Construction Of Small Molecular Fluorescent Probes For Norepinephrine Sensing And Imaging

Neural signaling in the brain consists of both electrical and chemical signaling,the latter of which relies on the release,transmission,and binding processes of neurotransmitters between synapses on time scales as low as milliseconds.Therefore,monitoring the dynamics of neurotransmitters at the molecular scale is essential for understanding the structure and function of neurons and the brain.Among them,norepinephrine(NE)is one of the key neurotransmitters in the vertebrate central nervous system,and its abnormal levels and transmission are closely associated with a range of neurodegenerative diseases,including Parkinson’s disease(PD),Alzheimer’s disease(AD),and depression.Although the importance of NE in various physiological and pathological processes is widely recognized,its highly specific,spatially,and temporally resolved dynamic monitoring remains a daunting challenge.Therefore,there is an urgent need to develop effective and sensitive methods to specifically detect NE.This thesis focuses on the key scientific issues of achieving high specificity,high sensitivity,and especially,high spatiotemporal resolution for monitoring the NE dynamics,and subsequent studies were conducted.In Chapter 1,the characteristics of neurotransmitters,especially norepinephrine,and their important role in the organism,as well as the association with various psychiatric and neurodegenerative disorders,are introduced.And the importance of analytical assays for norepinephrine is emphasized.Several common assays are then reviewed and their respective advantages and disadvantages are commented on.Some difficulties and challenges in the current norepinephrine assay are analyzed,and finally,the solutions and strategies of this thesis are proposed.In Chapter 2,a novel small molecular fluorescent probe BPS3 is designed and synthesized,bearing two phenyl pyridiniums linked by an alkyl chain,which guaranteed good water solubility and a flexible molecular conformation.The most noteworthy feature of the developed probe BPS3 is its ultrafast reaction kinetics,enabling NE detection within an impressively short time down to 100 ms,which is the most rapid small molecular probe reported by far for quantitative NE sensing and imaging.Through the combination of experimental and theoretical analyses,we revealed a unique dual acceleration mechanism,namely,molecular conformational folding and water bridging,which significantly reduced the activation energy of the reaction and increased the reaction rate by more than 10~5 and 10~3 times,respectively.Finally,we applied probe BPS3 to real-time imaging of NE fluctuations in living neurons under external chemical stimulation.Moreover,by two-photon fluorescence imaging of acute mouse brain tissue slices,we revealed the close correlations between downregulated NE levels and AD pathology as well as antioxidant therapy.In Chapter 3,a new small molecular fluorophore SPC is designed and synthesized,which bears a methiol substituted phenyl pyridinium and a S-p-tolyl thiocarbonate phenyl substituted pyridinium linked by a flexible alkyl chain.By introducing a host macrocycle cucurbit[8]uril(CB[8]),a supramolecular host-guest complex is self-assembled in aqueous solution,resulting in dual emission fluorescence.Therefore,a ratiometric fluorescence response is generated toward NE,which ensures accurate quantification of NE in living systems.The probe has a linear detection range of 0-400 nM and a detection limit of 1.2 nM,as well as ultra-fast response kinetics capable of detecting NE within 180 ms.Furthermore,the probe is applied to the ratiometric fluorescence imaging of NE releasing in living neurons under external chemical stimulation,which may help to reveal the physiological and pathological functions of NE.