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.

Reducing-End Functionalization of 2,5-Anhydro-d-mannofuranose-Linked Chitooligosaccharides by Dioxyamine: Synthesis and Characterization

“…Concerning the oximation conjugation, the reaction between COS-amf and PDHA was carried out with a large excess of the dioxyamine (10 molar eq./amf unit) in order to promote the coupling reaction with one oxyamine group only. Based on results of Baudendistel et al [20], the pH of the solution was kept constant at pH 5 during the reaction, firstly to solubilize COS-amf in the aqueous solvent, but also to ensure that the amine group of PDHA (pKa 4.2 [21]) is more nucleophilic than amine groups of COS-amf (pKa~6.2-7 [14]) towards the aldehyde group of the amf unit. In this condition of pH, the kinetic study of the reaction by 1 H NMR showed clearly: (i) the formation of the oxime group (CH=NO) characterized by two signals at 7.59 and 7.00 ppm for (E)-and (Z)-oxime protons, respectively [21] (Figure 1a); and (ii) the disappearance of the doublet signal at 5.10 ppm corresponding to the amf aldehyde group in its hydrated form (-CH(OH) 2 , below named gem diol group [16,17]; Figure S4 in Supplementary Materials).…”

“…Concerning the oximation conjugation, the reaction between COS-amf and PDHA was carried out with a large excess of the dioxyamine (10 molar eq./amf unit) in order to promote the coupling reaction with one oxyamine group only. Based on results of Baudendistel et al [20], the pH of the solution was kept constant at pH 5 during the Thanks to their high nucleophilicity in a large range of pH, oxyamines (pKa~4-5 [18]) have recently been described as a powerful chemo-selective tool for the reducing-end conjugation of various oligosaccharides in the development of new glycoconjugates [19,20]. Thus, dioxyamines conjugated to oligosaccharides could be particularly relevant precursors for the elaboration of advanced oligosaccharides-based materials.…”

The neoglycolipid (NGL) technology‐based microarrays and future prospects

“…This is, however, at the expense of sacrificing the natural O-glycan core monosaccharide. However, the predicted beta-configuration of the ring-closed form of oxime-linked Gal-NAc similar to GlcNAc would be a disadvantage for lectins and antibodies which recognize the GalNAc core in alpha-configuration [101]. There are several conditions for release of O-glycans under nonreductive conditions as reviewed by Chai and colleagues [100]; however, the yields of core-intact O-glycans (without peeling) are low.…”

Self‐Assembly in Water with N‐Substituted Imines

“…Even though oximes (‐C=N‐O‐) have a long history of being employed in the context of bioconjugation and soft materials, examples of oxime condensation for dynamic covalent self‐assembly are rare. One possible explanation is that the oxime bond is considered too inert to allow for dynamic error‐checking and self‐correcting.…”