WO2015081803A1 - 一类氟化硼络合二吡咯甲川荧光探针，其制备方法及应用 - Google Patents

一类氟化硼络合二吡咯甲川荧光探针，其制备方法及应用 Download PDF

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WO2015081803A1
WO2015081803A1 PCT/CN2014/092299 CN2014092299W WO2015081803A1 WO 2015081803 A1 WO2015081803 A1 WO 2015081803A1 CN 2014092299 W CN2014092299 W CN 2014092299W WO 2015081803 A1 WO2015081803 A1 WO 2015081803A1
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bclo
fluorescent probe
dipyrromethene
fluorescence
boron fluoride
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French (fr)
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樊江莉
朱浩
彭孝军
王静云
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大连理工大学
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Priority to US15/039,674 priority patent/US9862731B2/en
Priority to JP2016535729A priority patent/JP6275256B2/ja
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    • C—CHEMISTRY; METALLURGY
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    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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    • G01—MEASURING; TESTING
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    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom

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  • the invention relates to a fluorescent probe, a preparation method and a use thereof, and particularly relates to a boron fluoride complexed dipyrromethene fluorescent probe, a preparation method thereof and application thereof in the detection of hypochlorite.
  • Fluorescent probes have been widely used as functional dyes in various fields of science and technology, especially in the fields of life sciences, clinical medical diagnosis, and immunoassay detection.
  • fluorescent dyes boron fluoride complexed dipyrromethene (BDP) fluorescent dyes have high molar extinction coefficient, high fluorescence quantum yield, stable spectral properties, high photothermal, good chemical stability, small molecular weight and cytotoxicity.
  • BDP boron fluoride complexed dipyrromethene
  • hypochlorous acid As an important active oxygen in living organisms, hypochlorous acid (HClO) is physiologically mainly produced by the reaction of chloride ions and hydrogen peroxide under the action of myeloperoxidase. In the cellular environment, due to the strong oxidizing properties of hypochlorous acid/hypochlorite, it can kill foreign bacteria, prevent invasion, and regulate the cell life cycle. As we age, the concentration of hypochlorous acid in the human body will gradually increase. However, excessive hypochlorous acid/hypochlorite causes a series of diseases such as osteoarthritis, cardiovascular disease, etc. Therefore, the development of an effective method for monitoring hypochlorous acid has attracted more and more researchers' attention.
  • probes that can be used to detect hypochlorous acid/hypochlorite in aqueous solutions or organisms. These probes are mainly based on the strong oxidizing properties of hypochlorous acid, such as: oxidation mechanism of dibenzoyl hydrazide; oxidation of hydroxamic acid; mechanism of depurination; oxidation mechanism of p-methoxyphenol; mechanism of sulfur atom oxidation.
  • these probes generally have shortcomings such as long response time, low sensitivity, poor selectivity, and vulnerability to pH interference.
  • Pyrrole is an important raw material for the synthesis of BDP, and we have found that it can be selectively oxidized by hypochlorous acid to be insensitive to other reactive oxygen species (such as H 2 O 2 , ⁇ OH, etc.). Moreover, since pyrrole is aromatic and the unshared electron pair on the nitrogen participates in the formation of a cyclic large ⁇ bond, its ability to bind to protons is impaired, so that pH interference can be well avoided. In addition, the pyroelectricity of pyrrole makes it effective to produce photoinduced electron transfer (PET) effects on some fluorophores, thereby reducing background fluorescence and improving probe sensitivity.
  • PET photoinduced electron transfer
  • the present invention provides a class of boron fluoride complexed dipyrromethene fluorescent probes, , 4-dimethylpyrrole is a recognition group, and boron fluoride complexes dipyrromethene dye as a fluorophore, which improves probe selectivity (not interfered by other reactive oxygen species and pH) and sensitivity, accelerates response time, and has Good biological applications.
  • a type of boron fluoride complexed dipyrromethene fluorescent probe having the following structural formula I:
  • R 1 , R 2 and R 3 are each independently selected from H, C 1-8 alkyl, substituted or unsubstituted phenyl;
  • the substituted phenyl group is a phenyl group substituted by any one or more of the following groups: CN, COOH, NH 2 , NO 2 , OH, SH, C 1-6 alkoxy, C 1-6 alkyl Amino, C 1-6 amide, halogen or C 1-6 haloalkyl.
  • the method for preparing a boron fluoride complexed dipyrromethene fluorescent probe comprises the steps of: reacting a compound having the structural formula II with acryloyl chloride in a molar ratio of from 1 to 5:1, and then in the presence of a base And complexing with boron trifluoride to obtain the boron fluoride complexed dipyrromethene fluorescent probe;
  • the boron fluoride complexed dipyrromethene fluorescent probe of the present invention has the following remarkable features:
  • Figure 1 is the fluorescence intensity of the fluorescent probe compound BClO for different concentrations of hypochlorite in the BClO performance measurement experiment 1. Degree response graph. The concentration of the probe compound BClO was 1 ⁇ M, and the concentrations of sodium hypochlorite were 0, 1, 2, 3, 4, 5, 6, and 7 ⁇ M, respectively.
