Expedited Nuclear Magnetic Resonance Assignment of Small- to Medium-Sized Molecules with Improved HSQC-CLIP-COSY Experiments.

Resonance assignment is a pivotal step for any nuclear magnetic resonance (NMR) analysis, such as structure elucidation or the investigation of protein-ligand interactions. Both H-C heteronuclear single quantum correlation (HSQC) and H-H correlation spectroscopy (COSY) two-dimensional (2D) experiments are invaluable for H NMR assignment, by extending the high signal dispersion of C chemical shifts onto H resonances and by providing a high amount of through-bond H-H connectivity information, respectively. The recently introduced HSQC-CLIP(Clean In-Phase)-COSY method combines these two experiments, providing COSY correlations along the high-resolution C dimension with clean in-phase multiplets. However, two experiments need to be recorded to unambiguously identify COSY cross-peaks. Here, we propose novel variants of the HSQC-CLIP-COSY pulse sequence that edit cross-peak signs so that direct HSQC responses can be distinguished from COSY relay peaks, and/or the multiplicities of the C nuclei are reflected, allowing the assignment of all the peaks in a single experiment. The advanced HSQC-CLIP-COSY variants have the potential to accelerate and simplify the NMR structure-elucidation process of both synthetic and natural products and to become valuable tools for high-throughput computer-assisted structure determination.