Highly Sensitive Hyperpolarized Multiplexed Single-Scan 2D 13C−1H NMR of Complex Mixtures at Natural 13C Abundance

Abstract Hyperpolarization provides a major sensitivity boost in NMR spectroscopy, and dissolution Dynamic Nuclear Polarization (d-DNP) has emerged as a particularly well-suited approach to analyze complex mixtures at natural 13C abundance. However, it only allows single-scan NMR detection due to the irreversible nature of hyperpolarized magnetization decay in solution. This precludes the acquisition of heteronuclear 2D NMR spectra, which are highly needed to obtain information on the identity and concentration of mixture components. To overcome this issue, ultrafast (UF) 2D NMR has been the method of choice for the last two decades, but it suffers from limited resolution and sensitivity, which restrains its application to 13C-labeled or highly concentrated molecular mixtures. Herein, we propose a new concept coined Selective Multiplexed Acquisition by Sequential Harnessing (SMASH), which when combined with d-DNP allows us to retrieve 13C−1H correlations of small-molecule mixtures at low mM concentrations (in d-DNP juice) and natural 13C abundance. This new method improves the sensitivity of single-scan heteronuclear 2D NMR by a factor of 10 to 20 compared to UF experiments, thus considerably lowering the limit of detection of d-DNP-hyperpolarized 2D NMR experiments. The potential of SMASH NMR is illustrated on d-DNP hyperpolarized metabolite mixtures, showing that it will complement UF 2D NMR methods in the future to reveal the 13C−1H correlations of hyperpolarized small-molecule mixtures at 13C natural abundance.