WO2021087897A1 - 基于bodipy的高效实时生物硫醇荧光检测探针 - Google Patents

基于bodipy的高效实时生物硫醇荧光检测探针 Download PDF

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WO2021087897A1
WO2021087897A1 PCT/CN2019/116374 CN2019116374W WO2021087897A1 WO 2021087897 A1 WO2021087897 A1 WO 2021087897A1 CN 2019116374 W CN2019116374 W CN 2019116374W WO 2021087897 A1 WO2021087897 A1 WO 2021087897A1
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bod
fluoroboron
synthesis
dichloromethane
probe
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耿晋
何荣坤
连前进
关书
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6596—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having atoms other than oxygen, sulfur, selenium, tellurium, nitrogen or phosphorus as ring hetero atoms
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • 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

Definitions

  • the invention relates to a BODIPY-based high-efficiency real-time biological thiol fluorescence detection probe, and also relates to a preparation method and application of the probe, and belongs to the technical field of organic small molecule fluorescence probes.
  • fluorescence analysis technology has played an irreplaceable role in the field of biological analysis due to its high detection sensitivity, better selectivity, easy operation, low cost and other advantages.
  • the most critical part of the fluorescence analysis technology is the fluorescent dye.
  • the performance of the fluorescent dye directly affects the sensitivity and accuracy of the probe detection. Therefore, research on fluorescent dyes suitable for fluorescent labeling and development of excellent fluorescent probes will be crucial to the development of bioanalytical technology and life science research.
  • fluorescent dyes Due to the diversity of dye properties, different dyes show great differences in the biological detection and analysis process. Not all fluorescent dyes can be used as fluorophores for fluorescent probes and applied in the fields of biochemical analysis and biological detection.
  • the fluorescent dyes that have been successfully applied in the prior art include: coumarin-based fluorescent dyes, which have many advantages such as good light stability, large Stokes shift, good oil solubility, and relatively small molecular volume.
  • the main properties of coumarin dyes are The disadvantage is that the absorption and emission wavelength is too short, most of which are blue fluorescence, and the unsaturated lactone structure of the coumarin dye will undergo a hydrolysis reaction in the dilute lye, which limits its application.
  • Naphthimide dyes can interact with DNA and are an excellent DNA intercalator with high quantum yield and good photostability. They are very useful biomarkers. However, the naphthalimide dyes are lighter in color and have a low molar extinction coefficient, which limits their application. Fluorescein and rhodamine dyes, this type of fluorescent dyes are characterized by good water solubility, good light stability, relatively long absorption and emission wavelength, and high quantum yield; but due to the presence of amino groups and hydroxyl groups in the dye, the dye It is more sensitive to pH, and when used as ion or biomolecular probes, it has certain requirements for the pH of the working environment.
  • Cyanine dyes have been successfully used to detect DNA, nucleotides, proteins and other fluorescent probes.
  • the disadvantage of this type of dye is poor light stability.
  • the polymethine structure is prone to oxidative fracture under light, which leads to the decomposition of the dye.
  • polymethine The structure also causes the dye to accumulate easily, which affects the promotion of the dye in application.
  • BODIPY dyes are dyes with superior performance due to their good photochemical stability, high molar extinction coefficient, high fluorescence quantum yield, easy modification of molecular structure, and narrow half-peak width of the fluorescence spectrum. , It has many advantages such as good resistance to solvent polarity and pH, and is widely used in the fields of optoelectronic devices, biomarkers and fluorescent probes.
  • Biothiols mainly include cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), which play a vital role in biological systems.
  • the metabolism and transportation of thiol substances in biological systems are closely related to a series of important enzymes and proteins.
  • Abnormal intracellular thiol levels are associated with many diseases, such as white blood cell loss, psoriasis, liver damage and AIDS. Therefore, it is of great significance to design and synthesize fluorescent probes with high selectivity, high sensitivity and real-time rapid response to biological thiols.
  • the most significant feature of biothiols is that they contain strong nucleophilic sulfhydryl groups.
  • the currently reported biological thiol fluorescent probes are mainly based on the Michael addition reaction, metal complex replacement, cyclization addition to aldehydes, and disulfide bond reduction mechanisms.
  • the thiol probe based on the Michael addition reaction has attracted wide attention due to its high sensitivity and the ability to detect thiol under near-physiological conditions.
  • most of these types of probes have problems such as long response time to thiols, which generally range from a few minutes to several hours, which is not conducive to the real-time quantitative detection of thiols.
  • the main problem of the existing real-time response type biological thiol ratio fluorescent probe is that there is no organelle targeting function and the Stokes shift is small, which leads to serious fluorescence self-quenching and Rayleigh scattering, which cause detection errors.
  • an object of the present invention is to provide a ratio-type fluorescent probe for rapid and real-time detection of biological thiols.
  • the structural formula of the probe is a compound represented by the following formula (I):
  • R1 is selected from hydrogen, formula (III), and formula (IV);
  • R2 or R3 is selected from formula (II);
  • R4 or R5 is selected from methyl;
  • R4 or R5 is selected from hydrogen
  • the R7 is Cl.
  • the structure of the probe is selected from the following:
  • the R7 is Cl.
  • Another object of the present invention is to provide a method for preparing a ratio fluorescent probe for rapid and real-time detection of biological thiols, which includes the following steps:
  • the synthesis method of the step (1) includes: adding the intermediate dye fluoroboron dipyrromethene-simplified (BOD-J) and fluoroboron dipyrromethene to anhydrous N,N-dimethylformamide (DMF) -Alkyne (BOD-Q) or fluoroboron dipyrromethene-azid (BOD-D), and 3,5-dimethylbenzaldehyde, after deoxygenation, add piperidine and glacial acetic acid, stir at room temperature and place in microwave Reaction in the reactor.
  • BOD-J intermediate dye fluoroboron dipyrromethene-simplified
  • DMF N,N-dimethylformamide
  • BOD-Q alkyne
  • BOD-D fluoroboron dipyrromethene-azid
  • 3,5-dimethylbenzaldehyde 3,5-dimethylbenzaldehyde
  • the step (2) includes the dye fluoroboron dipyrromethoxine-aldehyde (BOD-JQ), fluoroboron dipyrromethoxine-acetylene aldehyde (BOD-QQ) and/or the dye fluoroboron dipyrromethoxine-azide (BOD-QQ). -DQ) synthesis;
  • the organelle is mitochondria or endoplasmic reticulum.
  • the method for synthesizing BOD-JQ, BOD-QQ or BOD-DQ in the above method includes: dissolving the dye BOD-JS, BOD-QS or BOD-DS in dichloromethane, and then adding 50-500 mesh Silica gel, rotary evaporate the solvent under reduced pressure, and place the mixture under the xenon lamp to illuminate. It can be observed that the mixture changes from dark green to purple-black. After the mixture is cooled to room temperature, it is extracted several times with dichloromethane, and the organic phases are combined.
  • the above method also includes the synthesis of intermediate dyes
  • the intermediate dyes are fluoroboron dipyrromethene-simplified (BOD-J), intermediate acetylenic aldehyde (QQ), fluoroboron dipyrromethene-alkynes (BOD-Q) , Intermediate bromoaldehyde (XQ), intermediate azide (DQ) and fluoroboron dipyrromethene-azid (BOD-D).
  • the organelle-targeting fluorescent dye synthesis step in the above method includes:
  • the filtrate was distilled under reduced pressure to remove excess organic solvent, and the obtained crude product was dissolved in a small amount of dichloromethane and then subjected to silica gel column chromatography, gradient elution for separation and purification, and finally powder was obtained; the organelles were mitochondria.
  • the organelle-targeting fluorescent dye synthesis step in the above method includes:
  • Another object of the present invention is to provide an application of a ratio-based fluorescent probe for rapid and real-time detection of biological thiols, the application being selected from one of the following (1)-(4):
  • the biological thiols are preferably cysteine (Cys), homocysteine (Hcy) and/or glutathione (GSH);
  • the biological thiol detection in the above application is carried out under alkaline conditions, preferably the pH is 7.2-8.0, and more preferably the pH is 7.4.
