Near-infrared fluorescent probe for selectively recognizing hydrogen sulfide as well as preparation method and application of near-infrared fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide as well as a preparation method and application of the near-infrared fluorescent probe. The near-infrared fluorescent probe for selectively identifying hydrogen sulfide, provided by the invention, realizes high-specificity identification of H2S, low detection limit and sensitive response of a near-infrared region through innovative molecular structure design, and has the characteristics of wide pH value application range and quick response at the same time; a new strategy is provided for accurate detection of the endogenous cell H2S in a complex biological system.

Description

Technical Field

[0001] The present invention relates to a near-infrared fluorescence probe, and particularly to a near-infrared fluorescence probe for selectively recognizing hydrogen sulfide, its preparation method and application, belonging to the technical field of chemistry and analytical detection. Background Art

[0002] In recent years, hydrogen sulfide (H2S), as the third gaseous signaling molecule, has attracted much attention for its important role in physiological and pathological processes. Its accurate detection is of great significance for disease diagnosis and mechanism research. Currently, the H2S detection technology based on fluorescence probes has become a research hotspot due to its high sensitivity, real-time property and spatial resolution. In the prior art, the H2S fluorescence probes are mainly based on the following design strategies: (1) Utilizing the reducibility of H2S to trigger the reduction of nitro or azide groups, inducing the release of fluorophores (such as probes based on rhodamine or fluorescein); (2) Initiating aromatic substitution reactions through the nucleophilicity of H2S (such as acrylate or sulfonate probes); (3) Based on the coordination of H2S with metal ions (such as copper ion chelating probes). However, these probes still have significant defects in practical applications. For example:

[0003] 1. Insufficient selectivity: Most probes are difficult to distinguish H2S from biological thiols (such as glutathione, cysteine), resulting in false positive signals. For example, acrylate-based probes are easily attacked by other thiols, reducing the detection specificity;

[0004] 2. Limited detection sensitivity: The detection limits of some probes are relatively high (>10 μM), making it difficult to meet the detection requirements of low-concentration H2S (nanomolar to micromolar levels) in physiological environments;

[0005] 3. Limited spectral range: Probes in the visible light region (400 - 650 nm) are easily interfered by the autofluorescence of biological tissues, affecting the penetration depth and signal-to-noise ratio of in vivo imaging;

[0006] 4. Slow response kinetics: Some probes require several hours to reach a stable signal, making it difficult to achieve dynamic monitoring;

[0007] 5. Significant pH dependence: Some probes are only effective under strongly acidic or alkaline conditions and cannot adapt to the physiological neutral environment (pH value of 7.4);

[0008] 6. Poor biocompatibility: Toxic groups introduced during the probe synthesis process may affect cell viability, limiting their in vivo applications.

[0009] Furthermore, although near-infrared (NIR) probes developed in recent years (such as probes based on hemicyanine or squarylium) reduce background interference through long-wavelength emission (650 - 900 nm), their synthesis process is complex, their photostability is poor, they are prone to photobleaching, their half-life is short, and their selectivity for H2S still needs to be improved, severely restricting their application in in vivo deep tissue imaging and long-term dynamic monitoring. Therefore, developing a long-term detection fluorescent probe with high selectivity, near-infrared response, rapid detection, and good biocompatibility is the key requirement to break through the bottleneck of existing technologies. Summary of the Invention

[0010] The main object of the present invention is to provide a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide and a preparation method thereof to overcome the deficiencies of the prior art.

[0011] Another object of the present invention is to provide the application of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide.

[0012] To achieve the foregoing invention objects, the technical solutions adopted by the present invention include:

[0013] An embodiment of the present invention provides a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide, which has a structure shown in formula (I):

[0014]

[0015] The near-infrared fluorescent probe for selectively recognizing hydrogen sulfide can selectively recognize hydrogen sulfide.

[0016] An embodiment of the present invention also provides a preparation method of a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide, which includes:

[0017] React a first compound with 2,4-dihydroxybenzaldehyde to obtain a second compound;

[0018] React the second compound with 4-pyridineacetonitrile to obtain a third compound;

[0019] React biotin with 4-aminobenzyl alcohol to obtain a fourth compound;

[0020] React the fourth compound with phosphorus tribromide to obtain a fifth compound;

[0021] React the third compound with the fifth compound to obtain a sixth compound;

[0022] React the sixth compound with 2,4-dinitrofluorobenzene to obtain a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide;

[0023] The first compound has the structure shown in formula (II), the second compound has the structure shown in formula (III), the third compound has the structure shown in formula (IV), the fourth compound has the structure shown in formula (V), the fifth compound has the structure shown in formula (VI), the sixth compound has the structure shown in formula (VII), and the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide has the structure shown in formula (I):

[0024]

[0025] An embodiment of the present invention also provides a near-infrared fluorescence probe for selectively recognizing hydrogen sulfide prepared by the foregoing preparation method.

[0026] An embodiment of the present invention also provides an application of the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide in detecting hydrogen sulfide.

[0027] Correspondingly, an embodiment of the present invention also provides a method for selectively recognizing hydrogen sulfide, which includes: contacting and reacting the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide with a system that may contain hydrogen sulfide to achieve selective recognition of hydrogen sulfide.

