Design,Synthesis And Imaging Analysis Of Fluorescent Probes For Neuroactive Molecules

Neuroactive molecules(e.g.,neurotransmitters,reactive oxygen species,ions,enzymes,etc.)play an irreplaceable role in both the production and transmission of neural signals in the brain.However,abnormal changes in the levels of these neuroactive molecules can lead to disturbances in neural signaling and induce central brain neurological disorders such as Alzheimer's disease,Parkinson's disease,and depression.Therefore,the realization of highly sensitive and selective quantitative analysis and dynamic distribution imaging of neuroactive molecules in the brain is of great physiological and pathological importance for exploring and understanding the pathogenesis of central brain neurological disorders.Many techniques have been developed for the detection and imaging of neuroactive molecules,among which fluorescence methods are widely used due to their high sensitivity,high spatial and temporal resolution,good biocompatibility,and non-invasiveness,but there are still great challenges to achieve real-time in situ acquisition of information on changes in neuroactive molecules!The main challenges are as follows:(1)The brain environment is complex,with many components,and neuroactive molecules(e.g.neurotransmitters,enzymes,etc.)are similar in structure and lack highly selective recognition ligands;(2)The correlation between neuroactive molecules is high,and there is an urgent need to develop probes for simultaneous detection of multiple substances to resolve the interactions between different neuroactive molecules;(3)The concentration distribution of neuroactive molecules in the brain spans a wide range(n M?m M)and is in a dynamic state of change,so there is an urgent need to design quantitative probes with high sensitivity and fast response time.This thesis focuses on the key scientific question of how to achieve real-time in situ access to information on changes in neuroactive molecules,with the following three research work:(1)The ratio-metric two-photon fluorescent probe TMF2P was designed and synthesized based on an intramolecular charge transfer-fluorescence resonance energy transfer(ICT-FRET)sensing mechanism with a high selectivity for monoamine oxidase-A(MAO-A),with a linear range of 1.0-21 m U m L^-1 and a detection limit of25 U m L^-1.The probe was successfully used for real-time imaging and quantitative analysis of MAO-A in neurons and tissues during ischemia.It was found that O₂^·-induced elevated MAO-A levels and activated mitochondrial TRPM2 channels,leading to Ca²⁺inward flow into mitochondria and ultimately neuronal death;furthermore,MAO-A and Ca²⁺levels were different in different brain regions,being higher in primary somatosensory cortex(S1BF)and hippocampal(CA1)regions than in caudate putamen(CPu)and thalamus(LD)regions.(2)The multisite recognition supramolecular fluorescent probe CN-DFP5 was designed and synthesized by analyzing the structural commonalities and differences in neurotransmitters to identify seven neurotransmitters.The boric acid-derived naphthalimide group in CN-DFP5 interacted with catechol-type neurotransmitters(Dopamine(DA),noradrenaline(NE),epinephrine(Ep)through condensation reaction;aldehyde-based coumarin derivative was coupled with monoamine-type neurotransmitters(Histamine(HA),5-hydroxytryptamine(5-HT),DA,NE,glutamate(Glu))by aldimine condensation reaction;and positive-charged neurotransmitter(Acetylcholine(Ach))was attracted by the electron-rich cavity of CN-DFP5 through electrostatic interaction.Therefore,the designed CN-DFP5 supramolecular sensor generated different fluorescence response patterns under two-photon excitation,obtained the corresponding two-channel fluorescence response signals,and after principal component analysis(PCA)statistical processing,achieved high-throughput analysis of seven neurotransmitters.In addition,the supramolecular fluorescence array sensing successfully achieved imaging analysis and high-throughput differentiation of seven neurotransmitters in living neurons.(3)Based on the host-guest recognition strategy of multi-locus hydrogen bonding synergy,a fully functional cysteamine derivatized pillar[5]arene(AFP5)was designed as a supramolecular"hydrogen bonding trap"with domain-limiting effect and assembled with the fluorescent indicator CMSP to form a host-guest fluorescent probe(AFP5-CMSP)for the highly selective and accurate quantitative analysis of Glu.The linear range of AFP5-CMSP for Glu detection was 1.0-60?M,with a limit of detection(LOD)of 55.2±0.1 n M and a response time of 5.8 s.AFP5-CMSP was successfully applied to the real-time imaging and quantification of Glu in neuronal and brain tissues.It was found that depression induced an increase Glu levels in mitochondria and further activated the mitochondrial ASIC1a channel,leading to Ca²⁺overload in mitochondria.Further brain section imaging studies revealed that the dynamics of Glu and Ca²⁺levels following depression induction were different in different brain regions,being significantly higher in CA1 and S1BF regions than in other brain regions.