  • Figure 1a is a fluorescence titration spectrum of sodium hypochlorite by BClO with an excitation wavelength of 480 nm
  • Figure 1b is a titration curve of BClO to sodium hypochlorite with an excitation wavelength of 480 nm.
  • Figure 2 is a sample of the fluorescent probe compound BClO in the BClO performance measurement experiment 2 for different concentrations of hypochlorite absorption titration test.
  • the concentration of the fluorescent probe compound BClO was 1 ⁇ M, and the concentrations of sodium hypochlorite were 0, 1, 2, 3, 4, 5, 6, and 7 ⁇ M, respectively.
  • Fig. 3 is a fluorescence selective histogram of the fluorescent probe compound BClO for different reactive oxygen species in Experiment 3 of BClO performance measurement.
  • the concentration of the fluorescent probe compound BClO was 1 ⁇ M, the concentration of sodium hypochlorite was 5 ⁇ M, and the concentration of other active oxygen was 10 ⁇ M.
  • Figure 4 is a graph showing the fluorescence intensity response of the fluorescent probe compound BClO to low concentration hypochlorite in the BClO performance measurement experiment 4.
  • the concentration of the fluorescent probe compound BClO was 1 ⁇ M, and the concentrations of sodium hypochlorite were 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 nM, respectively.
  • Fig. 5 is a graph showing the response time of the fluorescent probe compound BClO to the detection of sodium hypochlorite in the BClO performance measurement experiment 5.
  • the concentration of the fluorescent probe compound BClO was 1 ⁇ M, and the concentration of sodium hypochlorite added was 5 ⁇ M.
  • the abscissa is time (s) and the ordinate is fluorescence intensity.
  • Fig. 6 is a graph showing the fluorescence intensity response of the probe compound BClO to hypochlorite at different pH values in the experiment 6 of the BClO performance measurement.
  • the abscissa is pH and the ordinate is fluorescence intensity.
  • the concentration of the fluorescent probe compound BClO was 1 ⁇ M, and the concentration of sodium hypochlorite added was 5 ⁇ M.
  • the pH was adjusted with NaOH (1 M) and HCl (1 M).
  • Figure 7 is a fluorescent image of the probe compound BClO in human breast cancer (MCF-7) cells in BClO performance assay experiment 7.
  • the concentration of the fluorescent probe compound BClO was 1 ⁇ M.
  • a, b, and c respectively indicate fluorescence photographs in the 490 nm-550 nm band after addition of 0, 3, and 5 ⁇ M sodium hypochlorite in BClO-stained cells.
  • the excitation wavelength was 488 nm.
  • Figure 8 is a fluorescent image of the probe compound BClO in mouse macrophage (Raw 264.7) cells in the BClO performance assay experiment 8.
  • the concentration of the fluorescent probe compound BClO was 1 ⁇ M.
  • a indicates fluorescence staining of cells after addition of BClO;
  • b indicates that Raw 264.7 cells were cultured with 1 ⁇ g/mL of LPS (lipopolysaccharide) for 12 hours, and then incubated with 1 ⁇ g/mL of PMA (phorbol myristate) Fluorescence photographs of cells stained with BClO were added 1 hour later. Fluorescence in the 490 nm-550 nm band was acquired with an excitation wavelength of 488 nm.
  • the present invention provides a class of boron fluoride complexed dipyrromethene fluorescent probes having the structural formula I.
  • R 1 , R 2 and R 3 are each independently selected from H, C 1-8 alkyl, substituted or unsubstituted phenyl;
  • the substituted phenyl group is a phenyl group substituted by any one or more of the following groups: CN, COOH, NH 2 , NO 2 , OH, SH, C 1-6 alkoxy, C 1-6 alkyl Amino, C 1-6 amide, halogen or C 1-6 haloalkyl.
  • R 1 and R 3 are each independently selected from H or methyl, more preferably methyl;
  • R 2 is preferably hydrogen or ethyl, more preferably hydrogen.
  • the boron fluoride complexed dipyrromethene fluorescent probe of the present invention is BClO, and the structural formula is as follows:
  • the present invention further provides a method for preparing the boron fluoride complexed dipyrromethene fluorescent probe, comprising the steps of: reacting a compound having the structural formula II with acryloyl chloride in a molar ratio of from 1 to 5:1, and then in a base.
  • the boron fluoride complexed dipyrromethene fluorescent probe is obtained by complexing with boron trifluoride in the presence of the boron fluoride;
  • the compound having the structural formula II and the acryloyl chloride are prepared as the intermediate compound III: the molar ratio of the intermediate compound II to the acryloyl chloride can be from 0.1 to 1000:1, preferably from 0.5 to 100:1, more preferably from 0.5 to 10: 1, further preferably 1-5:1, most preferably 3:1, and a condensation reaction occurs to form a compound of the formula III.
  • the above reaction can be carried out in an aqueous organic solvent or an anhydrous organic solvent. It is preferably an anhydrous organic solvent.
  • the molar concentration of the pyrrole starting material in the solvent is from 0.012 to 0.4 mol/L.
  • Organic solvents include, but are not limited to, dichloromethane, tetrahydrofuran or acetonitrile.
