To evaluate the thermodynamic stability profiles of the new nitroxide scaffolds, we next assessed their redox behaviour by cyclic voltammetry (Figure ). Voltammograms of the nitroxides 5, 7 and 8 (20 µmol) in a 0.1 M solution of tetrabutylammonium hexafluorophosphate (TBAPF6) in acetonitrile were recorded using a three-electrode setup, with a 3 mm diameter glassy carbon electrode as the working electrode, platinum wire as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode (potentials were referenced to the ferrocene/ferrocenium redox pair) with 100 mV/s scan rate. Peak couples in the anodic region correspond to the reversible oxidation of the nitroxides to the corresponding oxoammonium cations. Exo-methylene nitroxides 7 and 8 exhibit lower oxidation potentials than the reference pyrrolinyl nitroxide 5, presumably due to the additional methylene carbon and absence of conjugation between the electron-withdrawing ester/acid functionality and the core nitroxide ring. The cathodic peaks arise from reduction of the nitroxides to hydroxylamines through a single-electron reduction (via aminoxylate anions) coupled with proton transfer. This process is quasi-reversible or irreversible under the conditions of the measurement, meaning the reduction potentials could only be estimated. It is known that the presence of functional groups that typically stabilize the reduced form result in a decrease in the reduction potential (i.e. less negative values). Surprisingly, however, the reduction potantial of exomethylene nitroxide 7 (-2.02 V) is lower than the reduction potential for pyrrolinyl nitroxide 5 (-2.28 V), despite the additional methylene unit between the electron-withdrawing functional group and the core nitroxide ring. Thus, the redox window for the new exo-methylene pyrrolidinyl nitroxides is than for pyrrolinyl nitroxides. The reduction potential of acid 8 is even lower (-1.51 V). We hypothesize that this anomaly can be explained by stabilization of the reduced (aminoxylate) form of nitroxide 8 by intramolecular hydrogen bonding with the carboxylic acid functionality. A similar effect has been observed in piperidinyl nitroxides. Beyond stability towards reduction, relaxation behaviour is a crucial consideration for the use of nitroxides for measurements and imaging in biological systems. We therefore next investigated the relaxation parameters Tm and T1 for the new exo-methylene pyrrolidine nitroxide 8. The phase memory time Tm is a measure of the decay rate of an electron spin Hahn echo, which is an important factor for the resolution and range of pulsed EPR experiments such as DEER. Spin echo decay curves were measured across a range of temperatures (Figure ), and from these the phase memory time Tm was extracted. Tm remains relatively stable at temperatures up to 160 K (Tm,50K = 4.15 µs and Tm,160K = 3.29 µs, which corresponds to 79% of Tm,50K). However, a significant change occurs at 180 K (Tm,180K = 0.85 µs), which may be due to increased dynamic motion as the water/glycerol glass softens. The change in spin echo dephasing rates (i.e. the reciprocal of Tm) across different temperatures for nitroxide 8 (Figure ) can also be compared with the reference nitroxides TEMPOL (12), TEEPOL (11), and 4-hydroxyspirocyclohexylpiperidine 1-oxyl (13). The advantages of nitroxides with spirocyclic α-substituents in terms of relaxation parameters are clearly illustrated, as the absence of the relaxation mechanism provided by rotatable methyl groups extends the useful temperature range of such nitroxides well above the usual cutoff of 50-70 K for 11 and 12. The longitudinal relaxation time T1 of the electron spin is a measure of the transfer of polarisation from the paramagnetic centre to the surrounding lattice. This process is crucial in techniques such as magic angle spinning dynamic nuclear polarisation (MAS-DNP) in solid-state NMR spectroscopy, where long T1 times can lead to loss of signal intensity by depolarisation. T1 is also an important consideration in the development of MRI contrast agents. The spin-lattice relaxation of nitroxide 8 was measured by inversion recovery at different temperatures (Figure ), from which numerical values for T1 were found to be 5.2 ms (50 K), 0.65 ms (120 K), and 0.12 ms (180 K). The logarithm of the spin-lattice relaxation rate (i.e. log10 (T1 -1 )) appears to show the expected linear dependence on temperature (Figure ).