成像细胞内巯基分子的新型荧光探针的研制
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摘要
动物体内的游离小分子物质诸如氨基酸、小肽链、单糖、激素、胆固醇及核苷酸等起着不亚于细胞器的重要作用。氨基酸是含有一个碱性氨基和一个酸性羧基的有机化合物,也是组成蛋白质的基本单元；单糖就是不能再水解的糖类,是构成各种二糖和多糖的分子的基本单位,为整个生命系统提供了能量；胆固醇的体内含量成为了现在评判健康状况的重要指标；激素含量的失调更会导致各种疾病；核苷酸是核糖核酸及脱氧核糖核酸的基本组成单位,是体内合成核酸的前身物。因此,探测细胞内各种游离小分子物质的含量及分布有着重要的生物学和药理学价值。然而,与各种细胞器不同,我们不能在光学显微镜甚至电子显微镜下直接观察到这些小分子。故人们发展了各种各样检测它们的方法,诸如高效液相色谱法、气相色谱-质谱分析方法、毛细管电泳分离分析方法、基于各种纳米粒子的等离子体谐振检测方法等。相比之下,基于荧光探针的光学探测方法具有可以原位观测、操作方便且对细胞损伤较小等优点,而得到了广泛的研究和发展。
分子功能通常与分子结构有着密不可分的联系,细胞内游离小分子的作用往往是由其化学结构决定的。硫醇类小分子含有亲核性很强的巯基,易与其它化合物进行亲核加成反应,与金属离子配位,且容易被氧化形成二硫键,也因此在细胞内起着其独特的作用。半胱氨酸(Cys)作为组成蛋白质的一种氨基酸,与蛋白质中的二硫键交联有着密不可分的联系,对形成蛋白的三维结构起着重要的作用。此外,半胱氨酸可以与细胞内微量的过渡金属离子有较强的配位作用,从而调节其浓度,避免了过多的重金属离子对细胞造成的损伤。研究表明,细胞内半胱氨酸浓度失调与许多疾病相关,例如风湿性关节炎、帕金森症、老年痴呆症、不良妊娠产出等。还原型谷胱甘肽(GSH)可以和其氧化态(GSSH)在细胞内保持着氧化还原平衡,还原型谷胱甘肽可以与细胞内的氧化性物质发生脱氢反应,自身转变为氧化态的同时还原了这些氧化性物质,因此维系了细胞内氧化还原稳态的平衡,防止了过多的氧化性物质在细胞内堆积。同型半胱氨酸在细胞内的浓度很低(只有5～15μM),但是它却是心血管疾病和老年痴呆症等许多疾病诊断的独立因子,特别是,近年来人们对同型半胱氨酸的重视程度大大增加,同型半胱氨酸的含量也被一些人认为是人体健康水平的重要指标之一。因此,检测细胞内各种巯基化合物的分布和浓度对疾病诊断、药物开发以及维持健康有着重要的意义。
如前文所提,巯基的强亲核性是其在生物体内产生特殊作用的基础,也同时为我们探测巯醇化合物指明了方法：将一个可以选择性的与巯基反应的基团与荧光团共轭相连就得到了一个巯醇的荧光探针分子,这样该探针分子进入细胞后可以选择性的与巯醇化合物反应,并给出荧光信号,从而实现原位的检测细胞中的巯醇化合物。如果该探测基团可以选择性的与特定巯醇反应,就可以成为一个专一识别特定巯醇的探针。到目前为止,研究者们已经合成并发展了各种各样的巯基探针,可以与谷胱甘肽,半胱氨酸和同型半胱氨酸等其中的一种、两种或所有巯醇反应,给出荧光信号变化。马来酰亚胺基团是最早用于识别细胞内巯醇总量的反应基团,基于这个识别基团,人们已经发展了具有各种光谱性质的巯基探针。Strongin课题组首先开创性的利用单醛来专一识别半胱氨酸和同型半胱氨酸,随后,以单醛为识别基团的半胱氨酸和同型半胱氨酸探针也得到了广泛的发展。由于半胱氨酸具有相对较强的反应活性,目前能专一识别半胱氨酸而同时不受同型半胱氨酸影响的荧光探针有了一定的发展,Jung课题组发展了具有查尔酮结构的化合物,它们通过与三种巯基化合物反应速率的不同专一性的识别半胱氨酸；我们课题组在之前的工作中也曾报道过两个可以专一识别半胱氨酸的探针AMl和CA1,这两个探针虽然以单醛为官能团,但是却不和同型半胱氨酸响应,这可以归结为半胱氨酸具有更高的反应活性。相反的,同型半胱氨酸的反应活性较弱,因此在同浓度下,专一识别同型半胱氨酸的探针尚不多见。Strongin课题组曾报道了在等浓度下可以筛选出同型半胱氨酸的探针。值得一提的是,细胞内各种巯醇的浓度差别很大,谷胱甘肽的细胞内浓度为1～10mM,半胱氨酸的细胞内浓度为30～200μM,同型半胱氨酸的细胞内浓度为5～15μM。因此若要专一性的识别半胱氨酸和同型半胱氨酸,还必须考虑较大浓度的谷胱甘肽的干扰,这为细胞内半胱氨酸或同型半胱氨酸的荧光探针设计提供了更大的难度。
在本文的工作中,我们设计和合成了9个小分子探针：以丙烯醛为识别基团的探针CB1和HOTA;以单醛为识别基团的探针]HMCA、BMCA和NMCA;以硝基丙烯或丙烯腈为识别基团的探针HMCN、HOVC、 HODVC和HOBVC。
