Preparation And Analytical Applications Of Fluorescent Probes For Recognition Of Pyrophosphate And Nucleosides Triphosphate

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.