Application of 15 N-Edited 1 H- 13 C Correlation NMR Spectroscopy─Toward Fragment-Based Metabolite Identification and Screening via HCN Constructs.

Many key building blocks of life contain nitrogen moieties. Despite the prevalence of nitrogen-containing metabolites in nature, 15 N nuclei are seldom used in NMR-based metabolite assignment due to their low natural abundance and lack of comprehensive chemical shift databases. However, with advancements in isotope labeling strategies, 13 C and 15 N enriched metabolites are becoming more common in metabolomic studies. Simple multidimensional nuclear magnetic resonance (NMR) experiments that correlate 1 H and 15 N via single bond 1 J NH or multiple bond 2-3 J NH couplings using heteronuclear single quantum coherence (HSQC) or heteronuclear multiple bond coherence are well established and routinely applied for structure elucidation. However, a 1 H- 15 N correlation spectrum of a metabolite mixture can be difficult to deconvolute, due to the lack of a 15 N specific database. In order to bridge this gap, we present here a broadband 15 N-edited 1 H- 13 C HSQC NMR experiment that targets metabolites containing 15 N moieties. Through this approach, nitrogen-containing metabolites, such as amino acids, nucleotide bases, and nucleosides, are identified based on their 13 C, 1 H, and 15 N chemical shift information. This approach was tested and validated using a [ 15 N, 13 C] enriched Daphnia magna (water flea) metabolite extract, where the number of clearly resolved 15 N-containing peaks increased from only 11 in a standard HSQC to 51 in the 15 N-edited HSQC, and the number of obscured peaks decreased from 59 to just 7. The approach complements the current repertoire of NMR techniques for mixture deconvolution and holds considerable potential for targeted metabolite NMR in 15 N, 13 C enriched systems.