[0028] Compared with the prior art, the present invention has at least the following advantages:

[0029] The near-infrared fluorescence probe for selectively recognizing hydrogen sulfide provided by the present invention realizes high-specificity recognition of H2S, low detection limit, and sensitive response in the near-infrared (650 - 710 nm) region through innovative molecular structure design. At the same time, it has a wide pH value adaptation range (7.0 - 10.0) and fast response (within 2 min) characteristics, providing a new strategy for the accurate detection of endogenous cellular H2S in complex biological systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is a schematic diagram of the response principle of the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide and hydrogen sulfide in a typical embodiment of the present invention;

[0032] Figure 2 It is the mass spectrum of compound 2 in Example 1 of the present invention;

[0033] Figure 3It is the mass spectrometry diagram of Compound 4 in Example 1 of the present invention;

[0034] Figure 4 It is the mass spectrometry diagram of Compound 5 in Example 1 of the present invention;

[0035] Figure 5 It is the mass spectrometry diagram of Compound 6 in Example 1 of the present invention;

[0036] Figure 6 It is the nuclear magnetic resonance spectrum of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide (i.e., Compound 7) in Example 1 of the present invention;

[0037] Figure 7 It is the absorption spectrum diagram before and after the reaction of the near-infrared fluorescent probe (10 μM) for selectively recognizing hydrogen sulfide with H2S in Example 1 of the present invention;

[0038] Figure 8 It is the fluorescence spectrum diagram after the reaction of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide with H2S in Test Example 1 of the present invention;

[0039] Figure 9 It is the fluorescence bar chart after the reaction of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide with H2S (100 μM) and other analytes (100 μM) in PBS buffer solution (pH = 7.4) in Test Example 1 of the present invention;

[0040] Figure 10 It is the fluorescence intensity change diagram before and after the reaction of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide with H2S at different pH values in Test Example 1 of the present invention;

[0041] Figure 11 It is the time response diagram of the reaction of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide with H2S in Test Example 1 of the present invention;

[0042] Figure 12 It is the linear relationship diagram between the fluorescence intensity of the near-infrared fluorescent probe (10 μM) for selectively recognizing hydrogen sulfide and the H2S concentration (0 - 20 μM) in PBS buffer solution (pH = 7.4) in Test Example 1 of the present invention;

[0043] Figure 13A and Figure 13B are respectively the exogenous H2S fluorescence imaging diagram and the fluorescence intensity quantification diagram in MCF-7 cells in Test Example 1 of the present invention;

[0044] Figure 14 It is the imaging ability result diagram of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide to H2S in a tumor mouse model in Test Example 1 of the present invention;

[0045] Figure 15 is Figure 14Quantitative graph of fluorescence intensity. Detailed implementation manners

[0046] In view of the deficiencies of the prior art, the inventors of this case have, through long-term research and a large number of practices, been able to propose the technical solution of the present invention, which mainly prepares a novel near-infrared fluorescence probe structure for selectively recognizing hydrogen sulfide through innovative molecular structure design. The following will further explain and illustrate the technical solution, its implementation process, principle, etc.

[0047] Specifically, as an aspect of the technical solution of the present invention, a near-infrared fluorescence probe for selectively recognizing hydrogen sulfide involved therein has a structure shown in formula (I):

[0048]

[0049] The near-infrared fluorescence probe for selectively recognizing hydrogen sulfide can selectively recognize hydrogen sulfide, and its response principle is as Figure 1 shown. After the probe reacts with hydrogen sulfide, a fluorescent mother nucleus is released.

[0050] In some embodiments, the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide exhibits excellent H2S response ability in a relatively wide pH range of 7.0 - 10.0.

[0051] In some embodiments, the response time of the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide is within 2 minutes. The near-infrared fluorescence probe of the present invention has the ability to rapidly detect H2S levels by fluorescence method.

[0052] In some embodiments, the detection limit of the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide is lower than 5 μM.

[0053] As another aspect of the technical solution of the present invention, a preparation method of a near-infrared fluorescence probe for selectively recognizing hydrogen sulfide involved therein includes:

[0054] React a first compound with 2,4-dihydroxybenzaldehyde to obtain a second compound;

[0055] React the second compound with 4-pyridineacetonitrile to obtain a third compound;

[0056] React biotin with 4-aminobenzyl alcohol to obtain a fourth compound;

[0057] React the fourth compound with phosphorus tribromide to obtain a fifth compound;

[0058] React the third compound with the fifth compound to obtain a sixth compound;

[0059] React the sixth compound with 2,4-dinitrofluorobenzene to obtain a near-infrared fluorescence probe for selectively recognizing hydrogen sulfide;

[0060] The first compound has the structure shown in formula (II), the second compound has the structure shown in formula (III), the third compound has the structure shown in formula (IV), the fourth compound has the structure shown in formula (V), the fifth compound has the structure shown in formula (VI), the sixth compound has the structure shown in formula (VII), and the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide has the structure shown in formula (I):

[0061]

[0062]

[0063] In some embodiments, the preparation method specifically includes: mixing N,N-dimethylformamide and a first solvent at -5 to 5 °C under a protective atmosphere, adding PBr3 and reacting until the solution turns milky white, and then adding cyclopentanone and carrying out a first reaction at room temperature for 10 to 15 h to obtain the first compound.