  • the organelle in the above application is endoplasmic reticulum and/or mitochondria.
  • the present invention has the following beneficial effects:
  • the present invention develops a ratio fluorescent probe for rapid and real-time detection of biological thiols.
  • Spectral test results show that the probe has the advantages of ratiometric response, high selectivity, high sensitivity, fast response time and large Stokes shift to biological thiols.
  • the probe Compared with most biological thiol fluorescent probes, the probe’s response time to biological thiols has the characteristics of real-time and rapid speed. This advantage enables it to meet the needs of high-throughput detection of biological thiols.
  • the ratio response mechanism of the probe can effectively compensate for the OFF/ON type.
  • this probe has organelle targeting functions, such as mitochondrial targeting: when the mitochondrial membrane potential in vitro is 180-200mV, the inner side is negatively charged, and it is negative in living cells and the body. The internal content is slightly lower, 130-150mV, and lipophilic cations are used for drug delivery.
  • organelle targeting functions such as mitochondrial targeting: when the mitochondrial membrane potential in vitro is 180-200mV, the inner side is negatively charged, and it is negative in living cells and the body. The internal content is slightly lower, 130-150mV, and lipophilic cations are used for drug delivery.
  • the most successful research is triphenylphosphine. Triphenylphosphine contains three benzene rings to increase the surface area of the molecule and form a delocalized positive charge.
  • the endoplasmic reticulum targeting the presence of the chlorine atom functional group realizes its specific binding with the chloride ion pump of the endoplasmic reticulum, thereby completing the positioning of the endoplasmic reticulum, and
  • the Stokes shift is larger, and the Stokes shift of the probe of the present invention also reaches 52 nm, which is about 13 nm higher than the maximum Stokes shift reported so far.
  • the color of the probe solution changes from pink to light yellow after encountering biological thiols in vitro, which can be used for visual naked eye detection. This also provides a new idea for the design of synthetic ratio-type biological thiol fluorescent probes.
  • Figure 1 The route of probe synthesis, specifically the synthesis of aldehyde BODIPY.
  • Figure 37 Reaction of Cys/Hcy with a probe based on an aldehyde group as a recognition group.
  • BODIPY fluoroboron dipyrromethene
  • the QQ synthesis route is shown in Figure 4.
  • p-hydroxybenzaldehyde (6.1g, 50mmol) and anhydrous potassium carbonate (9.7g, 70mmol)
  • anhydrous potassium carbonate (9.7g, 70mmol)
  • 3-bromopropyne 11.1mL, 100mmol
  • the solvent was distilled off under reduced pressure.
  • the crude product was dissolved in water and extracted with ethyl acetate (4 ⁇ 75 mL). The organic phases were combined and washed with saturated brine and water several times. After the organic phase was dried over anhydrous magnesium sulfate, the filtrate was filtered and distilled under reduced pressure to remove excess organic solvent.
  • the obtained crude product was dissolved in a small amount of dichloromethane and separated and purified by silica gel column chromatography (petroleum ether/chloroform as the eluent) gradient elution, and finally 7.8 g of light yellow powder was obtained, and the yield was 98%.
  • reaction was placed in an ice bath, and 6 mL of triethylamine and 6 mL of boron trifluoride ether were slowly added dropwise after 15 minutes, and stirring was continued for 2 hours. After the reaction is over, add 200 mL of water, shake vigorously for 5 min, collect the organic layer, and wash the organic layer with saturated saline and water three times in sequence. Subsequently, the organic layer was dried with anhydrous magnesium sulfate, and the filtrate was filtered and distilled under reduced pressure to remove excess organic solvent.
  • the obtained crude product was dissolved in a small amount of dichloromethane and separated and purified by silica gel column chromatography (petroleum ether/dichloromethane as the eluent) gradient elution, and finally 0.57 g of purple powder was obtained, and the yield was 24%.
  • BOD-QS The synthesis route of BOD-QS is shown in Figure 6.
  • the synthesis method and parameter conditions are the same as those of BOD-JS.
  • BOD-Q (113mg, 0.3mmol) is added to obtain golden yellow powder BOD-QS 178mg, yield 97% .
  • DQ (1.26g, 5.5mmol) and sodium azide (0.45g, 6.0mmol) were added to 40mL of anhydrous DMSO, and heated to reflux for 2h. After the reaction is completed and cooled, it is diluted with dichloromethane, then the organic phase is washed several times with saturated brine and water, and finally the organic layer is dried with anhydrous sodium sulfate. After filtration, the filtrate was distilled under reduced pressure to remove excess organic solvent.
  • the obtained crude product was dissolved in a small amount of dichloromethane and separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate as the eluent) gradient elution, and finally 0.82 g of light yellow powder was obtained, and the yield was 80%.
  • BOD-DS The synthesis route of BOD-DS is shown in Figure 10.
  • the synthesis method and parameters are the same as those of BOD-JS synthesis.
  • BOD-D (123mg, 0.3mmol) is added to obtain golden yellow powder BOD-QS 188mg, with a yield of 98%.
  • BOD-QQ The synthesis route of BOD-QQ is shown in Figure 15.
  • the synthesis method and its parameter conditions are the same as those of BOD-JQ synthesis.
  • BOD-QS (0.1 mmol, 61 mg) is added to obtain 37 mg of product with a yield of 72%.
  • the results of BOD-QQ characterization are shown in Figure 16-18:
  • BOD-DQ The synthesis route of BOD-DQ is shown in Figure 19, and its synthesis method and its parameter conditions are the same as those of BOD-JQ synthesis.
  • BOD-DS (0.1 mmol, 64 mg) is added to obtain 38 mg of product with a yield rate of 70%.
  • the BOD-DQ characterization results are shown in Figure 20-22:
  • BOD-PPh3 The synthetic route of BOD-PPh3 is shown in Figure 23.
  • BOD-QQ 50.8mg, 0.1mmol
  • (4-azidobutyl)triphenylphosphonium bromide (43.9mg, 0.1mmol) are dissolved
  • copper sulfate pentahydrate 25mg, 0.1mmol
  • sodium ascorbate (19.8mg, 0.1mmol
  • BOD-Cl The synthesis route of BOD-Cl is shown in Figure 27.
  • BOD-QQ 50.8 mg, 0.1 mmol
  • reaction was placed in an ice bath, and 20 mL of triethylamine and 20 mL of boron trifluoride ether were slowly added dropwise after 15 minutes, and stirring was continued for 2 hours. After the reaction is over, add 200 mL of water, shake vigorously for 5 min, extract with dichloromethane, collect the organic layer, and wash the organic layer with saturated brine and water three times in sequence. Subsequently, the organic layer was dried with anhydrous sodium sulfate, and the filtrate was filtered under reduced pressure to remove excess organic solvent.
  • the preparation method of the mother liquor Prepare the probe molecule BOD-JQ with tetrahydrofuran (THF) into a standard solution of 10 -3 mol L -1 and place it at 4°C for later use.
  • THF tetrahydrofuran
  • the sensitivity of the probe to GSH detection (R/R 0 ) is the largest when the pH is 7.4, so the pH value used in subsequent experiments is all 7.4.
  • the concentration of glutathione in the cell is 1-10 mM, so the probe can meet the detection requirements of glutathione in general biological systems.
  • BOD-JQ has a similar response effect to Cys and Hcy, although the concentration of Cys and Hcy in the cells is relatively low, generally 30-200 ⁇ M and 5-15 ⁇ M.
  • the probe BOD-JQ is a probe for detecting the total concentration of biological thiols in biological systems.
  • the total concentration of three common biological thiols is at the mM level, so the sensitivity of the probe can meet the requirements of biological systems in general biological systems. Demand for detection of mercaptans.
  • the concentration of the probe molecule is 5 ⁇ M, and 100 equivalents of AA are added to each sample. Ala, Arg, Glu, Gly, His, Leu, Phe, Trp, Val, Cys, Hcy and GSH, and measure the changes before and after the fluorescence spectrum.
