Synthesis And Characterization Of Monosubstituted Trityl Radicals

Objective:Triarylmethyl（trityl）radicals and their derivatives have been widely used for measurement of reactive oxygen species such as superoxide anion radical(O₂^·-)and assessment of tissue microenvironment（e.g.,p H,viscosity,intracellular O₂ levels）and can act as spin labels and hyperpolarizing agents due to their high biostability,sharp EPR singlet signal and long relaxation times.Although monosubstitution at the para position of trityl radicals is the main strategy for their derivatization,there is no report regarding substituent effect on their redox properties.In this work,several para-monosubstituted trityl radicals with different electron-withdrawing/donating groups were synthesized.Then,the reactions of these new trityl radicals and CT-03with various oxidoreductants including glutathione（GSH）,ascorbic acid（Asc）and O₂^·-were investigated.Moreover,in order to further interpret the substituent effect on the redox propreties of these trityl radicals,their redox potentials were also measured in phosphate buffer.This work will provide new routes for development of new para-monosubstituted trityl radicals with improved properties and further expand their biomedical and biophysical applications.Methods:Through amidation,esterification,triaryl carbanion nucleophilic substitution,Suzuki-Miyaura coupling and Sonogashira-Hagihara coupling reactions,five para-monosubstituted trityl radicals were synthesized,including electron-withdrawing ester/amide groups,neutral thioether group,electron-donating benzylamine group and conjugated phenyl group.Then,these compounds were separated and purified by column chromatography,recrystallization,high performance liquid chromatography（HPLC）and other methods.Thereafter,the molecular structure and physicochemical properties were well characterized by NMR,UV-Vis,cyclic voltammetry,and EPR.Moreover,the reactivity of these para-monosubstituted trityl radicals with GSH,Asc and O₂^·-were investigated and their redox potentials were measured by cyclic voltammetry.Finally,based on the above results,novel trityl-based biradicals were developed as O₂^·-probes.Results:（1）We have obtained several para-monosubstituted trityl radicals with various electron-withdrawing/donating groups,including ET-01（ester）,AT-01（amide）,TSA（thioether）,TGA（benzylamine）and TPA（phenyl）.（2）The redox properties of these trityl radicals are significantly different from each other.TPA is almost inert to GSH and Asc and exhibits excellent stability towards the reduction reactions,while TSA and TGA have good reactivity to O₂^·-.（3）The reduction stability experiments demonstrate that GSH is the main factor controlling the reduction stability of trityl radicals,while the effect of Asc is negligible.（4）The half-life of TPA in cell lysate is up to 24 hours,showing great potential for intracellular application.（5）The trityl-based biradicals can be used for the O₂^·-detection by measuring the increase of EPR signal intensity.Conclusions:This thesis aims at solving the issues of monosubstituted asymmetric trityl radicals in chemical synthesis and biological applications.The following results have been obtained:（1）In terms of synthesis,the new methods for the synthesis of monosubstituted trityl radicals have been developed,with easy purification,relatively high yield and good repeatability,which enables large-scale synthesis of mono-substituted trityl radicals;（2）The phenyl-substituted trityl radical TPA has excellent reduction stability,providing a new strategy for the development of stable trityl radicals;（3）GSH is the key factor controlling the bioreduction of trityl radicals.This work provides a new protocol for evaluation of the biostability of trityl radicals.（4）Moreover,our experiments have confirmed that a new detection strategy based on the signal growth mechanism can be achieved by using trityl-based biradicals as EPR probes(e.g.,O₂^·-probe).