探针CB1分子是以3-(4-吡啶乙烯基)-9-乙基咔唑为荧光母体,烯基醛为识别基团的荧光探针。值得一提的是,探针CBl分子可以先后和两分子的半胱氨酸或同型半胱氨酸发生两步反应,可以和一分子的谷胱甘肽发生加成反应。且CBl分子在乙醇和缓冲液的混合液中是弱荧光的,然而其和一分子各种巯基化合物的反应可以使其荧光强度大幅度提升,但在反应速率上有很大的差别：探针分子可以很快的完成和一分子半胱氨酸的反应(40min)；探针分子和同型半胱氨酸的反应速率要慢很多,需要24h左右才能够完成反应；探针分子和谷胱甘肽的反应速率极慢,只有在一周以后才会有明显的荧光增强。同时,探针分子还可以和第二分子的半胱氨酸或同型半胱氨酸发生反应,反应的结果是使探针分子的荧光恢复到弱荧光的状态：探针分子和半胱氨酸的第二分子反应在24h时几乎已经完全完成；探针分子和同型半胱氨酸的第二分子反应在一周时几乎完全完成。如此巧妙的两步反应速率使探针分子可以在三个时间窗口实现分别专一性检测半胱氨酸、同型半胱氨酸和谷胱甘肽的目的：40mmin时只有CB1-半胱氨酸体系具有强荧光,实现了半胱氨酸的检测；24h时,CB1-半胱氨酸体系的荧光变弱,只有CBl-同型半胱氨酸体系具有强荧光,实现了同型半胱氨酸的检测；一周后,CB1-半胱氨酸和CB1-同型半胱氨酸体系的荧光都变弱,这时CB1-GSH体系具有强荧光,实现了谷胱甘肽的专一性检测。考虑到CB1具有很好的选择性及靶向后的物质具有很好的双光子性能,我们利用该探针进行了宽场显微镜以及双光子显微镜的成像研究。
探针HOTA分子是以4-羟基苯乙烯基三苯胺为母体,具有和CB1相同的识别基团烯基醛,能够在加入半胱氨酸后短时间内高选择性的选择半胱氨酸,而同型半胱氨酸和谷胱甘肽几乎不发生干扰作用。特别是,细胞内谷胱甘肽的浓度几乎是半胱氨酸的四十多倍,在真实的细胞浓度下,谷胱甘肽依旧不会带来明显的干扰。因此我们利用探针HOTA进行了宽场显微镜下的成像研究,为了证实探针在细胞中的荧光信号来自半胱氨酸,我们利用谷胱甘肽合成酶的抑制剂BSO抑制了细胞内谷胱甘肽的合成,并进行了与正常细胞的对比成像实验,实验结果证明用BSO处理后的细胞与未处理的细胞荧光强度几乎相同,也证实了细胞中的荧光确实来自半胱氨酸。
探针分子HMCA、BMCA和NMCA都是以单醛为识别基团的荧光探针,由于醛基的存在,这三种分子在乙醇和缓冲液的混合体系中都是弱荧光的,三个分子都能和同型半胱氨酸和半胱氨酸反应,带来荧光强度的变化,与常报道的探针分子不同,它们对同型半胱氨酸具有更强的响应性,在同等浓度下是一个同型半胱氨酸的探针。HMCN分子是以硝基丙烯为识别基团的探针,探针分子本身是弱荧光的,它能够无差别的和各种巯醇小分子发生加成反应,反应后荧光强度得到了较大的提升。为了研究位阻效应对各种巯基化合物的响应性的影响,我们设计了探针分子HOVC、HODVC以及HOBVC,其中HOVC以丙烯腈为识别基团,HODVC是以不饱和丙二睛为识别基团,HOBVC是以不饱和苯乙腈为识别基团,它们的位阻效应依次增加,系统的研究将为以后的巯基探针分子的设计提供思路。
Low-weight bio-molecules, including amino acids, small peptides, monosaccharides, hormone and cholesterol, play roles as crucial as the organelles in cell. Amino acids are the basic units of various proteins. Monosaccharides are the main source of energy in cells. Level of cholesterol is now an important parameter for the physical condition. Therefore, the detection of the intracellular levels and distributions of various low-weight molecules are significant for biology and pharmacology. However, unlike the detection of the organelles, to visualize small intracellular molecules using optical microscopy or even the electron microscope is impossible. Consequently, many kinds of methods, such as high performance liquid chromatography, chromatography-mass