WO2023015795A1 - Ratiometric polysulfane fluorescent probe, and preparation method therefor and use thereof - Google Patents

Ratiometric polysulfane fluorescent probe, and preparation method therefor and use thereof Download PDF

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WO2023015795A1
WO2023015795A1 PCT/CN2021/137297 CN2021137297W WO2023015795A1 WO 2023015795 A1 WO2023015795 A1 WO 2023015795A1 CN 2021137297 W CN2021137297 W CN 2021137297W WO 2023015795 A1 WO2023015795 A1 WO 2023015795A1
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fluorescent probe
ratiometric
compound
preparation
hydrogen polysulfide
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Chinese (zh)
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龚萍
向春柏
蔡林涛
向晶晶
张鹏飞
罗媛
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中国科学院深圳先进技术研究院
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    • C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
    • C07D455/04—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing a quinolizine ring system condensed with only one six-membered carbocyclic ring, e.g. julolidine
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/64—Fluorescence; Phosphorescence
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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    • C09K2211/10—Non-macromolecular compounds
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    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom

Definitions

  • the invention relates to the technical field of molecular probes, in particular to a ratio-type hydrogen polysulfide fluorescent probe and its preparation method and application.
  • Reactive sulfur species are sulfur-containing molecules that have important regulatory functions in biological systems.
  • hydrogen sulfide (H 2 S) is an important endogenous gas transporter, which has been widely studied due to its participation in various physiological activities.
  • hydrogen polysulfide (H 2 S n ) has not attracted much attention.
  • some recent evidence suggests that hydrogen polysulfide is involved in an increasing number of physiological activities related to hydrogen sulfide. For example, polyhydrogen sulfide is more likely than hydrogen sulfide to activate TRAP1 channels and induce Ca influx in astrocytes.
  • ratiometric probes are plagued by aggregation-induced quenching (ACQ) in vivo, resulting in massive weak fluorescence and disruption of autofluorescence. Therefore, it is of great significance to develop ratiometric hydrogen polysulfide probes with high fluorescence intensity, fast response and high accuracy.
  • ACQ aggregation-induced quenching
  • the present invention proposes a ratiometric hydrogen polysulfide fluorescent probe and its preparation method and application.
  • the structural formula of the fluorescent probe is shown in formula (I).
  • the fluorescent probe uses 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile to react with a precursor compound to obtain a precursor with aggregation-induced luminescent properties body, and then attached to the electrophilic recognition group 2-fluoro-5-nitrobenzoate.
  • the present invention provides a ratiometric hydrogen polysulfide fluorescent probe, the structural formula of which is shown in formula (I):
  • R is, , , any of the.
  • the present invention also provides the preparation method of the ratio-type hydrogen polysulfide fluorescent probe, the fluorescent probe uses 2-(3-cyano-4,5,5-trimethylfuran-2(5H)- Methyl) malononitrile reacts with 4-(diethylamino) salicylaldehyde to obtain a precursor with aggregation-induced luminescent properties, and then connects the electrophilic recognition group 2-fluoro-5-nitrobenzoate.
  • the precursor compound is When, the intermediate compound is
  • the precursor compound is When, the intermediate compound is ;
  • the precursor compound is When, the intermediate compound is .
  • the basic compound is piperidine or pyridine; the first organic solvent is absolute ethanol, and the second organic solvent is anhydrous dichloromethane.
  • the reaction temperature of the step (1) is 60-100°C; the reaction time is 5-12 hours, preferably 10-12 hours.
  • the reaction temperature can ensure that the intermediate compound yield reaches 85%. If the reaction time is too short, the 85% yield of the fluorescent probe cannot be realized, and if the reaction time is too long, by-products will be generated.
  • the molar ratio of the precursor compound, the 2-fluoro-5-nitrobenzoic acid, the dicyclohexylcarbodiimide and the 4-dimethylaminopyridine is 1:(1-2.5 ): (1-2): (0.1-0.5), preferably 1:2:1.5:0.2; the intermediate compound, the 2-(3-cyano-4,5,5-trimethylfuran-
  • the molar ratio of 2(5H)-methylene)malononitrile to the basic compound is 1:1:0.5.
  • the yield of the fluorescent probe prepared by adopting the above ratio is high, up to 83%.
  • step (1) a step of post-processing the obtained product is also included, and the post-processing includes the following steps:
  • the concentrate was subjected to column chromatography to obtain the intermediate compound.
  • the present invention also provides the application of the ratio-type hydrogen polysulfide fluorescent probe in the qualitative or quantitative detection of hydrogen polysulfide in organisms.
  • the method includes the following steps: adding the sample to be tested into the ratiometric hydrogen polysulfide fluorescent probe solution, and then visually observing the color change and/or testing the change in ultraviolet absorption.
  • the solution of the ratiometric hydrogen polysulfide fluorescent probe is a mixed solution of the ratiometric hydrogen polysulfide fluorescent probe, water and an organic solvent.
  • the volume ratio of the water to the organic solvent is 99.5:5-99:10.
  • organic solvent is dimethylsulfoxide.
  • the ratiometric hydrogen polysulfide fluorescent probe of the present invention has the characteristics of aggregation-induced luminescence, and can emit strong fluorescence in the aggregated state, avoiding a large number of weak fluorescence and interruption of autofluorescence.
  • the ratiometric hydrogen polysulfide fluorescent probe of the present invention has a short response time and a fast response time of only 2 minutes.
  • the ratiometric hydrogen polysulfide fluorescent probe of the present invention performs self-calibration through the ratio change of the fluorescence intensity of two different emission wavelengths, which can effectively avoid the fluorescence quenching of traditional fluorescent molecules at high concentrations or the fluorescence intensity of a single wavelength is susceptible to The shortcomings of external factors such as concentration, temperature, pH value and instrument interference have effectively improved the accuracy and resolution of detection.
  • the ratiometric hydrogen polysulfide fluorescent probe of the present invention can realize a ratiometric linear response to hydrogen polysulfide in a certain concentration range, and can quantitatively detect hydrogen polysulfide in cells and living bodies.
  • Fig. 1 is the working principle figure of the ratio type hydrogen polysulfide fluorescent probe of the present invention
  • Figure 2 is a graph showing the ratio of the fluorescence intensity at 619 nm to the fluorescence intensity at 751 nm after incubation of 5 ⁇ M TCFPB-H 2 S n in the presence of different concentrations of hydrogen polysulfide for different times;
  • Fig. 3 is a change diagram of the fluorescence emission spectrum after adding different concentrations of hydrogen polysulfide to the 5 ⁇ M TCFPB-H 2 S n solution for incubation;
  • Fig. 4 is a linear graph of the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm of 5 ⁇ M TCFPB-H 2 S n solution and the concentration of hydrogen polysulfide;
  • Figure 5 is a graph showing the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm of a 5 ⁇ M TCFPB-H 2 Sn solution as a function of different pH values;
  • Figure 6 is a graph showing the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm after adding different competitor molecules to 5 ⁇ M TCFPB-H 2 S n solution;
  • Figure 7 is a photo of confocal laser microscopy imaging of 5 ⁇ M TCFPB-H 2 S n and HeLa cells incubated at 37°C for 20 minutes;
  • Fig. 8 is a diagram showing the change of the ratio of the fluorescence intensity of the green channel to the fluorescence intensity of the red channel in cells with different concentrations of polyhydrogen sulfide.
  • the invention provides a ratiometric hydrogen polysulfide fluorescent probe, the structural formula of which is shown in formula (I).
  • R is , , any of the.
  • the present invention also provides the preparation method of the fluorescent probe, the fluorescent probe is 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile React with the precursor compound to obtain a precursor with aggregation-induced luminescent properties, and then connect the electrophilic recognition group 2-fluoro-5-nitrobenzoate, the electrophilic recognition group 2-fluoro-5-nitrobenzene Formate is the hydrogen polysulfide recognition unit.
  • the reaction temperature is 60-100°C; the reaction time is 5-12 hours, preferably 10-12 hours.
  • the basic compound is piperidine or pyridine; the first organic solvent is absolute ethanol.
  • the molar ratio of the precursor compound, the 2-fluoro-5-nitrobenzoic acid, the dicyclohexylcarbodiimide and the 4-dimethylaminopyridine is 1: (1-2.5): ( 1-2):(0.1-0.5), preferably 1:2:1.5:0.2.
  • the precursor compound is When, the intermediate compound is
  • the precursor compound is When, the intermediate compound is ;
  • the precursor compound is When, the intermediate compound is .
  • This embodiment is prepared by a two-step method.
  • the precursor compound is 4-(diethylamino) salicylaldehyde, which is prepared according to the following route:
  • nuclear magnetic model is Bruker AVANCE 400, the test condition is room temperature, the NMR data is:
  • Mass spectrometry was performed on the purple intermediate compound prepared in Example 1.
  • the equipment model of the mass spectrometer was Orbitrap Fusion Tribrid mass spectrometer, and the test condition was room temperature.
  • the purple compound TCFPB-H 2 S n prepared in Example 1 was analyzed by NMR, and the purple compound TCFPB-H 2 S n is the hydrogen polysulfide fluorescent probe of the present invention.
  • the nuclear magnetic model is Bruker AVANCE 400, and the test condition is room temperature.
  • the resulting product is characterized by nuclear magnetic resonance, and it can be known that the structural formula of the ratiometric hydrogen polysulfide fluorescent probe prepared in this example is:
  • the intermediate compound can be obtained:
  • the structural formula of the ratio type hydrogen polysulfide fluorescent probe prepared by the present embodiment is:
  • the intermediate compound can be obtained:
  • Figure 2 is a diagram of the ratio change of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm after TCFPB-H 2 S n was incubated in a 95% PBS/DMSO mixed solution for different times in the presence of different concentrations of hydrogen polysulfide. It can be seen from Figure 2 , after incubating at 37°C for 2 minutes, the fluorescence intensity basically reached saturation, which indicated that the polyhydrogen sulfide probe responded quickly, only for 2 minutes.