  • the pyrrole starting material and the acryloyl chloride are condensed twice to form the active intermediate III. Since the product is unstable, the next reaction can be directly carried out without separation, and the end point of the reaction is judged by thin layer chromatography (TLC).
  • the reaction temperature should be controlled between 15 ° C and 120 ° C. Since the pyrrole molecule is liable to self-polymerize at a high temperature, a viscous brown substance is formed, thereby affecting the main reaction and greatly reducing the yield. When the reaction temperature is too low, it takes a long time to make the reaction sufficient. Therefore, the reaction temperature is preferably from 30 ° C to 100 ° C, further preferably from 30 ° C to 80 ° C, and most preferably from 40 ° C to 60 ° C.
  • the intermediate III obtained in the step 1 is reacted with a boron trifluoride compound, an organic base is added, and hydrogen fluoride is removed to form a product of the formula I.
  • the boron trifluoride compound to be added may be a gaseous boron trifluoride, a boron trifluoride diethyl ether complex or any other compound capable of releasing boron trifluoride at a normal temperature solution.
  • the above reaction temperature is -10 ° C - 100 ° C, preferably 0 ° C - 10 ° C; the addition of boron trifluoride compounds (such as gaseous boron trifluoride or boron trifluoride complex) requires the addition of an organic base to promote the reaction
  • the organic base is preferably triethylamine.
  • the method for purifying the boron fluoride complexed dipyrromethene fluorescent dye of the present invention is carried out by a conventional method, and is not particularly limited. Usually, after the reaction is completed, the solvent is distilled off. Preferably, the product is purified by column chromatography using petroleum ether/ethyl acetate as an eluent.
  • the resulting fluorescent dye can be recovered by separation and purification techniques well known in the art to achieve the desired purity.
  • aromatic substitution reaction examples include introduction of a nitro group with concentrated nitric acid, introduction of an acyl group under Friedel Crafts conditions using, for example, an acid halide and a Lewis acid (such as aluminum trichloride), and an alkyl halide and a Lewis acid (such as aluminum trichloride).
  • the alkyl group is introduced under the conditions of Friedel Crafts, and a halogen group is introduced.
  • modification examples include reduction of a nitro group to an amino group by, for example, catalytic hydrogenation with a nickel catalyst or heat treatment with iron in the presence of hydrochloric acid; oxidation of an alkylthio group to an alkylsulfinyl group or an alkylsulfonyl group.
  • alkyl as used herein includes both straight chain alkyl and branched alkyl groups.
  • a single alkyl group such as "propyl”
  • a single branched-chain alkyl group such as "isopropyl”
  • C 1-4 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl and the like. Similar rules apply to the other groups used in this specification.
  • the use of the boron fluoride complexed dipyrromethene fluorescent probe of the invention for detecting hypochlorite is particularly suitable for detecting hypochlorite in living cells.
  • 2,4-Dimethylpyrrole (2.8 g, 30 mmol) was added to a 500 mL single-necked flask containing 250 mL of dichloromethane, and then (0.9 g, 10 mmol) of acryloyl chloride was added, protected from light and nitrogen, and stirred at 50 ° C overnight. Under ice-cooling conditions, 10 mL of triethylamine and 10 mL of boron trifluoride diethyl ether were added dropwise, and stirring was continued for 1 hour. The solvent was evaporated under reduced pressure and the crystals were separated to afford white crystals (EtOAc).
  • the fluorescent probe compound BClO has high selectivity to sodium hypochlorite, and the addition of 5 ⁇ M sodium hypochlorite makes the fluorescence intensity of BClO significantly enhanced (100 times), while the addition of other active oxygen has no significant change.
  • the response to the nanomolar concentration of hypochlorite was evaluated using the compound BClO synthesized above.
  • Figure 5 is a time function of the detection of hypochlorite by the probe compound BClO.
  • concentration of the probe compound BClO was 1 ⁇ M
  • the excitation wavelength was 480 nm
  • the fluorescence intensity of BClO at 505 nm with time was recorded. It can be seen from Figure 5 that BClO responds very rapidly to hypochlorite and reaches equilibrium within one second.
  • the abscissa is time (seconds) and the ordinate is fluorescence intensity.
  • the response to pH was evaluated using the compound BClO synthesized above.
  • the pH was adjusted to about 4.0 in the PBS buffer (containing 10% by volume of ethanol as a dye cosolvent), and the excitation wavelength was 480 nm.
  • the alkali solution was added to slowly increase the pH to about 9.0, and the corresponding change in fluorescence intensity was recorded.
  • the probe compound BClO can be used for the detection of hypochlorite in this pH range.
  • the abscissa is pH and the ordinate is fluorescence intensity.
  • the pH was adjusted with NaOH (1 M) and HCl (1 M) in the experiment.
  • MCF-7 cells were cultured in DEME (invitrogen) with 10% FCS (invitrogen).
  • DEME invitrogen
  • FCS invitrogen
  • the cells were seeded in dedicated cell confocal culture dishes.
  • 1 ⁇ M of the probe compound BClO was added thereto, kept at 37 ° C and 5% CO 2 for 20 minutes, and then washed 3 times with a phosphate buffer solution for confocal imaging. Under the same conditions, 3 ⁇ M and 5 ⁇ M sodium hypochlorite were added separately, and confocal imaging was performed immediately.
