Construction Of Two-photon Fluorescent Biothiol Probes:Study Of Fluorescence Imaging

Biothiols mainly include glutathione?GSH?,cysteine?Cys?,and homocysteine?Hcy?.They play an important role in many physiological processes.Thiol molecules contain a strong nucleophilic sulfhydryl group,which is easy to nucleophilic addition with unsaturated double bonds,coordinate with metal ions,and participate in redox reactions.At the same time,as an amino acid for constituting a protein,it is related to the cross-linking of disulfide bonds in proteins,and play a vital role in the formation of three-dimensional structures of proteins.In addition,the balance of the intracellular biothiol content is associated with environmental stability in the living body.Many reserach have shown that the disorder of intracellular thiol concentrations could lead to many diseases such as rheumatoid arthritis,Parkinson's disease,and Alzheimer's disease.Therefore,detecting the distribution and concentration of various biothiols in cells is of great significance for disease diagnosis and drug delivery.Given this in mind,the fluoroboron dipyrrole?BODIPY?with excellent photophysical properties was used to design fluorescent probe.The conjugate systems were enlarged by introducing an aromatic ring structure at the 3.5 position?through Knoevenagel Condensation?and the 8-position to achieve near-infrared emission.Three reactive type fluorescent probes BODIPY-diONs,Cz-BDP-NBD and NBD-Se were designed and synthesized.It has been characterized by nuclear magnetic resonance(¹H NMR,¹³C NMR),and high resolution mass spectrometry?HRMS?.Not only the thiol detection and dual-channel ratio imaging of probes in vivo and in vitro were explored,strong two-photon fluorescence intensity and under the excitation of near-infrared laser were also found.The details are as follows:In chapter.2,a NIR emission two-photon fluorescent probe for specific detection of GSH under physiological condition?BODIPY-diONs?based on BODIPY has been synthesized.One and two-photon experiments was carried out.By introducing dual quenching group 2,4-dinitrobenzenesulfonyl?DNBS?enlarge the different reaction rate of biothiols with probe.The probe has some merits as follows:faster response time,low detection and excellent anti-interference ability.Under the sapphire pulsed laser's 800 nm irradiation,the two-photon excited fluorescence?TPEF?of probe BODIPY-diONs was turned on in presence of GSH.Furthermore,the cell imaging proved that the probe BODIPY-diONs is suitable for the detection of GSH in physiological media.In chapter.3,on the basis of BODIPY,a NIR emission two-photon fluorescent probe?Cz-BDP-NBD?for biothiols was prepared.Compared with BODIPY-diONs,electron-donating group Carbazole located in the meso-position of BODIPY and the conjugation system at the 3,5-position have been extended by a Knoevenagel condensation with p-hydroxybenzaldehyde,forming a D-A-D multi-arm fluorophore,which enhanced the two-photon absorption cross sections of Cz-BDP-NBD.The response time was shorter than BODIPY-diONs and detection limit is more sensitive.Besides,Cz-BDP-NBD can distinguish GSH and Hcy/Cys through two emission channel.Above all,this probe has been successfully applied to fluorescence ratio imaging of endogenous biothiols through two emission channel.In chapter.4,A fluorescence probe NBD-Se for detection of the redox cycles between HClO/ClO-and GSH has been accomplished via an aromatic nucleophilic substitution reaction.The reaction was using tetrahydrofuran as a solvent,potassium carbonate as base stirred at room temperature.The reaction was complete within 5 minutes with yields up to99.0%.The probe NBD-Se has the advantages of ultra-fast response,excellent solubility,low cytotoxicity and good light stability.Most importantly,the probe could be reduced by glutathione in the living body after being oxidized by hypochlorous acid in a physiological environment to realize the detection of the redox state in a physiological environment.The identification mechanism of the probe was verified by mass spectrometry and TD/TD-DFT calculation.The probe has a detection limit of 7.60 nM for hypochlorite.In addition,the probes have been applied to human hepatoma cells and zebrafish to detect hypochlorite and glutathione balances in vivo.