The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site‐directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in‐cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide‐functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm, and shows high stability against reduction. Using this label, the guanine‐nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.

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.

Pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS) offers several methods for measuring dipolar coupling constantsa nd thus the distance between electron spin centers. Up to now,P DS measurements have been mostly applied to spin centers whose g-anisotropies are moderatea nd therefore have an egligible effect on the dipolar coupling constants. In contrast, spin centers with large g-anisotropy yield dipolarc oupling constants that depend on the g-values.I nt his case, the usual methods of extracting distances from the raw PDSd ata cannot be applied. Here, the effect of the g-anisotropyo nP DS data is studied in detail on the example of the low-spin Fe 3 + ion. First, this effect is described theoretically,u sing the work of Bedilo andM aryasov (Appl. Magn. Reson. 2006,3 0, 683-702) as ab asis. Then, two knownF e 3 + /nitroxide compounds and one new Fe 3 + /trityl compound were synthesized and PDS measurements were carried out on them using am ethod called relaxation inducedd ipolarm odulation enhancement (RIDME). Based on the theoretical results, aR IDME data analysis procedure was developed, which facilitated the extraction of the inter-spin distance and the orientation of the inter-spin vector relative to the Fe 3 + g-tensor frame from the RIDME data. The accuracyo ft he determined distances and orientationsw as confirmed by comparison with MD simulations. This methodc an thus be appliedt ot he highly relevant class of metalloproteins with, for example, low-spin Fe 3 + ions.[a] Dr.Supporting information and the ORCID identification number(s) for the author(s) of this articlecan be found under: https://doi.org/10.

Organic radicals are usually highly reactive and short-lived species. In contrast, tetrathiatriarylmethyl radicals, the so-called trityl- or TAM-radicals, are stable and do survive over longer times even under in-cell conditions. In addition, they show strong EPR signals, have long phase memory times at room temperature, and are reporters on local oxygen and proton concentrations. These properties facilitated their use for magnetic resonance imaging, dynamic nuclear polarization, and spin-labeling EPR under in-cell conditions. Thus, synthetic approaches are required for functionalization of TAM radicals tailored to the desired application. However, most TAM derivatives reported in the literature are based on esterification of the Finland trityl, which is prone to hydrolysis. Here, we report on an approach in which TAM is site-selective iodinated and subsequently C–C cross-coupled to various building blocks in a modular approach. This yields conjugated trityl compounds such as a trityl attached to a porphyrin, an alkinyl functionalized trityl radical, and a strongly exchange-coupled trityl biradical. This synthesis approach thus has implications not only for magnetic resonance spectroscopy but also for the design of molecular magnets or quantum computing devices.

Photogenerated multi‐spin systems hold great promise for a range of technological applications in various fields, including molecular spintronics and artificial photosynthesis. However, the further development of these applications, via targeted design of materials with specific magnetic properties, currently still suffers from a lack of understanding of the factors influencing the underlying excited state dynamics and mechanisms on a molecular level. In particular, systematic studies, making use of different techniques to obtain complementary information, are largely missing. This work investigates the photophysics and magnetic properties of a series of three covalently‐linked porphyrin‐trityl compounds, bridged by a phenyl spacer. By combining the results from femtosecond transient absorption and electron paramagnetic resonance spectroscopies, we determine the efficiencies of the competing excited state reaction pathways and characterise the magnetic properties of the individual spin states, formed by the interaction between the chromophore triplet and the stable radical. The differences observed for the three investigated compounds are rationalised in the context of available theoretical models and the implications of the results of this study for the design of a molecular system with an improved intersystem crossing efficiency are discussed.