Fluorescent probe for detecting hydrogen sulfide as well as preparation method and use method of fluorescent probe
By constructing a fluorescent probe based on intramolecular charge transfer and photoinduced electron transfer systems, the problem of poor selectivity of existing probes was solved, enabling specific detection and highly sensitive quantitative analysis of hydrogen sulfide.
Patent Information
- Application Number
- CN202511199056.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-26
- Publication Date
- 2025-11-28
AI Technical Summary
Existing hydrogen sulfide fluorescent probes have poor selectivity and are easily interfered with by bio-thiols such as Cys and GSH, making it difficult to achieve specific recognition of hydrogen sulfide.
An intramolecular charge transfer (ICT) and photoinduced electron transfer (PET) system constructed using (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-diene-1-yl)malonitrile was used to form a thiol structure through nucleophilic attack by hydrogen sulfide, which then cleaved the ether bond to release the fluorophore and emitted red fluorescence.
It achieves specific detection of hydrogen sulfide, exhibiting excellent selectivity and sensitivity, and enables quantitative analysis at low concentrations.
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Figure CN121021383A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-diene-1-yl)malonadionitrile used as a hydrogen sulfide fluorescent probe, and its preparation and usage methods. Background Technology
[0002] Hydrogen sulfide (H2S) plays a crucial role in apoptosis and cellular redox homeostasis. In vivo, biogenic hydrogen sulfide is widely recognized as an important endogenous gaseous neurotransmitter in living systems, primarily produced through the degradation of cysteine (Cys) or homocysteine (Hcy) by three main enzymes: cysteine β-synthetase (CBS), cysteine γ-lyase (CSE), and 3-mercaptopyruvate thiotransferase (3-MST). It plays a vital role in physiological and pathological processes such as vascular function regulation, neurotransmission, inflammation, cell growth regulation, apoptosis, and cardiovascular protection. Hydrogen sulfide can also act as a cytoprotective agent, scavenging excess reactive oxygen species (ROS) and maintaining redox homeostasis and various life processes in various cell activity models. In mammals, excessive expression of hydrogen sulfide can lead to various diseases, including diabetes, tumors, cirrhosis, asthma, and myocardial damage. In the environment, low concentrations of hydrogen sulfide gas can irritate the eyes and throat and impair the sense of smell. Therefore, the development of analytical methods for detecting hydrogen sulfide in vivo with low toxicity has attracted significant attention.
[0003] Molecular fluorescent probe detection methods have advantages such as high selectivity, high stability, low toxicity, low cost, and no damage to samples, making them the most powerful tool for detecting cell and tissue levels.
[0004] Currently developed small-molecule fluorescent probes for detecting hydrogen sulfide mainly utilize reduced aryl azides, nucleophilic addition reactions based on the nucleophilicity of hydrogen sulfide, hydrogen sulfide-induced metal substitution, and disulfide exchange reactions. In the presence of hydrogen sulfide, the detection group in the probe molecule undergoes a specific reaction with hydrogen sulfide, altering the molecule's original PET or ICT effects, leading to changes in the probe molecule's fluorescence properties and achieving specific recognition of hydrogen sulfide.
