Synthesis And Bioimaging Application Of Aβ Protein Fluorescent Probes Based On Fluorescent Protein Chromophore

Alzheimer disease(AD)is an incurable,progressive neurodegenerative disorder that affects the health of tens of millions of people worldwide and also places a huge burden on families and society.Numerous attempts to decipher AD pathogenesis and discover effective treatments have all flopped,due to the facts that the pathogenesis of AD is not completely clear and AD patients often are not diagnosed until it is advanced.It is therefore critical to improve the diagnostic level of AD in the early stage.Green fluorescent protein consists of around 238 amino acids,it was isolated from Aequorea Victoria in the beginning and can emit green fluorescence under ultraviolet irradiation.The discovery and development of the green fluorescent protein provides us the possibility to get new insight of microscopic life.The structure of p-hydroxybenzylidine-imidazolinone(pHBDI),the fluorescent protein chromophore that causes fluorescent proteins to emit light,has also been attracting great attention from researchers.p-HBDI is a potential high-quality chemical fluorophore skeleton because of good biocompatibility,extremely low fluorescence background,large stokes shift,and good light stability.It can be chemically modified to be used for detection in various fields.In this dissertation,the fluorescent protein chromophore is used as a fluorescent template backbone for chemical modification,and Aβ peptide,the key peptide for early diagnosis and pathogenicity of AD,is used as the core target.Novel effective fluorescent probes based on the fluorescent protein chromophore for Aβ detection are developed and strategies for chemical modification of protein chromophores are studied.The primary contents of this thesis are as follows:(1)Based on the green fluorescent protein chromophore of p-HBDI,probe C25 was constructed to distinguish Aβ aggregates and lysosomal viscosities simultaneously and discriminately.Probe C25 is lysosome-targeting and can real-time monitor tiny variations of viscosity in lysosomes.It turns on fluorescence at 600 nm when imaging lysosomal viscosity.At the same time,probe C25 also has a good binding ability to Aβ aggregates.It can accurately image Aβ aggregates at the solution,cell and tissue levels.When detecting Aβ aggregates,it turns on fluorescence at 570 nm.Probe C25 also has the advantages of low fluorescence background,good light stability,low biological toxicity,and strong anti-interference ability,which provides a powerful tool for the early diagnosis and the development of new treatment methods of AD.(2)An anti-Kasha’s rule fluorescent protein chromophore derivative probe C17 with dual fluorescence emission was found.The molecule has two absorption peaks at 430 nm and 530 nm in different solvents.Probe C17 emits two fluorescent bands with maximum emission wavelengths at 480 nm and 620 nm.It can be utilized to detect Aβ oligomers in the green light region and Aβ oligomers and Aβ aggregates in the red light region.This probe can not only be used for the detection of Aβ oligomers,but also the qualitatively evaluation the relationship between Aβ oligomers and Aβ aggregates and AD,owning to its dual fluorescence emission properties.(3)The probe B2 was designed and synthesized based on the conformationally-locked fluorescent protein chromophore for the detection of Aβ oligomers.The design inspiration of B2 was from BD-Oligo,a reported Aβ oligomer probe.Conformationally-locked fluorescent protein chromophore was utilized to replaced BODIPY structure of BD-Oligo to improve optical imaging performance.Probe B2 has high binding ability to Aβ oligomers,and can sensitively and accurately detect Aβ oligomers in biological systems.(4)By replacing the phenolic hydroxyl group of p-HBDI with dimethylamino and introducing three electron-withdrawing groups into imidazolinone end,a total of six fluorescent probes in X series were obtained.The ultraviolet and fluorescence spectra revealed that strengthening electron donating ability of the phenolic hydroxyl position and improving the electron withdrawing ability of the imidazolinone end lead to a large redshift.Although no fluorescent probes targeting Aβ protein aggregates were found in tube test,further modification and optimization of this series of probes are still on-going in the laboratory.