  • the culture density of the cells was 2 ⁇ 10 5 cells/mL.
  • the instrument used for imaging was an Olympus FV1000-IX81 inverted microscope with a 100x oil mirror. The excitation light was excited at 488 nm and the 490-550 nm band was collected.
  • Figures 7a-c are photographs of live cell staining before and after the addition of BClO to sodium hypochlorite.
  • the probe compound BClO emits weak green fluorescence in the cells, which is due to the fact that MCF-7 cells contain a certain amount of reactive oxygen species, and the fluorescence intensity is fast after the addition of sodium hypochlorite.
  • the probe compound BClO is suitable for the detection of hypochlorous acid in living cells.
  • Raw264.7 cells were cultured in DEME (invitrogen) with 10% FCS (invitrogen). One day before the confocal fluorescence imaging experiment, the cells were seeded in dedicated cell confocal culture dishes. On the next day, 1 ⁇ M of the probe compound BClO was added thereto, kept at 37 ° C and 5% CO 2 for 20 minutes, and then washed 3 times with a phosphate buffer solution for confocal imaging. Under the same conditions, Raw264.7 cells were incubated with 1 ⁇ g/mL of LPS (lipopolysaccharide) for 12 hours, and then incubated with 1 ⁇ g/mL of PMA (phorbol myristate) for 1 hour, and then 1 ⁇ M was added thereto. The probe compound BClO was incubated at 37 ° C and 5% CO 2 for 20 minutes, and then washed 3 times with a phosphate buffer solution for confocal imaging.
  • LPS lipopolysaccharide
  • PMA phor
  • the culture density of the cells was 2 ⁇ 10 5 cells/mL.
  • the instrument used for imaging was an Olympus FV1000-IX81 inverted microscope with a 100x oil mirror. The excitation light was excited at 488 nm and the 490-550 nm band was collected.
  • Figures 8a-b are photographs of BClO staining of Raw264.7 cells before and after stimulation with LPS and PMA, respectively.
  • the probe compound BClO emits very weak green fluorescence in macrophages due to the low content of reactive oxygen species in Raw264.7 cells, and the fluorescence intensity after stimulation with LPS and PMA.
  • the probe compound BClO is suitable for the detection of endogenous hypochlorous acid in living cells.

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Abstract

一类具有如下通式I结构的氟化硼络合二吡咯甲川荧光探针，其制备方法及应用。所述的氟化硼络合二吡咯甲川荧光探针背景荧光低，加入次氯酸后，荧光增强快速明显，可达100倍；其荧光强度与次氯酸浓度在纳摩尔范围内呈现良好的线性关系，检测限为0.56纳摩尔；选择性好，对其他活性氧物质如H2O2，O2 -，TBHP，HO·，TBO·，1O2，NO·几乎无响应且在较宽范围内不受pH的干扰；可用于活细胞内次氯酸的检测。

Description

一类氟化硼络合二吡咯甲川荧光探针，其制备方法及应用 技术领域