  • Figure 3 is a graph showing the changes in fluorescence emission spectra after adding different concentrations of hydrogen polysulfide to 5 ⁇ M TCFPB-H 2 S n solution and incubating; it can be seen from Figure 3 that with the increase of the concentration of hydrogen polysulfide, the fluorescence intensity at 619nm Gradually enhanced, while the fluorescence intensity at 751nm gradually decreased.
  • Figure 5 is a graph showing the ratio change of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm in PBS/DMSO mixed solutions of different pH values for TCFPB-H 2 S n . It can be seen from Figure 5 that the probe TCFPB-H 2 S n itself has a very good Excellent pH stability, insensitive to pH value, the probe TCFPB-H 2 S n has a good response ability to hydrogen polysulfide in the range of pH 6.0 ⁇ 10.0.
  • Figure 6 is a diagram of the ratio change of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm after adding different competing molecules to the TCFPB-H 2 S n solution.
  • the fluorescence intensity ratio before and after the 2 S n reaction did not change significantly, indicating that TCFPB-H 2 S n can selectively recognize H 2 S n and is not easily interfered by other ions, and has strong specificity.
  • Example 7 The hydrogen polysulfide fluorescent probe of the present invention qualitatively and quantitatively detects hydrogen polysulfide in cells
  • HeLa cells were resuscitated and inoculated in RPMI 1640 medium containing 10% fetal bovine serum, cultured in an incubator at 37°C, 5% CO 2 , and 100% saturated humidity, and then cultured on 18mm coverslips for 24 hours before use.
  • Figure 7 is a laser confocal microscopic image of TCFPB-H 2 S n incubated with HeLa cells at 37°C for 5 minutes.
  • the light channels all showed weak fluorescent signals, but in the HeLa cells added with hydrogen polysulfide, the fluorescent signals of the red light channel gradually weakened, while the fluorescent signals of the green light channel increased significantly.
  • Figure 8 is a diagram of the ratio of the fluorescence intensity of the green channel to the fluorescence intensity of the red channel in cells with different concentrations of polysulfide hydrogen.
  • 2 S n can be used for ratiometric fluorescence imaging of intracellular hydrogen polysulfide.
  • the hydrogen polysulfide fluorescent probe of the invention can not only detect the hydrogen polysulfide qualitatively in cells and living bodies, but also realize quantitative detection.
  • the ratiometric hydrogen polysulfide fluorescent probe obtained in Example 4 and Example 5 was tested for response time, fluorescence titration, pH sensitivity, selectivity, and imaging conditions in human cervical cancer cells, and the obtained Result and embodiment 6 ⁇ 7 are similar.
  • the ratiometric hydrogen polysulfide fluorescent probe provided by the present invention has aggregation-induced luminescent characteristics and ratiometric response characteristics, and has a short response time, is insensitive to pH, and can qualitatively and quantitatively detect intracellular hydrogen polysulfide , and can respond and image cells through the fluorescence intensity ratio of two different emission wavelengths, which can effectively avoid the interference of a single wavelength from external factors such as temperature, pH, concentration and instruments, obtain higher imaging resolution, and prepare raw materials
  • the method is easy to obtain, has simple steps, is easy to operate, has high yield, is suitable for industrial production, and has broad application prospects.

Abstract

Disclosed are a ratiometric polysulfane fluorescent probe, and a preparation method therefor and the use thereof. The structural formula of the fluorescent probe is represented by formula (I). The fluorescent probe is obtained by reacting 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile with a precursor compound to obtain a precursor having an aggregation-induced emission characteristic, and then linking an electrophilic recognition group 2-fluoro-5-nitrobenzoate. The ratiometric polysulfane fluorescent probe has the advantages of easy-access synthetic raw materials, simple synthesis, a high yield of a target compound, ratiometric response and the like, avoids the defects that conventional fluorescent probes are not suitable for detection at high concentrations and single emission is prone to be interfered by external factors such as concentration, temperature, pH value, and instruments during the detection process, has high fluorescence intensity, fast response, a high resolution and a strong anti-interference capability, can be used for qualitative and quantitative detection of polysulfane in cells and living bodies, and has good prospects for application.

Description

一种比率型多硫化氢荧光探针及其制备方法和应用A ratio-type polyhydrogen sulfide fluorescent probe and its preparation method and application 技术领域technical field
本发明涉及分子探针技术领域，具体涉及一种比率型多硫化氢荧光探针及其制备方法和应用。The invention relates to the technical field of molecular probes, in particular to a ratio-type hydrogen polysulfide fluorescent probe and its preparation method and application.
背景技术Background technique
活性硫种（RSS）是一种含硫分子，在生物系统中具有重要的调节功能。其中硫化氢（H 2S）是一种重要的内源性气体传递体，由于参与多种生理活动而被广泛研究。作为硫化氢的一种氧化形式，多硫化氢（H 2S n）一直没有引起人们的注意。然而，最近的一些证据表明，多硫化氢参与了越来越多与硫化氢有关的生理活动。例如，多硫化氢比硫化氢更有可能激活TRAP1通道，诱导星形胶质细胞内Ca 2+内流。 Reactive sulfur species (RSS) are sulfur-containing molecules that have important regulatory functions in biological systems. Among them, hydrogen sulfide (H 2 S) is an important endogenous gas transporter, which has been widely studied due to its participation in various physiological activities. As an oxidized form of hydrogen sulfide, hydrogen polysulfide (H 2 S n ) has not attracted much attention. However, some recent evidence suggests that hydrogen polysulfide is involved in an increasing number of physiological activities related to hydrogen sulfide. For example, polyhydrogen sulfide is more likely than hydrogen sulfide to activate TRAP1 channels and induce Ca influx in astrocytes.
为了更好地了解多硫化氢在生物系统中的作用，开发新技术监测多硫化氢在生物系统中的作用至关重要。传统的检测多硫化氢的方法是紫外-可见分光光度法和质谱法。这些方法需要对样品进行预处理，使得在体内快速、准确地检测多硫化氢和实时成像具有挑战性。与传统分析方法相比，荧光探针具有灵敏度高、选择性好和非侵入性检测的优点。然而，大多数用于检测多硫化氢的荧光探针主要依靠单个波长荧光强度的变化，会受到探针浓度、温度、pH和仪器效率的干扰。此外，一些比率探针在体内被聚集诱导猝灭（ACQ）所困扰，导致大量弱荧光和自身荧光中断。因此，开发出荧光强度高、响应快、准确性高的比率型多硫化氢探针具有重要意义。To better understand the role of hydrogen polysulfide in biological systems, it is crucial to develop new techniques to monitor the role of hydrogen polysulfide in biological systems. The traditional methods for detecting hydrogen polysulfide are UV-Vis spectrophotometry and mass spectrometry. These methods require sample pretreatment, making rapid and accurate detection and real-time imaging of polyhydrogen sulfide in vivo challenging. Compared with traditional analytical methods, fluorescent probes have the advantages of high sensitivity, good selectivity and non-invasive detection. However, most fluorescent probes used to detect hydrogen polysulfide mainly rely on the change of fluorescence intensity at a single wavelength, which will be interfered by probe concentration, temperature, pH, and instrument efficiency. Furthermore, some ratiometric probes are plagued by aggregation-induced quenching (ACQ) in vivo, resulting in massive weak fluorescence and disruption of autofluorescence. Therefore, it is of great significance to develop ratiometric hydrogen polysulfide probes with high fluorescence intensity, fast response and high accuracy.
技术问题technical problem
针对现有技术中的缺陷，本发明提出了一种比率型多硫化氢荧光探针及其制备方法和应用。所述荧光探针的结构式如式（I）所示。所述荧光探针以2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈与前体化合物反应得到具有聚集诱导发光特性的前驱体，然后连接亲电识别基团2-氟-5-硝基苯甲酸酯所得。Aiming at the defects in the prior art, the present invention proposes a ratiometric hydrogen polysulfide fluorescent probe and its preparation method and application. The structural formula of the fluorescent probe is shown in formula (I). The fluorescent probe uses 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile to react with a precursor compound to obtain a precursor with aggregation-induced luminescent properties body, and then attached to the electrophilic recognition group 2-fluoro-5-nitrobenzoate.
技术解决方案technical solution
本发明提供一种比率型多硫化氢荧光探针，所述荧光探针的结构式如式（I）所示：The present invention provides a ratiometric hydrogen polysulfide fluorescent probe, the structural formula of which is shown in formula (I):
Figure dest_path_image001
，
Figure dest_path_image001
,
其中，R为，
Figure dest_path_image002
，
Figure dest_path_image003
，
Figure dest_path_image004
中的任意一种。 where R is,
Figure dest_path_image002
,
Figure dest_path_image003
,
Figure dest_path_image004
any of the.
本发明还提供所述的比率型多硫化氢荧光探针的制备方法，所述荧光探针以2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈与4-(二乙氨基)水杨醛反应得到具有聚集诱导发光特性的前驱体，然后连接亲电识别基团2-氟-5-硝基苯甲酸酯所得。The present invention also provides the preparation method of the ratio-type hydrogen polysulfide fluorescent probe, the fluorescent probe uses 2-(3-cyano-4,5,5-trimethylfuran-2(5H)- Methyl) malononitrile reacts with 4-(diethylamino) salicylaldehyde to obtain a precursor with aggregation-induced luminescent properties, and then connects the electrophilic recognition group 2-fluoro-5-nitrobenzoate.
