NOESY (Nuclear Overhauser Effect SpectroscopY) is an NMR experiment that can detect couplings between nuclei through spatial proximity (< 5 Å apart) rather than coupling through covalent bonds. The Nuclear Overhauser Effect (NOE) is the change in the intensity of the resonance of a nucleus upon irradiation of a nearby nucleus (about 2.5-3.5 Å apart). For example, when an RF pulse specifically irradiates a proton, its spin population is equalized and it can transfer its spin polarization to another proton and alter its spin population. The overall effect is dependent on a distance of r-6. NOESY uses a mixing time without pulses to accumulate NOEs and its counterpart ROESY (Rotating frame nuclear Overhauser Effect SpectroscopY) uses a series of pulses to accumulate NOEs. In NOESY, NOEs are positive when generated from small molecules, are negative when generated from large molecules (or molecules dissolved in a viscous solvent to restrict molecular tumbling), and are quite small (near zero) for medium-sized molecules. On the contrary, ROESY peaks are always positive, regardless of molecular weight. Both experiments are useful for determine proximity of nuclei in large biomolecules, especially proteins, where two atoms may be nearby in space, but not necessarily through covalent connectivity. Isomers, such as ortho-, meta-, and para-substituted aromatic rings, as well as stereochemistry, can also be distinguished through the use of an NOE experiment. Although NOESY and ROESY can generate COSY and TOCSY artifacts, respectively, those unwanted signals could be minimized by variations in the pulse sequences. Example NOESY and ROESY spectra are shown in Figure.
