【作者】 赵希娟；

【导师】 黄承志；

【作者基本信息】 西南大学 ， 分析化学， 2013， 博士

【摘要】 焦磷酸(PPi)和核苷三磷酸阴离子(NTP,其中N=A, C,G, U)在生命体的正常运转中发挥必不可少的作用,因此对它们的识别与检测具有非常重要的意义。目前文献报道的有效策略是利用金属配合物中金属离子和磷酸根的强配位作用实现焦磷酸和核苷三磷酸阴离子的识别与检测。但是很多作为荧光探针的金属配合物合成步骤繁琐,水溶性不好,且不能从众多的含磷酸根阴离子中特异性的识别某一种。因此,寻找合成方法简单、水溶性好、选择性好的荧光探针很有必要。本文试图通过简单的试剂制备一系列荧光探针,包括金属配合物、金属有机骨架和金属纳米簇,依次实现PPi和核苷三磷酸阴离子的高选择性识别与检测,具体的研究内容分为三部分。1.金属配合物识别与检测PPi这部分主要是利用金属离子和PPi的强配位作用,包括两个方面的内容：(1)利用竹红菌甲素(HA)的锌离子配合物实现了选择性识别PPi。锌离子与HA反应形成HA-Zn2+配合物,HA在604nm处的荧光发生猝灭,同时在629nm处产生一个新的荧光发射峰,但该荧光发射随着PPi的加入逐渐降低,而604nm处的荧光得以恢复,说明PPi和HA发生了配体的交换。其它的核苷酸阴离子如ATP, UTP, CTP, GTP, GDP和GMP等都不会引起HA-Zn2+产生类似的光谱变化,据此可以选择性识别PPi。布尔计算(Boolean calculations)显示,该体系可以成功用于设计分子逻辑门,且可以区分PPi和其它的核苷酸阴离子。该方法非常简单,不需要复杂的有机合成。(2)2,6-双-(2-苯咪唑)吡啶(bbimp)与铜离子形成的配合物可以用于选择性检测PPi。该配体分子bbimp只需一步合成,简单易得,它能和铜离子形成1：1的配合物,此时,bbimp的荧光发生猝灭。在PPi存在时,bbimp的荧光逐渐恢复,且恢复程度与PPi的浓度在一定范围内成线性关系,以此实现PPi的检测。选择性实验证明,bbimp-Cu2+配合物对PPi具有良好的选择性。相比之下,各核苷酸阴离子,如ATP, GTP, CTP, UTP, GDP等响应较小,其它的无机阴离子几乎无响应。2.金属有机骨架(MOFs)识别核苷三磷酸阴离子MOFs是一种新兴的基于有机配体和金属离子的配位作用形成的材料,在气体的储存和分离、药物的运载以及小分子的传感方面有了广泛的应用。本论文首次将MOFs用于核苷三磷酸阴离子的识别,解决了传统的金属配合物识别核苷三磷酸阴离子时选择性差的问题。(1)利用锌离子和对苯二甲酸构建的发光MOF—[Zn(BDC)(H2O)2]n实现了腺苷三磷酸阴离子(ATP)的选择性识别。该MOF的水分散体系在427.6nm处有明显的荧光发射。在ATP存在时,该发射峰发生红移,但GTP, CTP, UTP, PPi,磷酸根,醋酸根以及其它的无机阴离子等都不会引起[Zn(BDC)(H2O)2]n的荧光发射峰发生明显红移,说明该MOF对ATP的选择性很好。核磁共振和拉曼光谱证明,[Zn(BDC)(H2O)2]n与ATP之间既有π-π堆垛,又有配位作用,其中ATP中的腺嘌呤和磷酸根都参与了与锌离子的配位反应。相对于之前的金属配合物,引入了金属有机骨架,并以此建立了一种简单、选择性良好的识别ATP的方法。(2)基于铽离子的发光MOF用于高选择性传感胞苷三磷酸阴离子(CTP)。研究发现,2,3-吡嗪二羧酸和铽离子在水热条件下可以生成[Tb2(2,3-pzdc)2(ox)(H2O)2]n [2,3-pzdc2-=2,3-吡嗪二羧酸盐,ox2-=草酸根]。由于天线效应的存在,该MOF表现出强烈的Tb3+荧光发射。在CTP存在时,MOF的发射会逐渐被猝灭。GTP, UTP, ATP, PPi和其它无机阴离子,以及胞嘧啶、胞苷、CMP等对该MOF的荧光猝灭程度很小或者几乎没有猝灭,说明该发光MOF对CTP具有良好的选择性。机理研究表明,胞嘧啶和三磷酸基团是该选择性响应必不可少的两部分。实验进一步发现,与CTP反应后,该MOF的拉曼光谱和X-射线衍射(XRD)光谱都没有变化,说明,二者反应后没有明显的化学键生成,而且该MOF的结构也没有被破坏。氮气吸附实验证明该MOF的孔径很小,几乎无氮气吸附,通过计算得知,CTP的体积远大于氮气的体积,表明CTP不能进入该MOF的孔径,而是吸附在MOF的表面,通过阻止配体到Tb3+之间的天线效应引起了荧光的猝灭。3.发光金属纳米簇用于核苷三磷酸阴离子的识别有机配体和金属离子在一定条件下会发生配位反应,当条件改变时,二者也可能发生氧化还原反应。研究发现,室温下组氨酸和铜离子会发生配位反应,生成没有荧光的蓝色配合物。但该配合物在700C搅拌反应12小时后,溶液的颜色逐渐变成棕色,说明组氨酸和铜离子发生了氧化还原反应,铜离子被还原生成了铜纳米簇。用350nm的紫外光激发时,有较强的蓝绿色荧光发射；在GTP存在时,铜簇的荧光会发生一定程度的猝灭。与ATP, CTP和UTP相比,GTP对铜纳米簇的猝灭程度最大,其它的无机阴离子包括PPi、磷酸根、醋酸根等都不会猝灭铜簇的荧光,据此可实现GTP的选择性识别。该体系报道了一种一步法合成发光铜纳米簇的方法,不需要氮气保护,不需要模板分子：利用碱基还原性的差异,首次将发光铜簇用于核苷三磷酸阴离子的识别。总而言之,本论文建立了简单而高选择性的识别与检测PPi和核苷三磷酸阴离子的分析方法。除了金属配合物,还引入了两种新的荧光探针一一金属有机骨架和发光铜纳米簇,并成功用于含磷酸根阴离子的选择性识别中。本文解决了探针合成步骤繁琐以及选择性差的问题。实验证明,通过一步法合成的探针一样可以高选择性的识别含磷酸根阴离子；而且在选择性识别核苷三磷酸阴离子时,碱基的参与至关重要,这直接决定了体系的特异性。更多还原

【Abstract】 Pyrophosphate (PPi) and nucleosides triphosphate (NTP, N=A, C, G, U) play indispensable roles in the normal functions of organisms, and so recognition and detection of them become very significant. At present, the effective strategy reported for recognition and detection of these phosphate-containing anions is to utilize the strong coordination ability of the PPi with metal ion in the metal complex. However, many of the metal complexes, which have been reported to be fluorescent probes for PPi and nucleosides triphosphate, either need complicated synthesis steps, or have bad water-solubility. Also, it is noteworthy that most of the fluorescent probes can’t recognize a specific one among all the phosphate-containing anions owing to the same group of the phosphate. So, it is necessary to prepare simple and water-soluble fluorescent probes with good selectivity for a certain