进一步的，包括如下步骤：Further, the following steps are included:
（1）将前体化合物、2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈、碱性化合物和第一有机溶剂混合，在保护气氛下进行Knoevenagel反应，得到中间化合物；(1) mixing the precursor compound, 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile, the basic compound and the first organic solvent, Carry out Knoevenagel reaction under protective atmosphere, obtain intermediate compound;
（2）将所述中间化合物与2-氟-5-硝基苯甲酸、二环己基碳二亚胺、4-二甲氨基吡啶和第二有机溶剂混合，在保护气氛下进行缩合反应，得到所述比率型多硫化氢荧光探针；(2) Mix the intermediate compound with 2-fluoro-5-nitrobenzoic acid, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and a second organic solvent, and perform a condensation reaction under a protective atmosphere to obtain The ratiometric polyhydrogen sulfide fluorescent probe;
所述前体化合物为
Figure dest_path_image005
时，所述中间化合物为
Figure dest_path_image006
The precursor compound is
Figure dest_path_image005
When, the intermediate compound is
Figure dest_path_image006
所述前体化合物为
Figure dest_path_image007
时，所述中间化合物为
Figure dest_path_image008
； The precursor compound is
Figure dest_path_image007
When, the intermediate compound is
Figure dest_path_image008
;
所述前体化合物为
Figure dest_path_image009
时，所述中间化合物为
Figure dest_path_image010
。 The precursor compound is
Figure dest_path_image009
When, the intermediate compound is
Figure dest_path_image010
.
进一步的，所述碱性化合物为哌啶或吡啶；所述第一有机溶剂为无水乙醇，所述第二有机溶剂为无水二氯甲烷。Further, the basic compound is piperidine or pyridine; the first organic solvent is absolute ethanol, and the second organic solvent is anhydrous dichloromethane.
进一步的，所述步骤（1）反应的温度为60~100℃；反应时间为5~12个小时，优选为10~12小时。所述反应温度能够保证中间化合物产率达到85%。所述反应时间过短不能够实现所述荧光探针85%的产率，时间过长会生成副产物。Further, the reaction temperature of the step (1) is 60-100°C; the reaction time is 5-12 hours, preferably 10-12 hours. The reaction temperature can ensure that the intermediate compound yield reaches 85%. If the reaction time is too short, the 85% yield of the fluorescent probe cannot be realized, and if the reaction time is too long, by-products will be generated.
进一步的，所述前体化合物、所述2-氟-5-硝基苯甲酸、所述二环己基碳二亚胺和所述4-二甲氨基吡啶的摩尔比为1：（1-2.5）：（1-2）：（0.1-0.5），优选为1：2：1.5：0.2；所述中间化合物、所述2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈和所述碱性化合物的摩尔比为1：1：0.5。采用上述比例制备的所述荧光探针产率高，可达83%。Further, the molar ratio of the precursor compound, the 2-fluoro-5-nitrobenzoic acid, the dicyclohexylcarbodiimide and the 4-dimethylaminopyridine is 1:(1-2.5 ): (1-2): (0.1-0.5), preferably 1:2:1.5:0.2; the intermediate compound, the 2-(3-cyano-4,5,5-trimethylfuran- The molar ratio of 2(5H)-methylene)malononitrile to the basic compound is 1:1:0.5. The yield of the fluorescent probe prepared by adopting the above ratio is high, up to 83%.
进一步的，所述步骤（1）的Knoevenagel反应完成后，还包括将所得产物进行后处理的步骤，所述后处理包括以下步骤：Further, after the Knoevenagel reaction in the step (1) is completed, a step of post-processing the obtained product is also included, and the post-processing includes the following steps:
将反应后的液态混合物浓缩，得到浓缩物；Concentrating the reacted liquid mixture to obtain a concentrate;
将所述浓缩物进行柱层析，得到所述中间化合物。The concentrate was subjected to column chromatography to obtain the intermediate compound.
本发明还提供所述的比率型多硫化氢荧光探针在生物体内多硫化氢的定性或定量检测中的应用。The present invention also provides the application of the ratio-type hydrogen polysulfide fluorescent probe in the qualitative or quantitative detection of hydrogen polysulfide in organisms.
进一步的，包括如下步骤：将待测样品加入到比率型多硫化氢荧光探针溶液中，然后目测颜色变化和/或测试紫外吸收变化。Further, the method includes the following steps: adding the sample to be tested into the ratiometric hydrogen polysulfide fluorescent probe solution, and then visually observing the color change and/or testing the change in ultraviolet absorption.
进一步的，所述比率型多硫化氢荧光探针溶液为比率型多硫化氢荧光探针与水和有机溶剂的混合溶液。Further, the solution of the ratiometric hydrogen polysulfide fluorescent probe is a mixed solution of the ratiometric hydrogen polysulfide fluorescent probe, water and an organic solvent.
进一步的，所述水和所述有机溶剂的体积比为99.5：5~99：10。在上述测试条件的参数范围内，所述探针对多硫化氢有明显的检测识别效果，而在所述参数范围之外的测试条件下，所述探针对多硫化氢则无明显检测识别效果。Further, the volume ratio of the water to the organic solvent is 99.5:5-99:10. Within the parameter range of the above test conditions, the probe has an obvious detection and recognition effect on hydrogen polysulfide, while under the test conditions outside the parameter range, the probe has no obvious detection and recognition effect on hydrogen polysulfide Effect.
进一步的，所述有机溶剂为二甲基亚砜。Further, the organic solvent is dimethylsulfoxide.
有益效果Beneficial effect
综上，与现有技术相比，本发明达到了以下技术效果：In summary, compared with the prior art, the present invention achieves the following technical effects:
（1）本发明的比率型多硫化氢荧光探针具有聚集诱导发光特性，在聚集态时能发出较强的荧光，避免了大量弱荧光和自身荧光中断。(1) The ratiometric hydrogen polysulfide fluorescent probe of the present invention has the characteristics of aggregation-induced luminescence, and can emit strong fluorescence in the aggregated state, avoiding a large number of weak fluorescence and interruption of autofluorescence.
（2）本发明的比率型多硫化氢荧光探针响应时间短、速度快，仅为2min。(2) The ratiometric hydrogen polysulfide fluorescent probe of the present invention has a short response time and a fast response time of only 2 minutes.
（3）本发明的比率型多硫化氢荧光探针通过两个不同发射波长的荧光强度的比率变化进行自校正，可以有效避免传统荧光分子在高浓度下荧光猝灭或单一波长荧光强度易受浓度、温度、pH值及仪器等外界因素干扰的缺点，有效提高了检测的准确性和分辨率。(3) The ratiometric hydrogen polysulfide fluorescent probe of the present invention performs self-calibration through the ratio change of the fluorescence intensity of two different emission wavelengths, which can effectively avoid the fluorescence quenching of traditional fluorescent molecules at high concentrations or the fluorescence intensity of a single wavelength is susceptible to The shortcomings of external factors such as concentration, temperature, pH value and instrument interference have effectively improved the accuracy and resolution of detection.
（4）本发明的比率型多硫化氢荧光探针可以在一定浓度范围内对溶液中的多硫化氢实现比率型线性响应，能够定量检测细胞和活体内的多硫化氢。(4) The ratiometric hydrogen polysulfide fluorescent probe of the present invention can realize a ratiometric linear response to hydrogen polysulfide in a certain concentration range, and can quantitatively detect hydrogen polysulfide in cells and living bodies.
（5）本发明的比率型多硫化氢荧光探针的制备合成原料易得，步骤简单，容易操作，荧光探针的产率可达到83%，适合工业化生产。(5) Preparation of the ratiometric hydrogen polysulfide fluorescent probe of the present invention The synthetic raw materials are easy to obtain, the steps are simple, and the operation is easy. The yield of the fluorescent probe can reach 83%, which is suitable for industrial production.
附图说明Description of drawings
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.
图1为本发明的比率型多硫化氢荧光探针的工作原理图；Fig. 1 is the working principle figure of the ratio type hydrogen polysulfide fluorescent probe of the present invention;
图2为5μM的TCFPB-H 2S n在不同浓度的多硫化氢存在条件下孵化不同时间后619nm处荧光强度与751nm处荧光强度的比值变化图； Figure 2 is a graph showing the ratio of the fluorescence intensity at 619 nm to the fluorescence intensity at 751 nm after incubation of 5 μM TCFPB-H 2 S n in the presence of different concentrations of hydrogen polysulfide for different times;
图3为5μM的TCFPB-H 2S n溶液中加入不同浓度多硫化氢孵化后荧光发射光谱的变化图； Fig. 3 is a change diagram of the fluorescence emission spectrum after adding different concentrations of hydrogen polysulfide to the 5 μM TCFPB-H 2 S n solution for incubation;
图4为5μM的TCFPB-H 2S n溶液在619nm处荧光强度与751nm处荧光强度的比值与多硫化氢浓度的线性曲线图； Fig. 4 is a linear graph of the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm of 5 μM TCFPB-H 2 S n solution and the concentration of hydrogen polysulfide;
图5为5μM的TCFPB-H 2S n溶液619nm处荧光强度与751nm处荧光强度的比值随不同pH值变化图； Figure 5 is a graph showing the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm of a 5μM TCFPB-H 2 Sn solution as a function of different pH values;
图6为5μM的TCFPB-H 2S n溶液中分别加入不同竞争分子后619nm处荧光强度与751nm处荧光强度的比值变化图； Figure 6 is a graph showing the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm after adding different competitor molecules to 5μM TCFPB-H 2 S n solution;
图7为5μM的TCFPB-H 2S n与HeLa细胞37℃下孵化20min后的激光共聚焦显微成像照片； Figure 7 is a photo of confocal laser microscopy imaging of 5 μM TCFPB-H 2 S n and HeLa cells incubated at 37°C for 20 minutes;
图8为细胞中绿色通道荧光强度与红色通道荧光强度的比值随不同多硫化氢浓度的变化图。Fig. 8 is a diagram showing the change of the ratio of the fluorescence intensity of the green channel to the fluorescence intensity of the red channel in cells with different concentrations of polyhydrogen sulfide.
