Synthesis And Biological Activity Of Pyridostatin Derivatives Selectively Recognizing Double G-Quadruplex DNA

G-quadruplex DNA is a non-classical nucleic acid secondary structure formed by G-rich sequences under physiological conditions.It is widely present in the human genome region.Its formation and melting is involved in gene transcription,replication,translation regulation,chromosome maintenance and recombination.It has been proved that binders targeting G-quadruplexes can inhibit telomerase activity,which provides a new way for the development of anti-tumor drugs.However,a large number of G-rich sequences in the human genome can form G-quadruplex structures.So selective recognition towards a certain G-quadruplex has become a hot topic.In recent years,it has been reported that there are 150-300 nt single-stranded telomere DNA or RNA overhangs at the end of human telomeres.Though most of them can form monomeric G-quadruplexes,a small amount can form double or multimeric G-quadruplex structures.Therefore,design and synthesis of binders selectively recognizing and stabilizing double or multimeric G-quadruplexes will provide important insights into the structure and function of multimeric G-quadruplexes and the development of anti-tumor drugs with lower toxicity and side effects.In this paper,two series of compounds were designed and synthesized to selectively bind and fluorescently recognize human telomere double G-quadruplex DNA.In the first part of the study,three polyether-tethered bisquinoline PDS(pyridostatin)dimers have been designed and synthesized,and their interaction with human telomere double G-quadruplexes has been studied.Dimer 1c with the longest polyether linker exhibited higher binding affinity(Ka=23.2 ± 3.9 μM-1)towards mixed-type G2T1 than monomer PDS monomers and dimers la and lb with the shorter linkers,indicating that dimer 1c is an excellent mixed-type G2T1 binder.And dimer 1c also showed higher thermal stabilization(ΔTm=15.3℃)towards mixed-type G2T1 than monomer PDS and dimers la and 1b.In addition,dimer 1b with a medium-length polyether linker and monomer PDS showed higher binding affinities towards anti-parallel G2T1(Ka=40.2±4.5 and 49.2±5.9 μM-1 for 1b and PDS,respectively)than dimers la and 1c.Moreover,dimer 1b displayed 50-fold higher binding selectivity towards antiparallel G2T1 than antiparallel G1.Dimer 1b also showed high thermal stabilization towards antiparallel G2T1(ΔTm=29.5℃).These results indicate that,by adjusting the length of the polyether linker,the excellent double G-quadruplex binders,especially mixed-type G2T1 binders,can be gotten.Based on the good binding and thermal stabilization of dimer 1c towards mixed-type G2T1,it showed strong telomerase inhibition(IC50=3.6±0.7 μM).In the second part of the study,rhodamine-PDS conjugate 2 has been designed andsynthesized,and its fluorescent response,binding affinity and thermal stabilization towards double G-quadruplexes has been studied.Conjugate 2 displayed the stronger fluorescent response towards antiparallel G1 than mixed-type G2T1,mixed-type and antiparallel G2T1,c-kit 1,c-kit 2,c-myc,ds DNA and CT DNA.It has a detection sensitivity of 0.2 μM.In addition,it can induce the formation of mixed-type G-quadruplexes and display high thermal stabilization towards antiparallel G2T1(ΔTm=41℃).By the preliminary binding mode analysis,conjugate 2 bound G-quadruplexes through a mixed mode of π-π stacking and electrostatic interaction.In summary,we have found a series of binders and a fluorescent probe of double G-quadruplexes.These study provide new ways for the development of double G-quadruplex binders and probes to study their structures and functions and potential anticancer agents.