spectrometry and capillary electrophoresis methods plasmon resonance based on nanoparticles, have been developed for the detection of these molecules. Among these methods, optical measurements based on fluorescent probes exhibit many advantages, including convinence performance, low damage to intact cells and permission for visualization in situ, and thus has been widely studied and developed.
Molecular functions are usually tightly related to molecular structures, and the roles of small intracellular molecules are thought to be determined by their structures. Molecules with thiol group often exhibits high nucleophilic reaction activities, high ability to bind the metal ions and are easily oxidated to disulfide bond, and thus have distinct intracellular roles. Cysteine (Cys) is a basic amido acid for the synthesis of proteins, and was proved to be relative to the formation of the three dimension structures of proteins. Moreover, Cys could strongly bind to metal ions and balance their intracellular concentration, which protects the cell from damages caused by high concentration of heavy ions. According to many studies, abnormal levels of intracellular Cys are closely related to Parkinson's disease, Alzheimer's disease, adverse pregnancy outcomes and many other diseases. Intracellular redox glutathione (GSH) have a balance with its oxidation state (GSSG). GSH could be oxidated to GSSH by cellular oxidizing subjects, which limits the concentration of cellular oxides and prevents the stacking of these compounds. Intracellular concentration of homocysteine (Hcy) is rather low, only5-15μM. However, it was an independent factor for cardiovascular disease, Alzheimer's disease and many other diseases. Particularly, Hey has attracted more and more attentions and the level of Hey was considered as one of important parameters for disease. Therefore, determination of cellular levels and distributions of various bio-thiols is important for diagnosis, medicine development and keeping health.
