A Comparative Study of Nitroxide‐Based Biradicals for Dynamic Nuclear Polarization in Cellular Environments

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. Nitroxide‐based biradicals are increasingly being used as polarization agents for sensitivity‐enhanced NMR spectroscopy in cells. They are, however, susceptible to reduction in the cellular environment. Here, we evaluate the stability of three popular polarization agents, AMUPol, AsymPolPOK, and TOTAPOL in cellular lysates, and introduce a new polarization agent, POPAPOL, which exhibits improved properties under reducing conditions.