本发明涉及精细化工领域一类荧光探针、其制备方法和用途，尤其涉及一类氟化硼络合二吡咯甲川荧光探针，制备方法及其在次氯酸根检测方面的应用。
背景技术
荧光探针作为功能性染料在科学技术的各个领域取得了广泛应用，尤其在生命科学、临床医疗诊断、免疫分析检测等方面的研究成为热点。在众多荧光染料中，氟化硼络合二吡咯甲川(BDP)荧光染料具有摩尔消光系数较高、荧光量子产率高、光谱性质稳定、光热高、化学稳定性好、分子量小和细胞毒性较低等优点，作为生物分子荧光探针和成像荧光试剂等已被广泛应用。
作为生物体内一种重要的活性氧，次氯酸(HClO)在生理上主要是由氯离子和过氧化氢在髓过氧化酶催化作用下反应生成。在细胞环境内，由于次氯酸/次氯酸根的强氧化性，其可杀灭外来细菌，阻止入侵，调控细胞生命周期。随着年龄的增长，人体内次氯酸的浓度会逐渐的增加。但是过量的次氯酸/次氯酸根却会引发一系列疾病如骨关节炎，心血管疾病等，因此发展一种有效监测次氯酸的方法越来越受到研究者的关注。
近年来，相关研究人员开发出一系列可用于检测水溶液或生物体内的次氯酸/次氯酸根的荧光探针。这些探针主要基于次氯酸的强氧化性，如：联苯甲酰肼氧化机理；羟肟酸氧化；脱肟反应机理；对甲氧基酚氧化机理；硫原子氧化机理等。但是，这些探针普遍存在响应时间长，灵敏度不高，选择性差，易受pH干扰等缺点。吡咯是合成BDP的一种重要原料，同时我们发现它可以选择性地被次氯酸氧化而对其他活性氧(如H2O2，·OH等)不敏感。而且，由于吡咯具有芳香性且氮上的未共用电子对参与构成环状大π键，削弱了它与质子结合的能力，因此可以很好地避免pH的干扰。除此之外，吡咯的富电性使得其可以有效地对一些荧光团产生光诱导电子转移(PET)效应，从而降低背景荧光，提高探针灵敏度。
发明内容
为解决以上现有技术中次氯酸根荧光探针响应时间长、灵敏度不高、选择性差和易受pH干扰等缺点，本发明提供一类氟化硼络合二吡咯甲川荧光探针，以2,4-二甲基吡咯为识别基团，以氟化硼络合二吡咯甲川染料为荧光团，提高探针选择性(不受其他活性氧和pH干扰)和灵敏度，加快响应时间，并且具有良好的生物应用。
本发明的技术目的通过以下技术方案实现：
一类氟化硼络合二吡咯甲川荧光探针，具有如下结构通式I：
Figure PCTCN2014092299-appb-000001
通式I中：
R1，R2和R3各自独立地选自H、C1-8的烷基、取代或未取代苯基；
所述取代苯基为由以下一种或多种基团任意位置取代的苯基：CN、COOH、NH2、NO2、OH、SH、C1-6烷氧基、C1-6烷基氨基、C1-6酰胺基、卤素或C1-6卤代烷基。
本发明所述氟化硼络合二吡咯甲川荧光探针的制备方法，包括以下步骤：以具有结构通式II的化合物与丙烯酰氯按照摩尔比为1～5：1反应，然后在碱存在条件下与三氟化硼络合得到所述氟化硼络合二吡咯甲川荧光探针；
Figure PCTCN2014092299-appb-000002
本发明所述氟化硼络合二吡咯甲川荧光探针在检测次氯酸根中的应用。
本发明所述氟化硼络合二吡咯甲川荧光探针具有以下显著的特征：
①在水溶液中几乎无荧光，降低了检测过程中的荧光背景干扰；
②灵敏度高，在纳摩尔浓度次氯酸/次氯酸根存在下荧光有明显增强，并且荧光强度增加与次氯酸浓度呈现良好的线性关系；
③选择性好，对其他活性氧如H2O2，·OH等几乎无响应；
④响应迅速，在加入次氯酸后，几秒内其荧光强度即可达到平衡；
⑤在pH为4.0-9.0的范围内，其探针本身和对次氯酸的检测结果准确，不受pH的干扰；
⑥合成简便，产品易得。
附图说明
本发明附图7幅，
图1是BClO性能测定实验1中荧光探针化合物BClO对不同浓度的次氯酸根的荧光强 度响应图。探针化合物BClO的浓度为1μM，次氯酸钠浓度分别为0、1、2、3、4、5、6和7μM，测试体系为PBS缓冲溶液(含10％体积比的乙醇作为染料助溶剂，pH＝7.4)。图1a是BClO对次氯酸钠的荧光滴定光谱，激发波长为480nm；图1b是BClO对次氯酸钠的滴定曲线，激发波长为480nm。
图2是BClO性能测定实验2中荧光探针化合物BClO对不同浓度的次氯酸根吸收滴定测试。荧光探针化合物BClO的浓度为1μM，次氯酸钠浓度分别为0、1、2、3、4、5、6和7μM。
图3是BClO性能测定实验3中荧光探针化合物BClO对不同活性氧的荧光选择性柱状图。荧光探针化合物BClO的浓度是1μM，次氯酸钠的浓度为5μM，其他活性氧的浓度为10μM。
图4是BClO性能测定实验4中荧光探针化合物BClO对低浓度次氯酸根的荧光强度响应图。荧光探针化合物BClO的浓度为1μM，次氯酸钠浓度分别为0、1、2、3、4、5、6、7、8、9和10nM。
图5是BClO性能测定实验5中荧光探针化合物BClO对次氯酸钠检测的响应时间图。荧光探针化合物BClO的浓度为1μM，加入次氯酸钠的浓度为5μM。横坐标为时间(s)，纵坐标为荧光强度。
图6是BClO性能测定实验6中探针化合物BClO在不同pH值条件下对次氯酸根的荧光强度响应图。横坐标为pH，纵坐标为荧光强度。荧光探针化合物BClO的浓度为1μM，加入次氯酸钠的浓度为5μM。用NaOH(1M)和HCl(1M)调节pH。
图7是BClO性能测定实验7中探针化合物BClO在人乳腺癌(MCF-7)细胞中的荧光成像。荧光探针化合物BClO的浓度是1μM。a，b，c分别表示在被BClO染色的细胞中加入0、3和5μM次氯酸钠后采集490nm-550nm波段内的荧光照片。激发波长为488nm。通过双样本检验进行统计分析，以次氯酸钠为0μM时的荧光强度为标准。***P<0.001，误差条是指标准平均误差(n＝10)。
图8是BClO性能测定实验8中探针化合物BClO在小鼠巨噬(Raw 264.7)细胞中的荧光成像。荧光探针化合物BClO的浓度是1μM。a表示加入BClO后的细胞染色荧光照片；b表示将Raw 264.7细胞与1μg/mL的LPS(脂多糖)培养12小时，再与1μg/mL的PMA(十四酸佛波酯乙酸盐)孵育1小时后再加入BClO的细胞染色荧光照片。采集490nm-550nm波段内的荧光，激发波长为488nm。通过双样本检验进行统计分析，以只加入BClO时的荧光强度为标准。***P<0.001，误差条是指标准平均误差(n＝4)。