本发明的实施方式Embodiments of the present invention
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only It is an embodiment of a part of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.
本发明提供一种比率型多硫化氢荧光探针，其结构式如式（I）所示。The invention provides a ratiometric hydrogen polysulfide fluorescent probe, the structural formula of which is shown in formula (I).
Figure dest_path_image011
Figure dest_path_image011
其中，R为
Figure dest_path_image012
、
Figure dest_path_image013
、
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中的任意一种。 Among them, R is
Figure dest_path_image012
,
Figure dest_path_image013
,
Figure dest_path_image014
any of the.
本发明还提供所述荧光探针的制备方法，所述荧光探针以2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈与前体化合物反应得到具有聚集诱导发光特性的前驱体，然后连接亲电识别基团2-氟-5-硝基苯甲酸酯所得，亲电识别基团2-氟-5-硝基苯甲酸酯为多硫化氢识别单元。The present invention also provides the preparation method of the fluorescent probe, the fluorescent probe is 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile React with the precursor compound to obtain a precursor with aggregation-induced luminescent properties, and then connect the electrophilic recognition group 2-fluoro-5-nitrobenzoate, the electrophilic recognition group 2-fluoro-5-nitrobenzene Formate is the hydrogen polysulfide recognition unit.
包括如下步骤：Including the following steps:
（1）将前体化合物、2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈、碱性化合物和第一有机溶剂混合，在保护气氛下进行Knoevenagel反应，将所得产物进行后处理，将反应后的液态混合物浓缩，得到浓缩物；将所述浓缩物进行柱层析，得到中间化合物。反应的温度为60~100℃；反应时间为5~12个小时，优选为10~12小时。所述碱性化合物为哌啶或吡啶；所述第一有机溶剂为无水乙醇。所述前体化合物、所述2-氟-5-硝基苯甲酸、所述二环己基碳二亚胺和所述4-二甲氨基吡啶的摩尔比为1：（1-2.5）：（1-2）：（0.1-0.5），优选为1：2：1.5：0.2。(1) mixing the precursor compound, 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile, the basic compound and the first organic solvent, Carrying out Knoevenagel reaction under a protective atmosphere, post-processing the obtained product, concentrating the reacted liquid mixture to obtain a concentrate; subjecting the concentrate to column chromatography to obtain an intermediate compound. The reaction temperature is 60-100°C; the reaction time is 5-12 hours, preferably 10-12 hours. The basic compound is piperidine or pyridine; the first organic solvent is absolute ethanol. The molar ratio of the precursor compound, the 2-fluoro-5-nitrobenzoic acid, the dicyclohexylcarbodiimide and the 4-dimethylaminopyridine is 1: (1-2.5): ( 1-2):(0.1-0.5), preferably 1:2:1.5:0.2.
（2）将所述中间化合物与2-氟-5-硝基苯甲酸、二环己基碳二亚胺、4-二甲氨基吡啶和第二有机溶剂混合，在保护气氛下进行缩合反应，得到所述比率型多硫化氢荧光探针；所述第二有机溶剂为无水二氯甲烷。所述中间化合物、所述2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈和所述碱性化合物的摩尔比为1：1：0.5。(2) Mix the intermediate compound with 2-fluoro-5-nitrobenzoic acid, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and a second organic solvent, and perform a condensation reaction under a protective atmosphere to obtain The ratiometric hydrogen polysulfide fluorescent probe; the second organic solvent is anhydrous dichloromethane. The molar ratio of the intermediate compound, the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile and the basic compound is 1: 1:0.5.
所述前体化合物为
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时，所述中间化合物为
Figure dest_path_image016
The precursor compound is
Figure dest_path_image015
When, the intermediate compound is
Figure dest_path_image016
所述前体化合物为
Figure dest_path_image017
时，所述中间化合物为
Figure dest_path_image018
； The precursor compound is
Figure dest_path_image017
When, the intermediate compound is
Figure dest_path_image018
;
所述前体化合物为
Figure dest_path_image019
时，所述中间化合物为
Figure dest_path_image020
。 The precursor compound is
Figure dest_path_image019
When, the intermediate compound is
Figure dest_path_image020
.
实施例1  本发明的多硫化氢荧光探针（TCFPB-H 2S n）的制备 Example 1 Preparation of polyhydrogen sulfide fluorescent probe (TCFPB-H 2 S n ) of the present invention
本实施例采用两步法制备，前体化合物为4-(二乙氨基)水杨醛，按照如下路线进行制备：This embodiment is prepared by a two-step method. The precursor compound is 4-(diethylamino) salicylaldehyde, which is prepared according to the following route:
Figure dest_path_image021
Figure dest_path_image021
具体工艺为：The specific process is:
（1）在氮气保护下，准确称取199mg的4-(二乙氨基)水杨醛（前体化合物）和231mg的2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈溶于6mL无水乙醇，再往反应液中滴加28μL哌啶，在75℃回流搅拌过夜，当TLC监测反应已不再进行时停止反应，减压蒸馏除去溶剂，并经柱层析分离提纯，真空干燥后得到紫色固体318mg，即为中间化合物，产率为85％。(1) Under nitrogen protection, accurately weigh 199 mg of 4-(diethylamino) salicylaldehyde (precursor compound) and 231 mg of 2-(3-cyano-4,5,5-trimethylfuran- Dissolve 2(5H)-methylene)malononitrile in 6 mL of absolute ethanol, then add 28 μL of piperidine dropwise to the reaction solution, and stir at 75°C under reflux overnight, stop the reaction when the reaction is no longer monitored by TLC, reduce The solvent was distilled off under high pressure, separated and purified by column chromatography, and vacuum-dried to obtain 318 mg of a purple solid, which was an intermediate compound, with a yield of 85%.
（2）在氮气保护下，先准确称取93mg的2-氟-5-硝基苯甲酸和100mg中间化合物溶于6mL无水二氯甲烷中，接着再称取67mg的DCC和6mg DMAP加入反应液，室温搅拌5h。TLC监测反应完全后停止反应，减压蒸馏除去溶剂，并经柱层析分离提纯，真空干燥后得到紫色固体121mg，即为目标产物TCFPB-H 2S n，产率为83％。 (2) Under the protection of nitrogen, first accurately weigh 93mg of 2-fluoro-5-nitrobenzoic acid and 100mg of intermediate compounds and dissolve them in 6mL of anhydrous dichloromethane, then weigh 67mg of DCC and 6mg of DMAP to add to the reaction solution, stirred at room temperature for 5 h. The reaction was stopped after the completion of the reaction as monitored by TLC. The solvent was distilled off under reduced pressure, separated and purified by column chromatography, and dried in vacuo to obtain 121 mg of a purple solid, which was the target product TCFPB-H 2 S n , with a yield of 83%.
实施例2  中间化合物的鉴定Example 2 Identification of intermediate compounds
对实施例1制备得到的紫色中间化合物进行核磁分析，核磁型号为Bruker AVANCE 400，测试条件为室温，核磁共振数据为：Carry out nuclear magnetic analysis to the purple intermediate compound that embodiment 1 prepares, nuclear magnetic model is Bruker AVANCE 400, the test condition is room temperature, the NMR data is:
1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 8.24 (s, 1H), 7.70 (d, J = 9.2 Hz, 1H), 6.94 (d, J = 15.2 Hz, 1H), 6.45 (dd, J = 9.3, 2.4 Hz, 1H), 6.15 (d, J = 2.4 Hz, 1H), 3.46 (q, J = 7.1 Hz, 4H), 1.70 (s, 6H), 1.16 (t, J = 7.0 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 177.97, 175.75, 162.69, 154.55, 114.55, 113.67, 113.30, 112.47, 107.25, 97.67, 96.93, 49.26, 45.04, 26.43, 13.14。 1 H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 8.24 (s, 1H), 7.70 (d, J = 9.2 Hz, 1H), 6.94 (d, J = 15.2 Hz, 1H), 6.45 (dd, J = 9.3, 2.4 Hz, 1H), 6.15 (d, J = 2.4 Hz, 1H), 3.46 (q, J = 7.1 Hz, 4H), 1.70 (s, 6H), 1.16 (t, J = 7.0 Hz, 6H). 13 C NMR (101 MHz, DMSO-D6) Δ 177.97, 175.75, 162.69, 154.55, 114.55, 113.67, 113.30, 112.47, 107.25, 97.67.26, 45.04, 26.43, 13.14.
对实施例1制备得到的紫色中间化合物进行质谱分析，质谱分析仪的设备型号为Orbitrap Fusion Tribrid mass spectrometer，测试条件为室温，紫色中间化合物的高分辨质谱分析结果为：HRMS m/z calculated for C 22H 22N 4O 2Na:[ [M +Na]= 397.16405; found: 397.16354。说明中间化合物成功制备。 Mass spectrometry was performed on the purple intermediate compound prepared in Example 1. The equipment model of the mass spectrometer was Orbitrap Fusion Tribrid mass spectrometer, and the test condition was room temperature. The high-resolution mass spectrometry result of the purple intermediate compound was: HRMS m/z calculated for C 22 H 22 N 4 O 2 Na: [[M + Na]= 397.16405; found: 397.16354. It shows that the intermediate compound was successfully prepared.
