Stability of the polarization agent AsymPolPOK in intact and lysed mammalian cells.

Dynamic nuclear polarization (DNP) solid-state NMR enables detection of proteins at endogenous concentrations in cells through sensitivity enhancement from nitroxide biradical polarization agents. AsymPolPOK, a novel water-soluble asymmetric nitroxide biradical, offers superior sensitivity and faster build-up times compared to existing agents like AMUPol. Here, we characterize AsymPolPOK in mammalian HEK293 cells, examining its cellular distribution, reduction kinetics, and DNP performance. We demonstrate that electroporation achieves uniform cellular delivery of AsymPolPOK, including nuclear permeation, with no cytotoxicity at millimolar concentrations. However, the cellular environment rapidly reduces AsymPolPOK to its monoradical form, with one nitroxide center showing greater reduction resistance than the other. While AsymPolPOK maintains high DNP enhancements and short build-up times in lysates, its performance in intact cells depends critically on delivery method and exposure time to cellular constituents. Electroporation yields higher, more uniform enhancements compared to incubation, but prolonged exposure to the cellular environment diminishes DNP performance in both cases. These findings establish the potential of AsymPolPOK as a polarization agent for in-cell DNP NMR while highlighting the need for developing more bio-resistant polarization agents to further advance cellular structural biology studies.