具体实施方式
本发明提供一类具有结构通式I的氟化硼络合二吡咯甲川荧光探针。
Figure PCTCN2014092299-appb-000003
通式I中：
R1，R2和R3各自独立地选自H、C1-8的烷基、取代或未取代苯基；
所述取代苯基为由以下一种或多种基团任意位置取代的苯基：CN、COOH、NH2、NO2、OH、SH、C1-6烷氧基、C1-6烷基氨基、C1-6酰胺基、卤素或C1-6卤代烷基。
作为优选的实施方式，本发明所述氟化硼络合二吡咯甲川荧光探针的通式I中：R1和R3各自独立地选自H或甲基，更优选为甲基；
R2优选自氢或乙基，更优选为氢。
作为最优选的技术方案，本发明所述氟化硼络合二吡咯甲川荧光探针为BClO，结构式如下：
Figure PCTCN2014092299-appb-000004
本发明进一步提供所述氟化硼络合二吡咯甲川荧光探针的制备方法，包括以下步骤：以具有结构通式II的化合物与丙烯酰氯按照摩尔比为1～5：1反应，然后在碱存在条件下与三氟化硼络合得到所述氟化硼络合二吡咯甲川荧光探针；
Figure PCTCN2014092299-appb-000005
本发明所述的制备方法，更具体的实施方式如下：
Figure PCTCN2014092299-appb-000006
①具有结构通式II的化合物与丙烯酰氯制备中间体化合物III：中间体化合物II与丙烯酰氯的投料摩尔比可为0.1-1000：1，优选为0.5-100：1，更优选0.5-10：1，再优选1-5：1，最佳为3：1，二者发生缩合反应，生成结构通式为III的化合物。
以上反应可在含水的有机溶剂或无水的有机溶剂中进行。优选为无水的有机溶剂。吡咯原料在溶剂中的摩尔浓度为0.012-0.4mol/L。有机溶剂包括但不仅限于二氯甲烷、四氢呋喃或乙腈等。在反应过程中，吡咯原料与丙烯酰氯经两次缩合生成活性中间体III，由于产物不稳定，可不经分离直接进行下一步反应，反应过程通过薄层色谱(TLC)判断反应的终点。
反应温度应控制在15℃-120℃。由于吡咯分子在高温时很容易发生自聚合，生成粘稠状褐色物质，从而影响主反应，大大降低收率。而当反应温度太低时，则需要很长的时间来使反应充分。因此反应温度优选为30℃-100℃，进一步优选30℃-80℃，最优选为40℃-60℃。
②所述氟化硼络合二吡咯甲川荧光探针的合成：将步骤①中得到的中间体III与三氟化硼化合物反应，加入有机碱，脱掉氟化氢后形成产物式I化合物。加入的三氟化硼化合物可为气态三氟化硼、三氟化硼的乙醚络合物或其他任何能够在常温的溶液下释放出三氟化硼的化合物。
上述反应温度为-10℃-100℃，优选0℃-10℃；加入三氟化硼化合物(例如气态三氟化硼或三氟化硼的络合物)时需要加入有机碱来促进反应的进行，所述有机碱优选三乙胺。
本发明的氟化硼络合二吡咯甲川荧光染料的提纯方法采用常规方法，没有特别限制。通常，反应结束后，蒸去溶剂。优选用石油醚/乙酸乙酯作为洗脱液进行色谱柱分离提纯产物。
所得荧光染料可通过本领域公知的分离和纯化技术回收，以达到需要的纯度。
本发明中使用的各种原料均可市售获得，或者可通过本领域技术人员公知的方法或现有技术中公开的方法由本领域公知的原料简单地制备得到。
应认识到，本发明化合物中的各种环取代基有一些可在上述步骤进行之前或刚完成后，通过标准的芳族取代反应来引入或通过常规的官能团修饰来产生，这包括在本发明的方法步 骤方面。这种反应和修饰包括例如取代基通过芳族取代反应的引入、取代基的还原、取代基的烷基化和取代基的氧化。用于这些过程的试剂和反应条件是化学领域公知的。芳族取代反应的具体实例包括用浓硝酸引入硝基，用例如酰卤和路易斯酸(如三氯化铝)在Friedel Crafts条件下引入酰基，用烷基卤和路易斯酸(如三氯化铝)在Friedel Crafts条件下引入烷基，和引入卤素基团。修饰的具体实例包括通过例如用镍催化剂进行催化氢化或者用铁在盐酸存在下进行加热处理，将硝基还原成氨基；将烷硫基氧化成烷基亚磺酰基或烷基磺酰基。
除另有说明外，本文中使用的术语具有以下含义。
本文中使用的术语“烷基”包括直链烷基和支链烷基。如提及单个烷基如“丙基”，则只特指直链烷基，如提及单个支链烷基如“异丙基”，则只特指支链烷基。例如，“C1-4烷基”包括甲基、乙基、正丙基、异丙基、正丁基和叔丁基等。类似的规则也适用于本说明书中使用的其它基团。
本发明所述的氟化硼络合二吡咯甲川荧光探针在检测次氯酸根中的应用，尤其适用于活细胞中次氯酸根的检测。
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1
荧光探针化合物BClO的合成：
Figure PCTCN2014092299-appb-000007
将2,4-二甲基吡咯(2.8g，30mmol)加入装有250mL二氯甲烷的500mL单口烧瓶中，再加入(0.9g，10mmol)丙烯酰氯，避光，氮气保护，50℃搅拌过夜。冰浴条件下，滴加10mL三乙胺和10mL三氟化硼乙醚，继续搅拌1小时。减压蒸除溶剂，色谱柱分离得到橙色固体BClO(9.6％)。1H NMR(400MHz,CDCl3),δ:1.96(s,3H),2.17(s,3H),2.35(s,6H),2.53(s,6H),2.84(t,J＝8Hz,2H),3.17(t,J＝8Hz,2H),5.62(s,1H),6.04(s,2H),7.43(s,1H)；13C NMR(100MHz,CDCl3),δ:11.1,13.0,14.6,16.2,27.4,29.9,107.8,114.5,121.9,124.0,126.3,131.5,141.2,145.5,154.1ppm；TOF MS:m/z calcd for C21H27BF2N3 +[M+H]+:370.2216,found:370.2255.