实施例3  本发明的多硫化氢荧光探针的的鉴定Example 3 Identification of the polyhydrogen sulfide fluorescent probe of the present invention
对实施例1制备得到的紫色化合物TCFPB-H 2S n进行核磁分析，紫色化合物TCFPB-H 2S n即为本发明的多硫化氢荧光探针。核磁型号为Bruker AVANCE 400，测试条件为室温。通过核磁共振对所得产物进行表征，可知本实施例制备得到的比率型多硫化氢荧光探针的结构式为 The purple compound TCFPB-H 2 S n prepared in Example 1 was analyzed by NMR, and the purple compound TCFPB-H 2 S n is the hydrogen polysulfide fluorescent probe of the present invention. The nuclear magnetic model is Bruker AVANCE 400, and the test condition is room temperature. The resulting product is characterized by nuclear magnetic resonance, and it can be known that the structural formula of the ratiometric hydrogen polysulfide fluorescent probe prepared in this example is:
Figure dest_path_image022
Figure dest_path_image022
核磁共振数据为： 1H NMR (400 MHz, DMSO-d6) δ 8.87 (dd, J = 5.9, 2.9 Hz, 1H), 8.65 (dd, J = 8.0, 4.2 Hz, 1H), 8.15-8.07 (m, 2H), 7.77 (t, J = 9.5 Hz, 1H), 6.97-6.83 (m, 3H), 3.52 (q, J = 7.0 Hz, 4H), 1.67 (s, 6H), 1.17 (t, J = 7.0 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 178.08, 175.71, 166.68, 164.00, 153.26, 153.19, 144.15, 141.90, 131.85, 131.65, 128.56, 119.98, 119.73, 114.21, 113.82, 112.89, 111.35, 109.87, 105.89, 98.86, 91.85, 51.84, 45.00, 25.51, 13.01. The NMR data are: 1 H NMR (400 MHz, DMSO-d6) δ 8.87 (dd, J = 5.9, 2.9 Hz, 1H), 8.65 (dd, J = 8.0, 4.2 Hz, 1H), 8.15-8.07 (m , 2H), 7.77 (t, J = 9.5 Hz, 1H), 6.97-6.83 (m, 3H), 3.52 (q, J = 7.0 Hz, 4H), 1.67 (s, 6H), 1.17 (t, J = 7.0 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 178.08, 175.71, 166.68, 164.00, 153.26, 153.19, 144.15, 141.90, 131.85, 131.65, 128.56, 119.98, 119.73, 114.21, 113.82, 112.89, 111.35, 109.87, 105.89, 98.86, 91.85, 51.84, 45.00, 25.51, 13.01.
对实施例1制备得到的紫色化合物TCFPB-H 2S n进行质谱分析，质谱分析仪的设备型号为Orbitrap Fusion Tribrid mass spectrometer，测试条件为室温，紫色化合物TCFPB-H 2S n的高分辨质谱分析结果为：HRMS m/z calculated for C 29H 24N 5O 5FNa:[M +Na] =564.16592; found: 564.16553。以上结果表明本发明的多硫化氢荧光探针成功制备。 Perform mass spectrometry analysis on the purple compound TCFPB-H 2 S n prepared in Example 1. The equipment model of the mass spectrometer is Orbitrap Fusion Tribrid mass spectrometer, and the test condition is room temperature. High-resolution mass spectrometry analysis of the purple compound TCFPB-H 2 S n The result is: HRMS m/z calculated for C 29 H 24 N 5 O 5 FNa: [M + Na] = 564.16592; found: 564.16553. The above results indicated that the polyhydrogen sulfide fluorescent probe of the present invention was successfully prepared.
实施例4  本发明的多硫化氢荧光探针的制备和鉴定Example 4 Preparation and identification of polyhydrogen sulfide fluorescent probe of the present invention
其他条件和实施例1相同，仅将前体化合物替换为：
Figure dest_path_image023
，可得到中间化合物：
Figure dest_path_image024
，所述中间化合物的核磁共振数据为： 1H NMR (400 MHz, CDCl 3-d) δ 7.78-7.70 (m, 4H), 6.92 (dd, J = 7.5, 2.0 Hz, 2H), 6.85 (d, J = 2.0 Hz, 2H), 6.70 (s, 1H), 2.89 (s, 9H), 1.48 (s, 9H)； 13C NMR (101 MHz, CDCl 3-d) δ 156.21, 149.45, 142.05, 135.31, 135.23, 132.41, 129.16, 128.84, 126.02, 116.15, 115.61, 114.73, 102.89, 101.50, 78.77, 40.35, 27.22。所述中间化合物的高分辨质谱分析结果为：HRMS m/z calculated for C 20H 18N 4O 2Na:[ [M +Na]= 369.13275; found: 369.13254。以上结果表明中间化合物成功制备。 Other conditions are the same as in Example 1, only the precursor compound is replaced by:
Figure dest_path_image023
, the intermediate compound can be obtained:
Figure dest_path_image024
, the NMR data of the intermediate compound are: 1 H NMR (400 MHz, CDCl 3 -d) δ 7.78-7.70 (m, 4H), 6.92 (dd, J = 7.5, 2.0 Hz, 2H), 6.85 (d , J = 2.0 Hz, 2H), 6.70 (s, 1H), 2.89 (s, 9H), 1.48 (s, 9H); 13 C NMR (101 MHz, CDCl 3 -d) δ 156.21, 149.45, 142.05, 135.31 , 135.23, 132.41, 129.16, 128.84, 126.02, 116.15, 115.61, 114.73, 102.89, 101.50, 78.77, 40.35, 27.22. The high-resolution mass spectrometry analysis result of the intermediate compound is: HRMS m/z calculated for C 20 H 18 N 4 O 2 Na: [ [M + Na]= 369.13275; found: 369.13254. The above results indicated that the intermediate compound was successfully prepared.
通过核磁共振对所得产物进行表征，可知本实施例制备得到的比率型多硫化氢荧光探针的结构式为:
Figure dest_path_image025
，所述荧光探针的核磁共振数据为： 1H NMR (400 MHz, CDCl 3-d) δ 8.66(d, J = 2.0 Hz, 2H), 8.24 (dd, J = 7.5, 2.0 Hz, 2H), 7.56 (dd, J = 7.5, 1.1 Hz, 2H), 7.43-7.36 (m, 4H), 7.24 (d, J = 2.0 Hz, 2H), 7.18 (s, 1H), 6.96 (dd, J = 7.5, 2.0 Hz, 2H), 2.93 (s, 9H), 1.49 (s, 9H).; 13C NMR (101 MHz, CDCl 3-d) δ 164.96, 159.74, 156.78, 143.93, 141.65, 141.37, 137.21, 135.85, 129.04, 127.64, 127.03, 126.58, 125.72, 124.53, 118.25, 115.62, 114.97, 114.95, 114.13, 103.96, 88.49, 81.67, 40.52, 27.64。所述荧光探针的高分辨质谱分析结果为：HRMS m/z calculated for C 27H 20FN 5O 5Na:[ [M +Na]= 536.13462; found: 536.13496。以上结果表明本发明的多硫化氢荧光探针成功制备。 The obtained product is characterized by nuclear magnetic resonance, as can be known, the structural formula of the ratio type hydrogen polysulfide fluorescent probe prepared by the present embodiment is:
Figure dest_path_image025
, the nuclear magnetic resonance data of the fluorescent probe is: 1 H NMR (400 MHz, CDCl 3 -d) δ 8.66(d, J = 2.0 Hz, 2H), 8.24 (dd, J = 7.5, 2.0 Hz, 2H) , 7.56 (dd, J = 7.5, 1.1 Hz, 2H), 7.43-7.36 (m, 4H), 7.24 (d, J = 2.0 Hz, 2H), 7.18 (s, 1H), 6.96 (dd, J = 7.5 , 2.0 Hz, 2H), 2.93 (s, 9H), 1.49 (s, 9H).; 13 C NMR (101 MHz, CDCl 3 -d) δ 164.96, 159.74, 156.78, 143.93, 141.65, 141.37, 137.21, 135.85 , 129.04, 127.64, 127.03, 126.58, 125.72, 124.53, 118.25, 115.62, 114.97, 114.95, 114.13, 103.96, 88.49, 81.67, 40.52, 27.64. The high-resolution mass spectrometry analysis result of the fluorescent probe is: HRMS m/z calculated for C 27 H 20 FN 5 O 5 Na: [ [M + Na]= 536.13462; found: 536.13496. The above results indicated that the polyhydrogen sulfide fluorescent probe of the present invention was successfully prepared.
实施例5  本发明的多硫化氢荧光探针的制备和鉴定Example 5 Preparation and identification of polyhydrogen sulfide fluorescent probe of the present invention
其他条件和实施例1相同，仅将前体化合物替换为：
Figure dest_path_image026
，可得到中间化合物：
Figure dest_path_image027
，所述中间化合物的核磁共振数据为 1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 8.44 (s, 1H), 7.46 (s, 1H), 6.72 (d, J = 14.2 Hz, 1H), 3.41-3.34 (m, 4H), 2.66 (t, J = 6.2 Hz, 2H), 2.58 (d, J = 12.8 Hz, 2H), 1.84 (p, J = 6.1 Hz, 4H), 1.66 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ 177.98, 173.70, 157.58, 151.49, 118.01, 115.12, 114.22, 114.12, 113.99, 106.62, 97.01, 50.76, 49.96, 47.28, 27.08, 26.53, 21.43, 21.02, 20.40。所述中间化合物的高分辨质谱分析结果为：HRMS m/z calculated for C24H23N4O2:[ [M +H] += 399.18155; found: 399.18167。以上结果表明中间化合物成功制备。 Other conditions are the same as in Example 1, only the precursor compound is replaced by:
Figure dest_path_image026
, the intermediate compound can be obtained:
Figure dest_path_image027
, the nuclear magnetic resonance data of the intermediate compound is 1 H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 8.44 (s, 1H), 7.46 (s, 1H), 6.72 (d, J = 14.2 Hz, 1H), 3.41-3.34 (m, 4H), 2.66 (t, J = 6.2 Hz, 2H), 2.58 (d, J = 12.8 Hz, 2H), 1.84 (p, J = 6.1 Hz, 4H), 1.66 (s, 6H); 13 C NMR (101 MHz, DMSO-D6) Δ 177.98, 173.70, 157.58, 151.49, 118.01, 115.12, 114.22, 113.99, 106.62, 97.01, 49.96, 47.28, 27.08, 26.53 , 21.43, 21.02, 20.40. The high-resolution mass spectrometry analysis result of the intermediate compound is: HRMS m/z calculated for C24H23N4O2: [[M + H] + =399.18155; found: 399.18167. The above results indicated that the intermediate compound was successfully prepared.