As stated above, the strong nucleophilic reaction activity of thiols is the base of their distinctive intracellular roles. Simultaneously, this reaction activity also offers the methods for their detection:linking a thiol reactive functional group to a fluorophore could generate a fluorescent probe, which could react with thiols, give fluorescence responses and enable determination of intracellular thiols in situ. When this functional group reacts with specific thiols, this probe could have more specific selectivity. Up to now, many kinds of probes have been developed for the determination of GSH, Cys, Hey or total thiols. Cys and Hey were first detected by an aldehyde group by Strongin and co-workers. After that, many probes with an aldehyde as the receptor group have been synthesized and reported. Cys exhibits relatively stronger reactivity than other thiols, and thus the probes selectively detecting Cys have been developed. For example, Jung and co-workers have employed chalcone as probes for successfully detecting Cys over Hey and GSH. Our group has also reported two fluorescent probes containing aldehyde groups, AM1and CA1, which enables selective detection of Cys. As Hey has a weaker reactivity than Cys, fluorescent probes that could discriminate Hey remain rare so far. Zhang et al. and Strongin et al. have reported fluorescent probes for specific detection of Hey under equimolar measurements. It was worthwhile to notice that the intracellular concentrations of various thiols differ a lot. Cellular GSH has a rather higher concentration of1～10mM, while intracellular concentrations of Cys and Hey were30-200μM and5-15μM, respectively. Accordingly, large interferences from high concentration of GSH is bound to be considered for the detection of cellular Cys and Hey, which raises new challenge for the design of probes.
In this work, we have designed and synthesized9probes:CBl and HOTA with olefin aldehyde group; HMCA, BMCA and NMCA with aldehyde group; HMCN with the nitroolefin group; HOVC, HODVC and HOBVC with the vinyl cyanide group.
CBl is a fluorescent probe which exhibits3-(4-pyridinvinyl)-9-ethylcarbazole as the fluorophore, and olefin aldehyde as the receptor group. Particularly, a probe CBl molecule could react with two Cys molecules in two steps while could only react with one GSH molecule. CBl itself was weakly fluorescent in the mixture of ethanol and buffer solution, and its fluorescence could be largely enhanced after reaction with one thiol molecule. However, the reaction rates of CB1with one molecule of various thiols differ a lot:the reaction of CB1with one molecule of Cys could be completed in about40min, while the reaction was completed within24h of Hey and7days of GSH. Meanwhile, the product of CBl-Cys and CBl-Hcy could react with one more molecule of Cys and Hey, which could quench the fluorescence of the system. Fortunately, the second step reaction of Cys could be completed in24h, and this reaction of Hey could be completed in7days, which enables CBl a fluorescent probe for discriminative detection of Cys, Hey and GSH at three time windows. Only the system of CBl-Cys exhibits strong fluorescence at40min, while only the system of CBl-Hcy was strongly fluorescent at24h. After7days, the fluorescence of CBl-Cys and CBl-Hcy was quenched, and the system of CB1-GSH was emissive. Considering high selectivity and good two-photon excited fluorescent properties of CB1, the cell imaging experiments with CBl under wide-field microscopy and two-photon microscopy were successfully performed.
HOTA is a fluorescent probe which exhibits4-phenolvinyltriphenylamine as the fluorophore, and olefin aldehyde as the receptor unit. Probe HOTA has the same receptor unit as CB1, which could selectively react with Cys in a short response time. The selectivity is super-high and Hcy and GSH could hardly bring interferences. In particular, the concentration of intracellular GSH (1～10mM) is much higher than Cys (30～200μM), and probe HOTA could discriminate Cys over such high concentration of GSH. Consequently, cell imaging experiments were successfully performed with this probe. To testify the source of cellular emission, the inhibitor of GSH synthetase, BSO, has been used to prevent cellular synthesis of GSH before capturing cell images, and then these images were compared with the cells directly stained with HOTA. The experiment results showed that these two kinds of cells possess similar fluorescent intensity, which proved that cellular fluorescence was from the reaction of Cys and the probe.
