Design,Synthesis And Application Of Fluorescent Probes For ?-Glutamyl Transpeptidase And Vicinal Dithiol-Containing Proteins

?-Glutamyl transpeptidase?GGT?is an essential enzyme that primarily cleaves?-glutamyl groups in glutathione?GSH?or other?-glutamyl species,regulating intracellular GSH and cysteine?Cys?has an irreplaceable role in homeostasis,and its abnormal elevation is associated with various diseases such as cancer.However,most of the current probes are designed using a fluorescent light source with a sho rt excitation wavelength.Therefore,the development of a new long-wavelength fluorescent probes to detect and visualize mitochondrial GGT activity can provide more localized information to elucidate its physiological and pathological roles.Vicinal dithiol-containing proteins?VDPs?are key regulators of mitochondrial redox homeostasis,and mitochondrial redox balance is achieved primarily through interaction with reactive oxygen species and nitrogen.VDPs are also critical linking centers for stabilization of protein structures and have close association with protein functions.However,current probes have limited sensitivity and responsiveness to mitochondrial VDPs.Therefore,the development of a new fluorescent probe to detect and track VDPs,especially in mitochondria,is great significance for diagnosing redox-related diseases and elucidating their pathophysiology.This work mainly covers the following two parts:1.In view of the light damage caused by the short excitation wavelength of the traditional GGT fluorescent probe and the high autofluorescence background,we used the near-infrared amino semi-anthocyanin derivative?Bcy-NH₂?as the fluorophore and mitochondrial targeting group and designd the GGT fluorescent probe?Mito-Bcy-GGT?with near-infrared excitation and emission.Then the absorption spectrum and fluorescence spectrum were analyzed and the response of the probe to different analytes was screened under simulated physiological conditions in vitro.The results showed that the probe has a longer emission wavelength and has a good choice for GGT.However,other biomolecules do not substantially affect their fluorescent properties.It is used to display the activity of mitochondrial GGT in living cells because of its high sensitivity and spec ificity and strong penetrating ability.2.A new environmentally sensitive dye?ESD?was synthesized from1-?2-carboxyethyl?-2,3,3-trimethyl-3H-indol-1-ium/7-?dimethylamino?benzo[c][1,2,5]oxadiazole-4-carbaldehyde.ESD has an electron donor and acceptor structure that produces highly dipolar and distorted excited states with sensitive fluorescence responsiveness to microenvironmental viscosity and polarity.Based on the good responsiveness of ESD,we designed a new mitochondrial VDPs fluorescent probe?AMVF?with ESD as the fluorescent vector.Then,absorption spectroscopy and fluorescence spectroscopy were carried out and the response of the probe to different analytes was screened under simulated physiological conditions in vitro.The results showed that the probe exhibited specific mitochondrial targeting and red shift emission capability.It is also highly sensitive to reduced bovine serum albumin,which,to our knowledge,is the first VDPs fluorescent probe to display NIR emission,transient response,high on-fluorescence ratio and specific mitochondrial localization.