[0064] In some preferred embodiments, the molar ratio of N,N-dimethylformamide, PBr3 to cyclopentanone is 110 - 130:100 - 115:30 - 50.

[0065] Further, the first solvent may include any one or a combination of more of DCM, chloroform, carbon tetrachloride, THF, DMF, DMSO, DPM, ethyl acetate, etc., but is not limited thereto.

[0066] In some embodiments, the preparation method specifically includes: mixing the first compound with a second solvent, adding 2,4-dihydroxybenzaldehyde and cesium carbonate (as a catalyst), and carrying out a second reaction at room temperature for 10 to 15 h to obtain the second compound.

[0067] In some preferred embodiments, the molar ratio of the first compound, 2,4-dihydroxybenzaldehyde to cesium carbonate is 12 - 18:15 - 22:35 - 40.

[0068] Further, the second solvent may include any one or a combination of more of DMF, DMSO, THF, acetonitrile, acetone, etc., but is not limited thereto.

[0069] In some embodiments, the preparation method specifically includes: mixing the second compound, 4-pyridineacetonitrile, piperidine (as a catalyst) and a third solvent, and refluxing at 70 - 90 °C for a third reaction for 10 to 15 h to obtain the third compound.

[0070] In some preferred embodiments, the molar ratio of the second compound to 4-pyridineacetonitrile is 1 to 1.1:1 to 1.2.

[0071] Furthermore, the third solvent may include any one or a combination of more than one of ethanol (such as absolute ethanol), isopropanol, acetone, methanol, glycerol, propylene glycol, DMSO, THF, etc., but is not limited thereto.

[0072] In some embodiments, the preparation method specifically includes: mixing biotin with a fourth solvent, adding HATU (as a condensing agent) and reacting for 10 to 20 minutes, then adding DIPEA (N,N-diisopropylethylamine, as a catalyst) and 4-aminobenzyl alcohol, and performing a fourth reaction at room temperature for 3 to 8 hours to obtain a fourth compound.

[0073] Furthermore, the structural formula of the biotin is:

[0074] In some preferred embodiments, the molar ratio of biotin, HATU, DIPEA to 4-aminobenzyl alcohol is 1.8 to 2.2:1.8 to 2.2:2 to 2.5:2 to 2.5.

[0075] Furthermore, the fourth solvent may include any one or a combination of more than one of DMF, DMSO, THF, acetonitrile, acetone, etc., but is not limited thereto.

[0076] In some embodiments, the preparation method specifically includes: mixing the fourth compound with a fifth solvent at -5 to 5°C, then adding phosphorus tribromide and performing a fifth reaction at room temperature for 4 to 8 hours to obtain a fifth compound.

[0077] In some preferred embodiments, the molar ratio of the fourth compound to phosphorus tribromide is 0.2 to 0.4:0.5 to 0.8.

[0078] Furthermore, the fifth solvent may include any one or a combination of more than one of DCM, chloroform, carbon tetrachloride, THF, DMF, DMSO, DPM, ethyl acetate, etc., but is not limited thereto.

[0079] In some embodiments, the preparation method specifically includes: mixing the third compound, the fifth compound with a sixth solvent, and performing a sixth reaction at 70 to 90°C for 10 to 15 hours to obtain a sixth compound.

[0080] In some preferred embodiments, the molar ratio of the third compound to the fifth compound is 0.08 to 0.12:0.10 to 0.15.

[0081] Further, the sixth solvent may include any one or a combination of more of DMF, DMSO, THF, acetonitrile, acetone, etc., but is not limited thereto.

[0082] In some embodiments, the preparation method specifically includes: mixing a sixth compound, 2,4-dinitrofluorobenzene, triethylamine with a seventh solvent, and reacting at room temperature for 2 - 6 h to obtain a near-infrared fluorescence probe for selectively recognizing hydrogen sulfide.

[0083] In some preferred embodiments, the molar ratio of the sixth compound, 2,4-dinitrofluorobenzene to triethylamine is 0.04 - 0.06:0.05 - 0.08:0.4 - 0.6.

[0084] Further, the seventh solvent may include any one or a combination of more of DMF, DMSO, THF, acetonitrile, acetone, etc., but is not limited thereto.

[0085] As another aspect of the technical solution of the present invention, it also relates to a near-infrared fluorescence probe for selectively recognizing hydrogen sulfide prepared by the foregoing preparation method.

[0086] As another aspect of the technical solution of the present invention, it also relates to the application of the foregoing near-infrared fluorescence probe for selectively recognizing hydrogen sulfide in the detection of hydrogen sulfide (especially hydrogen sulfide in living cells).

[0087] Further, the hydrogen sulfide is derived from endogenous hydrogen sulfide in an organism.

[0088] Correspondingly, another aspect of the present invention relates to a method for selectively recognizing hydrogen sulfide, which includes:

[0089] Bringing the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide into contact with a system that may contain hydrogen sulfide and reacting to achieve selective recognition of hydrogen sulfide.

