To confirm that all peaks observed in the diagonal-free NOESY are actual NOE peaks and not artifacts, their assignment is indicated. They all correspond to proton

pairs which are close in space, like axial protons check details on the same side of the glucose ring (2–4 and 3–5) or neighboring protons (1–2, 1′–2′). The regular NOESY experiment ( Fig. 5a) was recorded with 32 scans per increment and the diagonal suppressed NOESY spectrum ( Fig. 5b) by using 256 scans per increment and otherwise identical parameters. To experimentally determine the signal/noise changes of the regular versus the spatially-selective, diagonal-suppressed NOESY spectrum, representative traces at the frequency 4.3 ppm for two short NOESY spectra recorded with the same acquisition parameters (number of scans, increments, receiver gain,

etc.) and processing scheme is shown in Fig. 6. As expected, for a selective pulse with an excitation bandwidth of ∼80 Hz and a 1.2 G/cm gradient the signal/noise ratio drops to about 2% of a regular NOESY spectrum. To evaluate the performance of the diagonal suppression scheme also on bigger, faster relaxing molecules, we acquired a diagonal suppressed NOESY spectrum of the 14 kDa protein lysozyme (3 mM) in D2O solution. As can be seen in Fig. 7, the presented approach leads to a complete removal of all diagonal peaks, while VEGFR inhibitor the cross peaks are unaffected. Both spectra were recorded with a mixing time of 150 ms and 8000 Hz spectral width in both dimensions. Sixty-four scans were acquired for the regular NOESY and 512 for the diagonal free version. The total duration of the pulse-sequence of the presented approach is not much longer than a regular NOESY. Only the first pulse is now 40 ms instead of the hard pulse and the diagonal suppression is technically the same as the typical solvent suppression. Therefore, any additional relaxation losses

of the diagonal-free spectrum, relative to the regular experiment, are minimal. When solvent suppression is needed in diagonal-free spectra, we use presaturation of the water signal before the first selective 90° pulse, rather than adding another excitation sculpting/watergate sequence prior to acquisition to keep relaxation losses Thiamet G to a minimum (see Supplementary Fig. S2). We have presented a generally applicable approach to obtain diagonal peak free homonuclear correlated spectra. It relies on the slice selective excitation during a weak gradient field. Signals that do not change the frequency during the mixing are removed by excitation sculpting right before the acquisition. Due to this spatially selective excitation the magnetic field is very uniform for each signal and therefore cancels most of the magnetic field inhomogeneities along the z-direction. However, as a result, the sensitivity is reduced compared to a regular spectrum.