[0005] The response mechanisms of reactive hydrogen sulfide fluorescent probes mainly include the following (see review articles: Haonan Li, Yuxi Fang, Junjie Yan, Xiangyu Ren, Chao Zheng, Bo Wu, Siyuan Wang, Zhanlin Li, Huiming Hua, Peng Wang, Dahong Li). Trends in Analytical Chemistry, 2021, 134, 116117.): 1) Hydrogen sulfide releases fluorophores after nucleophilic attack on recognition groups such as 2,4-dinitrophenyl (DNP), 7-nitrobenzo-2-oxa-1,3-diazole (NBD), and sulfonates; hydrogen sulfide also forms persulfide intermediates with thiol groups through nucleophilic attack on electrophilic carbons or sulfur centers, which subsequently initiate spontaneous intramolecular nucleophilic substitution reactions to release fluorophores. 2) Based on the reductive reduction mechanism of hydrogen sulfide to azides, this reaction reduces electron-withdrawing azide groups to electron-donating amino groups, changing the electron density and thus altering the fluorescence intensity. 3) Based on the response mechanism of CuS precipitation, Cu is introduced into the fluorophore framework. 2+ The complex portion consists of hydrogen sulfide and Cu. 2+ The formation of CuS precipitate causes a change in fluorescence intensity. However, these reported probes still suffer from poor selectivity, mainly because biothiols such as Cys and GSH can also undergo nucleophilic addition with the responding group. Two consecutive intermolecular and intramolecular affinity reactions can significantly improve the selectivity for hydrogen sulfide, avoid interference from other biothiols, and achieve specific recognition of hydrogen sulfide. Summary of the Invention
[0006] To overcome the aforementioned deficiencies in the prior art, this invention proposes a fluorescent probe (IPHS) for the quantitative detection of hydrogen sulfide. This invention enables the quantitative detection of trace amounts of hydrogen sulfide in a sample.
[0007] The core of this invention lies in using (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile to construct a classic intramolecular charge transfer (ICT) system and (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-dien-1-yl)malononitrile to construct a classic photoinduced electron transfer (PET) system. The probe, due to the presence of 2-chloro-5-nitropyridine, blocks the transition of electrons from the excited state back to the ground state, exhibiting the classic PET effect without fluorescence emission. In the presence of hydrogen sulfide, hydrogen sulfide first nucleophilically attacks the aldehyde group to form a thiol structure. Then, the thiol group undergoes intramolecular nucleophilic attack on the carbon atom at position 2 of the pyridine ring, forming a six-membered ring intermediate. This triggers the cleavage of the ether bond, releasing the fluorophore and creating a strong ICT effect, emitting red fluorescence. However, other biothiols cannot achieve two consecutive intermolecular and intramolecular nucleophilic substitutions, giving this probe excellent selectivity. For these reasons, this probe can specifically detect hydrogen sulfide.
[0008] The hydrogen sulfide fluorescent probe described in this invention is named IPHS, and its structural formula is shown in formula (I): The preparation method of the above fluorescent probe is as follows: (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malonitrile (1), 2-chloro-5-nitropyridine (2), and potassium carbonate (3) are dissolved in acetonitrile, reacted for a period of time, and after precipitation and filtration, (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-dien-1-ylidene)malonitrile (4), namely: IPHS.
[0009] Preferably, the molar ratio of (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonadionitrile, 2-chloro-5-nitropyridine, potassium carbonate, and acetonitrile is 1:1.2:1.5:304–60800; and the reaction temperature is 0–100 °C. o C, the reaction time is 1 to 24 hours.
[0010] Preferably, the molar ratio of (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonadionitrile, 2-chloro-5-nitropyridine, potassium carbonate, and acetonitrile is 1:1.2:1.5:486; the reaction temperature is 81°C. o C, the reaction time is 1 hour.
[0011] The reaction formula for preparing the above probe is as follows: The usage method of the above-mentioned hydrogen sulfide fluorescent probe is as follows: Step 1: Add the same concentration of the compound shown in formula (I) to phosphate buffer solutions (10 mM, pH = 7.4):ethanol = 1:1 (PBS:EtOH = 1:1) of different concentrations of hydrogen sulfide to prepare at least 5 standard solutions containing the compound shown in formula (I) with different hydrogen sulfide contents. The concentration of the compound represented by formula (I) in the standard solution shown is 1 nM to 10 μM; The hydrogen sulfide content in the standard solution shown is 0.1 nM to 1 mM; Step 2: Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 485 nm. Plot the hydrogen sulfide concentration on the x-axis and I on the y-axis. 644 Establish a standard curve with the vertical axis as the ordinate; I 644 This indicates the fluorescence emission intensity of the standard solution at a wavelength of 644 nm; Step 3: Add the compound shown in formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission spectrum under excitation light with an excitation wavelength of 485 nm, and calculate the hydrogen sulfide content of the sample to be tested based on the standard curve.