[0090] Further, the system containing hydrogen sulfide includes a living cell hydrogen sulfide system.

[0091] Further, the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide achieves selective recognition of hydrogen sulfide within a pH range of 7.0 - 10.0.

[0092] Verified, the near-infrared fluorescence probe for selectively recognizing hydrogen sulfide provided by the present invention realizes high-specificity recognition, low detection limit and sensitive response in the near-infrared region of H2S through innovative molecular structure design and optimization of the coupling mechanism between the reaction site and the fluorophore. At the same time, it has a wide pH adaptation range and rapid response characteristics, providing a new strategy for the accurate detection of endogenous cellular H2S in complex biological systems.

[0093] The technical solution of the present invention will be further explained and illustrated below in conjunction with several preferred embodiments. It is easy for those skilled in the art to understand that the embodiments are only helpful for understanding the present invention and should not be regarded as specific limitations on the present invention. However, those skilled in the art can easily understand that the specific material ratios, process conditions and their results described in the embodiments are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0094] In the following embodiments, the experimental materials used can be obtained from conventional biochemical reagent companies without special instructions.

[0095] Example 1

[0096] The preparation method of a near-infrared fluorescence probe for selectively recognizing hydrogen sulfide in this embodiment includes:

[0097] (1) Prepare the third compound, and the synthesis route is as follows:

[0098]

[0099] Synthesis of the first compound (i.e., compound 1): At 0 °C under N2 protection, N,N-dimethylformamide (120 mmol) was dissolved in DCM (40 ml), PBr3 (108 mmol) was added, and the reaction was carried out until the solution became milky white. Then cyclopentanone (40 mmol) was added and the reaction was stopped after reacting at room temperature for 12 h. The reaction was quenched with saturated sodium carbonate solution, and after extraction, column chromatography was carried out to obtain 5.3 g of a yellow liquid (yield 75%).

[0100] Synthesis of the second compound (i.e., compound 2): Compound 1 (15 mmol) was dissolved in DMF (20 ml), then 2,4-dihydroxybenzaldehyde (18 mmol) and cesium carbonate (37.5 mmol) were added, and the reaction was carried out at room temperature for 12 h. The insoluble impurities were removed by filtration, and after extraction and washing, column chromatography was carried out to obtain 1.4 g of a green solid (yield 43%). As Figure 2 shown, it is the mass spectrum of compound 2, and the data is HRMS(ESI-TOF) m / z: [M+H]+ calcd for C 14 H 10 O3 215.0703; found 215.0705.

[0101] Synthesis of the third compound (i.e., compound 3): Compound 2 (1 mmol) and 4-pyridineacetonitrile (1 mmol) were dissolved in absolute ethanol, refluxed at 80 °C for 12 h, cooled, and then filtered with ice ethanol to obtain compound 3 (yield 84%).

[0102] (2) Prepare the fifth compound, and the synthesis route is as follows:

[0103]

[0104] Synthesis of the fourth compound (i.e., compound 4): Dissolve biotin (2 mmol) in hot DMF (10 ml), then add HATU (2 mmol) and react for 15 min. After that, add DIPEA (2.2 mmol) and 4-aminobenzyl alcohol (2.2 mmol), and react at room temperature for 5 h. After the reaction is completed, add DCM and water. The system is layered and a solid precipitates. Filter by suction to obtain 448.3 mg of white solid (yield: 65%). Its mass spectrum is as shown in Figure 3 shown.

[0105] Synthesis of the fifth compound (i.e., compound 5): At 0 °C, dissolve compound 4 (0.3 mmol) in dry DCM (6 ml), then dropwise add phosphorus tribromide (0.6 mmol) and react at room temperature for 6 h. After the reaction is completed, add saturated sodium carbonate solution to make the solution neutral or weakly alkaline, and then filter by suction to obtain 99.6 mg of compound 5 (yield: 81%). Its mass spectrum is as shown in Figure 4 shown.

[0106] (3) Synthesis route of the parent nucleus and the probe:

[0107]

[0108] Synthesis of the sixth compound (i.e., compound 6): Dissolve compound 3 (0.1 mmol) and compound 5 (0.12 mmol) in dry DMF (2 ml), react at 80 °C for 12 h, then add EA and a black-purple solid precipitates. Filter by suction to obtain 59.4 mg of black-purple solid (yield: 90%). As shown in Figure 5 shown, it is the mass spectrum of compound 6. The data is HRMS(ESI-TOF) m / z: [M]+ calcd for C 37 H 36 N5O4S + 646.2483; found 646.2484.

[0109] Synthesis of the near-infrared fluorescent probe 152T (i.e., compound 7): Dissolve compound 6 (0.05 mmol), 2,4-dinitrofluorobenzene (0.06 mmol) and triethylamine (0.5 mmol) in dry DMF (2 ml), react at room temperature for 4 h, add EA to precipitate a solid, and filter by suction to obtain 28.7 mg of black solid (yield: 71%).

