Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions

Abstract

Ligation reactions at the anomeric center of carbohydrates have gained increasing importance in the field of glycobiology. Oxyamines are frequently used in labeling, immobilization, and bioconjugation of reducing carbohydrates. Herein, we present a systematic investigation of these ligation reactions under aqueous conditions. A series of four unprotected monosaccharides (glucose, N-acetylglucosamine, mannose, and 2-deoxyglucose) and one disaccharide (N,N'-diacetylchitobiose) was reacted with three primary and one secondary oxyamine. We monitored the concentrations of the starting materials and products by (1) H NMR spectroscopy and determined reaction times and equilibrium yields. Our experiments show that the outcome of the ligation reaction is not only dependent on the sugar and oxyamine used but also strongly on the reaction conditions. In the case of glucose, lowering the pH from 6 to 3 led to steadily increasing reaction rates, whereas the yields were decreasing at the same time. Variation of the temperature did not only influence the product ratio in equilibrium but can also have a strong impact on the equilibrium yield. In the case of reactions of a primary oxyamine, increased temperatures led to a higher proportion of acyclic products. Reaction of the secondary oxyamine with glucose unexpectedly led to lower yields at higher temperatures.