  • the concentration of the probe molecule is 5 ⁇ M, and 100 equivalents of GSH solution are added to each sample. And add 100 equivalents of AA, Ala, Arg, Glu, Gly, His, Leu, Phe, Trp and Val respectively, and measure the changes before and after the fluorescence spectrum to investigate the fluorescence spectrum response of the probe when GSH coexists with other substances.
  • the product was reasonably inferred by the change of the system spectrum after adding GSH solution, and the reaction product was identified by high-resolution mass spectrometry.
  • the sulfhydryl group of the biothiol can still selectively carry out Michael addition with the olefin of the recognition group or reduce the disulfide bond without interacting with the aldehyde group. Because the probe is very sensitive to the detection of sulfhydryl compounds, and there may be fewer products after the probe interacts with sulfhydryl groups, we have not obtained more intuitive evidence such as mass spectrometry to prove the mechanism.

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Abstract

一种快速实时检测生物硫醇的比率型荧光探针、制备方法及其应用。光谱测试结果表明，比率型荧光探针对生物硫醇具有比率型响应、高选择性、高灵敏度、响应时间快以及斯托克斯位移大等优点。比率型荧光探针对生物硫醇的响应时间与大多数生物硫醇荧光探针相比具有实时快速的特点，且能用于细胞以及细胞器的靶向成像，这些优点可使比率型荧光探针满足高通量检测生物硫醇的需求。

Description

基于BODIPY的高效实时生物硫醇荧光检测探针 技术领域
本发明涉及一种基于BODIPY的高效实时生物硫醇荧光检测探针，还涉及该探针的制备方法及应用，属于有机小分子荧光探针技术领域。
背景技术
随着生命科学研究的不断深入和发展，越来越多的精密生物分析手段应用到研究微观的生物体系来获取有效的研究现象和结果。例如放射性同位素标记法、高倍显微观察、电化学分析方法等等，但由于自身固有的缺陷，在实际应用中这些技术的局限性越来越突出。随着荧光分析检测技术的不断发展，荧光染料在DNA杂交测试、基因重组检测、肿瘤细胞早期诊断、免疫检测和各种生物活性离子以及生物体内各种自由基的分析检测等方面得到广泛应用。可以说，荧光分析技术以其较高的检测灵敏度、较好的选择性、易操作、低成本等优点，已在生物分析领域发挥着不可替代的作用。荧光分析技术最关键部分就是荧光染料，荧光染料性能的优劣直接影响探针检测的灵敏性及准确性。因此研究适用于荧光标识的荧光染料及开发优秀的荧光探针将对生物分析技术及生命科学研究的发展至关重要。
由于染料自身性能的多样性，导致不同染料在生物检测分析过程中表现出很大的差异，并不是所有的荧光染料都能用作荧光探针的荧光团而应用在生化分析与生物检测领域。现有技术中已成功应用的荧光染料有：香豆素类荧光染料，具有光稳定性较好、Stokes位移大、油溶性好、分子体积比较小等诸多优点，香豆素染料性能上的主要缺点是吸收发射波长过短，大多数都是蓝色的荧光，并且香豆素染料的不饱和内酯结构在稀碱液中会发生水解反应，对其的应用有很大的限制。萘酰亚胺类染料，能够与DNA相互作用，是一种优秀的DNA嵌入剂，量子产率较高，光稳定性好，是很有用的生物标记物。但是萘酰亚胺类染料的颜色较浅并且摩尔消光系数较低，对其应用有一定的限制。荧光素和罗丹明类染料，此类荧光染料的 特点是水溶性好，光稳定性较好，吸收发射波长相对较长，量子产率高；但由于染料中氨基和羟基的存在，使得该染料对pH值比较敏感，在应用作离子或者生物分子探针时，对工作环境酸碱性有一定的要求。菁染料，目前已成功引用于检测DNA、核苷酸、蛋白质等荧光探针，但是此类染料的缺点是光稳定性差，多甲川结构容易在光照下发生氧化断裂，导致染料分解，另外多甲川结构也导致染料容易发生聚积，影响了该染料在应用方面的推广。
然而，氟硼二吡咯甲川(BODIPY)类染料，是一种性能优越的染料，因其良好的光化学稳定性、高摩尔消光系数、高荧光量子产率、分子结构易于修饰、荧光光谱半峰宽窄、对溶剂极性和pH的耐受性好等众多优点而被广泛应用于光电器件、生物标记以及荧光探针等领域。
生物硫醇，主要包括半胱氨酸(Cys)、高半胱氨酸(Hcy)和谷胱甘肽(GSH)，它们在生物体系中扮演着至关重要的作用。生物体系中硫醇物质的新陈代谢与运输和一系列重要的酶和蛋白质密切相关。细胞内硫醇水平异常与许多疾病都有关联，如白细胞损失、牛皮癣、肝损伤和艾滋病等。因此设计合成对生物硫醇具有高选择性、高灵敏度和实时快速响应的荧光探针具有非常重要的意义。生物硫醇与其他氨基酸相比最为显著的特点是含有强亲核性的巯基。目前报道的生物硫醇荧光探针，主要基于迈克尔加成反应、金属络合物置换、与醛环化加成以及二硫键还原等机理。其中基于迈克尔加成反应的硫醇探针由于其灵敏度高，可在近生理条件下对硫醇进行检测等优点受到了广泛的关注。但该类探针大部分都存在着对硫醇响应时间较长等问题，一般从几分钟到几个小时不等，不利于对硫醇物质的实时定量检测。只有极少数探针对生物硫醇响应时间短，可用于生物硫醇的快速实时检测，但这些探针基本上是(关/开)OFF/ON型荧光探针。OFF/ON型荧光探针的信号表达容易受到外界因素的干扰而影响检测结果，比率型荧光探针由于以两个不同波长处的荧光强度比值作为自身校准参数，可以有效弥补OFF/ON型荧光探针的不足之处。因此设计和开发可以用于快速实时检测硫醇物质的比率型荧光探针具有很重要的现实意义。
现有的实时响应型生物硫醇比率型荧光探针存在的主要问题是没有细胞器靶向功能且斯托克斯位移小，导致严重的荧光自淬灭和瑞利散射而引起检测误差。
发明内容
为了解决上述现有技术中存在的问题，本发明的一个目的在于提供一种快速实时检测生物硫醇的比率型荧光探针，所述探针的结构式为如下式(I)所示化合物：
Figure PCTCN2019116374-appb-000001
其中R1选自氢、式(III)、式(IV)；R2或R3选自式(II)；R4或R5选自甲基；
所述式(II)如下：
Figure PCTCN2019116374-appb-000002
其中R4或R5选自氢；
所述式(III)如下：
Figure PCTCN2019116374-appb-000003
所述式(IV)如下：
Figure PCTCN2019116374-appb-000004
所述R7为Cl。优选地，所述的探针，其结构选自如下：
Figure PCTCN2019116374-appb-000005