通过核磁共振对所得产物进行表征，可知本实施例制备得到的比率型多硫化氢荧光探针的结构式为:
Figure dest_path_image028
，所述荧光探针的核磁共振数据为 1H NMR (400 MHz, DMSO-d6) δ 8.86 (dd, J = 6.1, 3.0 Hz, 1H), 8.70-8.61 (m, 1H), 8.13 (d, J = 15.5 Hz, 1H), 7.83-7.73 (m, 2H), 6.85 (d, J = 15.6 Hz, 1H), 3.43 (s, 4H), 2.79 (t, J = 6.2 Hz, 4H), 1.96-1.83 (m, 4H), 1.63 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 178.22, 174.96, 149.57, 148.59, 141.93, 132.21, 127.50, 121.21, 114.18, 113.91, 113.35, 113.21, 108.53, 98.47, 89.17, 50.58, 50.37, 49.72, 25.56, 21.27, 20.89, 20.03。高分辨质谱分析结果为：HRMS m/z calculated for C 31H 24FN 5O 5Na:[ [M +Na]= 588.16592; found: 588.16570。以上结果表明本发明的多硫化氢荧光探针成功制备。 The obtained product is characterized by nuclear magnetic resonance, as can be known, the structural formula of the ratio type hydrogen polysulfide fluorescent probe prepared by the present embodiment is:
Figure dest_path_image028
, the nuclear magnetic resonance data of the fluorescent probe is 1 H NMR (400 MHz, DMSO-d6) δ 8.86 (dd, J = 6.1, 3.0 Hz, 1H), 8.70-8.61 (m, 1H), 8.13 (d, J = 15.5 Hz, 1H), 7.83-7.73 (m, 2H), 6.85 (d, J = 15.6 Hz, 1H), 3.43 (s, 4H), 2.79 (t, J = 6.2 Hz, 4H), 1.96- 1.83 (m, 4H), 1.63 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 178.22, 174.96, 149.57, 148.59, 141.93, 132.21, 127.50, 121.21, 114.13, 113.31, 113.9 108.53, 98.47, 89.17, 50.58, 50.37, 49.72, 25.56, 21.27, 20.89, 20.03. The result of high-resolution mass spectrometry analysis is: HRMS m/z calculated for C 31 H 24 FN 5 O 5 Na:[ [M + Na]= 588.16592; found: 588.16570. The above results indicated that the polyhydrogen sulfide fluorescent probe of the present invention was successfully prepared.
实施例6  本发明的比率型多硫化氢探针(TCFPB-H 2S n)的性能测试 Example 6 Performance test of the ratiometric hydrogen polysulfide probe (TCFPB-H 2 S n ) of the present invention
以实施例1制备的荧光探针为测试对象，具体步骤如下：Taking the fluorescent probe prepared in Example 1 as the test object, the specific steps are as follows:
（1）比率型多硫化氢探针的响应时间测定：在2mL 95％PBS/DMSO混合溶液（pH值为7.4）中加入10μL TCFPB-H 2S n的DMSO溶液（1mM）得到5μM的TCFPB-H 2S n溶液，然后分别加入5μM、10μM和20μM的多硫化钠溶液，所得混合溶液在37℃下孵化不同的时间（30s、60s、90s、120s、180s、210s、140s、270s、300s），测定所述混合溶液的619nm处荧光强度与751nm处荧光强度的比值随孵化时间的变化情况。 (1) Determination of the response time of the ratiometric polyhydrogen sulfide probe: Add 10 μL TCFPB-H 2 S n DMSO solution (1 mM) to 2 mL 95% PBS/DMSO mixed solution (pH 7.4) to obtain 5 μM TCFPB- H2Sn solution, and then add 5 μM, 10 μM and 20 μM sodium polysulfide solution respectively, and the resulting mixed solution was incubated at 37°C for different times (30s, 60s, 90s, 120s, 180s, 210s, 140s, 270s, 300s) , measuring the variation of the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm of the mixed solution with the incubation time.
图2为TCFPB-H 2S n在不同浓度的多硫化氢存在条件下在95％PBS/DMSO混合溶液中孵化不同时间后619nm处荧光强度与751nm处荧光强度的比值变化图，由图2可知，在37℃下孵化2min后，荧光强度基本达到饱和，这说明多硫化氢探针响应较快，仅为2min。 Figure 2 is a diagram of the ratio change of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm after TCFPB-H 2 S n was incubated in a 95% PBS/DMSO mixed solution for different times in the presence of different concentrations of hydrogen polysulfide. It can be seen from Figure 2 , after incubating at 37°C for 2 minutes, the fluorescence intensity basically reached saturation, which indicated that the polyhydrogen sulfide probe responded quickly, only for 2 minutes.
（2）多硫化氢探针的荧光滴定测试：在2mL 95％PBS/DMSO混合溶液（pH值为7.4）中加入10μL TCFPB-H 2S n的DMSO溶液（1mM），得到5μM的TCFPB-H 2S n溶液，然后分别加入不同浓度的多硫化钠溶液(浓度范围为0～250μM)，在37℃下孵化5min后，测定加入不同浓度多硫化氢后所得溶液的荧光发射光谱（Ex＝575nm），并以619nm处荧光强度与751nm处荧光强度的比值为纵坐标、多硫化氢的浓度为横坐标建立TCFPB-H 2S n对多硫化氢检测的线性曲线。 (2) Fluorescence titration test of polyhydrogen sulfide probe: Add 10 μL TCFPB-H 2 S n DMSO solution (1 mM) to 2 mL 95% PBS/DMSO mixed solution (pH 7.4) to obtain 5 μM TCFPB-H 2 S n solution, and then add different concentrations of sodium polysulfide solution (concentration range is 0 ~ 250 μ M), after incubation at 37 ° C for 5 min, measure the fluorescence emission spectrum of the solution obtained after adding different concentrations of hydrogen polysulfide (Ex = 575nm ), and the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm as the ordinate, and the concentration of hydrogen polysulfide as the abscissa to establish a linear curve for the detection of hydrogen polysulfide by TCFPB-H 2 S n .
图3为在5μM的TCFPB-H 2S n溶液中加入不同浓度多硫化氢孵化后荧光发射光谱的变化图；根据图3可以看出，随着多硫化氢浓度的增大，619nm处荧光强度逐渐增强，而751nm处荧光强度逐渐下降。 Figure 3 is a graph showing the changes in fluorescence emission spectra after adding different concentrations of hydrogen polysulfide to 5 μM TCFPB-H 2 S n solution and incubating; it can be seen from Figure 3 that with the increase of the concentration of hydrogen polysulfide, the fluorescence intensity at 619nm Gradually enhanced, while the fluorescence intensity at 751nm gradually decreased.
图4为TCFPB-H 2S n溶液在619nm处荧光强度与751nm处荧光强度的比值与多硫化氢浓度的线性曲线图，所述线性曲线具体为Y＝0.04682X + 0.36183，荧光强度对于一氧化碳的浓度的线性响应在1～30μM（R 2＝99.3％）之间。由图4可以看出，荧光探针TCFPB-H 2S n可以在一定浓度范围内对溶液中的多硫化氢实现比率型线性响应。 Fig. 4 is a linear curve diagram of the ratio of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm of the TCFPB-H 2 S n solution and the concentration of hydrogen polysulfide, the linear curve is specifically Y=0.04682X+0.36183, and the fluorescence intensity is relative to that of carbon monoxide The linear response to concentration was between 1 and 30 μM (R 2 =99.3%). It can be seen from Figure 4 that the fluorescent probe TCFPB-H 2 S n can achieve a ratiometric linear response to hydrogen polysulfide in a certain concentration range.
（3）一多硫化氢探针对pH的敏感性测定：在2mL 95％PBS/DMSO混合溶液（pH值分别为3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0）中加入10μL TCFPB-H 2S n的DMSO溶液(1mM)，得到5μL TCFPB-H 2S n溶液，然后加入50μM的多硫化钠溶液，所得混合溶液在37℃下孵化5min后，测定所述混合溶液在619nm处荧光强度与751nm处荧光强度的比值随PBS缓冲溶液pH值的变化情况。 (3) Determination of the sensitivity of a polyhydrogen sulfide probe to pH: add 10 μL TCFPB to 2 mL 95% PBS/DMSO mixed solution (pH values are 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) -H 2 S n in DMSO solution (1mM), to obtain 5 μL TCFPB-H 2 S n solution, and then add 50 μM sodium polysulfide solution, after the resulting mixed solution was incubated at 37°C for 5 min, the measured value of the mixed solution at 619nm The ratio of the fluorescence intensity to the fluorescence intensity at 751nm varies with the pH value of the PBS buffer solution.