[0012] This invention has the following characteristics: 1) The fluorescent probe provided by this invention is an orange-yellow solid powder with a stable structure.
[0013] 2) The fluorescent probe provided by this invention has a solution that is sensitive to the concentration of hydrogen sulfide. As the concentration of hydrogen sulfide increases, its fluorescence in the test solution changes from weak fluorescence to bright red fluorescence under a 365 nm ultraviolet lamp.
[0014] 3) The fluorescent probe provided by this invention emits light at a wavelength of 644 nm after reacting with hydrogen sulfide. It is a fluorescence-on response, and the fluorescence intensity changes significantly before and after the reaction. This can greatly eliminate the influence of differences in detection conditions on the results and improve the sensitivity of the detection.
[0015] 4) The fluorescent probe provided by this invention has a linear relationship with hydrogen sulfide concentration and can be used for accurate measurement of hydrogen sulfide concentration.
[0016] 5) The open-type hydrogen sulfide probe based on (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-diene-1-yl)malonitrile provided by this invention has a good response to hydrogen sulfide solution, enabling sensitive quantitative detection of hydrogen sulfide in samples. It has the advantages of simple operation, low cost, sensitive response, and easy promotion and application. Attached Figure Description
[0017] Figure 1 : The proton NMR spectrum of the fluorescent probe IPHS.
[0018] Figure 2 Color response diagram of the fluorescent probe IPHS to hydrogen sulfide.
[0019] Figure 3 Fluorescence response of the fluorescent probe IPHS to hydrogen sulfide.
[0020] Figure 4 UV titration curve of the fluorescent probe IPHS against hydrogen sulfide, where the probe concentration is 10.0 μM.
[0021] Figure 5 The fluorescence titration curve of the fluorescent probe IPHS on hydrogen sulfide, with an excitation wavelength of 485 nm and a probe concentration of 10.0 μM.
[0022] Figure 6 The fluorescence response of the fluorescent probe IPHS to common biological thiols and cations is shown in the figure, with an excitation wavelength of 485 nm, a probe concentration of 10.0 μM, and an analyte concentration of 1000.0 μM. Detailed Implementation
[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0025] The compound numbers in the examples correspond to the numbers in the compounds described above.
[0026] Example 1: Synthesis of compound IPHS.
[0027] Synthesis of compound (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-diene-1-yl)malononitrile (4).
[0028] 100 mg (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohex-2,4-diene-1-yl)malonitrile (314 μmol), 60 mg 2-chloro-5-nitropyridine (2) (377 μmol), and 65 mg potassium carbonate were dissolved in 8 mL of acetonitrile and reacted at 81 °C for 1 hour. After cooling and filtration, 97 mg of compound (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-diene-1-yl)malonitrile (4) were obtained, with a yield of 70%.
[0029] 1 H NMR (400 MHz, Chloroform- d ) δ 10.16 (s, 1H), 8.99 (d, J = 2.8 Hz, 1H), 8.59 (dd, J = 9.0, 2.8 Hz, 1H), 8.09 (d, J = 2.3 Hz, 1H), 7.85 (dd, J = 8.5, 2.3 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.26 (d, J= 8.9 Hz, 1H), 7.15 – 7.03 (m,2H), 6.90 (s, 1H), 2.63 (s, 2H), 2.50 (s, 2H), 1.11 (s, 6H).
[0030] Example 2: Synthesis of compound IPHS.
[0031] Synthesis of compound (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-diene-1-yl)malononitrile (4).
[0032] 100 mg (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2,4-diene-1-yl)malonitrile (314 μmol), 60 mg 2-chloro-5-nitropyridine (2) (377 μmol), and 65 mg potassium carbonate were dissolved in 300 mL acetonitrile and reacted at 0 °C for 10 hours. After cooling and filtration, 97 mg of compound (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohexyl-2,4-diene-1-yl)malonitrile (4) were obtained, with a yield of 68%.