Figure PCTCN2019116374-appb-000006
所述R7为Cl。
本发明的另一目的在于提供一种快速实时检测生物硫醇的比率型荧光探针的制备方法，包括如下步骤：
(1)染料氟硼二吡咯甲川-简双(BOD-JS)、氟硼二吡咯甲川-炔双(BOD-QS)和氟硼 二吡咯甲川-叠双(BOD-DS)的合成；
(2)固相光化学合成α-甲酰化BODIPY衍生物；
(3)细胞器靶向荧光染料合成；
其中，所述步骤(1)的合成方法包括：向无水N,N-二甲基甲酰胺(DMF)中加入中间染料氟硼二吡咯甲川-简(BOD-J)、氟硼二吡咯甲川-炔(BOD-Q)或氟硼二吡咯甲川-叠(BOD-D)，以及3,5-二甲基苯甲醛，除氧后，加入哌啶和冰醋酸，室温搅拌后，置于微波反应器中反应。待反应结束后，将冷却的反应液倾入二氯甲烷中，并分别用饱和食盐水和水洗涤数次，随后用无水硫酸镁干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂，所得粗产物用少量二氯甲烷溶解后经硅胶柱层析，梯度洗脱进行分离提纯，最后得到BOD-JS、BOD-QS或BOD-DS粉末；
所述步骤(2)包括，染料氟硼二吡咯甲川-简醛(BOD-JQ)、氟硼二吡咯甲川-炔醛(BOD-QQ)和/或染料氟硼二吡咯甲川-叠醛(BOD-DQ)的合成；
所述步骤(3)中所述细胞器为线粒体或内质网。
优选地，上述方法中所述BOD-JQ、BOD-QQ或BOD-DQ的合成方法包括：将染料BOD-JS、BOD-QS或BOD-DS溶解于二氯甲烷中，然后加入50-500目硅胶，经减压旋转蒸发溶剂，并将混合物置于氙气灯下方处光照，可观察到混合物由墨绿色变为紫黑色，待混合物冷却至室温后，用二氯甲烷萃取数次，合并有机相，经减压蒸馏除去多余有机溶剂，所得粗产物用少量二氯甲烷溶解后经硅胶柱层析，梯度洗脱进行分离提纯，最后得到粉末。
优选地，上述方法还包括中间染料的合成，所述中间染料为氟硼二吡咯甲川-简(BOD-J)、中间体炔醛(QQ)、氟硼二吡咯甲川-炔(BOD-Q)、中间体溴醛(XQ)、中间体叠醛(DQ)和氟硼二吡咯甲川-叠(BOD-D)。
优选地，上述方法中所述细胞器靶向荧光染料合成步骤包括：
在氮气保护下，将BOD-QQ以及(4-叠氮丁基)三苯基溴化膦溶于混合溶剂中，随后加 入五水合硫酸铜和抗坏血酸钠，用锡箔纸包住避光，室温搅拌反应，待反应结束后，加入水，剧烈震荡，用二氯甲烷萃取，收集有机层，并将有机层用饱和食盐水和水依次洗涤数次，随后用无水硫酸钠干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂，所得粗产物用少量二氯甲烷溶解后经硅胶柱层析，梯度洗脱进行分离提纯，最后得到粉末；所述细胞器为线粒体。
优选地，上述方法中所述细胞器靶向荧光染料合成步骤包括：
在氮气保护下，将BOD-QQ以及1-叠氮-3-氯丙烷溶于混合溶剂中，随后加入五水合硫酸铜和抗坏血酸钠，用锡箔纸包住避光，室温搅拌反应；随后将反应置于冰浴中，缓慢滴加三乙胺和三氟化硼乙醚，继续搅拌；待反应结束后，加入水，剧烈震荡，用二氯甲烷萃取，收集有机层，并将有机层用饱和食盐水和水依次洗涤数次；随后用无水硫酸钠干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂；所得粗产物用少量二氯甲烷溶解后经硅胶柱层析，梯度洗脱进行分离提纯，最后得到粉末。
本发明的另一目的在于提供一种快速实时检测生物硫醇的比率型荧光探针的应用，所述应用选自如下(1)-(4)之一：
(1)在生物硫醇检测中的应用，所述生物硫醇优选为半胱氨酸(Cys)、高半胱氨酸(Hcy)和/或谷胱甘肽(GSH)；
(2)在细胞成像中的应用；
(3)在制备生物硫醇检测或细胞成像的试剂或试剂盒中的应用；
(4)在细胞器检测中的应用。
优选地，上述应用中所述生物硫醇检测是在偏碱性条件下进行，优选pH为7.2-8.0，更优选pH为7.4。
优选地，上述应用中所述细胞器为内质网和/或线粒体。
与现有技术相比，本发明具有以下有益效果：
本发明开发了一种快速实时检测生物硫醇的比率型荧光探针。光谱测试结果表明，该探针对生物硫醇具有比率型响应、高选择性、高灵敏度、响应时间快以及斯托克斯位移大等优点。探针对生物硫醇的响应时间与大多数生物硫醇荧光探针相比具有实时快速的特点，这一优点可使其满足高通量检测生物硫醇的需求。此外，与为数不多的几例实时响应生物硫醇荧光探针(OFF/ON型或(开/关)ON/OFF型)相比，探针的比率型响应机制可以有效弥补OFF/ON型或ON/OFF型荧光探针的信号表达容易受到外界因素的干扰而影响检测结果的缺点。与现有技术已报道的实时响应型比率型荧光探针相比，本探针具有细胞器靶向功能，如线粒体靶向：体外线粒体膜电位180～200mV时,内侧带负电，在活细胞和机体内略低,为130～150mV，利用亲脂阳离子进行药物递送，其中研究最为成功的是三苯基膦，三苯基膦含有三个苯环可增大分子表面积，并形成离域正电荷，可穿过线粒体双层疏水膜；又如内质网靶向：氯原子官能团的存在实现了其与内质网上氯离子泵的特异性结合，从而完成了对内质网的定位，且斯托克斯位移更大，本发明的探针的斯托克斯位移也达到了52nm，比目前报道的最大斯托克斯位移提高了约13nm。同时该探针溶液在体外遇到生物硫醇后颜色由粉红色变为淡黄色，可用于直观裸眼检测。这也为设计合成比率型生物硫醇荧光探针提供了新的思路。
附图说明
图1，探针合成路线图，具体为醛基BODIPY的合成。
图2，BOD-J合成路线。
图3，BOD-JS合成路线。
图4，QQ合成路线。
图5，BOD-Q合成路线。
图6，BOD-QS合成路线。
图7，XQ合成路线。
图8，DQ合成路线。
图9，BOD-D合成路线。
图10，BOD-DS合成路线。
图11，BOD-JQ合成路线。
图12，BOD-JQ  1H核磁谱图(CDCl 3)。
图13，BOD-JQ  13C核磁谱图(CDCl 3)。
图14，BOD-JQ高分辨质谱图。
图15，BOD-QQ合成路线。
图16，BOD-QQ  1H核磁谱图(CDCl 3)。
图17，BOD-QQ  13C核磁谱图(CDCl 3)。
图18，BOD-QQ高分辨质谱图。
图19，BOD-DQ合成路线。
图20，BOD-DQ  1H核磁谱图(CDCl 3)。
图21，BOD-DQ  13C核磁谱图(CDCl 3)。
图22，BOD-DQ高分辨质谱图。
图23，BOD-PPh3合成路线。
图24，BOD-PPh3  1H核磁谱图(DMSO-d6)。
图25，BOD-PPh3  13C核磁谱图(DMSO-d6)。
图26，BOD-PPh3高分辨质谱图。
图27，BOD-Cl合成路线。
图28，BOD-Cl  1H核磁谱图(CDCl 3)。
图29，BOD-Cl  13C核磁谱图(CDCl 3)。
图30，BOD-Cl高分辨质谱图。
图31，pH对BOD-JQ(5μM)加入500μM GSH前后荧光强度比值(I 544nm/I 596nm)的影响，R为加入GSH后I 544nm/I 596nm的比值，R 0为加入GSH前I 544nm/I 596nm的比值(λ ex＝510nm)。
图32，pH对BOD-JQ(5μM)加入500μM GSH前后荧光强度比值(I 544nm/I 596nm)相对值的影响，R为加入GSH后I 544nm/I 596nm的比值，R 0为加入GSH前I 544nm/I 596nm的比值(λ ex＝510nm)。
图33，探针分子BOD-JQ(5μM)随着GSH溶液的加入(0～1000μM)(a)紫外-可见吸收光谱和(b)荧光发射光谱变化图(λ ex＝510nm)。