[0110] As shown in Figure 6As shown, the nuclear magnetic resonance spectrum of the near-infrared fluorescent probe (i.e., compound 7) for selectively recognizing hydrogen sulfide prepared in this example is as follows. The data are: 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.92 (d, J = 2.7 Hz, 1H), 8.88 (d, J = 6.6 Hz, 2H), 8.50 (dt, J = 9.1, 2.3 Hz, 1H), 8.21 (s, 1H), 8.10 (d, J = 6.5 Hz, 2H), 7.92 (s, 2H), 7.60 (d, J = 7.3 Hz, 2H), 7.45 (dd, J = 9.2, 1.5 Hz, 1H), 7.40 (d, J = 8.2 Hz, 2H), 7.25 (s, 1H), 7.21 - 7.15 (m, 2H), 6.36 (d, J = 23.3 Hz, 2H), 5.62 (s, 2H), 4.30 - 4.24 (m, 1H), 4.10 (t, J = 6.1 Hz, 1H), 2.93 (s, 2H), 2.78 (dd, J = 12.5, 4.9 Hz, 2H), 2.54 (d, J = 12.5 Hz, 1H), 2.26 (d, J = 7.4 Hz, 2H), 1.58 - 1.30 (m, 7H). HRMS (ESI-TOF) m / z: [M] calcd for C 43 H 38 N7O8S + 812.2520; found 812.2513.

[0111] Example 2

[0112] The preparation method of a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide in this example includes:

[0113] Synthesis of the first compound (i.e., compound 1): At -5°C under N2 protection, N,N-dimethylformamide (110 mmol) was dissolved in THF (50 ml), PBr3 (100 mmol) was added, and the reaction was carried out until the solution turned milky white. Then cyclopentanone (30 mmol) was added, and the reaction was stopped after reacting at room temperature for 15 h. The reaction was quenched with saturated sodium carbonate solution, and after extraction, column chromatography was performed to obtain a yellow liquid (yield: 78%).

[0114] Synthesis of the second compound (i.e., compound 2): Compound 1 (12 mmol) was dissolved in DMSO (20 ml), then 2,4-dihydroxybenzaldehyde (15 mmol) and cesium carbonate (35 mmol) were added, and the reaction was carried out at room temperature for 10 h. The insoluble impurities were removed by filtration, and after extraction and washing, column chromatography was performed to obtain a green solid (yield: 42%).

[0115] Synthesis of the third compound (i.e., compound 3): Dissolve compound 2 (1 mmol) and 4-pyridineacetonitrile (1 mmol) in anhydrous isopropanol, reflux at 70 °C for 15 h, cool, add ice ethanol, and filter by suction to obtain compound 3 (yield: 85%).

[0116] Synthesis of the fourth compound (i.e., compound 4): Dissolve biotin (2 mmol) in hot DMSO (15 ml), add HATU (1.8 mmol) and react for 10 min, then add DIPEA (1.8 mmol) and 4-aminobenzyl alcohol (2 mmol), and react at room temperature for 3 h. After the reaction, add DCM and water, the system is layered and a solid precipitates, filter by suction to obtain a white solid (yield: 63%).

[0117] Synthesis of the fifth compound (i.e., compound 5): At -5 °C, dissolve compound 4 (0.2 mmol) in dry THF (6 ml), then dropwise add phosphorus tribromide (0.5 mmol) and react at room temperature for 8 h. After the reaction, add saturated sodium carbonate solution to make the solution neutral or weakly alkaline, and then filter by suction to obtain the compound (yield: 78%).

[0118] Synthesis of the sixth compound (i.e., compound 6): Dissolve compound 3 (0.08 mmol) and compound 5 (0.10 mmol) in dry DMSO (2 ml), react at 70 °C for 15 h, add EA, and a black-violet solid precipitates, filter by suction to obtain a black-violet solid (yield: 89%).

[0119] Synthesis of the near-infrared fluorescent probe 152T (i.e., compound 7): Dissolve compound 6 (0.04 mmol), 2,4-dinitrofluorobenzene (0.05 mmol) and triethylamine (0.4 mmol) in dry DMSO (2 ml), react at room temperature for 2 h, add EA to precipitate a solid, filter by suction to obtain a black solid (yield: 69%).

[0120] Example 3

[0121] The preparation method of a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide in this example includes:

[0122] Synthesis of the first compound (i.e., compound 1): Under the protection of N2 at 5 °C, dissolve N,N-dimethylformamide (130 mmol) in chloroform (60 ml), add PBr3 (115 mmol) and react until the solution turns milky white, then add cyclopentanone (50 mmol) and react at room temperature for 10 h to stop the reaction. Quench with saturated sodium carbonate solution, extract, and perform column chromatography to obtain a yellow liquid (yield: 75%).

[0123] Synthesis of the second compound (i.e., compound 2): Dissolve compound 1 (18 mmol) in THF (30 ml), then add 2,4-dihydroxybenzaldehyde (22 mmol) and cesium carbonate (40 mmol), and react at room temperature for 15 h. Filter off the insoluble impurities by suction, and obtain a green solid by extraction, washing, and column chromatography (yield: 45%).

