Synthesis Of Meso-substituted Pentamethine Cyanine Dyes And Study Of Their Interaction With G-quadruplex DNA

G-quadruplex DNA（G-DNA）is a non-canonical secondary nucleic acid structure which is prevalent at the telomeric ends of chromosomes and in the promoter regions of oncogenes.It has been shown to be closely associated with multiple diseases,including cancer and neurodegenerative diseases,and is one of the therapeutic targets of great interest.Many small molecule fluorescent probes have been developed for the detection of G-DNA structures in vivo.Among small molecule fluorescent probes,cyanine dyes with good optical properties and adjustable absorption/emission wavelengths are widely used in the field of biosensing.However,conventional cyanine dyes as“always on”probes,which inevitably produce background fluorescence signal,limiting their application range.Therefore,in this paper,to design specific fluorescent probes with high sensitivity and strong“turn-on”fluorescent for G-DNA,large sterically hindered substituents（heterocyclic-and phenyl-substitutions）were introduced into polymethine chain of pentamethine cyanine dye.Based on the synergistic effect of disaggregation induced emission（DIE）and inhibited twisting intramolecular charge transfer（TICT）,the meso-benzothiophene substituted probe A4 could realize sensitive recognition for G-quadruplex DNA and target G-DNA inside mitochondria.This paper mainly includes the following main contents:1.In this paper,a highly applicable synthetic strategy for synthesis of meso-substitued pentamethine cyanine dye was proposed.Using bromo malondiani hydrochloride as substrate,four meso-heterocyclic and three meso-phenyl-substituted pentamethine cyanine dyes were designed and synthesized by Suzuki coupling and then by the reaction with heterocyclic quaternary ammonium salt.The dyes were characterized by ¹H NMR,¹³C NMR,HRMS and single crystal X-ray diffraction.The single crystal structure of A5 shows that the meso-phenyl and parent cyanine dye framwork are not coplanar,and the dye has a twisted“T”shape structure.At the same time,the spectral properties of the dyes in five different solvents were studied.The results showed that the autofluorescence of all dyes after polymethine chain modificated were decreased due to the free rotation of the meso-substituents.In which the meso-benzothiophene substituted dye A4 had quenching fluorescence in aqueous solution due to H-aggregation.2.The interaction of dyes with G-DNA in solution was investigated by UV/Vis,fluorescence and CD spectra.Using the unsubstituted parent dye A0 as a control,c-myc G-DNA was first used for fluorescence screening of different dyes.The results showed the fluorescence of parent dye A0 was almost unchanged before and after adding c-myc.The meso-substituted dyes A1-A7 showed some increase in fluorescence upon addition of c-myc,but A4 showed the most intense fluorescence“turn-on”change due to autofluorescence differences.The interaction of A4 with different DNAs（G-DNA,ssDNA,dsDNA）was investigated by UV/Vis and fluorescence spectroscopic titration.It was found that A4 specifically recognized G-DNA structure through DIE,and the effect with c-myc was the most obvious,the fluorescence intensity was enhanced by 98-fold,and the lowest limit of detection（LOD）was 1.51 n M.3.The recognition mechanism and binding mode of A4 with G-DNA were investigated by density universal function（TD-DFT）theoretical calculation.TD-DFT theoretical calculations indicated that the dihedral angle between meso-substituent of A4and planar cyanine framework was twisted from 90.34°in the ground state（S₀）to 47.18°in the excited state（S₁）.The introduction of substituents increases the flexibility of the molecular structure of A4 and presented distorted intramolecular charge transfer（TICT）,making the dye fluorescence lower.At the same time,the mutually perpendicular structure,which enhanced the degree of H-aggregation of A4,was also sensitive to environmental changes.Molecular docking simulations indicated that A4 bounded at the5’end of c-myc in a terminal stacking mode,the whole molecule changed from the original vertical structure to a rigid planar structure.Therefor DIE plus the inhibited TICT enabled“turn-on”fluorescence of A4.4.The interaction of the probe with G-DNA at the cellular level was investigated by CCK-8 toxicity test,time-progressive imaging,co-localization imaging,DNase/RNase digestion and ligand competition experiments.The CCK-8 toxicity test showed that A4had higher toxicity to tumor cells,but very low or no toxicity to normal cells after long-term incubation,which has important guiding significance for targeted anticancer therapy.Time-progressive imaging was used to monitor the process of staining the cells with A4,and A4 can rapidly internalize into cells within 5 min.Colocalization imaging and DNase digestion experiments showed that A4 mainly stained G-DNA within mitochondria of cells.