Design, Synthesis And Imaging Applications Of Large Stokes Shift Mitochondrial-target Fluorescence Probes

Mitochondrion is a very important dynamic organelle among numerous organelles,which is called as the "power house" of cells.Mitochondrian plays an important role in various biological processes.Mitochondrial dysfunction will cause intrinsic apoptotic pathways and lead to a variety of neurodegenerative diseases including cancer,diabetes and Alzheimer's disease.Therefore,to further understand mitochondrial function,it is necessary to image mitochondria in vivo.However,most of fluorescent probes have the disadvantage of small Stokes shift.Fluorescence probes with small Stokes shift may cause self-quenching in the imaging processes,further resulting in measurement error.Therefore,fluorescent dyes with small Stokes shift are often detrimental to their intracellular fluorescence imaging.To avoid the above problems,it is necessary to construct fluorescent probes with large Stokes shift.Because ensuring the imaging accuracy is very important in the imaging field.Therefore,it is necessary to construct mitochondria-targetable fluorescent probes with large Stokes shift for biological imaging applications.In this paper,based on the current research status of mitochondria-targetable fluorescent probes,we successfully designed and synthesized a series of mitochondria-targetable fluorescent probes with the large Stokes shift using triphenylamine,carbazole,phenanthrenequinone imidazole and benzimidazole as the fluorescence platforms,respectively.We investigated their spectral properties and biological imaging applications.Firstly,we chose triphenylamine as the fluorescence platform to design and synthesize a mitochondrial targeted fluorescent probe CDPI with large Stokes shift.Spectral experiments showed that the probe CDPI had large Stokes shifts in various solvents(169-198 nm),and the maximum fluorescence emission value was at around 650 nm,in the red-emitting region.The results of cell imaging experiments showed that the probe had good cell membrane permeability and high co-localization coefficient(0.9)with the commercial Mitochondrial Tracker Red(MTR).Therefore,CDPI can image mitochondria in living cells.Secondly,a new mitochondria-targetable fluorescent probe for detecting H2O2 was designed and synthesized using carbazole as the fluorophore.A mitochondria site and a H2O2 site were then incorporated at the 3,6-positions,respectively.The probe CAI showed excellent optical properties,had high quantum yields and large Stokes shifts in various solvents(135-157 nm).The probe can detect H2O2 in aqueous solution and mitochondria of living RAW 264.7 cells with high sensitive and selectivity.Thirdly,we synthesized a series of fluorescent probes PIE1-PIE7 basing on phenanthrenequinone imidazole fluorophore core with different substituents.All of the fluorescent probes PIE1-PIE7 had high quantum yields.PIE2,which introduced diethyl aniline group on the phenanthrenequinone imidazole core,can be located on the lipid droplet in the living cells.Introduction of a tertiary amine group on the phenanthrenequinone imidazole core afforded a novel AIE-based fluorescent material PIE5.The probe PIE5 can selectively sense Pd(?)in aqueous solution.Modification of an indolium moiety on the same phenanthrenequinone imidazole core provided a new probe PIE6.The probe PIE6 could image mitochondrial pH in living cells based on two different sets of fluorescence signals.Introduction of pyridinium salt unit on the phenanthrenequinone imidazole core to provide PIE7,the probe PIE7 could stain the mitochondria of living cells.PIE6 and PIE7 had large Stokes shifts in various solutions.Fourthly,we designed and synthesized a small molecule fluorescent probe BISEI,using benzimidazole as the fluorophore.The probe BISEI had large Stokes shift,high fluorescence quantum yield and prominent photostability.Due to its excellent properties,the probe BISEI is successfully used to the mitochondrial and DNA imaging of cancer cells,tissues and zebrafish.For the living cell imaging,the images of BISEI showed distribution of the mitochondrial reticular structure,while the probe BISEI clustered in the nucleus of the fixed cells.For the tissue imaging,the 3D imaging results of the probe BISEI firstly exhibited distribution of nuclei in living tissues.For the in vivo imaging,the probe displayed a strong red fluorescence in the chest of zebrafish.