Conformational tuning improves the stability of spirocyclic nitroxides with long paramagnetic relaxation times

Funding: M.M.H. and A.-L.W. thank the Tromsø Research Foundation and UiT Centre for New Antibacterial Strategies (CANS) for a start-up grant (TFS project ID: 18_CANS). K.H.H. and S.G. thank the Research Council of Norway (No. 300769) and Sigma2 (No. nn9330k and nn4654k), and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 859910. J.E.L. thanks Drs Hassane El Mkami and Robert I. Hunter for technical assistance, the BBSRC (BB/T017740/1) and the Wellcome Trust (099149/Z/12/Z) for the Q-band EPR spectrometer, the Royal Society for a University Research Fellowship and Research Grant RG120645 for the benchtop spectrometer. B.A.L. thanks The Research Council of Norway for the Centre of Excellence and project grants (Grant Nos. 262695 and 274858). Nitroxides are widely used as probes and polarization transfer agents in spectroscopy and imaging. These applications require high stability towards reducing biological environments, as well as beneficial relaxation properties. While the latter is provided by spirocyclic groups on the nitroxide scaffold, such systems are not in themselves robust under reducing conditions. In this work, we introduce a strategy for stability enhancement through conformational tuning, where incorporating additional substituents on the nitroxide ring effects a shift towards highly stable closed spirocyclic conformations, as indicated by X-ray crystallography and density functional theory (DFT) calculations. Closed spirocyclohexyl nitroxides exhibit dramatically improved stability towards reduction by ascorbate, while maintaining long relaxation times in electron paramagnetic resonance (EPR) spectroscopy. These findings have important implications for the future design of new nitroxide-based spin labels and imaging agents. Publisher PDF