The efficacy in ¹H Overhauser dynamic nuclear polarization in liquids at ultralow magnetic field (ULF, B₀ = 92 ± 0.8 µT) and polarization field (B_p = 1–10 mT) was studied for a broad variety of 26 different spin probes. Among others, piperidine, pyrrolidine, and pyrroline radicals specifically synthesized for this study, along with some well-established commercially available nitroxides, were investigated. Isotope-substituted variants, some sterically shielded reduction-resistant nitroxides, and some biradicals were included in the measurements. The maximal achievable enhancement, E_max, and the radio frequency power, P_1/2, needed for reaching E_max/2 were measured. Physico-chemical features such as molecular weight, spectral linewidth, heterocyclic structure, different types of substituents, deuteration, and ¹⁵N-labeling as well as the difference between monoradicals and biradicals were investigated. For the unmodified nitroxide radicals, the E_max values correlate with the molecular weight. The P_1/2 values correlate with the spectral linewidth and are additionally influenced by the type of substituents neighboring the nitroxide group. The nitroxide biradicals with high intramolecular spin–spin coupling show low performance. Nitroxides enriched with ¹⁵N and/or ²H afford significantly higher |E_max| and require lower power to do so, compared to their unmodified counterparts containing at natural abundance predominantly ¹⁴N and ¹H. The results allow for a correlation of chemical features with physical hyperpolarization-related properties and indicate that small nitroxides with narrow spectral lines have clear advantages for the use in Overhauser dynamic nuclear polarization experiments. Perdeuteration and ¹⁵N-labeling can be used to additionally boost the spin probe performance.

Sterically shielded nitroxides of the pyrrolidine series have shown the highest resistance to reduction. Here we report the synthesis of new pyrrolidine nitroxides from 5,5-dialkyl-1-pyrroline N-oxides via the introduction of a pent-4-enyl group to the nitrone carbon followed by an intramolecular 1,3-dipolar cycloaddition reaction and isoxazolidine ring opening. The kinetics of reduction of the new nitroxides with ascorbate were studied and compared to those of previously published (1S,2R,3′S,4′S,5′S,2″R)-dispiro[(2-hydroxymethyl)cyclopentan-1,2′-(3′,4′-di-tert-butoxy)pyrrolidine-5′,1″-(2″-hydroxymethyl)cyclopentane]-1′-oxyl (1).

Site-directed spin labeling followed by investigation using Electron Paramagnetic Resonance spectroscopy is a rapidly expanding powerful biophysical technique to study structure, local dynamics and functions of biomolecules using pulsed EPR techniques and nitroxides are the most widely used spin labels. Modern trends of this method include measurements directly inside a living cell, as well as measurements without deep freezing (below 70 K), which provide information that is more consistent with the behavior of the molecules under study in natural conditions. Such studies require nitroxides, which are resistant to the action of biogenic reductants and have high spin relaxation (dephasing) times, T_m. (1R(S),5R(S),7R(S),8R(S))-1,8-bis(hydroxymethyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl is a unique nitroxide that combines these features. We have developed a convenient method for the synthesis of this radical and studied the ways of its functionalization. Promising spin labels have been obtained, the parameters of their spin relaxation T₁ and T_m have been measured, and the kinetics of reduction with ascorbate have been studied.

Activation of a hydroxyl group towards nucleophilic substitution via reaction with methanesulfonyl chloride or PPh₃-CBr₄ system is a commonly used pathway to various functional derivatives. The reactions of (5R(S),6R(S))-1-X-6-(hydroxymethyl)-2,2-dimethyl- 1-azaspiro[4.4]nonanes 1a–d (X = O·; H; OBn, OBz) with MsCl/NR₃ or PPh₃-CBr₄ were studied. Depending on substituent X, the reaction afforded hexahydro-1H,6H-cyclopenta[c]pyrrolo[1,2-b]isoxazole (2) (for X = O), a mixture of 2 and octahydrocyclopenta[c]azepines (4–6) (for X = OBn, OBz), or perhydro-cyclopenta[2,3]azeto[1,2-a]pyrrol (3) (for X = H) derivatives. Alkylation of the latter with MeI with subsequent Hofmann elimination afforded 2,3,3-trimethyl-1,2,3,4,5,7,8,8a-octahydrocyclopenta[c]azepine with 56% yield.

Modern trends in the development of structural biology make it relevant to study the structure of biomolecules under the conditions close to natural, i.e., directly in a living cell and at temperatures close to physiological ones. One of the promising technologies that allows approaching this ideal is the targeted introduction of spin labels (for example, nitroxides), followed by investigation using pulsed electron paramagnetic resonance (EPR). Today, the greatest stability in living systems is demonstrated by sterically hindered nitroxyl radicals of the pyrrolidine series with four ethyl groups at a paramagnetic centre, but their relaxation characteristics do not allow measurements by pulsed ESR methods at a temperature above 80 K. Nitroxides with spirocycloalkane fragments surrounding the nitroxyl group are suitable for measurements at higher temperatures, but they are rapidly reduced by the components of living systems. Pyrrolidine nitroxides with two spiro-(2-hydroxymethyl)cyclopentane moieties combine high resistance to reduction with high spin relaxation times at a temperature of 120 K and above. In this work, a hydrophilic chiral radical of this series - (1R,5S,7S,8R,12S,13S)-12,13-dihydroxy-1,8-bis(hydroxymethyl)-6-azadispiro[4.1.4.2]tridecane-6-oxyl was obtained and characterised by us for the first time.