| Objectives: Studies on structure and dynamic of proteins are crucial for understanding their physiological functions and interpreting the related mechanisms.The currently available techniques for structural determination of proteins mainly include X-ray crystallography,nuclear magnetic resonance(NMR)spectroscopy,cryo-electron microscopy(EM),and electron paramagnetic resonance(EPR).EPR coupled with the site-directed spin labeling(SDSL)method has been an indispensible technique to investigate the structure and dynamic of proteins,especially at physiological conditions.The principle of SDSL-EPR is based on the specific tag of spin labels on two cysteine groups of protein,followed by measuring the magnitude of dipolar-dipolar interaction between two spin labels by pulsed EPR spectroscopy.Then,the distance between these two cysteines is further extracted which contains the structural and dynamic information of protein.Among the available spin labels,triarylmethyl(trityl)radicals have accepted wide attention in the field of spin labeling owing to their outstanding EPR properties,long relaxation times and high biostability.However,their applications were limited by their relatively high hydrophobicity,relatively flexible linkage,low labeling efficiency and susceptibility of the resulting disulfide conjugates to thiols which are rich in cells.Based on these considerations,in the present thesis,we synthesized a series of Michael acceptor-based trityl spin labels.The reactivity and selectivity of these trityl spin labels to cysteine as well as the stability toward various oxidoreductants of the resulting disulfide conjugates with cysteine were studied.In addition,the effect of the electron-withdrawing substitutens on their spin labeling properties was also explored.Methods: The aldehyde-containing triarylmethanol was synthesized from 1,2,4,5-Tetra-tert-butylthiobenzene through 5 steps including ketalization,reduction and oxidation reactions.This triarylmethanol was further converted into the spin labels with various Michael acceptors by Wittig or Henry reactions,followed by acid treatments.The resulting spin labels were well characterized by EPR and the corresponding hyperfine splitting constants were also obtained by simulating their EPR spectra.The reactivity and selectivity of these trityl spin labels to thiol compounds as well as the stability toward various oxidoreductants of the resulting disulfide conjugates were studied by UV-Vis and EPR.Results: In this thesis,four spin label precursors with different Michael acceptors were synthesized and efficiently transformed by optimizing the synthetic methods into the corresponding spin labels.The Z and E stereoisomers of two spin labels were separated by HPLC.EPR results showed that the Z and E stereoisomers exhibited notably different hyperfine splitting constants.The electron-withdrawing substituents in the Michael acceptors exerted a crucial effect on the reactivity of these spin labels and only the nitro-substituted NMA had high reactivity to thiol compounds.UV-Vis and EPR results demonstrated that NMA was highly selective to thiols and the labeling efficiency between them was more than 97%.Moreover,the resulting conjugate was highly stable toward various oxidoreductants.Interestingly,UV-Vis and EPR spectra had distinct changes before and after the reaction of NMA with thiols,thus allowing these techniques to readily monitor the spin labeling reactions.Conclusions: Michael acceptor-based spin labels were designed and synthesized.The nitro-substituted spin label NMA was selected out which had high reactivity and selectivity to thiols.As a spin label,NMA overcame some issues of the currently available spin lablels and exhibited the following advantages: high labeling efficiency,short linker.Thus,NMA shows great potenital in the field of SDSL-EPR.Our present study opens up a new pathway for design and synthesis of novel spin labels with improved properties and would expand the applications of SDSL-EPR in structural and dynamic studies of proteins. |
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