19F electron nuclear double resonance (ENDOR) spectroscopy for distance measurements using trityl spin labels in DNA duplexes.

The combination of fluorine labeling and pulsed electron-nuclear double resonance (ENDOR) is emerging as a powerful technique for obtaining structural information about proteins and nucleic acids. In this work, we explored the capability of Mims 19F ENDOR experiments on reporting intermolecular distances in trityl- and 19F-labeled DNA duplexes at three electron paramagnetic resonance (EPR) frequencies (34, 94, and 263 GHz). For spin labeling, we used the hydrophobic Finland trityl radical and hydrophilic OX063 trityl radical. Fluorine labels were introduced into two positions of a DNA oligonucleotide. The results indicated that hyperfine splittings visible in the ENDOR spectra are consistent with the most populated interspin distances between 19F and the trityl radical predicted from molecular dynamic (MD) simulations. Moreover, for some cases, ENDOR spectral simulations based on MD results were able to reproduce the conformational distribution reflected in the experimental ENDOR line broadening. Additionally, MD simulations provided more detailed information about the melting of terminal base pairs of the oligonucleotides and about the configuration of the trityls relative to a DNA end.

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 19F, 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 19F 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 19F 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 19F-labeled human serum albumin (HSA).

Hyperfine interactions of narrow-line trityl radical with solvent molecules.

The electron nuclear dipolar interactions responsible for some dynamic nuclear polarization (DNP) mechanisms also are responsible for the presence formally in CW EPR spectra of forbidden satellite lines in which both the electron spin and a nuclear spin flip. Such lines arising from (1)H nuclei are easily resolved in CW EPR measurements of trityl radicals, a popular family of DNP reagents. The satellite lines overlap some of the hyperfine features from (13)C in natural abundance in the trityl radical, but their intensity can be easily determined by simple simulations of the EPR spectra using the hyperfine parameters of the trityl radical. Isotopic substitution of (2)H for (1)H among the hydrogens of the trityl radical and/or the solvent allows the dipolar interactions from the (1)H on the trityl radical and from the solvent to be determined. The intensity of the dipolar interactions, integrated over all the (1)H in the system, is characterized by the traditional parameter called reff. For the so-called Finland trityl in methanol, the reff values indicate that collectively the (1)H in the unlabeled solvent have a stronger integrated dipolar interaction with the unpaired electron spin of the Finland trityl than do the (1)H in the radical and consequently will be a more important DNP route. Although reff has the dimensions of distance, it does not correspond to any simple physical dimension in the trityl radical because the details of the unpaired electron spin distribution and the hydrogen distribution are important in the case of trityls.