Damien A. Reid

Development of Isoindoline Nitroxides for EPR Oximetry in Viable Systems

Nitroxides are widely used as biophysical probes to study molecular motion, intracellular oxygen, pH, transmembrane potential, and cellular redox metabolism, etc. They may be rapidly metabolized to hydroxylamines by cells, which limits their use in viable systems. In this study, we have characterized relevant properties in cells of several isoindoline nitroxides that have been prepared to have different physicochemical properties: 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) and its analogs 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO), 5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethyl isoindolin-2-yloxyl iodide (QATMIO) and 2-hydroxy-1,1,3,3-tetramethylisoindoline hydrochloride (TMIOH.HCI). The oxygen sensitivity and metabolic kinetics of these were compared in CHO cells under different oxygen tensions with 1-oxyl-2,2,6,6-tetramethyl-4-piperidione (Tempone) and 3-carboxyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (PCA). Cytotoxicity was evaluated by the measurement of oxygen consumption rates, trypan blue exclusion, and clone formation. TMIO and its analogues have a higher relative oxygen sensitivity than Tempone and PCA with the oxygen sensitivity in electron paramagnetic resonance (EPR) spectrometry in the order of: TMIO=TMIOH=CTMIO>QATMIO=Tempone<PCA. The rates of metabolism of these nitroxides are moderate and depend on oxygen concentration, ring type, ring substituent, and membrane permeation. These nitroxides have low cytotoxicity. The results indicate that TMIO and its analogues are potentially useful for EPR studies of viable systems, especially for oximetry.

ESR measurements of the partitioning of some new spin probes in n-octanol–water

The stable free radical 1,1,3,3‐tetramethylisoindolin‐2‐yloxyl (TMIO) has proved to be very suitable for use as a spin probe for a number of applications. Because it is soluble mainly in non‐polar liquids, there is a need for new derivatives that can be used in a variety of environments. This has been done by introducing substituents in the 5‐position of the aromatic ring, namely carboxyl (CTMIO), trimethylamino (TMTMIOI) and sodium sulphonate (NaTMIOS). An accurate ESR method was developed for the measurement of partition coefficients in n‐octanol–water. For comparison purposes the method was also applied to some Tempo derivatives. The effect of temperature on the rotational correlation times and the nitrogen‐14 hyperfine coupling constant of some of the spin probes was investigated. There is evidence for dimerization of CTMIO to form a biradical. Copyright

Electrospray ionization mass spectrometry of stable nitroxide free radicals and two isoindoline nitroxide dimers

Electrospray ionization (ESI) mass spectra were recorded for a range of substituted isoindoline nitroxides, two isoindoline nitroxide dimers and two piperidinyl nitroxides. In all cases the dominant molecular species arise from oxidation rather than protonation, an unusual process in ESI. Fragment ion spectroscopy was used to establish fragmentation mechanisms for the nitroxides under ESI conditions.