title: Sterically shielded spin labels for in-cell EPR spectroscopy: analysis of stability in reducing environment.
abstract: Electron paramagnetic resonance (EPR) spectroscopy is a powerful and widely used technique for studying structure and dynamics of biomolecules under bio-orthogonal conditions. In-cell EPR is an emerging area in this field; however, it is hampered by the reducing environment present in cells, which reduces most nitroxide spin labels to their corresponding diamagnetic N-hydroxyl derivatives. To determine which radicals are best suited for in-cell EPR studies, we systematically studied the effects of substitution on radical stability using five different classes of radicals, specifically piperidine-, imidazolidine-, pyrrolidine-, and isoindoline-based nitroxides as well as the Finland trityl radical. Thermodynamic parameters of nitroxide reduction were determined by cyclic voltammetry; the rate of reduction in the presence of ascorbate, cellular extracts, and after injection into oocytes was measured by continuous-wave EPR spectroscopy. Our study revealed that tetraethyl-substituted nitroxides are good candidates for in-cell EPR studies, in particular pyrrolidine derivatives, which are slightly more stable than the trityl radical.
authors.affiliation: University of Iceland, Department of Chemistry, Science Institute , Reykjavik , Iceland.
authors.forename: A P
authors.initials: AP
authors.lastname: Jagtap
authors.forename: I
authors.initials: I
authors.lastname: Krstic
authors.forename: N C
authors.initials: NC
authors.lastname: Kunjir
authors.forename: R
authors.initials: R
authors.lastname: Hänsel
authors.forename: T F
authors.initials: TF
authors.lastname: Prisner
authors.forename: S Th
authors.initials: ST
authors.lastname: Sigurdsson
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005609:Free Radicals / Q000737:chemistry*; D010084:Oxidation-Reduction; D013113:Spin Labels; D013237:Stereoisomerism
chemical_list: D005609:Free Radicals; D013113:Spin Labels
keywords: aminoxyl radical; nitroxide reduction; radical stability; spin labeling; trityl radical