PEGylation Effect Of Trityl Radical And Its Functionalization As Selective Thiol Probe

Objective:Recently,tetrathiatriarylmethyl(trityl)radicals have attracted considerable attention as spin probes for biological electron paramagnetic resonance(EPR)spectroscopy and imaging owing to their high biostability,sharp EPR singlet signals,high sensitivity,long relaxation times(> 10 ?s at 25 oC under anaerobic conditions).Much effort has focused on the optimization and functionalization of trityl radicals.For example,trityl radicals have been structurally modified to expand their applications for measurement of physiological parameters such as superoxide anion radical(O2·-),p H,thiol levels,as well as total redox status.Most recently,these radicals and their derivatives also have shown great promise as spin labels for distance measurements in proteins and nucleic acids by pulsed EPR spectroscopy at physiological temperature and as efficient polarizing agents for high-field dynamic nuclear polarization.Despite the fact that trityl radicals have excellent properties allowing for their wide applications in various research fields,their instability towards some oxidoreductants,short blood circulation lifetime and strong binding with albumins still limit their in vivo applications.Moreover,their applications for different physiological parameters are far from being optimized in terms of sensitivity and selectivity.Based on the above considerations,this thesis aims to improve the biocompatibility of trityl radicals and expand their applications for measurement of biothiols with enhanced sensitivity and specificity.Methods:(1)Instead of the previously reported divergent method,we utilized a completely convergent method,to efficiently synthesize PEGylated derivatives of the Finland trityl radical(CT-03)that contain different m PEG chain lengths(MW 350-2000)and numbers(3/9 chains).The physicochemical properties of these PEGylated trityl radicals including self-aggregation concentration,hydrophobicity,O2 sensitivity and binding with bovine serum albumin were investigated and compared.Their stability towards various oxidoreductants as well as in rat liver homogenate and whole blood were also studied.In addition,the deuterated derivatives d-TNP1 and d-TNP3 were also synthesized.The in vivo biocompatibility,pharmacokinetics of d-TNP3 and its application potential as oximetry probes were also evaluated in mice.(2)A sulfhydryl-reactive trityl methanethiosulfonate-MTST was synthesized through four steps.MTST can react rapidly with thiol compounds(e.g.,GSH and Cys)and the resulting conjugates exhibit distinct EPR spectra.Upon computer simulation of these EPR spectra,the characteristic EPR parameters such as composition,the corresponding hyperfine splitting constant and chemical exchange rate were obtained for each conjugate,and then these parameters can be used to identify these biothiols.Results:(1)PEGylation exerts an important effect on the physicochemical properties of trityl radicals.The dendritic PEGylated trityl radicals exhibit better water solubility,less susceptible self-aggregation,higher stability in various systems than the linear PEGylated analogues while both types of PEGylated trityl radicals share similar and negligible BSA binding properties.Moreover,the dendritic PEGylated trityl radicals are stable towards various biological oxidoreductants as well as in rat liver homogenate and whole blood.The deuterated derivatives d-TNP1 and d-TNP3 have sharp EPR singlet signals and high O2 sensitivity.Importantly,d-TNP3 exhibits excellent in vivo properties in mice including fast uptake,good biocompatiblity and prolonged kinetics with half-life time of > 10 hours.(2)The conjugates of MTST with GSH and Cys existed significantly different EPR spectra.The corresponding characteristic EPR parameters obtauned by computer simulation provide a theoretical basis and an effective method for the specific detection of different biothiols.Conclusion: The dendritic PEGylated trityl radicals exhibit excellent EPR properities including sharp EPR singlet signals and high O2 sensitivity,good biocompatiblity and enhanced in vivo applications.A methanethiosulfonate trityl radical shows distinctively distinct EPR fingerprints after reacting rapidly with different thiol compounds.The corresponding EPR parameters of those EPR spectra provide a theoretical basis and an effective method for the specific detection of different biothiols.Therefore,our present study provides a novel strategy for the development of new trityl radicals with improved properties,expanding biomedical applications of EPR spectroscopy and imaging.