Optimizing dissolution dynamic nuclear polarization

This article is a short review of some of our recent developments in dissolution dynamic nuclear polarization (d-DNP). We present the basic principles of d-DNP, and motivate our choice to step away from conventional approaches. We then introduce a modified d-DNP recipe that can be summed up as follows: (i) Using broad line polarizing agents to efficiently polarize H-1 spins. (ii) Increasing the magnetic field to 6.7 T and above. (iii) Applying microwave frequency modulation. (iv) Applying H-1-C-13 cross polarization. (v) Transferring hyperpolarized solution through a magnetic tunnel. (C) 2015 Elsevier Inc. All rights reserved

Cross Polarization for Dissolution Dynamic Nuclear Polarization Experiments at Readily Accessible Temperatures 1.2 < T < 4.2 K

Cross polarization can provide significant enhancements with respect to direct polarization of low-gamma nuclei such as C-13. Substantial gains in sample throughput (shorter polarization times) can be achieved by exploiting shorter build-up times tau(DNP)(H-1) < tau(DNP)(C-13). To polarize protons rather than low-gamma nuclei, nitroxide radicals with broad ESR resonances such as TEMPO are more appropriate than Trityl and similar carbon-based radicals that have narrow lines. With TEMPO as polarizing agent, the main Dynamic Nuclear Polarization (DNP) mechanism is thermal mixing (TM). Cross polarization makes it possible to attain higher polarization levels at 2.2 K than one can obtain with direct DNP of low-gamma nuclei with TEMPO at 1.2 K, thus avoiding complex cryogenic technology

Homonuclear decoupling for spectral simplification of carbon-13 enriched molecules in solution-state NMR enhanced by dissolution DNP

Complex overlapping multiplets due to scalar couplings (n)J(C-13, C-13) in fully C-13-enriched molecules can be simplified by polychromatic irradiation of selected spins. The signal intensities of the remaining non-irradiated signals are proportional to the concentrations, as shown in this work for the anomeric C-13 signals of the alpha- and beta-conformers of glucose. Homonuclear decoupling can therefore be useful for quantitative NMR studies. The resulting decoupled lineshapes show residual fine structures that have been investigated by means of numerical simulations. Simulations also show that homonuclear decoupling schemes remain effective despite inhomogeneous static fields that tend to hamper in cellulo and in vivo studies. Homonuclear decoupling schemes can be combined with dissolution DNP to obtain signal enhancements of more than four orders of magnitude. Polychromatic irradiation of selected spins does not cause significant losses of hyperpolarization of the remaining non-irradiated spins

Long-Lived States of Magnetically Equivalent Spins Populated by Dissolution-DNP and Revealed by Enzymatic Reactions

Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) offers a way of enhancing NMR signals by up to five orders of magnitude in metabolites and other small molecules. Nevertheless, the lifetime of hyperpolarization is inexorably limited, as it decays toward thermal equilibrium with the nuclear spin-lattice relaxation time. This lifetime can be extended by storing the hyperpolarization in the form of long-lived states (LLS) that are immune to most dominant relaxation mechanisms. Levitt and co-workers have shown how LLS can be prepared for a pair of inequivalent spins by D-DNP. Here, we demonstrate that this approach can also be applied to magnetically equivalent pairs of spins such as the two protons of fumarate, which can have very long LLS lifetimes. As in the case of para-hydrogen, these hyperpolarized equivalent LLS (HELLS) are not magnetically active. However, a chemical reaction such as the enzymatic conversion of fumarate into malate can break the magnetic equivalence and reveal intense NMR signals

Hyperpolarized Water to Study Protein-Ligand Interactions

The affinity between a chosen target protein and small molecules is a key aspect of drug discovery. Screening by popular NMR methods such as Water-LOGSY suffers from low sensitivity and from false positives caused by aggregated or denatured proteins. This work demonstrates that the sensitivity of Water-LOGSY can be greatly boosted by injecting hyperpolarized water into solutions of proteins and ligands. Ligand binding can be detected in a few seconds, whereas about 30 min is usually required without hyperpolarization. Hyperpolarized water also enhances proton signals of proteins at concentrations below 20 M so that one can verify in a few seconds whether the proteins remain intact or have been denatured

Cross polarization from H-1 to quadrupolar Li-6 nuclei for dissolution DNP

Cross polarization from protons to quadrupolar Li-6 nuclei is combined with dynamic nuclear polarization of protons at 1.2 K and 6.7 T using TEMPOL as a polarizing agent followed by rapid dissolution. Compared to direct Li-6 DNP without cross-polarization, a higher nuclear spin polarization P(Li-6) can be obtained in a shorter time. A double resonance H-1-Li-6 probe was designed that is equipped for Longitudinally Detected Electron Spin Resonance

