The sign of NOESY peaks

Occasionally NOESY spectra of small molecules show a mixture of positive and negative peaks. The negative peaks are often due to exchange between different magnetic environments. I recently came across a good example of this in the NOESY spectrum of 2,3-dihydroxybenzamide.

The sign of a NOESY crosspeak depends on the magnetic field, ω, used to record the spectrum and the correlation time, τ_c, or rate at which the molecule tumbles in solution. The correlation time depends mainly on the molecular weight. If ωτ_c < 1.12, which is likely for small molecules, then the NOESY crosspeaks are positive. For larger molecules like proteins, ωτ_c > 1.12 and NOESY crosspeaks are negative. The blue line on the graph below shows the theoretical maximum intensity of a NOESY crosspeak versus ωτ_c. The theoretical maximum for ROESY crosspeaks is shown in red.

NMR theory considers NOESY crosspeaks positive if they are opposite in sign to the diagonal. So to tell what sign your crosspeaks are, you should phase your NOESY spectra so that the diagonal peaks are negative. For small molecules this will give positive crosspeaks for all through space interactions. Negative crosspeaks will be due to exchange of the magnetic environment. This can be due to conformational exchange, or even a chemical reaction.

The structure of 2,3-dihydroxybenzamide is shown below, along with an expansion of the crosspeaks in its NOESY spectrum. Assignments are given along the top of the NOESY spectrum. The diagonal peaks have been phased to be negative and are shown in teal. The positive NOE crosspeaks in blue show interactions between H5 and H4, H5 and H6, and H6 to both NH protons. The two NH protons show a teal, negative crosspeak, indicating that these two protons exchange magnetic environments.

The two protons attached to the nitrogen in an amide group are well known to give separate peaks; one for the proton close to the carbonyl oxygen, and one for the more distant proton. This is due to partial double bond character of the C-N bond, which slows, but does not eliminate, rotation. Rotation about this bond exchanges the environments of the two nitrogen-bound protons and leads to the exchange peak seen in the NOESY spectrum.

In NOESY spectra of large molecules exchange peaks are not as obvious as in the spectrum above. This is because the through space crosspeaks are negative, the same as the exchange peaks. To identify exchange peaks a ROESY spectrum can be recorded. In the ROESY spectrum the exchange peaks will be negative while the through space interactions will give positive peaks.

Acknowledgements

Thanks to Carla Uranga and Marcelo Freire of the J. Craig Venter Institute for providing the sample of 2,3-dihydroxy benzamide.