Fluorescent Probe For Synergistic Detection Of Monoamine Oxidase And Its Oxidative Stress

As new biomarkers,monoamine oxidases（MAOs）play important roles in maintaining the homeostasis of biogenic amines which can catalyze the oxidation of biogenic amines to corresponding aldehydes with the generation of reactive oxygen species（ROS）.MAOs have two isoforms,MAO-A and MAO-B.MAO-A is considered to be a major factor of neuropsychiatric and depressive disorders.However,MAO-B is thought to be involved in several neurodegenerative diseases,such as Parkinson’s disease（PD）,Alzheimer’s disease（AD）and amyotrophic lateral sclerosis（ALS）.The changes of MAOs level in serum has been used to diagnose the liver disease in clinically.Therefore,to explore their distinct roles in different diseases,the selective detection of MAOs is essential.Herein,two new types of near-infrared（NIR）fluorescent probes,MitoCy-NH₂and MitoHCy-NH₂ are provided for ratiometric monitoring the levels of MAO-B and synergistic imaging of MAO-B and its contribution to oxidative stress in cells and in mice models.MitoCy-NMe₂ and MitoHCy-NMe₂ are used to monitor the MAOs activity and synergistic detection of MAOs and their oxidative stress in the cell and mouse models.These probes are composed of three moieties:heptamethine cyanine as fluorophore,substrate analogs as recognition group,and triphenylphosphonium cation as mitochondrial targeting group.The amine oxidation andβ-elimination reaction will release the fluorophore and then introduce an obvious fluorescence increase.And the color changes from green to blue.We have not only employed the photo-physical properties of fluorescent probes in vitro,but also defined its ability to detect in vivo.This paper is divided into the following sections:1.The synthesis and design of the probes.The methods and steps for the synthesis of fluorescent probes are described in detail and their structures are identified by nuclear magnetic resonance spectrum and mass spectrometry.2.The photo-physical properties,kinetics of enzymatic reaction and selectivity of the fluorescent probes were determined.Find the suitable experimental conditions in vivo.The detection of optimal excitation and emission wavelengths,the most suitable reaction conditions,and the reaction time were determined,providing a theoretical basis for subsequent biological experiments.3.The biological applications of fluorescent probes.（1）We evaluated the localization ability of the probes in the cell and the results suggested that our four probes can well locate on the mitochondria;（2）Cellular inhibitor experiments were carried out to verify the selectivity of the probes and our fluorescent probes had a good selectivity and can specifically detected the target product;（3）MitoCy-NH₂ and MitoHCy-NH₂ can offer the change of MAO-B levels and its contribution of ROS in H₂O₂-induced cell aging model and replicative senescence model and the results demonstrated that with the increase of aging,the level of MAO-B and oxidative stress were increased;（4）The activity change of MAO-B in aging mouse models were assessed by MitoCy-NH₂ and also evaluated therapeutic effects of pargyline and selegiline in mice models;（5）MitoCy-NMe₂ and MitoHCy-NMe₂ can provide the levels of MAOs in different types of liver cells and LPS-induced inflammatory liver cell.