Resolving the nanometer-scale structure of biomolecules in natural conditions still remains a challenging task. We report the first distance measurement in nucleic acid at physiological temperature using electron paramagnetic resonance (EPR). The model 10-mer DNA duplex has been labeled with reactive forms of triarylmethyl radicals and then immobilized on a sorbent in water solution and investigated by double quantum coherence EPR. We succeeded in development of optimal triarylmethyl-based labels, approach for site-directed spin labeling and efficient immobilization procedure that, working together, allowed us to measure as long distances as ~4.6 nm with high accuracy at 310 K (37 °C).

Tris(2,3,5,6-tetrathiaaryl)methyl cations, which were generated from the corresponding triarylmethanols in the presence of strong acids, underwent reaction with nucleophiles to give trityl radicals, as the product of a one-electron reduction of the carbocation. Depending on the nature of the nucleophile, the only byproducts were either diamagnetic quinone methides or asymmetrical monosubstituted trityl radicals. Herein, we report a protocol for the large-scale synthesis of the Finland trityl, which has the advantage of high overall yield and reproducibility.

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.

Triarylmethyl radicals (trityls, TAMs) represent a relatively new class of spin labels. The long relaxation of trityls at room temperature in liquid solutions makes them a promising alternative for traditional nitroxides. In this work we have synthesized a series of TAMs including perdeuterated Finland trityl (D36 form) , mono-, di-, and tri-ester derivatives of Finland-D36 trityl, deuterated form of OX63, dodeca-n-butyl homologue of Finland trityl, and triamide derivatives of Finland trityl with primary and secondary amines attached. We have studied room-temperature relaxation properties of these TAMs in liquids using pulsed Electron Paramagnetic Resonance (EPR) at two microwave frequency bands. We have found the clear dependence of phase memory time (Tm~T2) on magnetic field: room-temperature Tm values are ~1.5-2.5 times smaller at Q-band (34 GHz, 1.2 T) compared to X-band (9 GHz, 0.3 T). This trend is ascribed to the contribution from g-anisotropy that is negligible at lower magnetic fields but comes into play at Q-band. In agreement with this, while T1~Tm at X-band, we observe T1>Tm at Q-band due to increased contributions from incomplete motional averaging of g-anisotropy. In addition, the viscosity dependence shows that (1/Tm-1/T1) is proportional to the tumbling correlation time of trityls. Based on the analysis of previous data and results of the present work, we conclude that in general situation where spin label is at least partly mobile, X-band is most suitable for application of trityls for room-temperature pulsed EPR distance measurements.