HMCA, BMCA and NMCA were probes with an aldehyde as the receptor group. Due to PET process caused by the aldehyde group, these three probes were weakly fluorescent. The experiment results demonstrated that these probes could react with Cys and Hcy, which bring obvious fluorescent enhancement. Unlike other reported probes with aldehyde, these three probes have stronger interaction with Hcy. With equimolar measurements, HMCA, BMCA and NMCA were Hcy probes. According to the molecular structure of three kinds of thiols, the steric effect should be a key parameter for the selectivity. Therefore, we have designed three molecules HOVC, HODVC and HOBVC, to study the steric effects. HOVC has vinyl cyanide as a receptor unit, HODVC has an unsaturated malononitrile as a receptor unit and HOBVC bearing an unsaturated benzyl cyanide as a receptor unit. The three probes have rather different steric effects, and this study would provide inspiration for molecule design of probes for thiol detection.
分子功能通常与分子结构有着密不可分的联系,细胞内游离小分子的作用往往是由其化学结构决定的。硫醇类小分子含有亲核性很强的巯基,易与其它化合物进行亲核加成反应,与金属离子配位,且容易被氧化形成二硫键,也因此在细胞内起着其独特的作用。半胱氨酸(Cys)作为组成蛋白质的一种氨基酸,与蛋白质中的二硫键交联有着密不可分的联系,对形成蛋白的三维结构起着重要的作用。此外,半胱氨酸可以与细胞内微量的过渡金属离子有较强的配位作用,从而调节其浓度,避免了过多的重金属离子对细胞造成的损伤。研究表明,细胞内半胱氨酸浓度失调与许多疾病相关,例如风湿性关节炎、帕金森症、老年痴呆症、不良妊娠产出等。还原型谷胱甘肽(GSH)可以和其氧化态(GSSH)在细胞内保持着氧化还原平衡,还原型谷胱甘肽可以与细胞内的氧化性物质发生脱氢反应,自身转变为氧化态的同时还原了这些氧化性物质,因此维系了细胞内氧化还原稳态的平衡,防止了过多的氧化性物质在细胞内堆积。同型半胱氨酸在细胞内的浓度很低(只有5～15μM),但是它却是心血管疾病和老年痴呆症等许多疾病诊断的独立因子,特别是,近年来人们对同型半胱氨酸的重视程度大大增加,同型半胱氨酸的含量也被一些人认为是人体健康水平的重要指标之一。因此,检测细胞内各种巯基化合物的分布和浓度对疾病诊断、药物开发以及维持健康有着重要的意义。
如前文所提,巯基的强亲核性是其在生物体内产生特殊作用的基础,也同时为我们探测巯醇化合物指明了方法：将一个可以选择性的与巯基反应的基团与荧光团共轭相连就得到了一个巯醇的荧光探针分子,这样该探针分子进入细胞后可以选择性的与巯醇化合物反应,并给出荧光信号,从而实现原位的检测细胞中的巯醇化合物。如果该探测基团可以选择性的与特定巯醇反应,就可以成为一个专一识别特定巯醇的探针。到目前为止,研究者们已经合成并发展了各种各样的巯基探针,可以与谷胱甘肽,半胱氨酸和同型半胱氨酸等其中的一种、两种或所有巯醇反应,给出荧光信号变化。马来酰亚胺基团是最早用于识别细胞内巯醇总量的反应基团,基于这个识别基团,人们已经发展了具有各种光谱性质的巯基探针。Strongin课题组首先开创性的利用单醛来专一识别半胱氨酸和同型半胱氨酸,随后,以单醛为识别基团的半胱氨酸和同型半胱氨酸探针也得到了广泛的发展。由于半胱氨酸具有相对较强的反应活性,目前能专一识别半胱氨酸而同时不受同型半胱氨酸影响的荧光探针有了一定的发展,Jung课题组发展了具有查尔酮结构的化合物,它们通过与三种巯基化合物反应速率的不同专一性的识别半胱氨酸；我们课题组在之前的工作中也曾报道过两个可以专一识别半胱氨酸的探针AMl和CA1,这两个探针虽然以单醛为官能团,但是却不和同型半胱氨酸响应,这可以归结为半胱氨酸具有更高的反应活性。相反的,同型半胱氨酸的反应活性较弱,因此在同浓度下,专一识别同型半胱氨酸的探针尚不多见。Strongin课题组曾报道了在等浓度下可以筛选出同型半胱氨酸的探针。值得一提的是,细胞内各种巯醇的浓度差别很大,谷胱甘肽的细胞内浓度为1～10mM,半胱氨酸的细胞内浓度为30～200μM,同型半胱氨酸的细胞内浓度为5～15μM。因此若要专一性的识别半胱氨酸和同型半胱氨酸,还必须考虑较大浓度的谷胱甘肽的干扰,这为细胞内半胱氨酸或同型半胱氨酸的荧光探针设计提供了更大的难度。
在本文的工作中,我们设计和合成了9个小分子探针：以丙烯醛为识别基团的探针CB1和HOTA;以单醛为识别基团的探针]HMCA、BMCA和NMCA;以硝基丙烯或丙烯腈为识别基团的探针HMCN、HOVC、 HODVC和HOBVC。
