Enhanced Efficiency of ¹³C Dynamic Nuclear Polarization by Superparamagnetic Iron Oxide Nanoparticle Doping

The attainment of high NMR signal enhancements is crucial to the success of in vitro or in vivo hyperpolarized NMR or imaging (MRI) experiments. In this work, we report on the use of a superparamagnetic iron oxide nanoparticle (SPION) MRI contrast agent Feraheme (ferumoxytol) as a beneficial additive in ¹³C samples for dissolution dynamic nuclear polarization (DNP). Our DNP data at 3.35 T and 1.2 K reveal that the addition of 11 mM elemental iron concentration of Feraheme in trityl OX063-doped 3 M [1-¹³C] acetate samples resulted in a substantial improvement of ¹³C DNP signal by a factor of almost three-fold. Concomitant with the large DNP signal increase is the narrowing of the ¹³C microwave DNP spectra for samples doped with SPION. W-band electron paramagnetic resonance (EPR) spectroscopy data suggest that these two prominent effects of SPION doping on ¹³C DNP can be ascribed to the shortening of trityl OX063 electron <i>T</i>₁, as explained within the thermal mixing DNP model. Liquid-state ¹³C NMR signal enhancements as high as 20,000-fold for SPION-doped samples were recorded after dissolution at 9.4 T and 297 K, which is about three times the liquid-state NMR signal enhancement of the control sample. While the presence of SPION in hyperpolarized solution drastically reduces ¹³C <i>T</i>₁, this can be mitigated by polarizing smaller aliquots of DNP samples. Moreover, we have shown that Feraheme nanoparticles (∼30 nm in size) can be easily and effectively removed from the hyperpolarized liquid by simple mechanical filtration, and thus one can potentially incorporate an in-line filtration for these SPIONS along the dissolution pathway of the hyperpolarizer, a significant advantage over other DNP enhancers such as the lanthanide complexes. The overall results suggest that the commercially available and FDA-approved Feraheme is a highly efficient DNP enhancer that could be readily translated for use in clinical applications of dissolution DNP

Operando EPR for Simultaneous Monitoring of Anionic and Cationic Redox Processes in Li-Rich Metal Oxide Cathodes

Anionic redox chemistry offers a transformative approach for significantly increasing specific energy capacities of cathodes for rechargeable Li-ion batteries. This study employs operando electron paramagnetic resonance (EPR) to simultaneously monitor the evolution of both transition metal and oxygen redox reactions, as well as their intertwined couplings in Li₂MnO₃, Li_1.2Ni_0.2Mn_0.6O₂, and Li_1.2Ni_0.13Mn_0.54Co_0.13O₂ cathodes. Reversible O^2–/O₂^<i>n</i>– redox takes place above 3.0 V, which is clearly distinguished from transition metal redox in the operando EPR on Li₂MnO₃ cathodes. O^2–/O₂^<i>n</i>– redox is also observed in Li_1.2Ni_0.2Mn_0.6O₂, and Li_1.2Ni_0.13Mn_0.54Co_0.13O₂ cathodes, albeit its overlapping potential ranges with Ni redox. This study further reveals the stabilization of the reversible O redox by Mn and e^– hole delocalization within the Mn–O complex. The interactions within the cation–anion pairs are essential for preventing O₂^<i>n</i>– from recombination into gaseous O₂ and prove to activate Mn for its increasing participation in redox reactions. Operando EPR helps to establish a fundamental understanding of reversible anionic redox chemistry. The gained insights will support the search for structural factors that promote desirable O redox reactions

Transition Metal Doping Reveals Link between Electron <i>T</i>₁ Reduction and ¹³C Dynamic Nuclear Polarization Efficiency

Optimal efficiency of dissolution dynamic nuclear polarization (DNP) is essential to provide the required high sensitivity enhancements for <i>in vitro</i> and <i>in vivo</i> hyperpolarized ¹³C nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI). At the nexus of the DNP process are the free electrons, which provide the high spin alignment that is transferred to the nuclear spins. Without changing DNP instrumental conditions, one way to improve ¹³C DNP efficiency is by adding trace amounts of paramagnetic additives such as lanthanide (e.g., Gd³⁺, Ho³⁺, Dy³⁺, Tb³⁺) complexes to the DNP sample, which has been observed to increase solid-state ¹³C DNP signals by 100–250%. Herein, we have investigated the effects of paramagnetic transition metal complex R-NOTA (R = Mn²⁺, Cu²⁺, Co²⁺) doping on the efficiency of ¹³C DNP using trityl OX063 as the polarizing agent. Our DNP results at 3.35 T and 1.2 K show that doping the ¹³C sample with 3 mM Mn²⁺-NOTA led to a substantial improvement of the solid-state ¹³C DNP signal by a factor of nearly 3. However, the other transition metal complexes Cu²⁺-NOTA and Co²⁺-NOTA complexes, despite their paramagnetic nature, had essentially no impact on solid-state ¹³C DNP enhancement. W-band electron paramagnetic resonance (EPR) measurements reveal that the trityl OX063 electron <i>T</i>₁ was significantly reduced in Mn²⁺-doped samples but not in Cu²⁺- and Co²⁺-doped DNP samples. This work demonstrates, for the first time, that not all paramagnetic additives are beneficial to DNP. In particular, our work provides a direct evidence that electron <i>T</i>₁ reduction of the polarizing agent by a paramagnetic additive is an essential requirement for the improvement seen in solid-state ¹³C DNP signal

Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A‑Class Dye-Decolorizing Peroxidase from <i>Thermomonospora curvata</i>

Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases in which a catalytic distal aspartate is involved in H₂O₂ activation to catalyze oxidations under acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial <i>Tc</i>DyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds. On the basis of the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the <i>Tc</i>DyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in <i>Tc</i>DyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of the surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the cross-linking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class <i>Tc</i>DyP is minimal, possibly due to its extremely small solvent-accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding the DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin