gem‐Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility

Abstract

The availability of bioresistant spin labels is crucial for the optimization of site‐directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins’ fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin‐labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide‐ and maleimide‐functionalized spin labels based on the gem ‐diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem ‐dimethyl analogs by site‐directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the “true” biocompatibility of the gem ‐diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.

2-Butyl-2-tert-butyl-5,5-diethylpyrrolidine-1-oxyls: Synthesis and Properties

“…Nitroxides are broadly used as molecular probes and labels in biophysics, structural biology, and biomedical research [1][2][3][4][5][6]. Modern trends in these research fields, such as in-cell electron paramagnetic resonance (EPR)/pulsed electron-electron double resonance (PELDOR) experiments [7,8] and in vivo NMR and EPR imaging [9,10], require stable paramagnetic agents that can retain the radical center in live cells or tissues long enough for the measurements. Resistance of a nitroxide group bearing an unpaired electron to chemical reduction with low-molecular-weight antioxidants and enzymatic systems is crucial for these applications.…”

Site-Directed Spin Labeling of RNA with a Gem-Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability

“…In this regard, the usually used gem-dimethyl nitroxides have the disadvantage of being reduced within minutes. In contrast, gem-diethyl nitroxide or nitroxides with sterically even more demanding groups in the gem-position reveal increased redox stability [27,[40][41][42][43][44][45][46]]. Yet, protocols for spin-labeling DNA [47] or RNA [27] with gem-diethyl isoindoline nitroxides have been reported only rarely.…”

Unexpected one-pot formation of the 1H-6a,8a-epiminotricyclopenta[a,c,e][8]annulene system from cyclopentanone, ammonia and dimethyl fumarate. Synthesis of highly strained polycyclic nitroxide and EPR study

“…Sterically hindered nitroxides have high chemical stability [5] and can be used as spin labels to study biopolymers in cells [6]. The introduction of spirocyclic moieties has a smaller effect on the reduction rates of nitroxides than does the introduction of linear alkyl substituents.…”