Challenges in preparing, preserving and detecting para-water in bulk: overcoming proton exchange and other hurdles

Para-water is an analogue of para-hydrogen, where the two proton spins are in a quantum state that is antisymmetric under permutation, also known as singlet state. The populations of the nuclear spin states in para-water are believed to have long lifetimes just like other Long-Lived States (LLSs). This hypothesis can be verified by measuring the relaxation of an excess or a deficiency of para-water, also known as a "Triplet-Singlet Imbalance'' (TSI), i.e., a difference between the average population of the three triplet states T (that are symmetric under permutation) and the population of the singlet state S. In analogy with our recent findings on ethanol and fumarate, we propose to adapt the procedure for Dissolution Dynamic Nuclear Polarization (D-DNP) to prepare such a TSI in frozen water at very low temperatures in the vicinity of 1.2 K. After rapid heating and dissolution using an aprotic solvent, the TSI should be largely preserved. To assess this hypothesis, we studied the lifetime of water as a molecular entity when diluted in various solvents. In neat liquid H2O, proton exchange rates have been characterized by spin-echo experiments on oxygen-17 in natural abundance, with and without proton decoupling. One-dimensional exchange spectroscopy (EXSY) has been used to study proton exchange rates in H2O, HDO and D2O mixtures diluted in various aprotic solvents. In the case of 50 mM H2O in dioxane-d(8), the proton exchange lifetime is about 20 s. After dissolving, one can observe this TSI by monitoring intensities in oxygen-17 spectra of H2O (if necessary using isotopically enriched samples) where the AX(2) system comprising a "spy'' oxygen A and two protons X-2 gives rise to binomial multiplets only if the TSI vanishes. Alternatively, fast chemical addition to a suitable substrate (such as an activated aldehyde or ketone) can provide AX2 systems where a carbon-13 acts as a spy nucleus. Proton signals that relax to equilibrium with two distinct time constants can be considered as a hallmark of a TSI. We optimized several experimental procedures designed to preserve and reveal dilute para-water in bulk

Hybrid polarizing solids for pure hyperpolarized liquids through dissolution dynamic nuclear polarization

Hyperpolarization of substrates for magnetic resonance spectroscopy (MRS) and imaging (MRI) by dissolution dynamic nuclear polarization (D-DNP) usually involves saturating the ESR transitions of polarizing agents (PAs; e.g., persistent radicals embedded in frozen glassy matrices). This approach has shown enormous potential to achieve greatly enhanced nuclear spin polarization, but the presence of PAs and/or glassing agents in the sample after dissolution can raise concerns for in vivo MRI applications, such as perturbing molecular interactions, and may induce the erosion of hyperpolarization in spectroscopy and MRI. We show that D-DNP can be performed efficiently with hybrid polarizing solids (HYPSOs) with 2,2,6,6-tetramethyl-piperidine-1-oxyl radicals incorporated in a mesostructured silica material and homogeneously distributed along its pore channels. The powder is wetted with a solution containing molecules of interest (for example, metabolites for MRS or MRI) to fill the pore channels (incipient wetness impregnation), and DNP is performed at low temperatures in a very efficient manner. This approach allows high polarization without the need for glass-forming agents and is applicable to a broad range of substrates, including peptides and metabolites. During dissolution, HYPSO is physically retained by simple filtration in the cryostat of the DNP polarizer, and a pure hyperpolarized solution is collected within a few seconds. The resulting solution contains the pure substrate, is free from any paramagnetic or other pollutants, and is ready for in vivo infusion

Method for the nmr based determination of the affinity of drugs for a target protein

The document relates to a method for the determination of the binding properties of at least one first chemical compound to at least one second chemical compound between a free essentially dissociated state and a bound essentially associated state by using nuclear magnetic resonance (NMR) measurements, wherein at least one of the first chemical compound or the second chemical compound comprises two coupled 1/2 spins in inequivalent molecular sites, wherein the relaxation of a long-lived state of the spin pair is measured as a function of the concentration of at least one of said first chemical compound for the determination of the equilibrium constant (KD) between the free state and the bound state

Hyperpolarization of Deuterated Metabolites via Remote Cross-Polarization and Dissolution Dynamic Nuclear Polarization

In deuterated molecules such as [1-C-13]pyruvate-d(3), the nuclear spin polarization of C-13 nuclei can be enhanced by combining Hartmann-Hahn cross-polarization (CP) at low temperatures (1.2 K) with dissolution dynamic nuclear polarization (D-DNP). The polarization is transferred from remote solvent protons to the C-13 spins of interest. This allows one not only to slightly reduce build-up times but also to increase polarization levels and extend the lifetimes T-1(C-13) of the enhanced C-13 polarization during and after transfer from the polarizer to the NMR or MRI system. This extends time scales over which metabolic processes and chemical reactions can be monitored