Enhanced nuclear-spin hyperpolarization of amino acids and proteins via reductive radical quenchers

Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) has recently emerged as an effective tool for the hyperpolarization of aromatic amino acids in solution, either in isolation or within proteins. One factor limiting the maximum achievable signal-to-noise ratio in LC-photo-CIDNP is the progressive degradation of the target molecule and photosensitizer upon long-term optical irradiation. Fortunately, this effect does not cause spectral distortions but leads to a progressively smaller signal buildup upon long-term data-collection (e.g. 500 nM tryptophan on a 600 MHz spectrometer after ca. 200 scans). Given that it is generally desirable to minimize the extent of photodamage, we report that low-μM amounts of the reductive radical quenchers vitamin C (VC, i.e., ascorbic acid) or 2-mercaptoethylamine (MEA) enable LC-photo-CIDNP data to be acquired for significantly longer time than ever possible before. This approach increases the sensitivity of LC-photo-CIDNP by more than 100%, with larger enhancement factors achieved in experiments involving more transients. Our results are consistent with VC and MEA acting primarily by reducing transient free radicals of the NMR molecule of interest, thus attenuating the extent of photodamage. The benefits of this reductive radical-quencher approach are highlighted by the ability to collect long-term high-resolution 2D ¹H-¹³C LC-photo-CIDNP data on a dilute sample of the drkN SH3 protein (5 μM).