[0124] Synthesis of the third compound (i.e., compound 3): Dissolve compound 2 (1.1 mmol) and 4-pyridineacetonitrile (1.2 mmol) in anhydrous propylene glycol, reflux at 90 °C for 10 h, cool, add ice ethanol, and filter by suction to obtain compound 3 (yield: 86%).

[0125] Synthesis of the fourth compound (i.e., compound 4): Dissolve biotin (2.2 mmol) in hot THF (15 ml), then add HATU (2.2 mmol) and react for 20 min, then add DIPEA (2.5 mmol) and 4-aminobenzyl alcohol (2.5 mmol), and react at room temperature for 8 h. After the reaction is completed, add DCM and water, the system is layered and a solid precipitates, and filter by suction to obtain a white solid (yield: 64%).

[0126] Synthesis of the fifth compound (i.e., compound 5): At 5 °C, dissolve compound 4 (0.4 mmol) in dry THF (6 ml), then dropwise add phosphorus tribromide (0.8 mmol) and react at room temperature for 4 h. After the reaction is completed, add saturated sodium carbonate solution to make the solution neutral or weakly alkaline, and then filter by suction to obtain the compound (yield: 80%).

[0127] Synthesis of the sixth compound (i.e., compound 6): Dissolve compound 3 (0.12 mmol) and compound 5 (0.15 mmol) in dry THF (2 ml), react at 90 °C for 10 h, then add EA, and a black-violet solid precipitates, and filter by suction to obtain a black-violet solid (yield: 90%).

[0128] Synthesis of the near-infrared fluorescent probe 152T (i.e., compound 7): Dissolve compound 6 (0.06 mmol), 2,4-dinitrofluorobenzene (0.08 mmol), and triethylamine (0.6 mmol) in dry DMSO (2 ml), react at room temperature for 6 h, add EA to precipitate a solid, and filter by suction to obtain a black solid (yield: 70%).

[0129] Test Example 1

[0130] Taking the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide prepared in Example 1 as an example, the present inventors performed spectral determination on it, specifically including:

[0131] Weigh a certain amount of the solid of the near-infrared fluorescent probe 1152T for selectively recognizing hydrogen sulfide and dissolve it in DMSO to prepare 1.0×10 -3 mol·L-1 1152T stock solution. The H2S stock solution (l0.0×10 -3 mol·L -1 ) was prepared by dissolving a certain amount of solid NaHS in PBS. During the test, the DMSO content was 10%, and fluorescence tests were performed using PBS buffer (pH value 7.4, 1×). The concentration of 152T in the test solution was 10 μM, and the concentration of H2S was 100 μM. Spectral measurements in the solution were carried out using an excitation wavelength of 670.0 nm, and the emission wavelength range was 680 - 8220 nm. The slit width was set to 20 nm / 10 nm.

[0132] The test results are as follows:

[0133] I. Spectral study of fluorescence probe 152T

[0134] To study the sensing characteristics of fluorescence probe 152T towards H2S, the absorption spectra of fluorescence probe 152T before and after reaction with H2S were first studied. As Figure 7 shown, the absorption spectra of fluorescence probe 152T (10 μM) before and after reaction with H2S. The fluorescence probe has an obvious absorption peak at 530 nm. After adding H2S, a red shift in wavelength was observed, and a new absorption peak appeared at 680 nm, indicating that after adding H2S, it reacted with fluorescence probe 152T.

[0135] Subsequently, the fluorescence response of fluorescence probe 152T to different concentrations of H2S was studied. As Figure 8 shown is the fluorescence spectrum diagram of fluorescence probe 152T after reaction with H2S. In PBS buffer (pH = 7.4), the response of fluorescence probe 152T (10 μM) to different concentrations of H2S (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) (λex = 680 nm). The results show that fluorescence probe 152T itself has no fluorescence emission signal at 710 nm. However, with the increase in the concentration of H2S, the fluorescence emission signal at 710 nm shows a significant increasing trend with the increase in the concentration of H2S.

[0136] II. Study on the selectivity of the fluorescence probe

[0137] Good selectivity is a necessary factor for the application of the probe in organisms. Therefore, the present invention examined the response of probe 152T to H2S and other potential analytes. Figure 9The following is a fluorescence histogram of the reaction of probe 152T (10 μM) with H2S (100 μM) and other analytes (100 μM) in PBS buffer (pH = 7.4). These analytes include common cations, anions, biothiols, etc. (1. NaHS 2. Cys 3. GSH 4. Hcy 5. Na2S2O8 6. Ser 7. Br- / k + 8. His 9. Na2SO3 10. Mg 2+ / SO4 2- 11. H2PO4 - 12. Phe 13. Lys 14. NaCNS 15. Thr 16. Na2S2O3 17. Fe 3+ 18. CO3 2- 19. Na2S2O5 20. L-tert-leucine 21. Tyr 22. HCO3 - 23. Na2S2O4 24. Cl - / k + 25. ASP 26. Trp 27. Blank). Among these analytes, only when H2S is added does the fluorescent probe of the present invention show a significant fluorescence enhancement, while other analytes have little effect on the probe. It is worth mentioning that the fluorescent probe 152T can effectively distinguish H2S from biothiols (Cys, Hcy, GSH) that also have a mercapto group (-SH).