BClO性能测定实验1
荧光探针化合物BClO对次氯酸根荧光滴定实验
将1μM的探针化合物BClO加入到PBS缓冲液(含10％体积比的乙醇作为染料助溶剂，pH＝7.4)中，逐渐加入次氯酸钠溶液，使次氯酸根的浓度分别为0、1、2、3、4、5、6和7μM，记录响应的荧光强度，激发波长为480nm，测试结果显示于图1a和图1b中。从图中可以看出，随着次氯酸钠浓度的增加，探针化合物最大发射峰505nm处的荧光强度逐渐增强，次氯酸钠为5μM时达到饱和。
BClO性能测定实验2
荧光探针化合物BClO对次氯酸根吸收滴定实验
将1μM的探针化合物BClO加入到PBS缓冲液(含10％体积比的乙醇作为染料助溶剂，pH＝7.4)中，逐渐加入次氯酸钠溶液，使次氯酸根的浓度分别为0、1、2、3、4、5、6和7μM，记录吸收强度，测试结果显示于图2中。从图中可以看出，随着次氯酸钠浓度的增加，探针化合物的吸收光谱基本保持不变。
BClO性能测定实验3
荧光探针化合物BClO检测次氯酸根的选择性实验
使用上述合成的化合物BClO评价对次氯酸根和活性氧的选择性：将1μM的化合物BClO加到含5μM的次氯酸根或10μM的其他活性氧(H2O2，O2 -，TBHP，HO·，TBO·，1O2，NO·)的PBS缓冲液(含10％体积比的乙醇作为染料助溶剂，pH＝7.4)中，探针激发波长为480nm，探针发射波长505nm，记录相应的荧光强度，测试结果显示于图3中。从图中可以看到，荧光探针化合物BClO对次氯酸钠具有很高的选择性，5μM次氯酸钠的加入使得BClO的荧光强度明显增强(100倍)，而其他活性氧的加入则无明显变化。
BClO性能测定实验4
荧光探针化合物BClO检测次氯酸根的灵敏度
使用上述合成的化合物BClO评价对纳摩尔级浓度的次氯酸根的响应。将化合物BClO(1μM)分别加入到含有0、1、2、3、4、5、6、7、8、9和10nM浓度次氯酸钠的PBS缓冲液(含10％体积比的乙醇作为染料助溶剂，pH＝7.4)中，480nm激发，记录相应的荧光光谱和505nm处的强度，测试结果显示于图4中。从图中可以看出荧光探针化合物BClO在次氯酸根0-10nM的范围内荧光强度有明显增强，且荧光强度随次氯酸钠浓度变化呈现良好的线性关系(R2＝0.99724)。因此荧光探针化合物BClO可用于低浓度次氯酸钠的检测，且通过3σ/k计算得检测限为0.56nM。
BClO性能测定实验5
探针化合物BClO对次氯酸根检测的响应时间测试
图5是探针化合物BClO对次氯酸根检测的时间函数。探针化合物BClO的浓度为1μM，测试体系为PBS缓冲溶液(含10％体积比的乙醇作为染料助溶剂，pH＝7.4)，激发波长为480nm，记录BClO随时间在505nm处的荧光强度。从图5中可以看出BClO对次氯酸根的响应非常迅速，在1秒钟之内即可达到平衡。横坐标为时间(秒)，纵坐标为荧光强度。
BClO性能测定实验6
在探针化合物BClO对次氯酸根检测中pH的干扰研究
使用上述合成的化合物BClO评价对pH的响应，对于化合物BClO(1μM)在PBS缓冲液(含10％体积比的乙醇作为染料助溶剂)中调节pH值为4.0左右，激发波长为480nm，测定探针和加入5μM次氯酸钠后的荧光强度后，加入碱液，使pH值缓慢增大至9.0左右，记录相应的荧光强度变化，测试结果显示于图6中。从图中可以看出荧光探针化合物BClO在pH＝4.0-9.0的范围内，pH变化对探针本身和加入次氯酸钠后的荧光发射基本没有影响。因此探针化合物BClO可用于此pH范围内次氯酸根的检测。横坐标为pH，纵坐标为荧光强度。实验中用NaOH(1M)和HCl(1M)调节pH。
BClO性能测定实验7
探针化合物BClO在MCF-7细胞内检测不同浓度次氯酸根的研究
MCF-7细胞在DEME(invitrogen)中用10％的FCS(invitrogen)培养。共聚焦荧光成像实验前一天，细胞种在专用的细胞共聚焦培养皿中。第二天，向其中加入1μM的探针化合物BClO，保持在37℃及5％CO2条件下，孵育20分钟，然后用磷酸缓冲溶液冲洗3遍，进行共聚焦成像。同样条件下，再分别加入3μM和5μM的次氯酸钠，立即进行共聚焦成像。
