Measurement and Characterization of Hydrogen-Deuterium Exchange Chemistry Using Relaxation Dispersion NMR Spectroscopy.

One-dimensional heteronuclear relaxation dispersion NMR spectroscopy at 13C natural abundance successfully characterized the dynamics of the hydrogen-deuterium exchange reaction occurring at the Nε position in l-arginine by monitoring Cδ in varying amounts of D2O. A small equilibrium isotope effect was observed and quantified, corresponding to ΔG = -0.14 kcal mol-1. A bimolecular rate constant of kD = 5.1 × 109 s-1 M-1 was determined from the pH*-dependence of kex (where pH* is the direct electrode reading of pH in 10% D2O and kex is the nuclear spin exchange rate constant), consistent with diffusion-controlled kinetics. The measurement of ΔG serves to bridge the millisecond time scale lifetimes of the detectable positively charged arginine species with the nanosecond time scale lifetime of the nonobservable low-populated neutral arginine intermediate species, thus allowing for characterization of the equilibrium lifetimes of the various arginine species in solution as a function of fractional solvent deuterium content. Despite the system being in fast exchange on the chemical shift time scale, the magnitude of the secondary isotope shift due to the exchange reaction at Nε was accurately measured to be 0.12 ppm directly from curve-fitting D2O-dependent dispersion data collected at a single static field strength. These results indicate that relaxation dispersion NMR spectroscopy is a robust and general method for studying base-catalyzed hydrogen-deuterium exchange chemistry at equilibrium.