Construction Of Novel Asymmetric Arylimidazole Based Aggregation-induced Emission Probes For Visualization Of Cell Metabolism

Background:Cell metabolism is one of the frontiers of biomedical research,cell metabolism visualization is crucial for deciphering the pathogenesis of diseases and stimulating the development of efficient diagnosis and treatment strategies for diseases.Aggregation-induced emission based fluorescent molecular probes provide a powerful tool for in situ monitoring of cell metabolism due to their excellent photo stability and high sensitivity.Moreover,they are capable of overcoming the aggregation-induced quenching(ACQ)dilemma faced by traditional fluorescent probes in physiological environments to offer robust spatial and temporal dynamic information.The in-depth exploration of cell metabolism urgently requires the development of novel AIE molecular probes in order to empower the innovation of molecular tools.Objective:This study aims to develop new strategies to construct novel asymmetric arylimidazole-based AIE molecules and actively extend their applications in cell metabolism visualization and real-time monitoring.(1)Develop a novel strategy for constructing asymmetric arylimidazole based AIE fluorescent molecules to achieve the versatility of the fluorescent skeleton under mild conditions.(2)Design and develop a novel AIE probe based on asymmetric arylimidazole for monitoring leucine aminopeptidase(LAP).(3)Design and develop a novel AIE probe based on asymmetric arylimidazole for visualization and dynamic monitoring of autophagy.Methods:(1)To propose a new strategy based on the domino cyclization reaction mediated by carbon tetrabromide for the construction of AIE asymmetric arylimidazole derivatives.To explore the substrate scope of this strategy using asymmetric ketone and amidine substrates bearing more abundant sites.(2)To design and synthesize the first asymmetric arylimidazole-based AIE fluorescent probe ASSI-Leu for monitoring LPA utilizing the novel construction strategy.By elaborately introducing the 2-(2’-hydroxyphenyl)benzothiazole(HBT)into the 4-aryl position of the imidazole core to achieve the excited-state intramolecular proton transfer(ESIPT)characters.(3)De novo design of a novel AIE fluorescent probe tailored to autophagy visualization utilizing the novel construction strategy to integrate multiple functions into a single molecule.(4)To demonstrate the potential application value of these synthesized AIE probes in visualization of cell metabolism via optical properties study,in vitro and in vivo biological application research.Results:(1)The novel strategy was employed to directly create a smallfluorescent molecular library of 10 members based on asymmetric arylimidazoles at 4,5-positions of imidazole.The study of structural photophysical properties revealed the color tunability and multidirectional charge transfer characters.Through subsequent derivatization of substituents,AIE fluorescent molecules with longer emission wavelengths and larger Stokes shifts could be obtained.This strategy expanded structural diversity and provide unprecedented degrees of freedom.(2)The typical HBT skeleton was purposefully incorporated into the 4position to avail both AIE and ESIPT characters.And such design bestowed probe ASSI-Leu the self-calibration effect of ratiometric response via the protection/deprotection of hydroxyl group.Concomitantly,ASSI-Leu offered a large Stokes shift,appreciated biocompatibility,good sensitivity,outstanding selectivity and high signalto-noise ratio.Additionally,the in vivo imaging evaluation suggested that those favorable features enabled probe to real-time visualize the endogenous LAP in cisplatin-induced zebrafish acute liver injury model.These findings represent first implement of AIE probe in synergy with ratiometric behavior to track endogenous LAP,which may provide a considerable tool for auxiliary diagnosis of cell metabolism related diseases via interpreting critical enzymes.(3)ASMP-AP displayed a large stokes shift,great cell permeability and good biocompatibility.More importantly,ASMP-AP enabled a good linear response to pH,which derived from the fact that its aggregation state could be manipulated by the acidity.It was successfully applied for imaging autophagy in living cells and was proved capable of monitoringmitophagy.Moreover,this novel molecular tool with excellent physical and chemical properties was validated by ex vivo visualization of activated autophagy in drug-induced liver injury model.Interestingly,it provided a meaningful pharmacological insight that the melanin inhibitor 1-phenyl-2-thiourea(PTU)-induced autophagy was clearly presented in wild-type zebrafish.This is the first instance to visualize autophagy in zebrafish using a small-molecule probe with AIE characters,accurate lysosome targeting and simultaneous pH sensitivity.Ultimately,this novel fluorescence platform has great potential for in vivo translation to fuel autophagy research.Conclusions:In summary,this study devised a mild,facile,efficient,and scalable construction strategy served to obtain novel asymmetricarylimidazole based AIE fluorescent molecules.Furthermore,via the elaborate structural design with this proposed strategy,novel AIE fluorescent probes capable of real-time monitoring of cell metabolism related enzymes and metabolic pathways have been successfully obtained and implemented,providing new opportunities and possibilities for precisely visualizing and assessing cellular metabolism.