细胞的培养密度为2×105cells/mL。成像所用仪器为Olympus FV1000-IX81倒置显微镜，100倍油镜。激发光为488nm激发，收集490-550nm波段。
图7a-c分别是BClO加入次氯酸钠前后的活细胞染色照片。为了能够量化BClO在细胞内的荧光强度变化，我们从每张图中选取10个区域计算其相对荧光强度平均值，如图7d所示。从图中可以看出，在加入次氯酸钠之前，探针化合物BClO在细胞内发出微弱的绿色荧光，这是由于MCF-7细胞内含有一定量的活性氧物种，而加入次氯酸钠之后，其荧光强度快速明显增强，表明探针化合物BClO适用于活细胞内次氯酸的检测。
BClO性能测定实验8
探针化合物BClO在Raw264.7细胞内检测内源性次氯酸的研究
Raw264.7细胞在DEME(invitrogen)中用10％的FCS(invitrogen)培养。共聚焦荧光成像实验前一天，细胞种在专用的细胞共聚焦培养皿中。第二天，向其中加入1μM的探针化合物BClO，保持在37℃及5％CO2条件下，孵育20分钟，然后用磷酸缓冲溶液冲洗3遍，进行共聚焦成像。同样条件下，将Raw264.7细胞与1μg/mL的LPS(脂多糖)培养12小时，再与1μg/mL的PMA(十四酸佛波酯乙酸盐)孵育1小时，然后向其中加入1μM的探针化合物BClO，保持在37℃及5％CO2条件下，孵育20分钟，然后用磷酸缓冲溶液冲洗3遍，进行共聚焦成像。
细胞的培养密度为2×105cells/mL。成像所用仪器为Olympus FV1000-IX81倒置显微镜，100倍油镜。激发光为488nm激发，收集490-550nm波段。
图8a-b分别是Raw264.7细胞经LPS和PMA刺激前后的BClO染色照片。为了能够量化BClO在细胞内的荧光强度变化，我们从每张图中选取4个区域计算其相对荧光强度平均值，如图7c所示。从图中可以看出，探针化合物BClO在巨噬细胞内发出非常微弱的绿色荧光，这是由于Raw264.7细胞内的活性氧物种含量较低，而经LPS和PMA刺激之后，其荧光强度明显增强，表明探针化合物BClO适用于活细胞内内源性次氯酸的检测。

Claims (9)

  1. 一类氟化硼络合二吡咯甲川荧光探针，具有如下结构通式I：
    Figure PCTCN2014092299-appb-100001
    通式I中：
    R1、R2和R3各自独立地选自H、C1-8的烷基、取代或未取代苯基；
    所述取代苯基为由以下一种或多种基团任意位置取代的苯基：CN、COOH、NH2、NO2、OH、SH、C1-6烷氧基、C1-6烷基氨基、C1-6酰胺基、卤素或C1-6卤代烷基。
  2. 根据权利要求1所述的氟化硼络合二吡咯甲川荧光探针，其特征在于所述R1和R3各自独立地选自H或甲基。
  3. 根据权利要求2所述的氟化硼络合二吡咯甲川荧光探针，其特征在于所述R1和R3均为甲基。
  4. 根据权利要求1～3中任一权利要求所述的氟化硼络合二吡咯甲川荧光探针，其特征在于所述R2选自氢或乙基。
  5. 根据权利要求4所述的氟化硼络合二吡咯甲川荧光探针，其特征在于所述R2为氢。
  6. 权利要求1-5中任意一项权利要求所述的氟化硼络合二吡咯甲川荧光探针的制备方法，包括以下步骤：以具有结构通式II的化合物与丙烯酰氯按照摩尔比为1～5：1反应，然后在碱存在条件下与三氟化硼络合得到所述氟化硼络合二吡咯甲川荧光探针；
    Figure PCTCN2014092299-appb-100002
  7. 根据权利要求6所述的制备方法，其特征在于具有结构通式II的化合物与丙烯酰氯的反应摩尔比为3：1。
  8. 权利要求1-5中任意一项权利要求所述的氟化硼络合二吡咯甲川荧光探针在检测次氯酸根中的应用。
  9. 根据权利要求8所述的氟化硼络合二吡咯甲川荧光探针在检测次氯酸根中的应用，其特征在于所述氟化硼络合二吡咯甲川荧光探针用于活细胞中次氯酸根的检测。
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