图5为TCFPB-H 2S n在不同pH值的PBS/DMSO混合溶液619nm处荧光强度与751nm处荧光强度的比值变化图，由图5可知，探针TCFPB-H 2S n本身具有很好的pH稳定性，对pH值不敏感，探针TCFPB-H 2S n对多硫化氢的响应在pH值为6.0~10.0范围内具有很好的响应能力。 Figure 5 is a graph showing the ratio change of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm in PBS/DMSO mixed solutions of different pH values for TCFPB-H 2 S n . It can be seen from Figure 5 that the probe TCFPB-H 2 S n itself has a very good Excellent pH stability, insensitive to pH value, the probe TCFPB-H 2 S n has a good response ability to hydrogen polysulfide in the range of pH 6.0~10.0.
多硫化氢探针的选择性测试：在2mL 95％PBS/DMSO混合溶液（pH值为7.4）中加入10μL TCFPB-H 2S n的DMSO溶液（1mM），得到5μM的TCFPB-H 2S n溶液，分别加入250μM的其他离子，其中a:S 2O 5 2- ; b: ClO- ; c: H 2O 2; d: MnO 4 -; e: Mg 2+; f: K +; g: Cys; h:Fe 3+; i: H 2S; j: HSO 3-; k: SO 4 2-; l:HCy; m: I - ; n: OH ; o: ATP ; p: S 2O 8 2- ; q: GSH ; r: NO 3 -; s: blank; t: Na 2S 4。所得混合溶液分别在37℃下孵化5min，然后测定混合溶液619nm处荧光强度与751nm处荧光强度的比值的变化。 Selectivity test of polyhydrogen sulfide probe: Add 10 μL of TCFPB-H 2 S n in DMSO solution (1 mM) to 2 mL of 95% PBS/DMSO mixed solution (pH 7.4) to obtain 5 μM of TCFPB-H 2 S n solution, add 250μM other ions respectively, where a: S 2 O 5 2- ; b: ClO- ; c: H 2 O 2 ; d: MnO 4 - ; e: Mg 2+ ; f: K + ; g: Cys; h:Fe 3+ ; i: H 2 S; j: HSO 3- ; k: SO 4 2- ; l:HCy; m: I - ; n: OH ; o: ATP ; p: S 2 O 8 2- ; q: GSH ; r: NO 3 - ; s: blank; t: Na 2 S 4 . The resulting mixed solutions were incubated at 37° C. for 5 min, and then the change in the ratio of the fluorescence intensity at 619 nm to the fluorescence intensity at 751 nm of the mixed solution was measured.
图6为TCFPB-H 2S n溶液中分别加入不同竞争分子后619nm处荧光强度与751nm处荧光强度的比值变化图，由图6可知，除H 2S n外的其它生物分子与TCFPB-H 2S n反应前后的荧光强度比值均没有明显变化，说明TCFPB-H 2S n可以选择性的识别H 2S n，不易受其他离子的干扰，特异型强。 Figure 6 is a diagram of the ratio change of the fluorescence intensity at 619nm to the fluorescence intensity at 751nm after adding different competing molecules to the TCFPB-H 2 S n solution. The fluorescence intensity ratio before and after the 2 S n reaction did not change significantly, indicating that TCFPB-H 2 S n can selectively recognize H 2 S n and is not easily interfered by other ions, and has strong specificity.
实施例7 本发明的多硫化氢荧光探针定性和定量检测细胞内的多硫化氢Example 7 The hydrogen polysulfide fluorescent probe of the present invention qualitatively and quantitatively detects hydrogen polysulfide in cells
对人宫颈癌细胞（HeLa）内多硫化氢荧光成像情况进行测试，使用实施例1制备的荧光探针，具体步骤如下：The fluorescent imaging of hydrogen polysulfide in human cervical cancer cells (HeLa) was tested, using the fluorescent probe prepared in Example 1, and the specific steps were as follows:
HeLa细胞经过复苏接种于含10％胎牛血清的RPMI 1640培养基中，在37℃、5％CO 2、100％饱和湿度的培养箱中培养，然后在18mm盖玻片上培养24h，待用。 HeLa cells were resuscitated and inoculated in RPMI 1640 medium containing 10% fetal bovine serum, cultured in an incubator at 37°C, 5% CO 2 , and 100% saturated humidity, and then cultured on 18mm coverslips for 24 hours before use.
将培养后的HeLa细胞浸入含5μM TCFPB-H 2S n（实施例1制备得到）的培养基中，在37℃、5％CO 2、100％饱和湿度的培养箱中培养20min后，倒出培养基，用新鲜培养基清洗细胞3遍；加入2mL PBS溶液，实验组分别加入10μM、20μM、50μM的多硫化钠，在激光共聚焦荧光显微镜下观察，并用561nm作为激发光源，对其进行明场和暗场下拍照。 Immerse the cultured HeLa cells in the medium containing 5 μM TCFPB-H 2 S n (prepared in Example 1), culture them in an incubator at 37°C, 5% CO 2 , and 100% saturated humidity for 20 min, and pour out Culture medium, wash the cells 3 times with fresh medium; add 2mL PBS solution, add 10μM, 20μM, 50μM sodium polysulfide respectively in the experimental group, observe under the laser confocal fluorescence microscope, and use 561nm as the excitation light source to brighten it Take pictures in field and dark field.
图7为TCFPB-H 2S n与HeLa细胞37℃下孵化5min后的激光共聚焦显微成像照片，由图7可知，TCFPB-H 2S n在HeLa细胞中，不管是红光通道还是绿光通道都呈现弱的荧光信号，而在加入了多硫化氢的HeLa细胞中，红光通道荧光信号基本逐渐减弱，而绿光通道的荧光信号明显增强。 Figure 7 is a laser confocal microscopic image of TCFPB-H 2 S n incubated with HeLa cells at 37°C for 5 minutes. The light channels all showed weak fluorescent signals, but in the HeLa cells added with hydrogen polysulfide, the fluorescent signals of the red light channel gradually weakened, while the fluorescent signals of the green light channel increased significantly.
图8为细胞中绿色通道荧光强度与红色通道荧光强度的比值随不同多硫化氢浓度的变化图，可见TCFPB-H 2S n对细胞内多硫化氢的成像呈现浓度依赖关系，说明TCFPB-H 2S n可以对细胞内多硫化氢进行比率型荧光成像。本发明的多硫化氢荧光探针不仅能对细胞和活体内的多硫化氢进行定性检测，还可以实现定量的检测。 Figure 8 is a diagram of the ratio of the fluorescence intensity of the green channel to the fluorescence intensity of the red channel in cells with different concentrations of polysulfide hydrogen. 2 S n can be used for ratiometric fluorescence imaging of intracellular hydrogen polysulfide. The hydrogen polysulfide fluorescent probe of the invention can not only detect the hydrogen polysulfide qualitatively in cells and living bodies, but also realize quantitative detection.
按照实施例6~7的方法对实施例4和实施例5所得比率型多硫化氢荧光探针进行响应时间、荧光滴定、pH敏感性、选择性和人宫颈癌细胞内成像情况进行测试，所得结果和实施例6~7相似。According to the method of Examples 6-7, the ratiometric hydrogen polysulfide fluorescent probe obtained in Example 4 and Example 5 was tested for response time, fluorescence titration, pH sensitivity, selectivity, and imaging conditions in human cervical cancer cells, and the obtained Result and embodiment 6～7 are similar.
综合以上实施例，本发明提供的比率型多硫化氢荧光探针具有聚集诱导发光特性和比率型响应特性，且响应时间短，对pH值不敏感，能够定性和定量检测细胞内的多硫化氢，且能够通过两个不同发射波长的荧光强度比率进行响应和细胞成像，可有效避免单一波长易受温度、pH、浓度及仪器等外界因素的干扰，获得更高的成像分辨率，且制备原料易得，步骤简单，容易操作，产率高，适合工业化生产，具有广阔的应用前景。Based on the above examples, the ratiometric hydrogen polysulfide fluorescent probe provided by the present invention has aggregation-induced luminescent characteristics and ratiometric response characteristics, and has a short response time, is insensitive to pH, and can qualitatively and quantitatively detect intracellular hydrogen polysulfide , and can respond and image cells through the fluorescence intensity ratio of two different emission wavelengths, which can effectively avoid the interference of a single wavelength from external factors such as temperature, pH, concentration and instruments, obtain higher imaging resolution, and prepare raw materials The method is easy to obtain, has simple steps, is easy to operate, has high yield, is suitable for industrial production, and has broad application prospects.
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (12)

  1. 一种比率型多硫化氢荧光探针，其特征在于，所述荧光探针的结构式如式（I）所示：
    Figure dest_path_image001
    ,     A ratiometric polyhydrogen sulfide fluorescent probe, characterized in that the structural formula of the fluorescent probe is shown in formula (I):
    Figure dest_path_image001
    ,
    其中，R为，
    Figure dest_path_image002
    ，
    Figure dest_path_image003
    ，
    Figure dest_path_image004
    中的任意一种。     where R is,
    Figure dest_path_image002
    ,
    Figure dest_path_image003
    ,
    Figure dest_path_image004
    any of the.
  2. 权利要求1所述的比率型多硫化氢荧光探针的制备方法，其特征在于，所述荧光探针以2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈与前体化合物反应得到具有聚集诱导发光特性的前驱体，然后连接亲电识别基团2-氟-5-硝基苯甲酸酯所得。 The preparation method of ratio type hydrogen polysulfide fluorescent probe according to claim 1, is characterized in that, described fluorescent probe is with 2-(3-cyano group-4,5,5-trimethylfuran-2(5H )-methylene)malononitrile reacts with a precursor compound to obtain a precursor with aggregation-induced luminescent properties, and then connects an electrophilic recognition group 2-fluoro-5-nitrobenzoate.
