Separation of Intra- and Intermolecular NOEs through Simultaneous Editing and J-Compensated Filtering: A 4D Quadrature-Free Constant-Time J-Resolved Approach

The NMR methods used to characterize molecular interactions through NOEs rely on isotope editing/filtering strategies designed to select for/against 13C(15N)-bound proton spins. The filter efficiency depends critically on the optimal compensation for variations in J CH scalar coupling constants, which is effectively obtained by the currently available second- and third-order isotope filters but at the cost of losing all intramolecular NOEs. Here a new filtering/editing strategy based on quadrature-free constant-time J-spectroscopy is presented that achieves J CH-compensation and the simultaneous measurement of inter- and intramolecular NOEs between 13C-bound 1H spins. The J-resolved frequency dimension is incorporated in a semiconstant-time NOESY-HSQC experiment resulting in a 4D spectrum in which signals arising from protons bound to 13C (or 15N) are separated from those originating from protons bound to 12C (or 14N) nuclei. The filter performance does not depend on double tuning of delays or on empirical correlations. In addition, the filter efficiency is not limited by upper bounds on the J CH values. The large J-bandwidth and the substantial time-saving make the pulse sequence here outlined a valuable tool for investigating noncovalent molecular assemblies by NMR, especially in the context of fast structural elucidations required for the development of structural genomics.