[0033] Example 3: Synthesis of compound IPHS.
[0034] Synthesis of compound (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-diene-1-yl)malononitrile (4).
[0035] 100 mg (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2,4-diene-1-yl)malonitrile (314 μmol), 60 mg 2-chloro-5-nitropyridine (2) (377 μmol), and 65 mg potassium carbonate were dissolved in 1000 mL acetonitrile and reacted at 100 °C for 24 hours. After cooling and filtration, 97 mg of compound (E)-2-(3-(3-formyl-4-((5-nitropyridine-2-yl)oxy)styryl)-5,5-dimethylcyclohexyl-2,4-diene-1-yl)malonitrile (4) were obtained, with a yield of 67%.
[0036] Example 4: Color response of compound IPHS to hydrogen sulfide.
[0037] Prepare a 1 mM N,N-dimethylformamide stock solution of the fluorescent probe IPHS for detecting hydrogen sulfide described in this invention. Transfer 50 μL of this stock solution dropwise to a phosphate buffer solution (50% ethanol) containing a certain concentration of hydrogen sulfide, and dilute to 5 mL with the corresponding phosphate buffer solution (50% ethanol), so that the probe concentration in the test solution is 10.0 μM and the hydrogen sulfide concentration is 1000.0 μM, for color response testing. Figure 2 and Figure 3 As shown, after adding hydrogen sulfide solution, the color of the solution changed from pale yellow to red, and the fluorescence of the solution also changed from weak fluorescence to bright red fluorescence, indicating that the probe IPHS has a direct colorimetric response to hydrogen sulfide.
[0038] Example 5: Ultraviolet titration detection of compound IPHS by different concentrations of hydrogen sulfide.
[0039] Prepare a 1 mM N,N-dimethylformamide stock solution of the fluorescent probe IPHS for detecting hydrogen sulfide described in this invention. Transfer 50 μL of this stock solution and add it dropwise to phosphate buffer solutions (50% ethanol) of different concentrations of hydrogen sulfide, and then dilute to 5 mL with the corresponding phosphate buffer solutions (50% ethanol) to achieve a probe concentration of 10.0 μM and a hydrogen sulfide concentration ranging from 0 to 1300.0 μM in the test solution. Perform absorption spectroscopy measurements. Obtain the UV absorption curves for each system and establish a standard curve for absorbance versus hydrogen sulfide concentration. Figure 4 As shown, the absorbance at 500 nm increases continuously with increasing hydrogen sulfide concentration (A0). 500 ), indicating that the probe is in A 500 The changes are dependent on the concentration of hydrogen sulfide.
[0040] Example 6: Fluorescent titration detection of compound IPHS by different concentrations of hydrogen sulfide.
[0041] Prepare a 1 mM N,N-dimethylformamide stock solution of the fluorescent probe IPHS for detecting hydrogen sulfide described in this invention. Transfer 50 μL of this stock solution to different concentrations of hydrogen sulfide in phosphate buffer (50% ethanol), and dilute to 5 mL with the corresponding phosphate buffer (50% ethanol) to achieve a probe concentration of 10.0 μM and a hydrogen sulfide concentration of 0-1300.0 μM for fluorescence detection (λex = 485 nm, λem = 644 nm). Measure the fluorescence intensity in each system and establish a standard curve comparing fluorescence intensity with hydrogen sulfide concentration. Figure 5 As shown, with the increase of hydrogen sulfide concentration, the fluorescence intensity of the system (I) decreases. 644 As the concentration of hydrogen sulfide gradually increases, when the concentration reaches 1000.0 μM, the fluorescence intensity (IL) of the reaction system increases.644 It has reached its maximum value.
[0042] Example 7: Selectivity of compound IPHS for different common biothiols and cations.
