Design、Synthesis And Biological Studies Of Bis-catechol Derivatives As G-quadruplex Cross-linking Agents

Nucleic acids, which are the key genetic materials, play a crucial role in biological systems. DNA-targeted anti-cancer drug research has aroused wide interests as well as the regulation of DNA structure by using of small molecules which have very important theoretical and practical values.In this thesis, three compounds which containing bis(catechol) group were designed and synthesized. These compounds were proved high selectivity for G-quadruplex cross-linking property in oncogene promoters by using circular dichroism and denaturing polyacrylamide gel electrophoresis, and compound1for the best. Native polyacrylamide gel electrophoresisclearly demonstrated that compound1could specifically alkylate G-quadruplex DNA even in presence of large amount of its complementary DNA. MALDI-TOF-MS indicated a ratio of compound1:DNA to be1:1and DNase I degradation results clearly showed that the interaction between compound1and G-quadruplex is through oquinone-mediated guanine cross-linking.Further biological studies demonstrated its good inhibition activity against murine melanoma cells. Then DNA damage by compound1in the B16F1cells was characterized through alkaline comet assay which supported the tyrosinase-stimulated cross-linking mechanism in vivo. Then we studied c-myc gene expression upon incubation with compound1by using Western Blot, which indicated that when compound1could provide almost80%c-myc gene expression inhibition. Our systematic study of the mechanism of compound1’s selectively inhibition of malignant melanoma cells provided new ideas and methods for the malignant melanoma cells.To further investigate if G-quadruplex DNA was formed in vivo and as the target, a biotin labled derivative compound1a was synthesized and pull-down process toward chromosome DNAs combined with circular dichroism and high throughput deep sequencing were performed. CD test showed adirect evidence of G-quadruplex involving interaction between compound1a and chromosomal DNAs. Several simulated intracellular conditions, including X. laevis oocytes, Ficoll70and PEG-200with124bp long c-myc sequence was used to prove that KCl is the key factor for the formation of G-quadruplex, compound la just shows the pure cross-linking ability upon preformed G-quadruplexs. This was the first example of a G-quadruplex cross-linking agent that selectively targeted G-rich sequences in the promoter regions of the oncogenes in vivo and captured the G-quadruplex DNA structures from mammals cells.In summary, a series of potent G-quadruplex cross-linking agents were designed and synthesized, our strategy provided both valuable evidence of G-quadruplex structures in vivo and intense potential in anti-cancer therapy.