Ox‐SLIM: Synthesis of and Site‐Specific Labelling with a Highly Hydrophilic Trityl Spin Label

The combination of pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) with site‐directed spin labelling is a powerful tool in structural biology. Rational design of trityl‐based spin labels has enabled studying biomolecular structures at room temperature and within cells. However, most current trityl spin labels suffer either from aggregation with proteins due to their hydrophobicity, or from bioconjugation groups not suitable for in‐cell measurements. Therefore, we introduce here the highly hydrophilic trityl spin label Ox‐SLIM. Engineered as a short‐linked maleimide, it combines the most recent developments in one single molecule, as it does not aggregate with proteins, exhibits high resistance under in‐cell conditions, provides a short linker, and allows for selective and efficient spin labelling via cysteines. Beyond establishing synthetic access to Ox‐SLIM, its suitability as a spin label is illustrated and ultimately, highly sensitive PDS measurements are presented down to protein concentrations as low as 45 nm resolving interspin distances of up to 5.5 nm.