Fine optimization of a dissolution-DNP experimental setting for <sup>13</sup>C NMR of metabolic samples

Abstract. NMR based analysis of metabolite mixture provides crucial information on biological systems but mostly rely on 1D 1H experiments for maximizing sensitivity. However, strong peak overlap of 1H spectra often is a limitation for the analysis of inherently complex biological mixtures. Dissolution Dynamic Nuclear Polarization (d-DNP) improves NMR sensitivity by several orders of magnitude, which enables 13C NMR based analysis of metabolites at natural-abundance. We have recently demonstrated the successful introduction of d-DNP into a full untargeted metabolomics workflow applied to the study of plant metabolism. Here we describe the systematic optimization of d-DNP experimental settings for experiments at natural 13C abundance, and show how the resolution, sensitivity, and ultimately the number of detectable signals improve as a result. We have systematically optimized the parameters involved (in a semi-automated prototype d-DNP system, from sample preparation to signal detection, aiming at providing an optimization guide for potential users of such system who may not be experts in instrumental development). The optimization procedure makes it possible to detect previously inaccessible protonated-13C signals of metabolites at natural abundance with at least 4 times improved line shape and a high repeatability compared to a previously reported d-DNP enhanced untargeted metabolomic study. This extends the application scope of hyperpolarized 13C NMR at natural abundance and paves the way to a more general use of DNP-hyperpolarised NMR in metabolomics studies.