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

Many key building blocks of life contain nitrogen moieties. Despite the prevalence of nitrogen-containing metabolites in nature, ¹⁵N 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, ¹³C and ¹⁵N enriched metabolites are becoming more common in metabolomic studies. Simple multidimensional nuclear magnetic resonance (NMR) experiments that correlate ¹H and ¹⁵N via single bond ¹J_NH or multiple bond ^2-3J_NH couplings using heteronuclear single quantum coherence (HSQC) or heteronuclear multiple bond coherence are well established and routinely applied for structure elucidation. However, a ¹H-¹⁵N correlation spectrum of a metabolite mixture can be difficult to deconvolute, due to the lack of a ¹⁵N specific database. In order to bridge this gap, we present here a broadband ¹⁵N-edited ¹H-¹³C HSQC NMR experiment that targets metabolites containing ¹⁵N moieties. Through this approach, nitrogen-containing metabolites, such as amino acids, nucleotide bases, and nucleosides, are identified based on their ¹³C, ¹H, and ¹⁵N chemical shift information. This approach was tested and validated using a [¹⁵N, ¹³C] enriched Daphnia magna (water flea) metabolite extract, where the number of clearly resolved ¹⁵N-containing peaks increased from only 11 in a standard HSQC to 51 in the ¹⁵N-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 ¹⁵N, ¹³C enriched systems.