Targetable Tetrazine-Based Dynamic Nuclear Polarization Agents for Biological Systems.
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ABSTRACT:
SUBMITTER: Lim BJ
PROVIDER: S-EPMC7445144 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Herein, we investigate a novel set of polarizing agents-mixed-valence compounds-by theoretical and experimental methods and demonstrate their performance in high-field dynamic nuclear polarization (DNP) NMR experiments in the solid state. Mixed-valence compounds constitute a group of molecules in which molecular mobility persists even in solids. Consequently, such polarizing agents can be used to perform Overhauser-DNP experiments in the solid state, with favorable conditions for dynamic nuclear polarization formation at ultra-high magnetic fields.
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Project description:We demonstrate a 15-fold enhancement of solid-state NMR signals via dynamic nuclear polarization (DNP) based on a stable, naturally occurring radical in a protein: the flavin mononucleotide (FMN) semiquinone of flavodoxin. The line width of flavodoxin's EPR signal suggests that the dominant DNP mechanism is the solid effect, consistent with the field-dependent DNP enhancement profile. The magnitude of the enhancement as well as the bulk-polarization build-up time constant (?(B)) with which it develops are dependent on the isotopic composition of the protein. Deuteration of the protein to 85% increased the nuclear longitudinal relaxation time T(1n) and ?(B) by factors of five and seven, respectively. Slowed dissipation of polarization can explain the 2-fold higher maximal enhancement than that obtained in proteated protein, based on the endogenous semiquinone. In contrast, the long ?(B) of TOTAPOL-based DNP in nonglassy samples was not accompanied by a similarly important long T(1n), and in this case the enhancement was greatly reduced. The low concentrations of radicals occurring naturally in biological systems limit the magnitude of DNP enhancement that is attainable by this means. However, our enhancement factors of up to 15 can nonetheless make an important difference to the feasibility of applying solid-state NMR to biochemical systems. We speculate that DNP based on endogenous radicals may facilitate MAS NMR characterization of biochemical complexes and even organelles, and could also serve as a source of additional structural and physiological information.
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Project description:Nuclear magnetic resonance (NMR) spectroscopy is a powerful and popular technique for probing the molecular structures, dynamics and chemical properties. However the conventional NMR spectroscopy is bottlenecked by its low sensitivity. Dynamic nuclear polarization (DNP) boosts NMR sensitivity by orders of magnitude and resolves this limitation. In liquid-state this revolutionizing technique has been restricted to a few specific non-biological model molecules in organic solvents. Here we show that the carbon polarization in small biological molecules, including carbohydrates and amino acids, can be enhanced sizably by in situ Overhauser DNP (ODNP) in water at room temperature and at high magnetic field. An observed connection between ODNP 13C enhancement factor and paramagnetic 13C NMR shift has led to the exploration of biologically relevant heterocyclic compound indole. The QM/MM MD simulation underscores the dynamics of intermolecular hydrogen bonds as the driving force for the scalar ODNP in a long-living radical-substrate complex. Our work reconciles results obtained by DNP spectroscopy, paramagnetic NMR and computational chemistry and provides new mechanistic insights into the high-field scalar ODNP.
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Project description:We introduce a new family of highly efficient polarizing agents for dynamic nuclear polarization (DNP)-enhanced nuclear magnetic resonance (NMR) applications, composed of asymmetric bis-nitroxides, in which a piperidine-based radical and a pyrrolinoxyl or a proxyl radical are linked together. The design of the AsymPol family was guided by the use of advanced simulations that allow computation of the impact of the radical structure on DNP efficiency. These simulations suggested the use of a relatively short linker with the intention to generate a sizable intramolecular electron dipolar coupling/ J-exchange interaction, while avoiding parallel nitroxide orientations. The characteristics of AsymPol were further tuned, for instance with the addition of a conjugated carbon-carbon double bond in the 5-membered ring to improve the rigidity and provide a favorable relative orientation, the replacement of methyls by spirocyclohexanolyl groups to slow the electron spin relaxation, and the introduction of phosphate groups to yield highly water-soluble dopants. An in-depth experimental and theoretical study for two members of the family, AsymPol and AsymPolPOK, is presented here. We report substantial sensitivity gains at both 9.4 and 18.8 T. The robust efficiency of this new family is further demonstrated through high-resolution surface characterization of an important industrial catalyst using fast sample spinning at 18.8 T. This work highlights a new direction for polarizing agent design and the critical importance of computations in this process.
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