Exchange Phenomena in the Electron Paramagnetic Resonance Spectra of the Nitroxyl and Trityl Radicals: Multifunctional Spectroscopy and Imaging of Local Chemical Microenvironment

Abstract

This Feature overviews the basic principles of using stable organic radicals involved in reversible exchange processes as functional paramagnetic probes. We demonstrate that these probes in combination with electron paramagnetic resonance (EPR)-based spectroscopy and imaging techniques provide analytical tools for quantitative mapping of critical parameters of local chemical microenvironment. The Feature is written to be understandable to people who are laymen to the EPR field in anticipation of future progress and broad application of these tools in biological systems, especially in vivo, over the next years.

Site Selective and Efficient Spin Labeling of Proteins with a Maleimide-Functionalized Trityl Radical for Pulsed Dipolar EPR Spectroscopy

“…Since the introduction of 5 [46] many synthesis improvements [33,47,48,49,50] and derivatization strategies [44,49,50,51,52,53,54,55,56,57,58,59] as well as applications of trityl compounds in medicinal probing [60,61], imaging [62,63], as magnetic materials [64], and as spin labels in structural biology [28,33,35,36,37,43,44,45,65] have been reported. Recent examples for the trityl labeling of cysteine residues in proteins used butene ( 4 , Figure 1) [33] or methanethiosulfonate ( 6 and 7 , Figure 2) [33,35,36] derivatives of 5 to establish the bioconjugation via thioether bonds for in cell studies or via disulfide bonds for in vitro studies, respectively.…”

<em>In Vivo</em> EPR Assessment of pH, <em>p</em>O₂, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

“…Application of the particulate probe enables repeated measurements of tissue p O 2 for up to weeks after implantation 14,21 . Soluble probes demonstrate sensitivity to an extended number of the parameters beyond oxygen concentration 25 and provide an opportunity for spatial-resolved measurements 26 . In particular, an application of the multifunctional HOPE probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, p O 2 , and Pi in extracellular space.…”

Advances in the synthesis of nitroxide radicals for use in biomolecule spin labelling

“…8,10 The nitroxide is also influenced by local environment effects such as pH (which can lead to different protonation states of nitroxides such as 3), or oxygen concentration (due to spin exchange processes). 12 In pulsed EPR experiments such as DEER, the maximum measurable distance, and the resolution of the distance distributions, is limited by a parameter known as the phase memory time Tm, which should be as high as possible. This is the main reason DEER experiments are typically performed at cryogenic temperatures, and a major challenge for biomolecular applications of EPR spectroscopy is the development of spin labels that exhibit sufficiently long Tm values to enable measurements at room temperature.…”