ( Figure 4 ), verifying that the probe can detect endogenous hydrogen sulfide. To further demonstrate the probe's ability to detect endogenous peroxynitrite and to evaluate the correlation between hydrogen sulfide and peroxynitrite under oxidative stress conditions, we constructed an OGD / R model. As the oxidative stress time increased, the red channel fluorescence intensity gradually decreased, while the blue channel fluorescence intensity gradually increased, suggesting that under oxidative stress conditions, the level of peroxynitrite increased, which may be related to the cell's self-regulatory mechanism as a way to mitigate oxidative damage. These findings further confirm that BCH can effectively monitor endogenous and exogenous hydrogen sulfide and peroxynitrite levels in living cells.
[0048] Example 6 Fluorescence imaging of endogenous hydrogen sulfide and peroxynitrite in mice with ulcerative colitis using BCH fluorescent probe
[0049] Six-week-old male C57BL / 6 mice were used throughout the experiment. The mice were divided into three groups: the control group was given distilled water and had free access to it. The second group was induced with ulcerative colitis (UC) by ingestion of 3% DSS for 1 week. The third group was similarly induced with UC, and after the initial 1-week DSS treatment, 100 mg / kg of 5-aminosalicylic acid (5-ASA) was orally administered daily for 10 days. The mice were fasted for 12 h before imaging. Each group received an intraperitoneal injection of 200 μL of 0.2 mM BCH stock solution and maintained under 1% isoflurane anesthesia. Fluorescence imaging of the whole body and specific parts was performed at 0, 0.5, 1, 1.5 and 2 h using the AniView 600 imaging system. After in vivo fluorescence imaging, the mice were euthanized, and the brain, heart, liver, spleen, kidney and colon were removed for further imaging analysis. Fluorescence emission was recorded at 535-600 nm under 530 nm excitation, and dual-channel imaging of 430-500 nm under 425 nm excitation. like Figure 5 As shown, normal mice had weak fluorescence signals in the red channel. In contrast, in DSS-treated mice, the fluorescence intensity gradually increased, indicating that the probe was able to visualize the upregulation of hydrogen sulfide. Treatment with 5-aminosalicylic acid (5-ASA) significantly reduced the fluorescence intensity of UC mice, indicating that the drug reduced inflammation and reduced hydrogen sulfide levels. Biodistribution analysis of major organs and colon showed that the fluorescence signal in the DSS group was significantly higher than that in the normal group, which was consistent with the in vivo observations.
[0050] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.
Claims
1. A dual recognition fluorescent probe for sequential activation detection of hydrogen sulfide and peroxynitrite, characterized in that: The fluorescent probe has the following structural formula:
2. The method for preparing a fluorescent probe according to claim 1, wherein: The preparation method comprises the following steps: adding (E)-2-(benzo[d]thiazole-2-yl)-3-(7-hydroxycoumarin-3-yl)acrylonitrile to N,N-diisopropylethylamine and stirring, then adding 2,4-dinitrobenzenesulfonyl chloride and stirring to obtain the target probe BCH.
3. The preparation method according to claim 2, characterized in that: The molar ratio of the (E)-2-(benzo[d]thiazol-2-yl)-3-(7-hydroxycoumarin-3-yl)acrylonitrile to (N,N-diisopropylethylamine is 1:
2.
4. The preparation method according to claim 2, characterized in that: The molar ratio of the (E)-2-(benzo[d]thiazol-2-yl)-3-(7-hydroxycoumarin-3-yl)acrylonitrile to 2,4-dinitrobenzenesulfonyl chloride is 1:
4.
5. The preparation method according to claim 2, characterized in that: When the target probe is generated by the reaction, the reaction conditions are: first, stirring the reaction at 0°C for 0.2 to 1 hour, and then reacting at room temperature for 2 to 10 minutes.
6. The preparation method according to claim 2, characterized in that: After the target probe is generated by the reaction, the product is separated and purified by vacuum filtration.
7. Use of the fluorescent probe as claimed in claim 1 for sequential activation and detection of hydrogen sulfide and peroxynitrite in cells and organisms.