Synthesis And Characterization Of Water-soluble Trityl Radicals

Objective:Tetrathiatriarylmethyl?trityl,TAM?radicals have found wide applications as spin probes/labels for electron paramagnetic resonance?EPR?spectroscopy and imaging.CT-03 and OX063 are two typical representatives of trityl radicals.The large-scale synthesis of CT-03 has been reported in the literature,so the structural modification and functional derivation of trityl radicals are mainly based on the parent core of CT-03.Nevertheless,the high hydrophobicity of CT-03 and its derivatives limits their biomedical and biophysical applications.For example,CT-03has strong hydrophobic interaction with proteins such as albumin.Moreover,due to the relatively high hydrophobicity of the TAM core and high pK_a?⁴.0?of its carboxylate groups,CT-03 is subject to self-aggregation or precipitation especially in the compartments with low dielectric constants?e.g.,biomembranes?.Therefore,CT-03 and its derivatives exhibit the in vivo toxicity,limiting the application of CT-03and its analogues in vivo.In addition,the high hydrophobicity of CT-03-based spin labels results in protein precipitation after labeling and reduces the accuracy of protein distance measurement.Therefore,the high hydrophilicity of trityl radicals is very important for its biomedical applications.Although trityl radical OX063 has high hydrophilicity,its synthesis has great challenges and has not been accomplished by the research community.So far,its synthetical methods have only been reported in patent literature and are not repeatable.In view of this,the aim of this dissertation is to develop new water-soluble trityl radicals.By optimizing the experimental conditions,the efficient synthesis of water-soluble trityl radicals has been realized.By comparing the physicochemical properties of CT-03 and OX063,the application potential of new water-soluble trityl radicals in magnetic resonance fields is clarified.Methods:This dissertation consists of two parts:?1?Aromatic monomers were synthesized from 1,2,4,5-tetra?tert-butyl thio?benzene via condensation,reduction and protection to the corresponding alcohols.The stable tert-butyl and TBS?tert-butyl dimethyl chlorosilane?ethers were selected as hydroxyl protection groups.Two routes were used to synthesize the key intermediate triphenylmethanol.Then,the triphenylmethanol triesters were synthesized by reaction with excessive dimethyl carbonate in the presence of n-BuLi.Finally,TFA was used to deprotect the four tert-butyl ether bonds and convert the trityl alcohol into the trityl carbocation.The trityl radical TFO was obatined by reduction of the carbocation using SnCl₂,followed by the hydrolysis by LiOH.The sensitivity of TFO to oxygen,albumin binding,oil-water partition coefficient,solubility,pKa and concentration-dependent EPR line broadening were studied by EPR and UV-Vis spectroscopy.?2?Accordin to the results from the first project,we synthesized the triarylmethanol derivative of OX063from the corresponding aryl monomer and optimized the experimental conditions:deprotonated reagents,chelating agents,reaction temperature,reaction time and equivalents.Results:?1?In this study,the efficient synthesis of the trityl radical TFO with tetrahydroxyl groups on the side chains was achieved.The results showed that the side chain substituents did not affect the oxygen sensitivity of TFO?4.2 mG/%O₂?.The low oil-water partition coefficient?logP,-1.17?,high solubility?154 mM in PBS?and relatively high self-aggregation concentration?3.0 mM?of TFO indicate its high water solubility and weak self-aggregation tendency.More importantly,the EPR signal of TFO did not change in the presence of high concentration BSA?1 mM?.The above results show that TFO has excellent physicochemical properties.?2?Based on results obtained from the synthesis of TFO,the optimized route for synthesis of the triarylmethanol derivative,the key intermediate of OX063,was studied.Conclusion:?1?In this study,a novel trityl radical TFO with high water solubility was synthesized.TFO retains narrow EPR linewidth,high oxygen sensitivity,no binding with albumin and low self-aggregation tendency.Therefore,TFO will have wide application potential in EPR and magnetic resonance-related fields.This study also provides a new strategy for the synthesis of new hydrophilic trityl radicals.?2?The key intermediate of 0X063 was synthesized,which provided the possibility for further efficient synthesis of OX063.