探针CB1分子是以3-(4-吡啶乙烯基)-9-乙基咔唑为荧光母体,烯基醛为识别基团的荧光探针。值得一提的是,探针CBl分子可以先后和两分子的半胱氨酸或同型半胱氨酸发生两步反应,可以和一分子的谷胱甘肽发生加成反应。且CBl分子在乙醇和缓冲液的混合液中是弱荧光的,然而其和一分子各种巯基化合物的反应可以使其荧光强度大幅度提升,但在反应速率上有很大的差别：探针分子可以很快的完成和一分子半胱氨酸的反应(40min)；探针分子和同型半胱氨酸的反应速率要慢很多,需要24h左右才能够完成反应；探针分子和谷胱甘肽的反应速率极慢,只有在一周以后才会有明显的荧光增强。同时,探针分子还可以和第二分子的半胱氨酸或同型半胱氨酸发生反应,反应的结果是使探针分子的荧光恢复到弱荧光的状态：探针分子和半胱氨酸的第二分子反应在24h时几乎已经完全完成；探针分子和同型半胱氨酸的第二分子反应在一周时几乎完全完成。如此巧妙的两步反应速率使探针分子可以在三个时间窗口实现分别专一性检测半胱氨酸、同型半胱氨酸和谷胱甘肽的目的：40mmin时只有CB1-半胱氨酸体系具有强荧光,实现了半胱氨酸的检测；24h时,CB1-半胱氨酸体系的荧光变弱,只有CBl-同型半胱氨酸体系具有强荧光,实现了同型半胱氨酸的检测；一周后,CB1-半胱氨酸和CB1-同型半胱氨酸体系的荧光都变弱,这时CB1-GSH体系具有强荧光,实现了谷胱甘肽的专一性检测。考虑到CB1具有很好的选择性及靶向后的物质具有很好的双光子性能,我们利用该探针进行了宽场显微镜以及双光子显微镜的成像研究。
探针HOTA分子是以4-羟基苯乙烯基三苯胺为母体,具有和CB1相同的识别基团烯基醛,能够在加入半胱氨酸后短时间内高选择性的选择半胱氨酸,而同型半胱氨酸和谷胱甘肽几乎不发生干扰作用。特别是,细胞内谷胱甘肽的浓度几乎是半胱氨酸的四十多倍,在真实的细胞浓度下,谷胱甘肽依旧不会带来明显的干扰。因此我们利用探针HOTA进行了宽场显微镜下的成像研究,为了证实探针在细胞中的荧光信号来自半胱氨酸,我们利用谷胱甘肽合成酶的抑制剂BSO抑制了细胞内谷胱甘肽的合成,并进行了与正常细胞的对比成像实验,实验结果证明用BSO处理后的细胞与未处理的细胞荧光强度几乎相同,也证实了细胞中的荧光确实来自半胱氨酸。
探针分子HMCA、BMCA和NMCA都是以单醛为识别基团的荧光探针,由于醛基的存在,这三种分子在乙醇和缓冲液的混合体系中都是弱荧光的,三个分子都能和同型半胱氨酸和半胱氨酸反应,带来荧光强度的变化,与常报道的探针分子不同,它们对同型半胱氨酸具有更强的响应性,在同等浓度下是一个同型半胱氨酸的探针。HMCN分子是以硝基丙烯为识别基团的探针,探针分子本身是弱荧光的,它能够无差别的和各种巯醇小分子发生加成反应,反应后荧光强度得到了较大的提升。为了研究位阻效应对各种巯基化合物的响应性的影响,我们设计了探针分子HOVC、HODVC以及HOBVC,其中HOVC以丙烯腈为识别基团,HODVC是以不饱和丙二睛为识别基团,HOBVC是以不饱和苯乙腈为识别基团,它们的位阻效应依次增加,系统的研究将为以后的巯基探针分子的设计提供思路。
Low-weight bio-molecules, including amino acids, small peptides, monosaccharides, hormone and cholesterol, play roles as crucial as the organelles in cell. Amino acids are the basic units of various proteins. Monosaccharides are the main source of energy in cells. Level of cholesterol is now an important parameter for the physical condition. Therefore, the detection of the intracellular levels and distributions of various low-weight molecules are significant for biology and pharmacology. However, unlike the detection of the organelles, to visualize small intracellular molecules using optical microscopy or even the electron microscope is impossible. Consequently, many kinds of methods, such as high performance liquid chromatography, chromatography-mass spectrometry and capillary electrophoresis methods plasmon resonance based on nanoparticles, have been developed for the detection of these molecules. Among these methods, optical measurements based on fluorescent probes exhibit many advantages, including convinence performance, low damage to intact cells and permission for visualization in situ, and thus has been widely studied and developed.
Molecular functions are usually tightly related to molecular structures, and the roles of small intracellular molecules are thought to be determined by their structures. Molecules with thiol group often exhibits high nucleophilic reaction activities, high ability to bind the metal ions and are easily oxidated to disulfide bond, and thus have distinct intracellular roles. Cysteine (Cys) is a basic amido acid for the synthesis of proteins, and was proved to be relative to the formation of the three dimension structures of proteins. Moreover, Cys could strongly bind to metal ions and balance their intracellular concentration, which protects the cell