[0138] III. Study of pH value

[0139] The influence of pH value is another factor for evaluating the performance of the fluorescent probe. Subsequently, the present invention studied the fluorescence characteristics of the fluorescent probe 152T before and after reacting with H2S under different pH value conditions. The results are as Figure 10 shown in the fluorescence intensity change diagram of the fluorescent probe before and after reacting with H2S, showing excellent H2S response ability in a relatively wide range of pH = 7.0 - 10.0. The probe has strong fluorescence under neutral and alkaline conditions because under physiological conditions, most of H2S exists in the form of HS-, which has stronger nucleophilicity, and the fluorescent probe has good fluorescence response signals in a relatively wide pH range, suitable for the detection of H2S under physiological environment (pH value is 7.4).

[0140] IV. Study of response time

[0141] The present invention also studied the fluorescence intensity change of the fluorescent probe 152T with H2S (100 μM) within 60 min. As Figure 11As shown, it is the time response diagram of the fluorescence probe reacting with H2S. In a PBS buffer solution (pH = 7.4), the change in fluorescence intensity over time after the reaction of the fluorescence probe 152T (10 μM) with H2S (40 μM) is shown. The results indicate that the probe reaches a plateau at around 30 minutes, and the fluorescence remains stable thereafter, and the probe responds rapidly. The above experiments show that the fluorescence probe of the present invention has the ability to rapidly detect H2S levels by fluorescence method.

[0142] V. Study on the detection limit

[0143] Figure 12 The linear relationship between the fluorescence intensity of the near-infrared fluorescence probe 152 (10 μM) that selectively recognizes hydrogen sulfide and the H2S concentration (0 - 20 μM) in a PBS buffer solution (pH = 7.4) was calculated. The correlation coefficient R between the two was obtained 2 = 0.9905. This result shows that there is an excellent linear relationship between the fluorescence intensity of the fluorescence probe and the H2S concentration (0 - 20 μM). At the same time, through experiments and calculations, the detection limit (LOD = 3σ / k) of the fluorescence probe for H2S was obtained as 1.44 μM. These results show that the fluorescence probe 152T can detect H2S well.

[0144] VI. Study on exogenous H2S cell imaging

[0145] After co-incubating breast cancer cells (MCF-7) with the fluorescence probe 152T (10 μM) in a culture dish for 30 minutes, imaging was performed. As Figure 13A shown, it is the fluorescence imaging diagram of exogenous H2S in MCF-7 cells. Among them, A represents the NaHS group: the cells were first incubated with NaHS (100 μM) for 30 minutes, and then co-incubated with the fluorescence probe 152T (10 μM) for 30 minutes; B represents the GSH group: the cells were incubated with GSH (100 μM) for 30 minutes, and then the fluorescence probe 152T (10 μM) was added and co-incubated for 30 minutes; C represents the control group: the cells were incubated with 152T (10 μM) for 30 minutes. D represents the blank group: confocal imaging was directly performed on the cells. The results show that due to the addition of exogenous sodium hydrosulfide in group A, the fluorescence intensity is higher than the other three groups. The fluorescence intensity in group B after adding GSH is almost the same as that in group C, and there is no fluorescence in group D. The fluorescence probe of the present invention can respond well to exogenous hydrogen sulfide. Figure 13B It is the fluorescence intensity quantification diagram of each group.

[0146] VII. Study on in vivo imaging of the fluorescence probe

[0147] Based on excellent cell imaging experiments, the present invention also explored the imaging ability of fluorescent probe 152T for H2S in a tumor mouse model. A tumor mouse model was established by implanting cancer cells into nude mice. After intratumoral injection of fluorescent probe 152T into the tumor-bearing mice, strong fluorescence could be observed at the tumor site, and the fluorescence intensity increased with time, and remained stable within 5 h. These results indicate that fluorescent probe 152T can image H2S in tumors, and the results are as Figure 14 shown (Ex = 660 nm, Em = 710 nm), and the fluorescence intensity quantitative map is as Figure 15 shown.

[0148] In addition, the inventors of this case also conducted experiments under other conditions listed in this specification by referring to the manner of the examples, and achieved the same technical effects as well.

[0149] It should be understood that the above examples are only for illustrating the technical concept and characteristics of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe for selectively recognizing hydrogen sulfide, characterized in that, The near-infrared fluorescent probe has a structure shown in formula (I): The near-infrared fluorescent probe for selectively recognizing hydrogen sulfide can selectively recognize hydrogen sulfide.

2. The near-infrared fluorescence probe for selectively recognizing hydrogen sulfide according to claim 1, wherein: The near-infrared fluorescent probe for selectively recognizing hydrogen sulfide has the ability to respond to H2S within the pH range of 7.0 - 10.0; and / or, the response time of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide is within 2 min; and / or, the detection limit of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide is lower than 5 μM.

