The Flexibility of Oligosaccharides Unveiled Through Residual Dipolar Coupling Analysis

Abstract

The intrinsic flexibility of glycans complicates the study of their structures and dynamics, which are often important for their biological function. NMR has provided insights into the conformational, dynamic and recognition features of glycans, but suffers from severe chemical shift degeneracy. We employed labelled glycans to explore the conformational behaviour of a β(1-6)-Glc hexasaccharide model through residual dipolar couplings (RDCs). RDC delivered information on the relative orientation of specific residues along the glycan chain and provided experimental clues for the existence of certain geometries. The use of two different aligning media demonstrated the adaptability of flexible oligosaccharide structures to different environments.

Synthetic Approaches to Break the Chemical Shift Degeneracy of Glycans

“…A collection of linear β(1‐6)‐linked hexaglucosides, bearing one 13 C‐labeled Glc unit in different positions of the chain, granted access to J ‐coupling values, supporting the helical model predicted by MD (Figure 1B) [14] . This collection was further expanded to include oligomers with two 13 C‐labeled Glc units, providing geometrical information on the relative orientation of the Glc residues along the glycan backbone, measured via 13 C‐ 1 H residual dipolar couplings (RDCs) (Figure 1B) [27] . A detailed NMR analysis using different aligning media demonstrated the high flexibility of these oligomers.…”

“…[14] This collection was further expanded to include oligomers with two 13 C-labeled Glc units, providing geometrical information on the relative orientation of the Glc residues along the glycan backbone, measured via 13 C-1 H residual dipolar couplings (RDCs) (Figure 1B). [27] A detailed NMR analysis using different aligning media demonstrated the high flexibility of these oligomers.…”

“… [41] Synthesis of site‐specific 19F‐labeling can also be combined with 13 C‐labeling to provide additional geometrical information for conformational analysis. [27] As drawback, the synthesis of 19 F‐labeled glycans can be challenging, often demanding different protocols depending on the position(s) to be labeled on the glycan residue. [ 42 , 43 ] The enzymatic incorporation of 19 F‐labeled monosaccharides in glycoconjugates is an interesting avenue to access complex glycan probes, [44] but 19 F‐monomers are not always accepted by enzymes.…”

“…Simple spectra analysis offers the possibility of studying events in real‐time, with potential applications for in vivo studies, [40] or to monitor enzymatic reactions [41] . Synthesis of site‐specific 19F‐labeling can also be combined with 13 C‐labeling to provide additional geometrical information for conformational analysis [27] . As drawback, the synthesis of 19 F‐labeled glycans can be challenging, often demanding different protocols depending on the position(s) to be labeled on the glycan residue [42,43] .…”

Recent Developments in Solid-Phase Glycan Synthesis

“…21 53 13 C-labelled­ monosaccharides have also been tested to generate useful NMR probes. 52 56 Key to their successful implementation in SPGS is the stability of these moieties during the entire assembly process. Particular attention should be paid when implementing modifications that could influence the glycosylation step, either by lowering the BB reactivity or by influencing its stereochemical outcome (e.g., deoxyfluorination).…”