Removal of ²H-decoupling sidebands in ¹³CHD₂ ¹³C-CEST profiles

A unique aspect of NMR is its capacity to provide integrated insight into both the structure and intrinsic dynamics of biomolecules. Chemical exchange phenomena that often serve as probes of dynamic processes in biological macromolecules can be quantitatively investigated with chemical exchange saturation transfer (CEST) experiments. ²H-decoupling sidebands, however, always occur in the profiles of ¹³CHD₂ ¹³C-CEST experiments when using the simple CW (continuous wave) method, which may obscure the detection of minor dips of excited states. Traditionally, these sidebands are manually eliminated from the profiles before data analysis by removing experimental points in the range of ²H-decoupling field strength ±50 Hz away from the major dips of the ground state on either side of the dips. Unfortunately, this may also eliminate potential minor dips if they overlap with the decoupling sidebands. Here, we developed methods that use pseudo-continuous waves with variable RF amplitudes distributed onto ramps for ²H decoupling. The new methods were thoroughly validated on Bruker spectrometers at a range of fields (¹H frequencies of 600, 700, and 850 MHz, and 1.1 GHz). By using these methods, we successfully removed the sidebands from the NMR profiles of ¹³CHD₂ ¹³C-CEST experiments.