Benzothiazole-substituted Benzofuroquinolinium As New Fluorescent Ligands For G-quadruplex DNA

Nucleic acids containing G-rich sequences can form stable secondary four-stranded structures called G-quadruplex, which have important biological functions. Many researchers have been engaged in the study of the existence, distribution, and dynamic procedure of G-quadruplex in recent decade. They have obtained some preliminary evidence and developed a series of antitumor compounds targeting for G-quadruplex. However, the structure and function of G-quadruplex in body have not fully elucidated. G-quadruplex fluorescent ligands have many advantages including excellent G-quadruplex selectivity, strong optical property and good biocompatibility, which provide a significant tool for detection of the existence, distribution and biological functions of G-quadruplex. Therefore, developing G-quadruplex fluorescent ligands with good performance has a significant meaning.Benzofuroquinolmium, which can interact with G-quadruplex by π-stacking, is a G-quadruplex ligand derived from the structure of cryptolepine. However, the ligand had no fluorescent response in the presence of G-quadruplexes, which limited the application of detection of G-quadruplexes. Meanwhile, our group conjugated the benzothiazole fragment of TO (signaling unit) to benzofuroquinolinium (binding scaffold) and formed a novel fluorescent probe CYTO. Studies showed that the fluorescent intensity of CYTO was enhanced remarkably upon binding to human telomere G-quadruplex DNA. However, it had poor water solubility and selectivity to G-quadruplexes. Based on our previous modification experience, we introduced different side chains in the benzothiazole group of CYTO and synthesized a series of derivatives 2a-f to apply in cell imaging and study their selectivity, water-solubility, sensitivity, binding capacity and fluorescence quantum yield as G-quadruplex fluorescent ligands.The main contents include:1) A review on the research and development of G-quadruplex fluorescent ligands. This part briefly described the structural characteristics and biological functions of G-quadruplex DNA, and foused on the design ideas of G-quadruplex fluorescent ligands, and summarized the development of several kinds of G-quadruplex fluorescent ligands2) Design and synthesis of G-quadruplex fluorescent ligands. This part summarized the results of the previous study in our group, based on that we proposed design ideas and synthetic methods of compouds, and designed and synthesized 6 new benzothiazole-substituted benzofuroquinolinium derivatives.3) The application of fluorescent ligands in the detection of G-quadruplex DNA. This part used the fluorescent ligands to detect the G-quadruplex in solution system, gel system, cell system. The results showed that the G-quadruplex could be well detected by the fluorescent ligands in three systems. Moreover, fluorescence spectroscopy and UV spectroscopy were used to further explore the limits of detection and fluorescence quantum yield of the fluorescent ligands. The results showed that the sensitivity and solubility of fluorescent ligands had been further improved. Although the fluorescence quantum yields showed a slight decline, the decline was not obvious and these compounds still had good fluorescence properties.4) Study on the interaction between fluorescent ligands and G-quadruplex. UV spectroscopy results revealed that the whole ligand could interact with G-tetred by π-stackin. Fluorescence titration experiments were carried out among different DNA. The results showed that such conjugates had excellent selectivity to G-quadruplex. Fluorescence spectroscopy and FRET experiments showed that the binding capacity (Kd= 4.96～8.62× 105M-1) and stabilizing capacity (△Tm=7～25℃) had a good enhancement when ligands combined G-qudruplex DNA. We speculated that it might be the result of the interaction between amino aliphatic side chain and G-quadruplex grooves.