Synthesis,DNA Interaction And The Living Cell Imaging Of G-quadruplex DNA-targeted Benzothiazole Derivatives

With the discover of the function of G-quadruplexes DNA in vivo,the design of small molecule fluorescent probes that can selectively detect G-quadruplexes has become a research hotspot at home and abroad.In this paper,we synthesized two new D–?–A benzothiazole derivatives by analyzing the property-structure relationship of the compounds.The interaction of compounds with DNAs,toxicity and imaging of compounds in living cells were studied through a variety of experimental methods.The main work contents and research results are as follows:(1)The target compounds 1 and 2 were synthesized with 2-methylbenzothiazole as the parent by simple modification,and the structures of the compounds were characterized by ¹H-NMR,¹³C-NMR and HRMS.(2)A series of experiments were conducted to study the interaction between the compounds and DNAs.UV-Vis absorption and fluorescence spectra show that compounds1 and 2 could interact with different secondary structures of DNA,and both compounds have high affinity to the promoter G4(c-kit,c-myc and bcl-2).In the fluorescence titration experiment,the addition of G-quardurplex make a significant enhanced of the fluorescence of the compounds,such as 1 combined with c-kit causes a fluorescence enhancement of 135 times and 2 combined with c-myc makes 14 times,while there is no obvious change after binding with double-stranded and i-motif DNA which indicated that1 and 2 are turn-on G-quadruplex fluorescent probe,moreover,the combination of the promoter G-quardurplex with 2 make a redshifts about 50 nm of the fluorescence,suggesting 2 is a promoter G-quadruplex fluorescent probe.Then,1 and 2(12.5 eq.)increase the T_mof DNA about 8.0?and 2.5?in FRET-melting experiments,indicating that 1 and 2 stabilize the G-quadruplexes DNA structure and 1 stabilize the G-quadruplexes DNA more effectively.CD experiments show that the compounds do not change the conformation of DNA,but compounds could induce promoter G-rich sequences to form G-quadruplex structures to a certain extent.ITC experiments show that the binding of the compounds to DNA are enthalpy-driven.One enthalpy-driven strong binding and two entropy-driven weak binding between 2 and c-myc may be mainly due to the cause hydrophobic force after the groove combination.The N indicated that the binding ratio of two compounds with the bcl-2 and c-kit is 2:1 and 1:1 respectively,presumably one or two compound molecules interaction with G-tetrad by the end of the accumulation.For 1 to c-myc G-quadruplex,the binding ratio of 3:1 may mean that there is still one small molecule bound to the side loop.(3)MTT assay show that both 1 and 2 could inhibit the proliferation of Hep G2 cells to a certain extent,with IC₅₀values of 3.23 and 38.9?M,respectively,while have week effect on non-cancer cells and 1 has a stronger inhibitory ability.Confocal fluorescence imaging and DNase and RNase digestion indicate that 1 mainly distributes in the cytoplasm of living cells and interacts with DNA.The current results provide some useful information for design of fluorescence probes targeting G4 in the living cell.