[0043] Prepare a 1 mM N,N-dimethylformamide stock solution of the fluorescent probe IPHS for detecting hydrogen sulfide described in this invention. Prepare 10 mM solutions of various bio-thiols and cations to be tested as backup. Transfer 50 μL of this stock solution to different phosphate buffer solutions (50% ethanol) for testing small molecules, and dilute to 5 mL with the corresponding phosphate buffer solutions (50% ethanol) to achieve a probe concentration of 10.0 μM and a molecule concentration of 1000.0 μM in the test solution for fluorescence detection (λex = 485 nm, λem = 644 nm). Measure the fluorescence intensity in each system and establish a fluorescence intensity (IL) measurement method. 644 A bar chart showing the relationship between the analytes and the various test objects. (e.g.) Figure 6 As shown, other common analyte biothiols and cations have almost no effect on the fluorescence of the IPHS probe.
Claims
1. A fluorescent probe for detecting hydrogen sulfide, characterized in that: Its molecular formula C 25 H 20 N4O4, abbreviated as IPHS, has the structural formula (I). 。 2. The method for preparing a fluorescent probe for detecting hydrogen sulfide according to claim 1, characterized in that, The synthesis steps are as follows: (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile, 2-chloro-5-nitropyridine, and potassium carbonate were dissolved in acetonitrile. After reacting for a period of time, (E)-2-(3-(3-formyl-4-((5-nitropyridin-2-yl)oxy)styryl)-5,5-dimethylcyclohex-2,4-dien-1-yl)malonitrile, i.e., IPHS, was obtained.
3. The method for preparing a fluorescent probe for detecting hydrogen sulfide according to claim 1, characterized in that: The molar ratio of (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonadionitrile, 2-chloro-5-nitropyridine, potassium carbonate, and acetonitrile is 1:1.2:1.5:304–60800; the reaction temperature is 0–100 °C. o C, the reaction time is 1 to 24 hours.
4. The method for preparing a fluorescent probe for detecting hydrogen sulfide according to claim 1, characterized in that: i) The molar ratio of (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonadionitrile, 2-chloro-5-nitropyridine, potassium carbonate, and acetonitrile is 1:1.2:1.5:486; the reaction temperature is 81°C. o C, the reaction time is 1 hour.
5. A method for using a fluorescent probe for detecting hydrogen sulfide; characterized in that: 1) Add the same concentration of the compound shown in formula (I) to phosphate buffer solutions and ethanol solutions of hydrogen sulfide with different concentrations, with the molar ratio of the phosphate buffer solution and ethanol solution of hydrogen sulfide being 1:1, to prepare at least 5 standard solutions containing the compound shown in formula (I) with different hydrogen sulfide contents. The concentration of the compound represented by formula (I) in the standard solution shown is 1 nM to 10 μM; The hydrogen sulfide content in the standard solution shown is 0.1 nM to 1 mM; 2) Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 485 nm. Plot the hydrogen sulfide concentration on the x-axis and I on the y-axis. 644 Establish a standard curve with the vertical axis as the ordinate; I 644 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 644 nm; 3) Add the compound shown in formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; The fluorescence emission spectrum of the sample was measured under excitation light with an excitation wavelength of 485 nm, and the hydrogen sulfide content of the sample was calculated based on the standard curve.
6. The method for preparing a fluorescent probe for detecting hydrogen sulfide according to claim 1, characterized in that: In step i), the molar ratio of (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-ethylene)malonadionitrile, 2-chloro-5-nitropyridine, and potassium carbonate is 1:1.2:1.5; the amount of acetonitrile is 5–1000 mL; and the reaction temperature is 0–100 °C. o C, the reaction time is 1 to 24 hours.
7. The method for preparing a fluorescent probe for detecting hydrogen sulfide according to claim 1, characterized in that: i) The molar ratio of (E)-2-(3-(3-formyl-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile, 2-chloro-5-nitropyridine, and potassium carbonate is 1:1.2:1.5; acetonitrile is 8 mL; the reaction temperature is 81 °C. o C, the reaction time is 1 hour.