Novel Sample Formulations for Pure and Persistent Hyperpolarized Solutions via Dissolution Dynamic Nuclear Polarization

Nuclear Magnetic Resonance (NMR) spectroscopy allows one to study and analyze the structure, motions and interactions of a broad variety of molecules. However, this technique has a major inconvenience: its low sensitivity, which therefore often results in the use of highly concentrated samples in order to observe tiny signals. As recently as 2003, a new technique known as dissolution-DNP or d-DNP was invented by Ardenkjaer-Larsen et al. to overcome this drawback and to get more intense NMR signals in solution with enhancements factors larger than 10'000. This method consists essentially in mixing paramagnetic species (radicals) with samples containing the metabolites to be analyzed. These mixtures are then rapidly frozen at very low temperatures (T = 1.2 â 4.2 K) in liquid helium and, by applying a proper microwave irradiation, the polarization of the electrons can be transferred to nuclei such as 1H or 13C. This allows one to build up and store the enhanced magnetization of these nuclei and then to dissolve the samples by injecting a superheated solvent via a dissolution stick. The resulting hyperpolarized solution can be finally transferred to an NMR spectrometer where the signals can be recorded. In the first two chapters of this thesis, the principles of NMR are introduced and the theory of DNP is explained in some detail. The dissolution equipment used in our laboratory and its different parts are shown, in particular the DNP polarizer where the transfer of polarization from electrons to nuclei occurs, the microwave source that is connected to the polarizer, and the dissolution system itself, which comprises the dissolution stick and the dissolution transfer line. Another chapter is dedicated to the optimization of our DNP setup in order to achieve the highest possible polarization before the dissolution process. Several radicals are tested under carefully controlled conditions to identify the best suited for our DNP system. Furthermore, the modulation of the microwave frequency has been optimized in order to enhance the polarization transfer. Finally, a number of dissolution experiments are presented that relate to different projects that have been carried out in the course of this thesis. The extent of the polarization can be determined accurately by looking directly at the hyperpolarized NMR spectrum. Filterable polymers containing suitable radical moieties have been synthesized in order to obtain pure hyperpolarized solutions. A final chapter outlines future applications and projects that can benefit from dissolution Dynamic Nuclear Polarization

Measuring absolute spin polarization in dissolution-DNP by Spin PolarimetrY Magnetic Resonance (SPY-MR)

Dynamic nuclear polarization at 1.2 K and 6.7 T allows one to achieve spin temperatures on the order of a few millikelvin, so that the high-temperature approximation (Delta E < kT) is violated for the nuclear Zeeman interaction Delta E = gamma B(0)h/(2 pi) of most isotopes. Provided that, after rapid dissolution and transfer to an NMR or MRI system, the hyperpolarized molecules contain at least two nuclear spins I and S with a scalar coupling J(IS), the polarization of spin I (short for 'investigated') can be determined from the asymmetry A(S) of the multiplet of spin S (short for 'spy'), provided perturbations due to second-order (strong coupling) effects are properly taken into account. If spin S is suitably discreet and does not affect the relaxation of spin I, this provides an elegant way of measuring spin polarizations 'on the fly' in a broad range of molecules, thus obviating the need for laborious measurements of signal intensities at thermal equilibrium. The method, dubbed Spin PolarimetrY Magnetic Resonance (SPY-MR), is illustrated for various pairs of C-13 spins (I, S) in acetate and pyruvate. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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 para-Ethanol

We show that an imbalance between the populations of singlet (S) and triplet (T) states in pairs of magnetically equivalent spins can be generated by dissolution dynamic nudear polarization: In partly deuterated ethanol ((CD3CH2OD)-C-13), this T/S imbalance can be transferred by cross-relaxation to observable, enhanced signals of protons and coupled C-13

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

Drug Screening Boosted by Hyperpolarized Long-Lived States in NMR

Transverse and longitudinal relaxation times (T1ρ and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1(1H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak “spy ligands” can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput

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

An automated system for fast transfer and injection of hyperpolarized solutions

Dissolution dynamic nuclear polarization (dDNP) has become a hyperpolarization method of choice for enhancing nuclear magnetic resonance (NMR) signals. Nuclear spins are polarized in solid frozen samples (in a so-called polarizer) that are subsequently dissolved and transferred to an NMR spectrometer for high sensitivity detection. One of the critical challenges of dDNP is that it requires both a fast transfer to limit nuclear spin relaxation losses as well as stability to guarantee high resolution (no bubbles nor turbulences). Here we describe the design, construction and performances of such a transfer and injection system, that features a 5 m/s speed and sub-Hz spectral resolution upon arrival at the detection spot. We demonstrate the use of such a system for inter-magnet distances of up to 10 m