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

Abstract

EPR spectroscopy is an increasingly useful analytical tool to probe biomolecule structure, dynamic behaviour, and interactions. Nitroxide radicals are the most commonly used radical probe in EPR experiments, and many methods have been developed for their synthesis, as well as incorporation into biomolecules using site-directed spin labelling. In this Tutorial Review, we discuss the most practical methods for the synthesis of nitroxides, focusing on the tunability of their structures, the manipulation of their sidechains into spin labelling handles, and their installation into biomolecules.

Isoindoline‐Based Nitroxides as Bioresistant Spin Labels for Protein Labeling through Cysteines and Alkyne‐Bearing Noncanonical Amino Acids

“…In addition to such shielding effects by α‐substituents, it is known that nitroxides are more stable if they are embedded in pyrrolidines (Figure E, F) as compared to piperidines . Spin labels based on isoindolines (Figure A, B) have the potential to exhibit even higher stability than pyrrolidine‐based spin labels . Isoindoline‐based nitroxides have been used for RNA labeling and an improved stability was shown for tetraethyl‐shielded relative to tetramethyl‐shielded isoindoline‐labels .…”

In‐Cell EPR: Progress towards Structural Studies Inside Cells

“…By far, the largest family of spin labels in SDSL‐EPR spectroscopy are nitroxide radicals, with gadolinium(III) complexes and trityl radicals being interesting alternatives. One of the main advantages of nitroxide spin labels is their high sensitivity to the local environment, which, in the SDSL‐EPR approach, is used to obtain structural and dynamic information on the biomolecule on which the nitroxide is attached …”

“…In this manuscript, when possible, we will point up the reactive group properties of the depicted spin labels, but we will not discuss them in depth. For an exhaustive discussion we suggest an interesting and detailed review recently appeared …”

The Hunt for the Closed Conformation of the Fruit‐Ripening Enzyme 1‐Aminocyclopropane‐1‐carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study

“…Site‐directed spin‐labeling (SDSL) followed by electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful tool to probe the conformations and dynamics of proteins . This technique relies on the covalent insertion of one (or several) stable paramagnetic probe(s) at a selected site of a protein, often a cysteine residue, introduced by site‐directed mutagenesis.…”