Fast Spatially Encoded 3D NMR Strategies for ¹³C‑Based Metabolic Flux Analysis

The measurement of site-specific ¹³C enrichments in complex mixtures of ¹³C-labeled metabolites is a powerful tool for metabolic flux analysis. One of the main methods to measure such enrichments is homonuclear ¹H 2D NMR. However, the major limitation of this technique is the acquisition time, which can amount to a few hours. This drawback was recently overcome by the design of fast COSY experiments for measuring specific ¹³C-enrichments, based on single-scan 2D NMR. However, these experiments are still limited by overlaps because of¹H–¹³C splittings, thus limiting the metabolic information accessible for complex biological mixtures. To circumvent this limitation, we propose to tilt the ¹H–¹³C coupling into a third dimension via fast-hybrid 3D NMR methods combining the speed of ultrafast 2D NMR with the high resolution of conventional methods. Two strategies are described that allow the acquisition of a complete 3D J-resolved-COSY spectrum in 12 min (for concentrations as low as 10 mM). The analytical potentialities of both methods are evaluated on a series of ¹³C-enriched glucose samples and on a biomass hydrolyzate obtained from <i>Escherichia coli</i> cells. Once optimized, the two complementary experiments lead to a trueness and a precision of a few percent and an excellent linearity. The advantages and drawbacks of these approaches are discussed and their potentialities are highlighted