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.

Keywords::

• nitroxide reduction
• aminoxyl radical
• radical stability
• spin labeling
• trityl radical

Acknowledgements

We thank Dr. S. Jonsdottir for assistance in collecting analytical data for structural characterization of compounds.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by the Icelandic Research Fund (120001021), the Deutsche Forschungsgemeinschaft (SFB 902, Molecular principles of RNA-based regulation) and by a doctoral fellowship to A. P. Jagtap from the University of Iceland Research Fund.

Supplementary material available online

Supplementary Figures 1–4 and Table I.