from damages caused by high concentration of heavy ions. According to many studies, abnormal levels of intracellular Cys are closely related to Parkinson's disease, Alzheimer's disease, adverse pregnancy outcomes and many other diseases. Intracellular redox glutathione (GSH) have a balance with its oxidation state (GSSG). GSH could be oxidated to GSSH by cellular oxidizing subjects, which limits the concentration of cellular oxides and prevents the stacking of these compounds. Intracellular concentration of homocysteine (Hcy) is rather low, only5-15μM. However, it was an independent factor for cardiovascular disease, Alzheimer's disease and many other diseases. Particularly, Hey has attracted more and more attentions and the level of Hey was considered as one of important parameters for disease. Therefore, determination of cellular levels and distributions of various bio-thiols is important for diagnosis, medicine development and keeping health.
As stated above, the strong nucleophilic reaction activity of thiols is the base of their distinctive intracellular roles. Simultaneously, this reaction activity also offers the methods for their detection:linking a thiol reactive functional group to a fluorophore could generate a fluorescent probe, which could react with thiols, give fluorescence responses and enable determination of intracellular thiols in situ. When this functional group reacts with specific thiols, this probe could have more specific selectivity. Up to now, many kinds of probes have been developed for the determination of GSH, Cys, Hey or total thiols. Cys and Hey were first detected by an aldehyde group by Strongin and co-workers. After that, many probes with an aldehyde as the receptor group have been synthesized and reported. Cys exhibits relatively stronger reactivity than other thiols, and thus the probes selectively detecting Cys have been developed. For example, Jung and co-workers have employed chalcone as probes for successfully detecting Cys over Hey and GSH. Our group has also reported two fluorescent probes containing aldehyde groups, AM1and CA1, which enables selective detection of Cys. As Hey has a weaker reactivity than Cys, fluorescent probes that could discriminate Hey remain rare so far. Zhang et al. and Strongin et al. have reported fluorescent probes for specific detection of Hey under equimolar measurements. It was worthwhile to notice that the intracellular concentrations of various thiols differ a lot. Cellular GSH has a rather higher concentration of1～10mM, while intracellular concentrations of Cys and Hey were30-200μM and5-15μM, respectively. Accordingly, large interferences from high concentration of GSH is bound to be considered for the detection of cellular Cys and Hey, which raises new challenge for the design of probes.
