Design And Synthesis Of Novel Fluorescent Probes And Their Bioimaging Application

The activity of many kinds of acid hydrolases in lysosomes reached the best state in acid environment,and the p H value of microenvironment in lysosomes was 3.8-5.0.Lysosomes play important roles in endocytosis,autophagy,plasma membrane repair and oxidative stress.It has been reported that the quantity of lysosomes in tumor cells is significantly more than that in normal cells and the size is larger.When the lysosomes in tumor cells release cathepsin to the cytoplasm,it will promote the development of tumor cells.Lysosomes have become a pharmacological target for selectively destroying cancer cells.Therefore,it is very important to monitor the p H value of lysosomes in cancer cells in real time to study the role of lysosomes in tumor invasion and metastasis.In this thesis,three lysosomal-targeting p H fluorescent probes with large Stokes shift,high fluorescence quantum yield and good cell membrane permeability were designed and prepared,and their structures were characterized.In combination with fluorescence confocal microscopy,the probes were used to visualize the p H fluctuation in lysosomes.The specific research works as follows:Chapter 1: p H fluorescent probes and its application are reviews.Firstly,we outline the functions and monitoring significance of intracellular p H.Then we introduced the typical mechanism of recognition of fluorescent probe.The development and application of subcellular organelles targeted p H fluorescent probes were emphasically summarized.Chapter 2: Two novel ratiometric fluorescent probes(MCDI and MCBI)based on ICT mechanism were synthesized by one-step condensation via ethylene bridging(benzo)indole and carbazole derivatives for lysosomal p H detection.MCDI and MCDBI are strongly p H dependent,and their emission ratios are highly sensitive to lysosomal acidity.Ratiometric probes can be used to quantitatively and accurately determine,and can eliminate the interference caused by instruments,probe concentration and photobleaching.Furthermore,the probes have good optical properties,such as large Stocks shift,high fluorescence quantum yield,high sensitivity,good optical stability and reversibility.Co-localization study of MCDBI and MCDI with Lyso Tracker Green DND-26 were successfully realized.The probes wree applied to detect the ratio of p H fluctuation in living cells.All the findings show that MCDBI and MCDI were suitable for the monitoring of lysosome p H.Chapter 3: A ratiometric fluorescent probe(BIMC)based on ICT mechanism was synthesized from carbazole as two-photon fluorescent group and benzimidazole as proton binding site using Knoevenagle condensation reaction.BIMC emits blue fluorescence in neutral or alkaline media.Under acidic conditions,BIMC can exhibit pronounced p H-dependent red-shift in both absorption and emission spectra upon benzimidazole group N atoms protonation.Furthermore,BIMC shows significantly large Stokes shifts and high quantum yield can greatly reduce the excitation interference and effectively overcome the auto-fluorescence of biological samples.The p Ka value was determined by the sigmoidal fitting of the ratiometric emission ratio yielding a p Kavalue of 4.46,which is suitable for the detection of lysosomal p H.In two-photon optical mode,BIMC has a large two-photon action cross-section values,the TP fluorescene p H titrations yielding a p Ka value of 4.38.The co-localization studies of BIMC with Lyso Tracker Red DND-99 shown that BIMC merged well with Lyso Tracker Red with Pearson's co-localization coefficient,A = 0.905.These findings reliably suggest that BIMC is selective targeting to lysosomes in live cells.BIMC can visually monitor the fluctuation of lysosome p H,and two-photon imaging results in rat liver tissues show that BIMC can visually distinguish cancer tissues from normal tissues.It provides a new analytical method for lysosome-associated physiological and pathological processes.Chapter 4: A fluorescent probe(EMCDI)based on intramolecular charge transfer p H-regulated spiropyran switching ring was synthesized from2-hydroxycarbazole and indole derivatives.Its structure was characterized byMS and NMR spectra.Under acidic conditions,the closed-form spiropyran was transformed to open-form merocyanine derivative.The fluorescence p H titration experiments showed that EMCDI exhibited obvious emission enhancement effect with the p H values decrease from 10.0 to 5.0.EMCDI has high sensitivity,good selectivity for H+ and optical stability.Imaging of He La cells show that EMCDI can penetrate the cell membrane and has been successfully applied to real-time monitoring of intracellular p H changes during autophagy.