The long and the long of it

A novel NMR technique has measured the largest distance between two atomic nuclei using NMR, demonstrating that tritium magic angle spinning NMR could be a promising tool for structural applications in the biological and material sciences.

Alexander Yuen, Olivier Lafon, Thibault Charpentier, Myriam Roy, Francine Brunet, Patrick Berthault, Dimitrios Sakellariou, Bruno Robert, Sylvie Rimsky, Florence Pillon, Jean-Christophe Cintrat and Bernard Rousseau of Commissariat à l'Energie Atomique in France (iBiTecS and IRAMIS in Gif sur Yvette) and LBPA, CNRS, ENS in Cachan) provide details in the Journal of the American Chemical Society.

The team explains that solid-state NMR spectroscopy can be a powerful technique for determining the structure of ligands bound to amorphous macromolecular systems, such as an insoluble or membrane protein where X-ray crystallography is not possible, by providing key interatomic distances.

"Knowledge of this conformation is of key importance in drug design," the team explains. While long-range distances can be determined using beacons such as fluorine-19 this is limited to 8 angstrom separations. Paramagnetic spin labels can open this distance up to 20 angstroms. However, both techniques require chemical modifications of the ligand that ultimately distort the relationship between small molecule and protein, for instance, a problem best avoided when attempting to understand a protein-drug interaction.

Isotopic labelling using with carbon-13 and nitrogen-15 do not involve structural modifications, but their reach is a mere 5 to 6 angstroms. "Most of the conformational changes when a ligand binds its receptor involve variation of long-range interatomic distances," the team says, which means isotopic labelling with these nuclei is irrelevant. For example, the anticancer drug paclitaxel (commonly known as Taxol) is involved in conformational changes at distances above 10 angstroms.

The researchers have now investigated the selective labelling of specific locations within the ligand using tritium without requiring any structural modifications of the molecule. "Modern synthetic protocols can furnish a variety of selectively labelled materials with tritium atoms at precise locations," the team explains. Indeed, prices of ready labelled compounds are on a par with those labelled with carbon-13 and nitrogen-15.

The tritium nucleus has a spin of a half and a high gyromagnetic ratio and so, the team reasoned, MAS experiments should allow them to accurately measure the long-range distances in a ligand-protein system labelled with these nuclei. The team points out that the background levels of tritium are negligible so there is no interference from its natural presence.

To test the potential of this approach, the researchers designed small molecules rigid enough to obtain tritium-tritium distances that would not fluctuate. They made a range of compounds with distances of 4.31, 6.0, 9.4, and 13.8 angstroms. "For each NMR analysis, a powder mixture of 50 mCi (0.15 to 0.3 mg) of the tritiated compound, 40 mg of its unlabelled form, and 0.2 wt % of paramagnetic salt Cu(NO3)2 was prepared (the latter being added to shorten relaxation times)," the team expains. They have now used this approach to obtain the largest NMR distance ever measured between two nuclei, 14.4 angstroms +/- 2.2 angstroms.

To make the approach more generally applicable for samples with larger tritium chemical shift distributions or shorter relaxation times, it will be necessary to modify the experimental conditions, adopting higher spinning frequency, smaller sample volumes, novel pulse sequences, for instance.

"Tritium has already proven useful in liquid-state NMR where it has helped elucidate stereochemical and mechanistic aspects of small-molecule chemistry over the past few decades," the team adds. As such they suggest it could become a powerful tool in solid-state NMR. The approach might now allow drug molecules to be studied at otherwise inaccessible binding sites in insoluble or membrane proteins. The team is currently investigating the bioactive conformations of the microtubule-bound paclitaxel using this approach. 

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