Application of W-band 19F electron nuclear double resonance (ENDOR) spectroscopy to distance measurement using a trityl spin probe and a fluorine label

Recently, Marina Bennati and coworkers (M. Bennati et al., Angew. Chem., Int. Ed., 2020, 59, 373-379., M. Bennati et al., J. Magn. Reson., 2021, 333, 107091) proposed to use electron nuclear double resonance (ENDOR) spectroscopy in the W-band for a pair of labels, nitroxide and F-19, for measurements of short (0.5-1.0 nm) distances in biomolecules. In our paper, we investigated the suitability of high-field ENDOR spectroscopy in the W-band for pairs of triarylmethyl and fluorine labels using five newly synthesized model compounds. It is shown that the application of strong magnetic fields allows distinguishing nuclear frequencies of F-19 and protons with sufficient resolution. On the one hand, in contrast to nitroxides, for triarylmethyl radicals, it is not necessary to obtain spectra in different orientations owing to low g-factor anisotropic and long electron spin relaxation times of triarylmethyls. On the other hand, the size of the triarylmethyl radical is substantially larger than that of nitroxide and comparable with measured distances. We theoretically analyzed the suitability of the dipole-dipole approach for triarylmethyl to be used in a F-19 ENDOR experiment and determined limitations of this approach. Finally, for comparison, we performed paramagnetic relaxation enhancement (PRE) NMR on the same compounds. In addition, we applied this approach to study the process of a thiol exchange between molecules of triarylmethyl-labeled and F-19-labeled human serum albumin (HSA).

Automated summary

In this study, the suitability of high-field ENDOR spectroscopy in the W-band for triarylmethyl and fluorine label pairs was investigated using newly synthesized model compounds. The results showed that strong magnetic fields allowed for distinguishing nuclear frequencies of F-19 and protons with sufficient resolution. The study also applied this approach to investigate the thiol exchange process between triarylmethyl-labeled and F-19-labeled human serum albumin molecules.