Sterically shielded spin labels for in-cell EPR spectroscopy: Analysis of stability in reducing environment

TLDR

EPR spectroscopy is widely used to study biomolecule structure and dynamics, but in‑cell EPR is limited by the reducing cellular environment that converts nitroxide spin labels to diamagnetic forms. The study aimed to identify radicals most suitable for in‑cell EPR by systematically evaluating substitution effects across five radical classes, including piperidine, imidazolidine, pyrrolidine, isoindoline nitroxides, and the Finland trityl radical. Thermodynamic reduction parameters were measured by cyclic voltammetry, while reduction rates in ascorbate, cellular extracts, and oocyte injections were monitored using continuous‑wave EPR spectroscopy. Tetraethyl‑substituted nitroxides, especially pyrrolidine derivatives, proved to be the most stable candidates for in‑cell EPR, outperforming the trityl radical.

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.