图34，(a)探针分子BOD-JQ(5μM)随着GSH溶液的加入(0～1000μM)其荧光强度比值(I 544nm/I 596nm)与GSH浓度关系曲线(b)探针分子BOD-JQ(5μM)荧光强度比值(I 544nm/I 596nm)对不同GSH浓度(0～70μM)的拟合曲线(λ ex＝510nm)。
图35，探针BOD-JQ(5μM)在不同物质(500μM)存在时的荧光强度比值(I 544nm/I 596nm)(λ ex＝510nm)。
图36，探针BOD-JQ(5μM)在不同物质(500μM)存在时对GSH响应的荧光强度比值(I 544nm/I 596nm)(λ ex＝510nm)。
图37，基于醛基为识别基团的探针与Cys/Hcy的反应。
图38，探针BOD-JQ(5μM)与Cys、Hcy和GSH响应的荧光光谱图(500μM，λ ex＝510nm)。
具体实施方式
以下通过具体实施例对本发明作进一步详细说明，以使本领域技术人员能够更好地理解本发明并予以实施，但实施例并不作为本发明的限定。
以下实施例中所使用的实验方法如无特殊说明，均为常规方法。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1 实时检测生物硫醇的比率型荧光探针的合成
采用新型固相光化学合成技术合成了氟硼二吡咯甲川(BODIPY)类荧光染料，该系列染料对生物硫醇具有快速实时响应的优点，且为比率型荧光探针。该探针的合成路线图，如附图1所示，醛基BODIPY的合成。具体包括如下：
1.中间染料氟硼二吡咯甲川-简(BOD-J)的合成和表征
BOD-J合成路线如图2所示，在氮气保护下，将苯甲酰氯(2.8g，0.02mol)和2,4-二甲基吡咯(3.8g，0.04mol)用注射器加入到150mL氮气饱和的无水DCM的250mL圆底烧瓶中，用锡箔纸包住避光，室温搅拌过夜。随后将反应置于冰浴中，15min后缓慢滴加20mL三乙胺和20mL三氟化硼乙醚，继续搅拌2h。待反应结束后，加入200mL水，剧烈震荡5min，收集有机层，并将有机层用饱和食盐水和水依次洗涤三遍。随后用无水硫酸镁干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂。所得粗产物用少量二氯甲烷溶解后经硅胶柱层析(石油醚/二氯甲烷为洗脱剂)梯度洗脱进行分离提纯，最后得到碧绿色粉末2.64g，产率：42％。
1H NMR(500MHz,CDCl 3)δ7.50–7.46(m,3H),7.28(dd,J＝7.4,2.0Hz,2H),5.98(s,2H),2.56(s,6H),1.37(s,6H)。
2.染料氟硼二吡咯甲川-简双(BOD-JS)的合成和表征
BOD-JS合成路线如图3所示，向5mL无水N,N-二甲基甲酰胺(DMF)中加入染料BOD-J (97mg,0.3mmol)和3,5-二甲基苯甲醛(160mg，1.2mmol)，除氧30min后，加入100μL哌啶和100μL冰醋酸，室温搅拌5min后，置于微波反应器中，在150W功率下反应45min。待反应结束后，将冷却的反应液倾入入50mL二氯甲烷中，并分别用饱和食盐水和水洗涤数次。随后用无水硫酸镁干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂。所得粗产物用少量二氯甲烷溶解后经硅胶柱层析(石油醚/二氯甲烷为洗脱剂)梯度洗脱进行分离提纯，最后得到棕褐色粉末154mg，产率：93％。
1H NMR(500MHz,CDCl 3)δ7.72(d,J＝16.3Hz,2H),7.50(dd,J＝4.8,2.3Hz,3H),7.32(dd,J＝7.2,2.1Hz,2H),7.26(s,4H),7.21(d,J＝16.3Hz,2H),6.97(s,2H),6.63(s,2H),2.37(s,12H),1.44(s,6H)。
3.中间体炔醛(QQ)的合成和表征
QQ合成路线如图4所示，往250mL无水丙酮中加入对羟基苯甲醛(6.1g,50mmol)和无水碳酸钾(9.7g，70mmol)，加热回流30min，然后加入3-溴丙炔(11.1mL，100mmol)，继续加热回流反应2h。反应结束后减压蒸馏除去溶剂，将粗产品溶于水中并用乙酸乙酯(4×75mL)萃取，合并有机相，并用饱和食盐水和水洗涤数次。有机相在无水硫酸镁干燥后，过滤后滤液经减压蒸馏除去多余的有机溶剂。所得粗产物用少量二氯甲烷溶解后经硅胶柱层析(石油醚/三氯甲烷为洗脱剂)梯度洗脱进行分离提纯，最后得到淡黄色粉末7.8g，产率：98％。
1H NMR(500MHz,CDCl 3)δ9.91(s,1H),7.86(d,J＝8.9Hz,2H),7.10(d,J＝8.7Hz,2H),4.79(d,J＝2.4Hz,2H),2.57(s,1H)。
4.中间染料氟硼二吡咯甲川-炔(BOD-Q)的合成和表征
BOD-Q合成路线如图5所示，在氮气保护下，将QQ(1g，6.2mmol)溶于150mL氮气饱和无水DCM，随后在氮气氛围中将2,4-二甲基吡咯(1.2g，12.6mmol)用注射器注入。加入100μL三氟乙酸，溶液有无色变为棕红色，用锡箔纸包住反应体系避光，室温搅拌过夜。 加入DDQ(1.4g，6.2mmol)后继续避光室温搅拌45min。随后将反应置于冰浴中，15min后缓慢滴加6mL三乙胺和6mL三氟化硼乙醚，继续搅拌2h。待反应结束后，加入200mL水，剧烈震荡5min，收集有机层，并将有机层用饱和食盐水和水依次洗涤三遍。随后用无水硫酸镁干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂。所得粗产物用少量二氯甲烷溶解后经硅胶柱层析(石油醚/二氯甲烷为洗脱剂)梯度洗脱进行分离提纯，最后得到紫红色粉末0.57g，产率：24％。
1H NMR(500MHz,CDCl 3)δ7.20(d,J＝8.7Hz,2H),7.09(d,J＝8.8Hz,2H),5.97(s,2H),4.76(d,J＝2.4Hz,2H),2.55(s,7H),1.42(s,6H)。
5.染料氟硼二吡咯甲川-炔双(BOD-QS)的合成和表征
BOD-QS合成路线如图6所示，其合成方法及其参数条件与BOD-JS的合成相同，投入BOD-Q(113mg，0.3mmol),得到金黄色粉末BOD-QS 178mg，产率97％。
1H NMR(500MHz,CDCl3)δ7.71(d,J＝16.3Hz,2H),7.25(s,3H),7.23(t,J＝3.0Hz,2H),7.19(s,1H),7.13–7.07(m,2H),6.96(s,2H),6.63(s,2H),4.78(d,J＝2.4Hz,2H),2.57(s,1H),2.37(s,12H),1.49(s,6H).
13C NMR(126MHz,CDCl3)δ158.15(s),152.77(s),142.08(s),138.56(s),138.26(s),136.75(s),136.42(s),133.67(s),130.92(s),129.72(s),128.20(s),125.51(s),118.76(s),117.82(s),115.59(s),78.10(s),75.93(s),56.07(s),21.29(s),14.85(s).
6.中间体溴醛(XQ)的合成与表征
XQ合成路线如图7所示，往40mL无水DMF中加入对羟基苯甲醛(2.44g,20mmol)和无水碳酸钾(5.53g，40mmol)，加热回流30min，然后加入1,2-二溴乙烷(7.43g，40mmol)，继续加热回流反应6h。反应结束后将反应液倾入冰水混合物中，过滤得到淡黄色粉末。将粉末重新溶解于二氯甲烷中，并经无水硫酸钠干燥，过滤干燥得到淡黄色产物1.26g，产率30％。
1H NMR(400MHz,CDCl 3)δ9.92(s,1H),7.87(d,J＝8.8Hz,2H),7.04(d,J＝8.7Hz,2H),4.40(t,J＝6.2Hz,2H),3.69(t,J＝6.2Hz,2H).