  3. 根据权利要求2所述的制备方法，其特征在于，包括如下步骤： The preparation method according to claim 2, is characterized in that, comprises the steps:
    （1）将所述前体化合物、所述2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈、碱性化合物和第一有机溶剂混合，在保护气氛下进行Knoevenagel反应，得到中间化合物；(1) The precursor compound, the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile, the basic compound and the first Organic solvents are mixed, and a Knoevenagel reaction is carried out under a protective atmosphere to obtain an intermediate compound;
    （2）将所述中间化合物与所述2-氟-5-硝基苯甲酸、二环己基碳二亚胺、4-二甲氨基吡啶和第二有机溶剂混合，在保护气氛下进行缩合反应，得到所述比率型多硫化氢荧光探针；(2) Mix the intermediate compound with the 2-fluoro-5-nitrobenzoic acid, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and a second organic solvent, and perform a condensation reaction under a protective atmosphere , to obtain the ratiometric polyhydrogen sulfide fluorescent probe;
    所述前体化合物为
    Figure dest_path_image005
    时，所述中间化合物为
    Figure dest_path_image006
    The precursor compound is
    Figure dest_path_image005
    When, the intermediate compound is
    Figure dest_path_image006
    所述前体化合物为
    Figure dest_path_image007
    时，所述中间化合物为
    Figure dest_path_image008
    ；     The precursor compound is
    Figure dest_path_image007
    When, the intermediate compound is
    Figure dest_path_image008
    ;
    所述前体化合物为
    Figure dest_path_image009
    时，所述中间化合物为
    Figure dest_path_image010
    。     The precursor compound is
    Figure dest_path_image009
    When, the intermediate compound is
    Figure dest_path_image010
    .
  4. 根据权利要求3所述的制备方法，其特征在于，所述碱性化合物为哌啶或吡啶；所述第一有机溶剂为无水乙醇；所述第二有机溶剂为无水二氯甲烷。 The preparation method according to claim 3, wherein the basic compound is piperidine or pyridine; the first organic solvent is absolute ethanol; and the second organic solvent is anhydrous dichloromethane.
  5. 根据权利要求3所述的制备方法，其特征在于，所述步骤（1）反应的温度为60~100℃；反应时间为5~12个小时，优选为10~12小时。 The preparation method according to claim 3, characterized in that the reaction temperature of the step (1) is 60-100°C; the reaction time is 5-12 hours, preferably 10-12 hours.
  6. 根据权利要求3所述的制备方法，其特征在于，所述前体化合物、所述2-氟-5-硝基苯甲酸、所述二环己基碳二亚胺和所述4-二甲氨基吡啶的摩尔比为1：（1-2.5）：（1-2）：（0.1-0.5），优选为1：2：1.5：0.2； The preparation method according to claim 3, wherein the precursor compound, the 2-fluoro-5-nitrobenzoic acid, the dicyclohexylcarbodiimide and the 4-dimethylamino The molar ratio of pyridine is 1:(1-2.5):(1-2):(0.1-0.5), preferably 1:2:1.5:0.2;
    所述中间化合物、所述2-(3-氰基-4,5,5-三甲基呋喃-2(5H)-亚甲基)丙二腈和所述碱性化合物的摩尔比为1：1：0.5。The molar ratio of the intermediate compound, the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile and the basic compound is 1: 1:0.5.
  7. 根据权利要求3所述的制备方法，其特征在于，所述步骤（1）的Knoevenagel反应完成后，还包括将所得产物进行后处理的步骤，所述后处理包括以下步骤： The preparation method according to claim 3, characterized in that, after the Knoevenagel reaction in the step (1) is completed, the step of post-processing the obtained product is also included, and the post-processing includes the following steps:
    将反应后的液态混合物浓缩，得到浓缩物；Concentrating the reacted liquid mixture to obtain a concentrate;
    将所述浓缩物进行柱层析，得到所述中间化合物。The concentrate was subjected to column chromatography to obtain the intermediate compound.
  8. 权利要求1所述的比率型多硫化氢荧光探针在生物体内多硫化氢的定性或定量检测中的应用。 The application of the ratiometric hydrogen polysulfide fluorescent probe according to claim 1 in the qualitative or quantitative detection of hydrogen polysulfide in organisms.
  9. 根据权利要求8所述的应用，其特征在于，包括如下步骤： The application according to claim 8, characterized in that, comprising the steps of:
    将待测样品加入到比率型多硫化氢荧光探针溶液中，然后目测颜色变化和/或测试紫外吸收变化。Add the sample to be tested into the ratiometric hydrogen polysulfide fluorescent probe solution, and then visually observe the color change and/or test the change in ultraviolet absorption.
  10. 根据权利要求9所述的应用，其特征在于，所述比率型多硫化氢荧光探针溶液为比率型多硫化氢荧光探针与水和有机溶剂的混合溶液。 The application according to claim 9, characterized in that the ratio-type hydrogen polysulfide fluorescent probe solution is a mixed solution of the ratio-type hydrogen polysulfide fluorescent probe, water and an organic solvent.
  11. 根据权利要求10所述的应用，其特征在于，所述水和所述有机溶剂的体积比为99.5：5~99：10。 The application according to claim 10, characterized in that the volume ratio of the water to the organic solvent is 99.5:5-99:10.
  12. 根据权利要求10所述的应用，其特征在于，所述有机溶剂为二甲基亚砜。 The application according to claim 10, characterized in that the organic solvent is dimethyl sulfoxide.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102482504A (en) * 2009-08-24 2012-05-30 独立行政法人情报通信研究机构 Second-order non-linear optical compound and non-linear optical element comprising same
CN110511742A (en) * 2019-09-04 2019-11-29 中南大学 A kind of near infrared fluorescent probe of specific recognition hydrogen polysulfide
CN113582950A (en) * 2021-08-10 2021-11-02 中国科学院深圳先进技术研究院 Ratio type hydrogen polysulfide fluorescent probe and preparation method and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107188876B (en) * 2017-04-10 2019-08-09 台州学院 It is a kind of for detecting the synthesis and application of the near infrared fluorescent probe of hydrogen polysulfide
CN112159377B (en) * 2019-12-11 2023-07-11 渤海大学 Near infrared emission and simultaneous identification H 2 S、HSO 3- Fluorescent probe of (2) and use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102482504A (en) * 2009-08-24 2012-05-30 独立行政法人情报通信研究机构 Second-order non-linear optical compound and non-linear optical element comprising same
CN110511742A (en) * 2019-09-04 2019-11-29 中南大学 A kind of near infrared fluorescent probe of specific recognition hydrogen polysulfide
CN113582950A (en) * 2021-08-10 2021-11-02 中国科学院深圳先进技术研究院 Ratio type hydrogen polysulfide fluorescent probe and preparation method and application thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
LI CHUNBIN, JIANG GUOYU, LIU XIANG, LAI QINGFANG, KANG MIAOMIAO, WANG DONG, ZHANG PENGFEI, WANG JIANGUO, TANG BEN ZHONG: "An easily available ratiometric AIE probe for nitroxyl visualization in vitro and in vivo", MATERIALS CHEMISTRY FRONTIERS, vol. 5, no. 4, 23 February 2021 (2021-02-23), pages 1817 - 1823, XP093034698, DOI: 10.1039/D0QM00995D *
LIU CHUNRONG, CHEN WEI, SHI WEN, PENG BO, ZHAO YU, MA HUIMIN, XIAN MING: "Rational Design and Bioimaging Applications of Highly Selective Fluorescence Probes for Hydrogen Polysulfides", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, vol. 136, no. 20, 21 May 2014 (2014-05-21), pages 7257 - 7260, XP093034700, ISSN: 0002-7863, DOI: 10.1021/ja502968x *
XIANG CHUNBAI, LI CHUNBIN, XIANG JINGJING, LUO YUAN, PENG JIAOFENG, DENG GUANJUN, WANG JIANGUO, KOLEMEN SAFACAN, LI HONGCHUN, ZHAN: "An easily available lysosomal-targeted ratiometric fluorescent probe with aggregation induced emission characteristics for hydrogen polysulfide visualization in acute ulcerative colitis", MATERIALS CHEMISTRY FRONTIERS, vol. 5, no. 20, 11 October 2021 (2021-10-11), pages 7638 - 7644, XP093034701, DOI: 10.1039/D1QM01043C *
ZHANG, CHANGLI ET AL.: "A BODIPY-based ratiometric probe for sensing and imaging hydrogen polysulfides in living cells", SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY, vol. 223, 19 June 2019 (2019-06-19), XP085811763, ISSN: 1386-1425, DOI: 10.1016/j.saa.2019.117295 *
ZHAO LIMING, SUN QIAN, SUN CHUANZHI, ZHANG CHANGLI, DUAN WENZENG, GONG SHUWEN, LIU ZHIPENG: "An isophorone-based far-red emitting ratiometric fluorescent probe for selective sensing and imaging of polysulfides", JOURNAL OF MATERIALS CHEMISTRY. B, ROYAL SOCIETY OF CHEMISTRY, GB, vol. 6, no. 43, 7 November 2018 (2018-11-07), GB , pages 7015 - 7020, XP093034695, ISSN: 2050-750X, DOI: 10.1039/C8TB01813H *
ZHONG KELI, CHEN LIN, PAN YONGXIN, YAN XIAOMEI, HOU SHUHUA, TANG YIWEI, GAO XUE, LI JIANRONG, TANG LIJUN: "A colorimetric and near-infrared fluorescent probe for detection of hydrogen sulfide and its real multiple applications", SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY, ELSEVIER, AMSTERDAM, NL, vol. 221, 1 October 2019 (2019-10-01), AMSTERDAM, NL, pages 117135, XP093034703, ISSN: 1386-1425, DOI: 10.1016/j.saa.2019.117135 *

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