Design,Synthesis And Application Of Hemicyanine-Based Fluorescent Probes For Detection Of Small Bioactive Molecules

Compared with traditional detection methods,fluorescent probes have excellent properties such as simple operation,good selectivity,high sensitivity,low toxicity,real-time monitoring,and simple and economical.It has been used as a useful tool to facilitate the analysis of target components and has received increasing attention from researchers.To date,a wide variety of fluorescent probe molecules have been used in the field of pharmaceutical and environmental science.Intracellular pH is an important parameter for cell proliferation,function,and survival.Under normal physiological conditions,subcellular organelles must maintain a specific pH to maintain the individual characteristics of the biological process.For example,the lysosomes have an internal pH in the range of 4.5 to 5.5,which can stably maintain the optimal activity of hydrolase in the degradation of unwanted biomolecules such as proteins,nucleic acids,polysaccharides,lipids,and damaged organelles.In contrast,mitochondria require an alkaline environment(approximately pH 8.0)to maintain normal operation.Biothiols,such as cysteine(Cys),homocysteine(Hey)and glutathione(GSH),are involved in many biological processes and play a key role in maintaining proper redox status of biological systems.Cys and Hcy are important biomolecules required for detoxification,immunity,and growth and delay of cell and tissue aging in living systems,abnormal levels of Cys with slow growth,decolorization,liver damage,muscle and fat loss,skin damage and cancer,etc.Hcy has been implicated in various types of vascular and renal diseases and is considered a risk factor for cardiovascular diseases and Alzheimer's disease.Reduced glutathione(GSH)is the most abundant biomarker of intracellular non-protein thiol(1-10 m M)and oxidative stress.GSH has been shown to play a key role in controlling oxidative stress to maintain cells growth and functional redox homeostasis.Considering the important roles of biothiols as well as the pH values in the cellular biological and physiological process,the detection of biologically active molecules related to diseases is of urgent practical significance for the early diagnosis and preventive treatment of diseases.Herein,we firstly present a hemicyanine-based probe 1 for detecting pH changes in mitochondria during mitochondrial-associated autophagy.Due to the balance between phenol and benzoquinone,phenolic hydroxyl groups introduce dithizone fluorescence changes,which may change the conjugated system.Probe 1 exhibits strong orange fluorescence in alkaline medium,whereas the fluorescence emission becomes green when treated with an increasing acidity gradient.This probe selectively accumulates in the mitochondria and overlaps with the reference Mito-Tracker Green FM.More importantly,fluorescence imaging of cells deprived of nutrients showed that they can monitor changes in intracellular pH during mitochondria-associated mitochondrial autophagy.Therefore,this work provides a promising possibility to explore autophagy in cells,and it is expected that Probe 1 can serve as a useful tool for image organelles to respond to changes in pH during physiological and pathological processes.Next,we designed and synthesized the probe 2 with good photochemical and photophysical properties and favourable biocompatibility.The experimental results demonstrated that the cyanine-based probe 2 possessed highly selective for distinguishing Cys from the structurally and functionally similar to amino acids and thiols.The high specificity for Cys is based on their respective thioether adducts that influence the rate of kinetics of cyclization reactions in different molecules.In addition,probe 2 has been successfully applied to the detection of intracellular Cys by cellular fluorescence imaging of cancer cells.Thereofore,based on the characteristics of high selectivity and good biocompatibility,probe 2 would be of great practical value for biological applications.