7.中间体叠醛(DQ)的合成和表征
DQ合成路线如图8所示，往40mL无水DMSO中加入DQ(1.26g，5.5mmol)和叠氮化钠(0.45g，6.0mmol)，加热回流2h。待反应结束并冷却后用二氯甲烷稀释，随后用饱和食盐水和水将有机相洗涤数次，最后用无水硫酸钠干燥有机层。过滤后滤液经减压蒸馏除去多余的有机溶剂。所得粗产物用少量二氯甲烷溶解后经硅胶柱层析(石油醚/乙酸乙酯为洗脱剂)梯度洗脱进行分离提纯，最后得到淡黄色粉末0.82g，产率：80％。
1H NMR(500MHz,CDCl 3)δ9.90(s,1H),7.93–7.73(m,2H),7.03(d,J＝8.7Hz,2H),4.26–4.20(m,2H),3.72–3.62(m,2H).
8.中间染料氟硼二吡咯甲川-叠(BOD-D)的合成和表征
BOD-D合成路线如图9所示，其合成方法及其参数条件与BOD-Q合成相同，投入DQ(1.9g，10mmol)，得到亮黄色针状产物HOD-D818mg，产率：20％。
1H NMR(500MHz,CDCl 3)δ7.21–7.17(m,2H),7.06–7.01(m,2H),5.98(s,2H),4.23–4.18(m,2H),3.68–3.63(m,2H),2.55(s,6H),1.43(s,6H).
9.染料氟硼二吡咯甲川-叠双(BOD-DS)的合成和表征
BOD-DS合成路线如图10所示，其合成方法及其参数条件与BOD-JS合成相同，投入BOD-D(123mg，0.3mmol),得到金黄色粉末BOD-QS 188mg，产率98％。
1H NMR(500MHz,CDCl 3)δ7.71(d,J＝16.3Hz,2H),7.26–7.25(m,2H),7.24(s,1H),7.23(s,2H),7.20(s,1H),7.05(d,J＝8.7Hz,2H),6.97(s,2H),6.63(s,2H),4.24–4.20(m,2H),3.69–3.64(m,2H),2.37(s,12H),1.50(s,6H).
13C NMR(126MHz,CDCl 3)δ158.82(s),152.75(s),142.11(s),138.63(s),138.29(s), 136.75(s),136.41(s),133.69(s),130.95(s),129.80(s),127.91(s),125.51(s),118.76(s),117.82(s),115.12(s),66.98(s),50.25(s),21.29(s).
固相光化学合成α-甲酰化BODIPY衍生物
10.染料氟硼二吡咯甲川-简醛BOD-JQ的合成和表征
BOD-JQ合成路线如图11所示，将55.6mg染料BOD-JS溶解于10mL二氯甲烷中，然后加入2g 200目硅胶，经减压旋转蒸发溶剂，并将混合物置于100W氙气灯下方10cm处光照40min。可观察到混合物由墨绿色变为紫黑色。待混合物冷却至室温后，用二氯甲烷萃取数次，合并有机相，经减压蒸馏除去多余有机溶剂，所得粗产物用少量二氯甲烷溶解后经硅胶柱层析(石油醚/三氯甲烷为洗脱剂)梯度洗脱进行分离提纯，最后得到淡黄色粉末31.3mg，产率：69％。BOD-JQ表征结果如图12-14所示：
理论分子量：454.20280，FT-MS：477.19202(M+Na +)。
1H NMR(500MHz,CDCl 3)δ10.36(s,1H),7.73(d,J＝16.3Hz,1H),7.57–7.52(m,3H),7.43(d,J＝16.2Hz,1H),7.35–7.32(m,2H),7.28(s,2H),7.06(s,1H),6.81(d,J＝8.8Hz,2H),2.37(s,6H),1.52(s,3H),1.41(s,3H).
13C NMR(126MHz,CDCl 3)δ184.46(s),161.08(s),148.14(s),143.48(s),143.22(s),141.58(s),138.69(s),137.00(s),135.30(s),134.22(s),133.39(s),132.76(s),129.53(d,J＝15.3Hz),127.92(s),126.35(s),121.01(s),120.12(s),117.88(s),21.23(s),15.27(s),14.14(s).
11.染料氟硼二吡咯甲川-炔醛(BOD-QQ)的合成和表征
BOD-QQ合成路线如图15所示，其合成方法及其参数条件与BOD-JQ合成相同，投入BOD-QS(0.1mmol，61mg)，得到产物37mg，产率72％。BOD-QQ表征结果如图16-18所示：
理论分子量：508.21337，FT-MS：531.20259(M+Na +)。
1H NMR(500MHz,CDCl 3)δ10.35(s,1H),7.73(d,J＝16.1Hz,1H),7.44(d,J＝16.2Hz,1H),7.28(s,2H),7.24(s,2H),7.15(d,J＝8.4Hz,2H),7.06(s,1H),6.81(d,J＝9.1Hz,2H),4.79(d,J＝2.0Hz,2H),2.58(s,1H),2.37(s,6H),1.58(s,3H),1.47(s,3H).
13C NMR(126MHz,CDCl 3)δ184.47(s),160.98(s),158.53(s),148.12(s),143.41(s),143.19(s),141.42(s),138.69(s),136.96(s),135.30(s),132.74(s),129.28(s),127.14(s),126.34(s),120.95(s),120.11(s),117.89(s),115.94(s),77.87(s),76.08(s),56.08(s),31.93(s),29.67(s),29.37(s),22.70(s),21.23(s),15.49(s),14.36(s),14.13(s).
12.染料氟硼二吡咯甲川-叠醛(BOD-DQ)的合成和表征
BOD-DQ合成路线如图19所示，其合成方法及其参数条件与BOD-JQ合成相同，投入BOD-DS(0.1mmol，64mg)，得到产物38mg，产率70％。BOD-DQ表征结果如图20-22所示：
理论分子量：539.23041，FT-MS：562.21963(M+Na +)。
1H NMR(500MHz,CDCl 3)δ10.35(s,1H),7.73(d,J＝16.3Hz,1H),7.43(d,J＝16.2Hz,1H),7.28(s,2H),7.24(s,2H),7.11–7.07(m,2H),7.06(s,1H),6.81(d,J＝9.2Hz,2H),4.26–4.19(m,2H),3.71–3.64(m,2H),2.37(s,6H),1.58(s,3H),1.48(s,3H).
13C NMR(126MHz,CDCl 3)δ184.47(s),161.00(s),159.24(s),148.11(s),143.43(s),143.18(s),141.43(s),138.69(s),137.42(s),136.93(s),135.30(s),133.73(s),132.75(s),129.41(s),126.93(s),126.34(s),120.97(s),120.09(s),117.88(s),115.43(s),67.05(s),50.22(s),31.94(s),29.67(s),22.71(s),21.23(s),15.55(s),14.42(s),14.14(s).
细胞器靶向荧光染料合成
13.线粒体靶向氟硼二吡咯甲川-三苯基膦(BOD-PPh3)的合成
BOD-PPh3合成路线如图23所示，在氮气保护下，将BOD-QQ(50.8mg，0.1mmol) 和(4-叠氮丁基)三苯基溴化膦(43.9mg，0.1mmol)溶于混合溶剂中(14mL，CHCl 3:EtOH:H 2O＝12:1:1，v/v)，随后加入五水合硫酸铜(25mg，0.1mmol)和抗坏血酸钠(19.8mg，0.1mmol)，用锡箔纸包住避光，室温搅拌反应24h。待反应结束后，加入200mL水，剧烈震荡5min，用二氯甲烷萃取，收集有机层，并将有机层用饱和食盐水和水依次洗涤三遍。随后用无水硫酸钠干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂。所得粗产物用少量二氯甲烷溶解后经硅胶柱层析(二氯甲烷/甲醇为洗脱剂)梯度洗脱进行分离提纯，最后得到碧绿色粉末37.9mg，产率：40％。BOD-PPh3表征结果如图24-26所示：
理论分子量：947.2947，FT-MS：868.3759(M–Br -)。
1H NMR(400MHz,DMSO-d6)δ10.19(s,1H),8.24(s,1H),7.97(d,J＝16.3Hz,1H),7.91(ddt,J＝8.8,5.6,2.9Hz,3H),7.79(s,13H),7.58(s,1H),7.43(s,2H),7.33(s,2H),7.28(s,2H),7.16(s,1H),6.87(s,1H),5.20(s,2H),4.49(s,2H),4.38(s,2H),2.36(s,6H),2.04(s,4H),1.58(s,3H),1.45(s,3H).
