Design?Synthesis And Application Of Novel Organic Fluorescence Probes And Biorthogonal Probes

With the rapid development of science,the occurrence of various diseases has been proved to be related to abnormal metabolism of enzymes and physiological,and the research of new drugs that can specifically regulate en zyme activity and physiological processes has become a research hotspot in contemporary medicine.It is an important responsibility for analytical chemistry to develop the new and efficient methods for the detection of bioactive enzymes and physiological p rocesses.Due to the advantages of high sensitivity,good selectivity,simple operation,good data reproducibility,the rational design of organic fluorescent probes for the detection of bioactive enzymes and physiological processes has become a research h otspot in the field of biology and chemistry.It is still of great theoretical and practical significance to develop new organic fluorescent probes for the detection of bioactive enzymes and physiological processes.So in this paper,on the basis of consul ting relevant literature and skills learning,and with combination the existing research conditions of the laboratory,a series of new organic fluorescent probes were designed and synthesized,and the analytical abilities of these probes for detecting bioactive enzymes and physiological processes was comprehensively and systematically studied.The main contents were summarized as following:?1?In this chapter,a novel TPE fluorescent probe,trimethyl locked quinone caged Acedan?Q₃CA-P?,is developed for hNQO1 detection and imaging in living cells and tissues.The probe was designed by conjugating the trimethyl lock quinine substrate moiety to Acedan via an acetamidum linker.The results showed Q₃CA-P displays over25-fold enhancement in fluorescence intensity toward hNQO1 with a Stokes shift over100 nm in one-photon excitation and exhibits a very low detection limit of 5.6 ng/mL.The imaging experiments performed in tumour cells and tissue slices by Q ₃CA-P demonstrate that Q₃CA-P could image the endogenous hNQO1 with high selectivity and sensitivity with a TPE probing depth of 120?m.Thus,our probe may have a great opportunity for cancer diagnosis and image-guided surgery.?2?In this chapter,we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues.The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker.Upon deacetylation by HDAC,the probe undergoes a rapid self-immolative intramolecular cyclization reaction,producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore.The probe is shown to exhibit high sensitivity,high specificity and fast response for HDAC detection in vitro.Imaging studies reveal that the probe can directly visualize and monitor HDAC activity in living cells.Moreover,the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to130?m.This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.?3?In this chapter,we develop the development of the first near infrared?NIR?turn-on fluorescence probe?NIR-PAP?for detecting and imaging PAP in living cells.The probe is prepared by reacting a cysteine-proline dipeptide to an acryloylated NIR fluorophore via a facile thiol-Michael addition reaction.NIR-PAP exhibits a dynamic response toward PAP in the range of 0.02-2.5 U/mL with an estimated limit of detection of 0.013 U/mL.In vitro studies also reveal that the probe also displays high specificity and robust responses toward PAP in physiological pH and temperature conditions.Moreover,NIR-PAP is successfully introduced to detect and differentiate PAP activity in four different cell lines via both confocal fluorescence imaging and flow cytometry.Therefore,our probe may hold a great promise in diagnosing pathogen infectious diseases and screening therapeutic drugs in vivo.?4?In this chapter,based on the advantages of biorthogonal reaction,a mitochondrial-targeted probe?Mito-Tz?and a lysosome-targeted probe?Lyso-TCO?were designed for the imaging of the fusion process of autophagy and lysosome in mitochondrial autophagy.Mito-Tz can specifically target mitochondria due to its cationic structure and its fluorescence was quenched by effect of tetrazine structure on rhodamine.Moreover,Lyso-TCO structure has acid response ability and can specifically target lysosomes,and the modified s-TCO can react with Mito-Tz to activate fluorescence signal.The experimental results also show that these two probes exhibit excellent organelle targeting properties and can aggregate in lysosomes and mitochondria respectively under the normal cell culture conditions.When mitochondria autophagy occurs,mitochondria are consumed by autophagy and finally fuse with lysosomes,resulting in the meeting and reacting of Mito-Tz and Lyso-TCO,thus activation of fluorescence signal and detection of the fusion process of autophagy and lysosomes during mitochondrial autophagy are realised.The experimental results show that the designed probes have the advantages of fast response speed,strong anti-interference ability and high specificity.The membrane fusion process of autophagy and lysosome in mitochondria autophagy of HeLa,HepG2 and MCF-7 cells both can be imaged and detected by theser two probes,which proves that this design concept has a great application prospect in imaging research of physiological processes.