Two-dimensional exchange NMR spectroscopy in fractionally deuterated molecules: Separation of exchange and cross-relaxation processes by proton dilution

The separation of cross-relaxation and chemical exchange is exploited by the difference in dependence on the degree of deuteration. The build-up rate of a normalized cross-peak from a proton-diluted spectrum is independent of the dilution for an exchange cross-peak, whereas it is proportional to the proton concentration for a cross-relaxation cross-peak. The method is demonstrated experimentally on the side chain NH₂ protons of glutamine in aqueous solution with variable H₂O/D₂O ratio. The cross-relaxation between the non-equivalent NH₂ protons is caused by their close proximity, whereas the chemical exchange is caused by the rotation of the whole NH₂ group around the CN chemical bond. At 275 K and 500 MHz, in a fully protonated system, their cross-relaxation and chemical exchange rates are the same, 0.20 s^-1. Hence, in a standard, either laboratory or rotating frame, exchange experiment the two processes cancel each other, leading to a false conclusion that neither chemical exchange nor cross-relaxation takes place. The separation of the two processes is of interest in the study of water-macromolecule interactions, in which the labile protons from a macromolecule exhibit simultaneous cross-relaxation and chemical exchange towards water protons. The short residence time of water molecules near the macromolecule precludes the use of alternative methods.