Construction Of Molecular Fluorescent Probes Based On New Recognition Groups

Small molecule biothiols such as cysteine(Cys),homocysteine(Hcy),and glutathione(GSH)play many crucial roles in biological systems.For example,Cys deficiency is involved in many syndromes such as skin lesions,liver damage,edema and slow growth in children.At elevated levels in plasma,Hcy is a risk factor for Alzheimer's disease,folate and cobalamin(vitamin B12)deficiencies,and cardiovascular diseases.Plasma total Hcy concentrations have also been linked to birth defects and cognitive impairment in the elderly.GSH serves many cellular functions,including maintenance of intracellular redox activities,xenobiotic metabolism,intracellular signal transduction,and gene regulation.Moreover,its level is known to be directly linked to many diseases,including cancer,Alzheimer's,and cardiovascular disease.Thiophenol,as a kind of very useful organic compound,plays a vital role in organic synthesis and is widely used in preparing pharmaceuticals,agrochemicals,and various industrial products.However,thiophenol is a kind of highly toxic and pollutant compound.In addition,prolonged exposure to thiophenol can cause a series of serious health problems,central nervous system damage,increased respiration,muscle weakness,hind limb paralysis,and even death.Being a strong oxidant and good nucleophile,Peroxynitrite(ONOO-)can react with different biomolecules,including proteins,transition-metal-containing enzyme centers,lipids,and nucleic acids,eventually contributing to cell death.Meanwhile,ONOO-has been implicated in a growing list of diseases,such as cardiovascular diseases,neurodegenerative diseases,diabetes,and cancer.Moreover,ONOO-is also reported to display protective activities in vivo by functioning as a cytotoxic effector against invading pathogens and as a signaling molecule.In view of the physiological functions of thiols and ONOO-as well as the physiological toxicity of thiophenol,monitoring their concentrations in the living system is very important for understanding the functions/toxicity mechanism and the pathogenesis of related diseases.Fluorescence imaging based on small-molecule fluorescent probes has emerged as an efficient methodology to visualize the spatial and temporal distribution of biomolecules in biological specimens due to its real-time,sensitive,and noninvasive characteristics.A typical fluorescent probe is constructed by incorporating the specific recognition unit into a classic fluorophore platform,such as cyanine,fluorescein,and resorufin,etc.Generally,the recognition unit mainly accounts for the reactivity and specificity of fluorescent probes.Therefore,we expect to develop a high sensitive and selective molecular fluorescent probes with new recognition unit for qualitative and quantitative detection of the above active species.This article mainly includes the following two aspects:(1)We implemented a developmental program to identify novel reaction-based fluorescent probes via a combinatorial strategy.In this process,pyrimidine moieties were investigated and optimized as unique recognition units for thiols for the first time through a parallel synthesis in combination with rapid screening process.As a result,Res-Biot and Flu-Pht were identified as optimal fluorescent probes for biothiol and thiophenol respectively.Their favorable characteristics and superior applicability have been well demonstrated in both chemical and biological contexts.In particular,Res-Biot enables the direct visualization of biothiol fluctuations during oxidative stress and cell apoptosis.Meanwhile,Flu-Pht is competent to visualize thiophenols without the interference from endogenous biothiols in living cells.(2)In this section,we identify the isatin moiety as a novel recognition unit specific for ONOO-.Integration of this unique moiety into naphthalene fluorophore afforded a fluorescence “off-on” probe,BNI-PN,for highly specific and sensitive detection of ONOO-.BNI-PN exhibited weak fluorescence emission at 445 nm.When ONOO-was added,the emission wavelength shifted to 498 nm,and the fluorescence intensity enhanced.The probe selectively reacts to ONOO-,while the other active molecules in cells,such as ROS,RNS and RSS do not.In addition,taking advantage of the two photon emission property of BNI-PN,endogenous ONOO-in living cells were visualized by BNI-PN by a two-photon fluorescent microscope.In this paper,three new fluorescent probes for the detection of thiols,thiophenol and ONOO-were developed by using new recognition groups.And the selectivity,sensitivity,response mechanism and biological application of the probes were studied deeply.This study provides robust analytical tools for the corresponding biomolecules for bioanalytical and biomedical applications.