3. A preparation method of a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide, characterized in that, Including: Reacting a first compound with 2,4-dihydroxybenzaldehyde to obtain a second compound; Reacting the second compound with 4-pyridineacetonitrile to obtain a third compound; Reacting biotin with 4-aminobenzyl alcohol to obtain a fourth compound; Reacting the fourth compound with phosphorus tribromide to obtain a fifth compound; Reacting the third compound with the fifth compound to obtain a sixth compound; Reacting the sixth compound with 2,4-dinitrofluorobenzene to obtain a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide; The first compound has a structure shown in formula (II), the second compound has a structure shown in formula (III), the third compound has a structure shown in formula (IV), the fourth compound has a structure shown in formula (V), the fifth compound has a structure shown in formula (VI), the sixth compound has a structure shown in formula (VII), and the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide has a structure shown in formula (I):

4. The preparation method according to claim 3, characterized in that, Including: Under the condition of -5 to 5 °C and in a protective atmosphere, mixing N,N-dimethylformamide and a first solvent, adding PBr3 and reacting until the solution turns milky white, then adding cyclopentanone and carrying out a first reaction at room temperature for 10 - 15 h to obtain a first compound; Preferably, the molar ratio of N,N-dimethylformamide, PBr3 to cyclopentanone is 110 - 130:100 - 115:30 - 50; Preferably, the first solvent includes any one or a combination of more than one of DCM, chloroform, carbon tetrachloride, THF, DMF, DMSO, DPM, ethyl acetate; and / or, the preparation method includes: mixing the first compound with a second solvent, adding 2,4-dihydroxybenzaldehyde and cesium carbonate and carrying out a second reaction at room temperature for 10 - 15 h to obtain a second compound; Preferably, the molar ratio of the first compound, 2,4-dihydroxybenzaldehyde to cesium carbonate is 12 - 18:15 - 22:35 - 40; Preferably, the second solvent includes any one or a combination of more than one of DMF, DMSO, THF, acetonitrile, acetone; and / or, the preparation method includes: mixing the second compound, 4-pyridineacetonitrile, piperidine and a third solvent, and refluxing at 70 - 90 °C for a third reaction for 10 - 15 h to obtain a third compound; Preferably, the molar ratio of the second compound to 4-pyridineacetonitrile is 1 - 1.1:1 - 1.2; Preferably, the third solvent includes any one or a combination of more than one of ethanol, isopropanol, acetone, methanol, glycerol, propylene glycol, DMSO, and THF.

5. The preparation method according to claim 3, characterized in that, Comprising: Mix biotin with a fourth solvent, add HATU and react for 10 - 20 min, then add DIPEA and 4-aminobenzyl alcohol, and carry out a fourth reaction at room temperature for 3 - 8 h to obtain a fourth compound; Preferably, the molar ratio of biotin, HATU, DIPEA to 4-aminobenzyl alcohol is 1.8 - 2.2:1.8 - 2.2:2 - 2.5:2 - 2.5; Preferably, the fourth solvent includes any one or a combination of more than one of DMF, DMSO, THF, acetonitrile, and acetone; And / or, the preparation method includes: at -5 to 5 °C, mix the fourth compound with a fifth solvent, then add phosphorus tribromide and carry out a fifth reaction at room temperature for 4 - 8 h to obtain a fifth compound; Preferably, the molar ratio of the fourth compound to phosphorus tribromide is 0.2 - 0.4:0.5 - 0.8; Preferably, the fifth solvent includes any one or a combination of more than one of DCM, chloroform, carbon tetrachloride, THF, DMF, DMSO, DPM, and ethyl acetate.

6. The preparation method according to claim 3, characterized in that, Comprising: Mix the third compound, the fifth compound with a sixth solvent, and carry out a sixth reaction at 70 - 90 °C for 10 - 15 h to obtain a sixth compound; Preferably, the molar ratio of the third compound to the fifth compound is 0.08 - 0.12:0.10 - 0.15; Preferably, the sixth solvent includes any one or a combination of more than one of DMF, DMSO, THF, acetonitrile, and acetone.

7. The preparation method according to claim 3, wherein Comprising: Mix the sixth compound, 2,4-dinitrofluorobenzene, triethylamine with a seventh solvent, and react at room temperature for 2 - 6 h to obtain a near-infrared fluorescent probe for selectively recognizing hydrogen sulfide; Preferably, the molar ratio of the sixth compound, 2,4-dinitrofluorobenzene to triethylamine is 0.04 - 0.06:0.05 - 0.08:0.4 - 0.6; Preferably, the seventh solvent includes any one or a combination of more than one of DMF, DMSO, THF, acetonitrile, and acetone.

8. A near-infrared fluorescent probe for selectively recognizing hydrogen sulfide prepared by the preparation method according to any one of claims 3 - 7.

9. Use of the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide according to any one of claims 1 - 2, 8 in detecting hydrogen sulfide; preferably, the hydrogen sulfide is derived from endogenous hydrogen sulfide in organisms.

10. A method for selectively recognizing hydrogen sulfide, characterized in that, Comprising: Contact and react the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide according to any one of claims 1 - 2, 8 with a system that may contain hydrogen sulfide to achieve selective recognition of hydrogen sulfide; Preferably, the system containing hydrogen sulfide includes a live cell hydrogen sulfide system; Preferably, the near-infrared fluorescent probe for selectively recognizing hydrogen sulfide achieves selective recognition of hydrogen sulfide within a pH range of 7.0 - 10.0.

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