The Design And Synthesis Of Novel Fluorescent Probes For Lysosome Targetable Imaging Applications

Organic small molecule fluorescent probes have made great progress in detecting various metalions,anions and small biomolecules due to their high sensitivity,high selectivity,high resolution and real-time positioning.Fluorescence imaging technology based on small molecule fluorescent probes has attracted many people's attention because of its low cost,non-destructive sample,high temporal and spatial resolution,and long-term observation.In the past two decades,a wide variety of functionalized fluorescent probes have been reported and are widely used in fluorescence sensing and bioimaging applications.However,there are still some problems and hot spots to be solved in the field of fluorescent probe research.For example,the development of near-infrared dyes including near-infrared absorption and fluorescence emission is of great significance for high-through imaging;development of fluorescent dyes with good photostability and chemical stability is more conducive to long-term imaging observations in vivo;Fluorescent probes(including subcellular organ localization,fluorescence detection,drug release,etc.)provide a powerful tool for medical and life sciences.The lysosome is an acidic organelle(pH 4.5-5.5)in eukaryotic cells,which contains a variety of digestive enzymes with high catalytic activity at pH 5.As a degradation chamber for eukaryotic cells,the main function of lysosomes is to recover damaged organelles and digest nucleic acids,polysaccharides,fats and proteins.In addition,lysosomes are highly dynamic and they constantly change their internal morphology and spatial distribution.Tracking their movements and morphological changes in real time not only helps to understand their working status,but also provides an effective means to prevent and diagnose lysosomal dysfunction-related diseases.Lysosomes in various analytes such as pH,viscosity,various metal ions,anions,small biomolecules,etc.,their concentration and distribution levels are closely related to the physiological functions of lysosomes.Abnormal concentrations of these analytes may be indicative of various lysosomal-related diseases,including inflammation,silicosis,lysosomal storage diseases,cancer,neurodegenerative diseases,and the like.therefore,the development of highly selective and highly sensitive fluorescent probes for monitoring the concentration levels and distribution of various analytes in lysosomes is significant.This highly efficient real-time observation technique is very helpful in studying lysosomal-related Physiological and pathological processes provide a theoretical basis for disease prevention and early diagnosis.In this paper,based on the targeted lysosomal functionalization,we constructed a novel long-wavelength emission rhodamine analog fluorescent probe for the detection of lysosomal pH,hypochlorous acid and selenocysteine.The research work of the thesis mainly includes the following three parts:1.In Chapter 2,by reducing the size of the typical lysosomal targeting group 4-(2-aminoethyl) morpholine,N-aminomorpholine is used as a rhodamine fluorescent regulatory switch and targeted solution.The enzymatic group,we designed the rhodamine analog probe Ly-HN2AM for imaging observation of lysosomal pH changes,and the reported 4-(2-aminoethyl)morpholine modified probe Ly-HN2AEM In contrast,the new probe Ly-HN2AM is more susceptible to H~+binding to form a stable five-membered ring chelate(pKa 5.35)with significantly improved sensitivity at pH values from 4.79 to 6.07.The new probe was successfully applied to visually detect lysosomal-related pH changes,and it has good photostability and low cytotoxicity,indicating its potential application value for targeted localization of lysosomal bioimaging.2.In the third chapter,combined with our previous research,based on the synthesis of dye HN4 with long-wavelength fluorescence emission,the lysosomal target was synthesized for the first time by modifying the morpholine group that can target lysosomes.To the functional HN4 derivative HN4M,and the dye,a thiolactone rhodamine analog probe HN4MS was constructed.In general,the classical rhodamine spiro derivative probe is sensitive to pH and is generally not used as a lysosomal targeting probe for the detection of analytes.Interestingly,our probe HN4MS showed strong acid resistance and high sensitivity,high selectivity and fast response to hypochlorous acid at lysosomal pH.In addition,the probe has good two-photon fluorescence properties,and a near-infrared two-photon laser source(800 nm)can be used as the excitation light to emit a long-wavelength fluorescent signal(632 nm),which can better avoid spontaneous in the physiological matrix.Fluorescence interference and self-absorption effects.It has been proved by experiments that the probe HN4MS can successfully visualize HClO in living cells and living tissues.