In this work, we have designed and synthesized9probes:CBl and HOTA with olefin aldehyde group; HMCA, BMCA and NMCA with aldehyde group; HMCN with the nitroolefin group; HOVC, HODVC and HOBVC with the vinyl cyanide group.
CBl is a fluorescent probe which exhibits3-(4-pyridinvinyl)-9-ethylcarbazole as the fluorophore, and olefin aldehyde as the receptor group. Particularly, a probe CBl molecule could react with two Cys molecules in two steps while could only react with one GSH molecule. CBl itself was weakly fluorescent in the mixture of ethanol and buffer solution, and its fluorescence could be largely enhanced after reaction with one thiol molecule. However, the reaction rates of CB1with one molecule of various thiols differ a lot:the reaction of CB1with one molecule of Cys could be completed in about40min, while the reaction was completed within24h of Hey and7days of GSH. Meanwhile, the product of CBl-Cys and CBl-Hcy could react with one more molecule of Cys and Hey, which could quench the fluorescence of the system. Fortunately, the second step reaction of Cys could be completed in24h, and this reaction of Hey could be completed in7days, which enables CBl a fluorescent probe for discriminative detection of Cys, Hey and GSH at three time windows. Only the system of CBl-Cys exhibits strong fluorescence at40min, while only the system of CBl-Hcy was strongly fluorescent at24h. After7days, the fluorescence of CBl-Cys and CBl-Hcy was quenched, and the system of CB1-GSH was emissive. Considering high selectivity and good two-photon excited fluorescent properties of CB1, the cell imaging experiments with CBl under wide-field microscopy and two-photon microscopy were successfully performed.
HOTA is a fluorescent probe which exhibits4-phenolvinyltriphenylamine as the fluorophore, and olefin aldehyde as the receptor unit. Probe HOTA has the same receptor unit as CB1, which could selectively react with Cys in a short response time. The selectivity is super-high and Hcy and GSH could hardly bring interferences. In particular, the concentration of intracellular GSH (1～10mM) is much higher than Cys (30～200μM), and probe HOTA could discriminate Cys over such high concentration of GSH. Consequently, cell imaging experiments were successfully performed with this probe. To testify the source of cellular emission, the inhibitor of GSH synthetase, BSO, has been used to prevent cellular synthesis of GSH before capturing cell images, and then these images were compared with the cells directly stained with HOTA. The experiment results showed that these two kinds of cells possess similar fluorescent intensity, which proved that cellular fluorescence was from the reaction of Cys and the probe.
HMCA, BMCA and NMCA were probes with an aldehyde as the receptor group. Due to PET process caused by the aldehyde group, these three probes were weakly fluorescent. The experiment results demonstrated that these probes could react with Cys and Hcy, which bring obvious fluorescent enhancement. Unlike other reported probes with aldehyde, these three probes have stronger interaction with Hcy. With equimolar measurements, HMCA, BMCA and NMCA were Hcy probes. According to the molecular structure of three kinds of thiols, the steric effect should be a key parameter for the selectivity. Therefore, we have designed three molecules HOVC, HODVC and HOBVC, to study the steric effects. HOVC has vinyl cyanide as a receptor unit, HODVC has an unsaturated malononitrile as a receptor unit and HOBVC bearing an unsaturated benzyl cyanide as a receptor unit. The three probes have rather different steric effects, and this study would provide inspiration for molecule design of probes for thiol detection.
引文
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