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

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