Assessing the Onset of Calcium Phosphate Nucleation by Hyperpolarized Real-Time NMR.

We report an experimental approach for high-resolution real-time monitoring of transiently formed species occurring during the onset of precipitation of ionic solids from solution. This is made possible by real-time nuclear magnetic resonance (NMR) monitoring using dissolution dynamic nuclear polarization (D-DNP) to amplify signals of functional intermediates and is supported by turbidimetry, cryogenic electron microscopy, and solid-state NMR measurements. D-DNP can provide drastic signal improvements in NMR signal amplitudes, permitting dramatic reductions in acquisition times and thereby enabling us to probe fast interaction kinetics such as those underlying formation of prenucleation species (PNS) that precede solid-liquid phase separation. This experimental strategy allows for investigation of the formation of calcium phosphate (CaP)-based minerals by 31P NMR-a process of substantial industrial, geological, and biological interest. Thus far, many aspects of the mechanisms of CaP nucleation remain unclear due to the absence of experimental methods capable of accessing such processes on sufficiently short time scales. The approach reported here aims to address this by an improved characterization of the initial steps of CaP precipitation, permitting detection of PNS by NMR and determination of their formation rates, exchange dynamics, and sizes. Using D-DNP monitoring, we find that under our conditions (i) in the first 2 s after preparation of oversaturated calcium phosphate solutions, PNS with a hydrodynamic radius of Rh ≈ 1 nm is formed and (ii) following this rapid initial formation, the entire crystallization processes proceed on considerably longer time scales, requiring >20 s to form the final crystal phase.

A DNP-hyperpolarized solid-state water NMR MASER: observation and qualitative analysis.

We present observations of an NMR MASER (microwave amplification by stimulated emission of radiation) of hyperpolarized 1H nuclei by dynamic nuclear polarization (DNP) at 1.2 K and in a magnetic field of 6.7 T. The sustained maser pulses originate from the interplay between radiation damping (RD) due to the large 1H magnetization, and the remagnetization to a negative value by the DNP process. NMR signals lasting for several tens of seconds are thus observed on an ensemble of dipolar-coupled nuclear spins. Magnetization dynamics are analyzed in terms of the combined Bloch-Maxwell and Provotorov (BMP) equations for RD and DNP. Insight into the long time evolution of the magnetization is provided by a theoretical analysis of this nonlinear dynamical system, and by fitting the NMR signal to a simplified version of the BMP equations.

Sample Ripening through Nanophase Separation Influences the Performance of Dynamic Nuclear Polarization.

Mixtures of water and glycerol provide popular matrices for low-temperature spectroscopy of vitrified samples. However, they involve counterintuitive physicochemical properties, such as spontaneous nanoscopic phase separations (NPS) in solutions that appear macroscopically homogeneous. We demonstrate that such phenomena can substantially influence the efficiency of dynamic nuclear polarization (DNP) by factors up to 20 % by causing fluctuations in local concentrations of polarization agents (radicals). Thus, a spontaneous NPS of water/glycerol mixtures that takes place on time scales on the order of 30-60 min results in a confinement of polarization agents in nanoscopic water-rich vesicles, which in return affects the DNP. Such effects were found for three common polarization agents, TEMPOL, AMUPol and Trityl.

Rates of Chemical Reactions Embedded in a Metabolic Network by Dissolution Dynamic Nuclear Polarisation NMR.

The isomerisation of 6-phosphogluconolactones and their hydrolyses into 6-phosphogluconic acid form a non enzymatic side cycle of the pentose-phosphate pathway (PPP) in cells. Dissolution dynamic nuclear polarisation can be used for determining the kinetic rates of the involved transformations in real time. It is found that the hydrolysis of both lactones is significantly slower than the isomerisation process, thereby shedding new light onto this subtle chemical process.

Communication: Dissolution DNP reveals a long-lived deuterium spin state imbalance in methyl groups.

We report the generation and observation of long-lived spin states in deuterated methyl groups by dissolution DNP. These states are based on population imbalances between manifolds of spin states corresponding to irreducible representations of the C3v point group and feature strongly dampened quadrupolar relaxation. Their lifetime depends on the activation energies of methyl group rotation. With dissolution DNP, we can reduce the deuterium relaxation rate by a factor up to 20, thereby extending the experimentally available time window. The intrinsic limitation of NMR spectroscopy of quadrupolar spins by short relaxation times can thus be alleviated.

Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water.

Hyperpolarized water can selectively enhance NMR signals of rapidly exchanging protons in osteopontin (OPN), a metastasis-associated intrinsically disordered protein (IDP), at near-physiological pH and temperature. The transfer of magnetization from hyperpolarized water is limited to solvent-exposed residues and therefore selectively enhances signals in 1 H-15 N correlation spectra. Binding to the polysaccharide heparin was found to induce the unfolding of preformed structural elements in OPN.

Dissolution dynamic nuclear polarization of deuterated molecules enhanced by cross-polarization.

We present novel means to hyperpolarize deuterium nuclei in 13CD2 groups at cryogenic temperatures. The method is based on cross-polarization from 1H to 13C and does not require any radio-frequency fields applied to the deuterium nuclei. After rapid dissolution, a new class of long-lived spin states can be detected indirectly by 13C NMR in solution. These long-lived states result from a sextet-triplet imbalance (STI) that involves the two equivalent deuterons with spin I = 1. An STI has similar properties as a triplet-singlet imbalance that can occur in systems with two equivalent I = 12 spins. Although the lifetimes TSTI are shorter than T1(Cz), they can exceed the life-time T1(Dz) of deuterium Zeeman magnetization by a factor of more than 20.