Aurélien Bornet

Boosting Dissolution Dynamic Nuclear Polarization by Cross Polarization

The efficiency of dissolution dynamic nuclear polarization can be boosted by Hartmann-Hahn cross polarization at temperatures near 1.2 K. This enables high throughput of hyperpolarized solutions with substantial gains in buildup times and polarization levels. During dissolution and transport, the (13)C nuclear spin polarization P((13)C) merely decreases from 45 to 40%.

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

Significance Hyperpolarization by dissolution dynamic nuclear polarization can dramatically enhance signal intensities in MRI and NMR, notably for metabolic tracers for imaging and diagnosis. It is applicable to a variety of substrates for in vivo imaging and chemistry but requires the use of contaminants (glassing agents and free radicals) that may interact with cells and proteins and can have potential side effects. These contaminants can sometimes be eliminated by precipitation followed by filtration or solvent extraction, but these methods are substrate-specific, are usually time-consuming, and typically result in signal loss. Here, production of pure hyperpolarized liquids free of contaminants is shown by a simple wetting–polarization–filtration sequence for a solid silica matrix containing homogeneously distributed persistent radicals. 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.

A magnetic tunnel to shelter hyperpolarized fluids

To shield solutions carrying hyperpolarized nuclear magnetization from rapid relaxation during transfer through low fields, the transfer duct can be threaded through an array of permanent magnets. The advantages are illustrated for solutions containing hyperpolarized (1)H and (13)C nuclei in a variety of molecules.

Boosting the Sensitivity of Ligand−Protein Screening by NMR of Long-Lived States

A new NMR method for the study of ligand-protein interactions exploits the unusual lifetimes of long-lived states (LLSs). The new method provides better contrast between bound and free ligands and requires a protein-ligand ratio ca. 25 times lower than for established T(1ρ) methods, thus saving on costly proteins. The new LLS method was applied to the screening of inhibitors of urokinase-type plasminogen activator (uPA), which is a prototypical target of cancer research. With only 10 μM protein, a dissociation constant (K(D)) of 180 ± 20 nM was determined for the strong ligand (inhibitor) UK-18, which can be compared with K(D) = 157 ± 39 nM determined by the established surface plasmon resonance method.

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.

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 μM so that one can verify in a few seconds whether the proteins remain intact or have been denatured.

Hyperpolarization of deuterated metabolites via remote cross-polarization and dissolution dynamic nuclear polarization.

In deuterated molecules such as [1-(13)C]pyruvate-d3, the nuclear spin polarization of (13)C 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 (13)C spins of interest. This allows one not only to slightly reduce build-up times but also to increase polarization levels and extend the lifetimes T1((13)C) of the enhanced (13)C 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.

Hyperpolarized NMR of plant and cancer cell extracts at natural abundance

Natural abundance (13)C NMR spectra of biological extracts are recorded in a single scan provided that the samples are hyperpolarized by dissolution dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell extracts can also be obtained in a single scan. Hyperpolarized NMR of extracts opens many perspectives for metabolomics.

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.

Long-lived coherences for line-narrowing in high-field NMR.

2010 Elsevier B.V. All rights reserved.Contents1. Introduction . . . 832. The tailored Hamiltonian. . . . . . . . . . . . 843. Definition of long-lived coherences . . . 854. Relaxation of long-lived coherences . . . 855. Designing suitable experiments . . . . . . 866. Long-lived coherences in a small molecule . . . . . . . . . . . . . . . . 867. Long-lived coherences in a protein. . . . 878. Simultaneous excitation of several long-lived coherences . . . . 879. Conclusions. . . . 88Acknowledgements . . . . . . . . . . . . . . . . 89Appendix A . . . . . 89References . . . . 90