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 th e 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 demo nstrated experimentally on the side chain NH2 protons of glutamine in aqueo us solution with variable H2O/D2O ratio. The cross-relaxation between the n on-equivalent NH2 protons is caused by their close proximity, whereas the c hemical exchange is caused by the rotation of the whole NH2 group around th e CN chemical bond. At 275K and 500 MHz, in a fully protonated system, thei r cross-relaxation and chemical exchange rates are the same, 0.20 s(-1). He nce, in a standard, either laboratory or rotating frame, exchange experimen t the two processes cancel each other, leading to a false conclusion that n either chemical exchange nor cross-relaxation takes place. The separation o f the two processes is of interest in the study of water-macromolecule inte ractions, in which the labile protons from a macromolecule exhibit simultan eous cross-relaxation and chemical exchange towards water protons. The shor t residence time of water molecules near the macromolecule precludes the us e of alternative methods.