Pure Shift 2D NMR with Water Suppression for Structural and Dynamic Analysis of Flexible Molecules

Abstract Achieving high-resolution proton NMR spectra in aqueous media remains a major challenge for structurally complex and conformationally flexible molecules, where homonuclear scalar couplings and limited chemical-shift dispersion lead to severe spectral congestion. Here, we introduce two multidimensional pure-shift pulse sequences, Presat-PSYCHE-TOCSY-ES and NOESY-Presat-PSYCHE-NOESY-ES, that combine broadband homonuclear decoupling with efficient dual solvent suppression. This approach enables ultrahigh-resolution two-dimensional correlation spectroscopy in biologically relevant aqueous conditions while preserving spectral purity and sensitivity. Applied to the diamagnetic analog of a commercial contrast agent, these experiments combined with molecular modeling allow unambiguous discrimination and assignment of coexisting diastereoisomers that remain otherwise unresolved in conventional spectra. Moreover, the application of our approach to the crystallophore LuXo4 yields pure-shift EXSY experiments that provide a quantitative access to the slow conformational exchange process, in agreement with a ligand-arm rotational mechanism as identified by DFT. Overall, this work establishes water-compatible two-dimensional pure-shift NMR as a general platform for simultaneously probing stereochemistry, structure, and dynamics in broad range of flexible chemical species, including supramolecular or bioinorganic systems.