phosphate-containing anion. Here, we obtain a series of fluorescent probes through very simple synthesis steps including metal complexes, metal-organic frameworks and metal nanoclusters, and make the selective recognition and detection of PPi and a certaion nucleoside triphosphate come true. The main contents of this research include three sections.Section one focuses on the recognition and detection of PPi with metal complexes, using the strong coordination ability of metal ions and PPi. The details are listed as follows: 1. We introduce a hypocrellin A-Zn(II) complex (HA-Zn2+) for highly selective recognition of PPi. The fluorescence emission of HA at604nm can be effectively quenched by Zn2+, accompanied with a new emission at629nm owing to the formation of the HA-Zn2+complex. Upon the addition of PPi, the emission at629nm is gradually quenched with the recovery of the emission at604nm. Other nucleosides phosphate-containing anions including ATP, UTP, CTP, GTP, GDP and GMP have no such spectral actions as PPi. On the basis of this, selective recognition of PPi is achieved. On the other hand, the recognition system can be successfully applied to design logic gates, and can distinguish PPi from the other anions. This method is very simpe, and needs no complicated synthesis.2. The copper(II) complex of2,6-bis(2-benzimidazolyl)pyridine (bbimp) can be used to selective detection of PPi. The ligand of bbimp is easy to be obtained with only one synthesis step, and can be complexed with Cu2+by the molar ratio of1:1. The fluorescence of bbimp is quenched by Cu2+once the complex of bbimp-Cu2+is formed. In the presence of PPi, the fluorescence will be recovered gradually. The degree of the fluorescence recovery has a linear relationship with PPi in the range of3-90u.mol/L. This detection method has good selectivity for PPi over other nucleotide anions such as ATP, GTP, CTP, UTP, and GDP as well as some other inorganic anions.Section two investigates the application of metal-organic frameworks (MOFs) to the recognition and detection of nucleosides triphosphate.MOF is a new kind of material based on the coordination of organic ligands and metal ions, which is widely used in gas storage and separation, drug delivery and sensing of small molecules. Here, we for the first time use MOFs to recognize nucleosides triphosphate, solving the problem of poor selectivity when traditional metal complexes are used.1. Highly selective recognition of adenosine5’-triphosphate (ATP) is successfully achieved with a luminescent MOF of [Zn(BDC)(H2O)2]n (BDC=1,4-benzenedicarboxylate). The MOF dispersed in water shows an obvious fluorescence emission centered at427.6nm. In the presence of ATP, there will be a remarkable redshift of the emission wavelength. While guanosine5’-triphosphate (GTP), cytidine5’-triphosphate (CTP), uridine5’-triphosphate (UTP), PPi, PO43-and CH3COO-as well as some other inorganic anions can’t induce such spectral change as ATP, indicating the good selectivity of [Zn(BDC)(H2O)2]n for ATP.’HNMR,31P NMR and Raman spectra indicate that π-π stacking interactions and the coordination of Zn2+with both adenine and the phosphate group are involved in the interaction of [Zn(BDC)(H2O)2]n with ATP. In contrast to the metal complex, we introduce a MOF, and establish a simple method for selective recognition of ATP.2. We