Indirect Detection of Hydroxy Proton Exchange Through Deuterium-Induced ¹³C-NMR Isotope Shifts

Abstract

Hydroxy groups are abundant on glycans and stabilize interactions with binding partners by forming intra- and inter-hydrogen bond networks. Information on the hydroxy proton and its bonding could be very useful for understanding glycan structure, their interactions, and their dynamics. Unfortunately, hydroxy protons are overlooked in NMR analyses because samples are typically measured in D₂O. Furthermore, their rapid exchange with water protons makes direct NMR detection difficult. We have developed a dynamic ¹³C-NMR technique employing deuterium-induced ¹³C-NMR isotope shifts, which provides indirect observation of exchangeable hydroxy protons through neighboring ¹³C signals. In the presence of both H₂O and D₂O and a slow exchange environment, a doublet of each isotopomer, ¹³C-OH and ¹³C-OD, is observed. A fast exchange environment makes each isotopomer indistinguishable and a sharp single signal appears at the average chemical shift. Thus, the ¹³C-line shape is highly dependent on the OH/D exchange rate, which, in turn, reflects the presence/absence/strength of hydrogen bonding. This simple ¹³C-NMR strategy is suitable for analyzing extremely weak interactions including bonds between carbohydrates and those involved in metal coordination.