Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pK_a = 3, Δa_N = 0.055 mT and Δa_H = 0.075 mT.

Triarylmethyl radicals (TAMs) have recently emerged as highly effective polarizing agents in dynamic nuclear polarization (DNP) under viscous conditions, enabling substantial hyperpolarization via the solid-effect (SE) DNP mechanism even at room temperature. A comparable, though less pronounced, enhancement was observed for BDPA radicals embedded in phosphocholine-based lipid bilayers. Given the increasing interest in elucidating the structure and dynamics of biopolymers and their high-molecular-weight assemblies—such as cell membranes—this study focuses on the design, synthesis, and characterization of paramagnetic agents tailored for DNP-based structural biology. To this end, we synthesized a series of TAM derivatives functionalized with lipophilic substituents and characterized their magnetic resonance properties, including isotropic hyperfine interaction (HFI) constants on carbon nuclei and electron spin relaxation times (T₁ and T_m) at low temperatures (80 K). Echo-detected EPR spectra and electron spin echo envelope modulations (ESEEM) were recorded for novel TAM incorporated into liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). These low-temperature measurements revealed that the radicals are localized either at the liposome surface or within the lipid bilayer, ensuring optimal accessibility to water molecules. Crucially, the presence of a single cholesterol moiety provides strong noncovalent anchoring within the hydrophobic core of the bilayer. Guided by these findings, we identify an amphiphilic TAM bearing a single cholesterol group and polar carboxyl functionalities as a highly promising candidate for DNP applications in membrane biology, combining efficient polarization transfer, bilayer integration, and aqueous accessibility.