introduce a terbium(III)-organic framework with2,3-pyrazinedicarboxylic acid (2,3-H2pzdc) as the ligand, which is highly selective for sensing cytidine5’-triphosphate (CTP). The MOF of [Tb2(2,3-pzdc)2(ox)(H2O)2]n can be prepared through hydrothermal reaction of2,3-H2pzdc and Tb(NO3)3-5H2O in water. Owing to the antenna-effect, the MOF shows strong emission features of Tb3+. Upon the addition of CTP, the emission will be gradually quenched. However, GTP, UTP, ATP, PPi and some other inorganic anions, as well as cytosine, cytidine and CMP induce small or even little quenching extent, which indicates that cytosine and the group of triphosphate are two indispensable parts in the highly selective sensing process. Furthermore, we observe that after interacting with CTP, the Raman and powder X-ray diffraction (PXRD) spectra of [Tb2(2,3-pzdc)2(ox)(H2O)2]n don’t show any changes, indicating that no new chemical binding species has formed between this MOF and CTP, and the structure of the MOF keeps stable. N2adsorption-desorption experiment show that the pores of the MOF are too small to fit N2to diffuse through it. On the other hand, CTP, as calculated in vacuum, is much larger than N2. So CTP can’t easily enter the pores of [Tb2(2,3-pzdc)2(ox)(H2O)2]n, and is able to adsorb on the surface architecture of the MOF. Close contact of CTP and MOF will reduce the antenna-effect, leading to the luminescence quenching.Section three mainly studies the application of luminescent metal nanoclusters to the recognition of nucleosides triphosphate.Sometimes, metal ions can be complexed by organic ligands. At other times, metal ions can be reduced by organic ligands. Here, at room temperature, histidine and can form nonfluorescent complex. When refluxed for12h at70℃, and the color of the solution changed from blue to brown gradually, indicating the formation of Cu NCs. With the excitation wavelength of350.0nm, the as-synthesized Cu NCs exhibit strong luminescence. In the presence of GTP, the emission of Cu NCs is gradually quenched. Compared to ATP, CTP and UTP, GTP induces the largest quenching extent. And some other inorganic anions such as P2O74-, PO43-, and CH3COO-have no such quenching effect. Based on this, selective recognition of GTP is successfully achieved. Here, we report a very simple method to synthesize water-soluble luminescent Cu NCs through only one step reaction. Neither nitrogen atmosphere nor a template molecule is needed. By using the difference of the bases, we for the first time apply the luminescent Cu NCs to selective recognition of a certain nucleoside triphosphate.In summary, we establish a series of simple methods for selective recognition and detection of PPi and nucleosides triphosphate. Apart from metal complex, two new fluorescent probes, metal-organic framework and luminescent copper nanoclusters are introduced, and successfully applied to the selective recognition and detection of the phosphate-containing anions. In this work, the problems of complicated synthesis and poor selectivity are solved. The experimental results show that the probes obtained by one step can also recognize a specific phosphate-containing anion. Furthermore, we find that the base plays an indispensable role in the selective recognition of nucleosides triphosphate, which directly determines the specificity of the probe.更多还原