Tailoring 1H Spin Dynamics in Small Molecules via Supercooled Water: A Promising Approach for Metabolite Identification and Validation

Metabolic mixtures are often analyzed via NMR spectroscopy as it provides a metabolic profile without sample alteration in a noninvasive manner. These mixtures however tend to be very complex and demonstrate considerable spectral overlap resulting in assignments that are sometimes ambiguous given the range of current NMR methods available. De novo molecular identification in these mixtures is generally accomplished using chemical shift information and J-coupling based experiments to determine spin connectivity information, but these techniques fall short when a molecule of interest contains nonrelaying centers. A method is presented here that enhances intramolecular spatial interactions via supercooled water and uses the resulting spatial correlations to edit mixtures. This is accomplished by utilizing nuclear Overhauser effect spectroscopy (NOESY) at subzero temperatures in capillaries to enhance NOE and provide more complete spin systems. This technique is applied to a standard mixture of three known molecules in D2O with overlapping resonances and is further demonstrated to assign molecules in a worm tissue extract. The current method proves to be a powerful complement to existing methods such as total correlation spectroscopy (TOCSY) to expand the range of molecules that can be assigned in situ without physical separation of mixtures.