Dynamic nuclear polarization (DNP) is a powerful tool to enhance the NMR signals of molecules by transferring polarization from unpaired electron spins to nuclei through microwave irradiation. The resulting signal enhancements can enable the analysis of samples that have previously been intractable by NMR spectroscopy, including proteins, nucleic acids, and metabolites in cells. To carry out DNP, the sample is doped with a polarization agent, a biradical containing two nitroxide moieties. DNP applications in cells, however, present significant challenges as nitroxides are often susceptible to the reducing cellular environment. Here, we introduce a novel polarization agent, POPAPOL, that exhibits increased lifetimes under reducing conditions. We also compare its bioresistance and DNP performance with three popular, commercially available polarization agents. Our work indicates that pyrrolidine-based nitroxides can outperform piperidine-based nitroxides in cellular environments, and that future polarization agent designs must carefully balance DNP performance and stability for cellular applications.
Keywords: EPR spectroscopy; NMR spectroscopy; dynamic nuclear polarization; in cell NMR spectroscopy; nitroxide radical stability.
© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.