In order to assign resonances to H-bonded protons by proton-NMR, chemists in teaching laboratories often employ the inclusion of deuterium by exchange with deuterium oxide. In the case of secondary amides, where vicinal coupling to H-bonded protons is often observed, this deuterium exchange offers an opportunity to selectively decouple protons alpha to the N-atom. However, due to slow exchange with deuterium oxide D2O, this method of chemical exchange is not ideal, especially when the resulting HOD resonance obscures other key resonances in the spectrum. In this study, three N-benzoyl amino acids, from alanine, glycine, and valine, were synthesized and deuterated using chemical exchange with D2O. Their structures were confirmed by 1H-NMR spectra in DMSO-d6 solvent. Benzoyl glycine and alanine were synthesized and deuterated successfully, showing reasonable splitting and presence of a substantial HOD peak. However, the deuterium exchange of benzoyl valine was less effective due to slower exchange of the amide NH presumably due to higher steric hindrance. To resolve the incomplete deuterium exchange by D2O, we instead implemented homonuclear decoupling as opposed to a chemical exchange. The decoupling pulse sequence was employed at the chemical shift of the amide proton. In order to maintain proton integration ratios, careful control of pulse lengths and attenuation values were required due to NOE from the decoupling pulse. Following the irradiation of the amide proton with an attenuation value of 70 dB at 8.4 ppm, the signal of the suppressed proton was absent. The success of this experiment was justified in the consequential decoupling of the proton belonging to the adjacent secondary carbon of the carboxylic acid as evident in a change of splitting patterns. This work demonstrates an alternative, non-chemical technique used in a teaching laboratory in order to assign all proton resonances and elucidate the splitting patterns in N-benzoylamino acids. By direct irradiation of the secondary amide proton as part of the acquisition of the 1D 1H-NMR spectra, along with solvent and water suppression, cleanly decoupled spectra were obtained by students for comparison with their unmodified 1H-NMR spectra.

Elucidating N-Benzoylamino acid proton coupling assignments by selective homonuclear decoupling and chemical exchange

ACS Spring Meeting 2021

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