13C NMR(101MHz,DMSO-d6)δ183.56,174.73,161.71,159.50,149.09,145.57,142.66,142.08,139.03,137.66,136.45,135.55,135.44,135.41,134.09,133.99,133.79,133.36,130.79,130.67,130.11,129.93,126.47,126.06,125.26,122.69,120.23,119.30,119.22,118.44,118.36,117.11,116.04,61.71,56.48,55.39,49.87,48.61,35.58,31.75,30.85,30.67,29.55,29.49,29.33,29.30,29.20,29.16,29.04,27.02,25.58,22.56,21.25,20.31,19.81,19.46,19.25,19.03,15.64,14.42,14.29.
14.内质网靶向氟硼二吡咯甲川-氯(BOD-Cl)的合成
BOD-Cl合成路线如图27所示，在氮气保护下，将BOD-QQ(50.8mg，0.1mmol)和1-叠氮-3-氯丙烷(11.9mg，0.1mmol)溶于混合溶剂中(10mL，DMSO:CH 2Cl 2＝1:1，v/v)，随后加入五水合硫酸铜(25mg，0.1mmol)和抗坏血酸钠(19.8mg，0.1mmol)，用锡箔纸包住避光，室温搅拌反应24h。随后将反应置于冰浴中，15min后缓慢滴加20mL三乙胺和 20mL三氟化硼乙醚，继续搅拌2h。待反应结束后，加入200mL水，剧烈震荡5min，用二氯甲烷萃取，收集有机层，并将有机层用饱和食盐水和水依次洗涤三遍。随后用无水硫酸钠干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂。所得粗产物用少量二氯甲烷溶解后经硅胶柱层析(二氯甲烷/甲醇为洗脱剂)梯度洗脱进行分离提纯，最后得到碧绿色粉末28.2mg，产率：45％。BOD-Cl表征结果如图28-30所示：
理论分子量：627.2384，FT-MS：650.2287(M+Na +)。
1H NMR(400MHz,CDCl 3)δ10.35(s,1H),7.78–7.67(m,2H),7.43(d,J＝16.2Hz,1H),7.27(s,2H),7.25–7.21(m,2H),7.16(d,J＝8.4Hz,2H),7.06(s,1H),6.81(d,J＝6.7Hz,2H),5.28(s,2H),4.61(t,J＝6.6Hz,2H),3.55(t,J＝6.0Hz,2H),2.44(p,J＝6.2Hz,2H),2.37(s,6H),1.57(s,3H),1.46(s,3H).
13C NMR(101MHz,CDCl 3)δ184.45,161.00,159.23,148.13,143.46,143.09,141.42,138.67,137.41,136.84,135.28,133.74,132.74,129.35,126.81,126.33,123.60,120.98,120.09,117.83,116.52,115.68,62.00,47.18,41.09,32.46,29.71,29.35,21.23,15.51,14.38.
实施例2 探针性能及其应用——光谱测试用溶液的配置以及测试方法
以下实验如未特殊注明，皆以生物体内含量最多的GSH作为生物硫醇物质代表进行实验。
母液的配制方法：将探针分子BOD-JQ用四氢呋喃(THF)配置成10 -3mol L -1的标准溶液，放置于4℃备用。
1.pH影响的测定——pH选择
配置一系列浓度为0.2mol L -1的醋酸-醋酸钠缓冲溶液、磷酸氢二钠-磷酸二氢钠缓冲溶液、磷酸氢二钾-磷酸二氢钾缓冲溶液(PBS)、硼酸-硼砂缓冲溶液以及碳酸氢钠-碳酸钠缓冲溶液。然后分别将DMSO、水以及这些溶液按14:5:1体积进行混溶，然后取4mL该系列混合溶 剂，并各加入20μL母液，探针终浓度为5μM，测其荧光光谱，观察探针分子随着pH的变化荧光光谱的变化。并分别测试加入100当量的GSH溶液后，观察探针分子与GSH反应产物随着pH的变化荧光光谱的变化。
结果如图31所示，当pH在3.66～7.4范围时，探针分子在544nm处和596nm处荧光强度比值R 0(I 544nm/I 596nm)基本保持不变。当pH值在7.4～11.01范围时，探针分子荧光强度比值R 0(I 544nm/I 596nm)先略微增强而后下降。当加入GSH后探针荧光强度比值R(I 544nm/I 596nm)在pH小于6.72的范围内基本不变，在pH在6.72～8.35之间，探针分子荧光强度比值R 0(I 544nm/I 596nm)，随着pH的增大而增大。此后随着pH的升高其值基本保持不变。这些结果表明，该探针对GSH的检测在偏碱性条件下最为有利。
如图32所示，在不同pH值下，探针对GSH检测的灵敏度(R/R 0)在pH为7.4时其值最大，故后续实验所用pH值皆为7.4。
2.BOD-JQ对GSH的荧光检测
取20μL母液溶于4mL DMSO:H 2O:PBS(14:5:1，pH＝7.4，浓度为0.2mol L -1)，探针终浓度为5μM。用移液枪逐渐加入一定体积的GSH溶液(控制最终加入浓度总体积<75μL)，混合均匀，立即扫相应的紫外-可见吸收光谱。(因为探针对GSH的响应时间极快，1s以内即可反应完全)。荧光光谱图由类似方法扫得。
结果如图33中的浓度滴定曲线可以看出，在DMSO:H 2O:PBS＝14:5:1(PBS缓冲溶液浓度为0.2mol L -1，pH＝7.4)的测试体系中(探针终浓度为5μM)，随着GSH溶液的加入，探针分子的最大吸收波长570nm处吸光度逐渐下降，并在519nm处生成一个新峰，溶液颜色由粉红色变为橙黄色。与此同时探针在596nm处的荧光逐渐减弱，而在544nm处的荧光则逐渐增强，在紫外灯的照射下，溶液由呈现出强烈的橙红色荧光变为黄绿色荧光。我们做了I 544nm/I 596nm荧光强度比值与GSH浓度变化曲线，如图34所示。我们通过线性拟合发现，加入GSH后，I 544nm/I 596nm荧光强度比值与GSH浓度在4～70μM之间呈非常好的线 性关系(R 2＝0.997)，据此可以定量检测低浓度的GSH。
对没有加入GSH的探针BOD-JQ空白溶液(空白样)的荧光比值(I 544nm/I 596nm)进行6次测试，计算出测量标准偏差为0.0057。则BOD-JQ的检测下限可以根据如下公式计算：
LOD＝K×SD/S
其中K＝3；SD为空白样的标准偏差；S为拟合曲线的斜率。
LOD＝K×SD/S＝3×0.0057/0.00961＝1.8μM，也即检测下限LOD为1.8μM。一般而言细胞中谷胱甘肽的浓度为1～10mM，因此该探针可以满足一般生物体系中谷胱甘肽的检测要求。此外由图34可知，BOD-JQ对Cys和Hcy具有类似的响应效果，虽然细胞中Cys和Hcy的浓度较低，一般为30～200μM和5～15μM。但探针BOD-JQ是检测生物体系中生物硫醇总浓度的探针，一般而言三个常见生物硫醇物质的总浓度在mM级别，因此该探针的灵敏度可满足一般生物体系中生物硫醇的检测需求。
3.选择性实验
在DMSO:H 2O:PBS(14:5:1，pH＝7.4，浓度为0.2mol L -1)的测试溶液中，探针分子浓度为5μM，每个样品中分别加入100当量的AA，Ala，Arg，Glu，Gly，His，Leu，Phe，Trp，Val，Cys，Hcy和GSH，并测定荧光光谱前后的变化。
