Search for: All records

Sensitive in-cell distance measurements in proteins using pulsed-Electron Spin Resonance (ESR) require reduction-resistant and cleavage-resistant spin-labels. Among the reduction-resistant moieties, the hydrophilic trityl core known as OX063 is promising due to its long phase-memory relaxation time (T_m). This property leads to a sufficiently intense ESR signal for reliable distance measurements. Furthermore, the T_m of OX063 remains sufficiently long at higher temperatures, opening the possibility for measurements at temperatures above 50 K. In this work, we synthesized deuterated OX063 with a maleimide linker (mOX063-d24). We show that the combination of the hydrophilicity of the label and the maleimide linker enables high protein labeling that is cleavage-resistant in-cells. Distance measurements performed at 150 K using this label are more sensitive than the measurements at 80 K. The sensitivity gain is due to the significantly short longitudinal relaxation time (T_1) at higher temperatures, which enables more data collection per unit of time. In addition to in vitro experiments, we perform distance measurements in Xenopus laevis oocytes. Interestingly, the T_m of mOX063-d24 is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d24 provides highly sensitive distance measurements both in vitro and in-cells. 

ABSTRACT PurposeElectron paramagnetic resonance oxygen imaging (EPROI) techniques have the ability to quantify the partial pressure of oxygen (pO₂), in vivo, through quantitative measurement of T_1emaps of the trityl radical,OXO71.OXO71‐basedEPROIhas seen several applications in small animal imaging studies, but translation to humans will benefit from optimization ofEPROIusing large animal subjects that are anatomically and physiologically similar to humans. The goal of this study was to characterize pharmacokinetics and tissue distribution following an intravenous administration ofOXO71in rhesus macaques. MethodsA 92 μmol/kg OXO71 dose, equivalent to doses used in studies with other animal species, was administered as a bolus injection over a 10‐min period in 3 animals. Plasma and urine were sampled over the subsequent 2 h. A second equivalent dose was then rapidly administered, animals were euthanized, and OXO71 distribution was assessed in harvested tissues by EPR imaging. ResultsPharmacokinetics of plasma OXO71 determined by EPR spectroscopy showed biphasic clearance, characterized by distinct initial and terminal half‐lives of 12.4 ± 1.2 min and 67.7 ± 8.9 min, respectively. Some variability in trityl concentration was observed between organs, but appreciable concentration was found for all tissues except the brain. ConclusionNo adverse physiological effects of the infusions were observed, consistent with a low toxicity of OXO71. These findings indicate EPROI using OXO71 is safe in primates, and provide information related to the dose and time constraints for performing EPROI in other large animal species including humans.