Triarylmethyl (TAM) radicals are widely used in Electron Paramagnetic Resonance (EPR) spectroscopy as spin labels and in EPR imaging as spin probes for oxymetry. One of the key advantages of TAMs is extremely narrow EPR line, especially in case of deuterated analogues (~5 μT). Another advantage is their slow spin relaxation even at physiological temperatures allowing, in particular, application of pulsed dipolar EPR methods for distance measurements in biomolecules. In this paper a large series of TAM radicals and their deuterated analogues is synthesized, and corresponding spectroscopic parameters including C hyperfine constants are obtained for the first time. The negligible dependence of C hyperfine constants on solvent, as well as on structure and number of substituents at para-C atoms of aromatic rings, has been found. In addition, we have demonstrated that C signals at natural abundance can be employed for successful room-temperature distance measurements using Pulsed Electron Double Resonance (PELDOR or DEER).

In vivo oximetry by pulsed electron paramagnetic resonance is based on measurements of changes in electron spin relaxation rates of probe molecules, such as the triarylmethyl radicals. A series of experiments was performed at frequencies between 250 MHz and 1.5 GHz to assist in the selection of an optimum frequency for oximetry. Electron spin relaxation rates for the triarylmethyl radical OX063 as a function of radical concentration, salt concentration, and resonance frequency were measured by electron spin echo 2-pulse decay and 3-pulse inversion recovery in the frequency range of 250 MHz-1.5 GHz. At constant OX063 concentration, 1/T decreases with increasing frequency because the tumbling dependent processes that dominate relaxation at 250 MHz are less effective at higher frequency. 1/T also decreases with increasing frequency because 1/T is a significant contribution to 1/T for trityl radicals in fluid solution. 1/T-1/T, the incomplete motional averaging contribution to 1/T, increases with increasing frequency. At constant frequency, relaxation rates increase with increasing radical concentration due to contributions from collisions that are more effective for 1/T than 1/T. The collisional contribution to relaxation increases as the concentration of counter-ions in solution increases, which is attributed to interactions of cations with the negatively charged radicals that decrease repulsion between trityl radicals. The Signal-to-Noise ratio (S/N) of field-swept echo-detected spectra of OX063 were measured in the frequency range of 400 MHz-1 GHz. S/N values, normalized by √Q, increase as frequency increases. Adding salt to the radical solution decreased S/N because salt lowers the resonator Q. Changing the temperature from 19 to 37 °C caused little change in S/N at 700 MHz. Both slower relaxation rates and higher S/N at higher frequencies are advantageous for oximetry. The potential disadvantage of higher frequencies is the decreased depth of penetration into tissue.

In conventional pulsed magnetic resonance suppression of unwanted signals is achieved by changing pulse phases with respect to the reference signal and spin magnetization phase. This method is called phase cycling. An alternative approach is suggested to separate the unwanted signals from the spin echo by using magnetic field modulation. Precession frequency of the spins, and therefore phases of free indication decays and echo signals, can be controlled by the selection of modulation parameters. This enables phase cycling. Since the signal is detected in the presence of the changing magnetic field, which drives spin precession, the echo signal is frequency-modulated. Numerical transformation into an accelerating reference frame associated with the Larmor frequency restores the signal to a form that would have been observed in the absence of modulation. The suggested phase cycling method is analyzed in detail for the two pulse spin echo case.

The Transition Path Sampling (TPS) method is a powerful technique for studying rare events in complex systems, that allows description of reactive events in atomic detail without prior knowledge of reaction coordinates and transition states. We have applied TPS in combination with a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) method to study the enzyme human purine nucleoside phosphorylase (hPNP). This enzyme catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to generate the corresponding purine base and (deoxy)ribose 1-phosphate. Hundreds of reactive trajectories were generated. Analysis of this transition path ensembles provides insight into the detailed mechanistic dynamics of reaction in the enzyme. Our studies have indicated a reaction mechanism involving the cleavage of the N-ribosidic bond to form transition states with substantial ribooxacarbenium ion character, that is then followed by conformational changes in the enzyme and the ribosyl group leading to migration of the anomeric carbon of the ribosyl group toward phosphate to form the product ribose 1-phosphate. This latter process is crucial in PNP, because several strong H-bonds form between active site residues in order to capture and align the phosphate nucleophile. Calculations of the commitment probability along reactive paths demonstrated the presence of a broad energy barrier at the transition state. Analysis of these transition state structures showed that bond-breaking and bond-forming distances are not a good choice for the reaction coordinate, but that the pseudorotational phase of the ribose ring is also a significant variable.