经荧光光谱仪测定了探针BOD-JQ对不同物质的荧光响应，我们选择了常见的氨基酸和抗坏血酸来考察探针的选择性。实验结果从图35可以看出：相对于未加硫醇分子时的荧光光谱，Cys，Hcy和GSH的加入带来了明显的荧光变化，而其他氨基酸和抗坏血酸的加入基本不会对探针的荧光性能造成影响。这说明探针BOD-JQ对生物硫醇分子具有非常好的选择性。
4.干扰性实验
在DMSO:H 2O:PBS(14:5:1，pH＝7.4，浓度为0.2mol L -1)的测试溶液中，探针分子浓度为5μM，每个样品中都加入100当量GSH溶液，并分别加入100当量的AA，Ala， Arg，Glu，Gly，His，Leu，Phe，Trp和Val，并测定荧光光谱前后的变化，考察探针在GSH与其他物质共存时其荧光光谱响应情况。
为了进一步考察探针BOD-JQ对生物硫醇检测的干扰性，我们选择GSH为生物硫醇类物质的代表，在其他不同物质共存的条件下测试了探针BOD-JQ对GSH的检测能力，其结果如图3.8所示。从图36可以看出，其他物质共存时并不会明显影响探针BOD-JQ对GSH的响应，因此，探针BOD-JQ作为生物硫醇分子的荧光探针在检测时具有较好的抗干扰能力。
5.BOD-JQ检测机理验证
通过对加入GSH溶液后体系光谱的变化情况对产物进行合理的推断，并通过高分辨质谱对反应产物进行鉴定。
根据之前的荧光光谱滴定实验中体系荧光蓝移的现象，据此可以推断分子的共轭程度应该有所减小。BOD-JQ上存在着一个芳香醛基，基于醛基可以构建硫醇类物质的荧光探针。但基于醛基的硫醇类物质荧光探针能选择性的检测半胱氨酸/高半胱氨酸，而对谷胱甘肽则不会有响应。这是因为巯基与醛基作用后邻近的氨基会继续与其作用，最后生成稳定的五(六)元环，而谷胱甘肽的邻近位置则没有氨基，不能进行响应，其反应机理如图37所示。此外由图38可知，三个硫醇分子与探针作用后，其荧光光谱几乎一致，因此应该是基于相同响应机理。结合光谱实验我们推断该探针检测机理应该是基于巯基与BODIPY衍生物5-位苯乙烯基上的烯烃发生迈克尔加成反应。有文献报道在探针有芳香醛和识别基团共存时，生物硫醇的巯基仍可选择性地与识别基团的烯烃进行迈克尔加成或者还原二硫键而不与醛基发生作用。由于该探针对巯基化合物的检测很灵敏，且探针与巯基作用后生成的产物可能较少等原因，我们并没有得到质谱等更为直观的证据证明该机理。
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。

Claims (10)

  1. 一种快速实时检测生物硫醇的比率型荧光探针，所述探针的结构式为如下式(I)所示化合物：
    Figure PCTCN2019116374-appb-100001
    其中R1选自氢、式(III)、式(IV)；R2或R3选自式(II)；R4或R5选自甲基；
    所述式(II)如下：
    Figure PCTCN2019116374-appb-100002
    其中R4或R5选自氢；
    所述式(III)如下：
    Figure PCTCN2019116374-appb-100003
    所述式(IV)如下：
    Figure PCTCN2019116374-appb-100004
    所述R6为Cl。
  2. 根据权利要求1所述的探针，其结构选自如下：
    Figure PCTCN2019116374-appb-100005
    Figure PCTCN2019116374-appb-100006
    Figure PCTCN2019116374-appb-100007
    所述R7为Cl。
  3. 权利要求1或2所述的探针的制备方法，包括如下步骤：
    (1)染料氟硼二吡咯甲川-简双(BOD-JS)、氟硼二吡咯甲川-炔双(BOD-QS)和氟硼二吡咯甲川-叠双(BOD-DS)的合成；
    (2)固相光化学合成α-甲酰化BODIPY衍生物；
    (3)细胞器靶向荧光染料合成；
    其中，所述步骤(1)的合成方法包括：向无水N,N-二甲基甲酰胺(DMF)中加入中间染料氟硼二吡咯甲川-简(BOD-J)、氟硼二吡咯甲川-炔(BOD-Q)或氟硼二吡咯甲川-叠(BOD-D)，以及3,5-二甲基苯甲醛，除氧后，加入哌啶和冰醋酸，室温搅拌后，置于微波反应器中反应。待反应结束后，将冷却的反应液倾入二氯甲烷中，并分别用饱和食盐水和水洗涤数次，随后用无水硫酸镁干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂，所得 粗产物用少量二氯甲烷溶解后经硅胶柱层析，梯度洗脱进行分离提纯，最后得到BOD-JS、BOD-QS或BOD-DS粉末；
    所述步骤(2)包括，染料氟硼二吡咯甲川-简醛(BOD-JQ)、氟硼二吡咯甲川-炔醛(BOD-QQ)和/或染料氟硼二吡咯甲川-叠醛(BOD-DQ)的合成；
    所述步骤(3)中所述细胞器为线粒体或内质网。
  4. 根据权利要求3所述的方法，所述BOD-JQ、BOD-QQ或BOD-DQ的合成方法包括：将染料BOD-JS、BOD-QS或BOD-DS溶解于二氯甲烷中，然后加入50-500目硅胶，经减压旋转蒸发溶剂，并将混合物置于氙气灯下方处光照，可观察到混合物由墨绿色变为紫黑色，待混合物冷却至室温后，用二氯甲烷萃取数次，合并有机相，经减压蒸馏除去多余有机溶剂，所得粗产物用少量二氯甲烷溶解后经硅胶柱层析，梯度洗脱进行分离提纯，最后得到粉末。
  5. 根据权利要求3所述的方法，还包括中间染料的合成，所述中间染料为氟硼二吡咯甲川-简(BOD-J)、中间体炔醛(QQ)、氟硼二吡咯甲川-炔(BOD-Q)、中间体溴醛(XQ)、中间体叠醛(DQ)和氟硼二吡咯甲川-叠(BOD-D)。
  6. 根据权利要求3所述的方法，所述细胞器靶向荧光染料合成步骤包括：
    在氮气保护下，将BOD-QQ以及(4-叠氮丁基)三苯基溴化膦溶于混合溶剂中，随后加入五水合硫酸铜和抗坏血酸钠，用锡箔纸包住避光，室温搅拌反应，待反应结束后，加入水，剧烈震荡，用二氯甲烷萃取，收集有机层，并将有机层用饱和食盐水和水依次洗涤数次，随后用无水硫酸钠干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂，所得粗产物用少量二氯甲烷溶解后经硅胶柱层析，梯度洗脱进行分离提纯，最后得到粉末；所述细胞器为线粒体。
  7. 根据权利要求3所述的方法，所述细胞器靶向荧光染料合成步骤包括：
    在氮气保护下，将BOD-QQ以及1-叠氮-3-氯丙烷溶于混合溶剂中，随后加入五水合硫酸铜和抗坏血酸钠，用锡箔纸包住避光，室温搅拌反应；待反应结束后，加入水，剧烈震荡， 用二氯甲烷萃取，收集有机层，并将有机层用饱和食盐水和水依次洗涤数次；随后用无水硫酸钠干燥有机层，过滤后滤液经减压蒸馏除去多余的有机溶剂；所得粗产物用少量二氯甲烷溶解后经硅胶柱层析，梯度洗脱进行分离提纯，最后得到粉末。
  8. 权利要求1或2所述的探针的应用，所述应用选自如下(1)-(4)之一：
    (1)在生物硫醇检测中的应用，所述生物硫醇优选为半胱氨酸(Cys)、高半胱氨酸(Hcy)和/或谷胱甘肽(GSH)；
    (2)在细胞成像中的应用；
    (3)在制备生物硫醇检测或细胞成像的试剂或试剂盒中的应用；
    (4)在细胞器检测中的应用。
  9. 根据权利要求8所述的应用，所述生物硫醇检测是在偏碱性条件下进行，优选pH为7.2-8.0，更优选pH为7.4。
  10. 根据权利要求8所述的应用，所述细胞器为内质网和/或线粒体。
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