NMR analysis and molecular dynamics conformation of α-1,6-linear and α-1,3-branched isomaltose oligomers as mimetics of α-1,6-linked dextran.

The conformational preferences of several α-1,6-linear and α-1,3-branched isomalto-oligosaccharides were investigated by NMR and MD-simulations. Right-handed helical structure contributed to the solution geometry in isomaltotriose and isomaltotetraose with one nearly complete helix turn and stabilizing intramolecular hydrogen bonds in the latter by MD-simulation. Decreased helix contribution was observed in α-1,3-glucopyranosyl- and α-1,3-isomaltosyl-branched saccharide chains. Especially the latter modification was predicted to cause a more compact structure consistent with literature rheology measurements as well as with published dextranase-resistant α-1,3-branched oligosaccharides. The findings presented here are significant because they shed further light on the conformational preference of isomalto-oligosaccharides and provide possible help for the design of dextran-based drug delivery systems or for the targeted degradation of capsular polysaccharides by dextranases in multi-drug resistant bacteria.

Ultraviolet photodissociation of fondaparinux generates signature antithrombin-like 3-O-sulfated -GlcNS3S6S- monosaccharide fragment (Y3/C3).

The 3-O sulfate-modified -GlcNS3S6S- monosaccharide in heparin and heparan sulfate glycosaminoglycans (HSGAGs) is a relatively rare yet important modification that facilitates HSGAG-antithrombin binding and subsequent anticoagulant activity. Detecting this modification in complex HSGAG mixtures is a longstanding goal to identify novel 3-O-sulfated HSGAG-protein interactions with biologically significant functions. Tandem mass spectrometry has been applied to HSGAG structural analysis but is limited by the fact that traditional collision-induced dissociation techniques (e.g., CID, HCD) results in extensive sulfate loss prior to generating structurally informative glycosidic and cross-ring fragments. In the present study, we investigated the potential of ultraviolet photodissociation (UVPD) to generate structurally informative fragments from the synthetic heparin mimetic, fondaparinux, under electrospray conditions commensurate with hydrophilic interaction liquid chromatography (HILIC). The two predominant un-adducted precursors, [Fonda-2H+]2- and [Fonda-3H+]3-, were subjected to UVPD, CID, and HCD on an Orbitrap Fusion Lumos Tribrid mass spectrometer and the resulting fragmentation spectra directly compared. Close inspection of the UVPD data identified a unique peak at m/z 417.9425 that matched the Y3/C3 double glycosidic fragment of fondaparinux (i.e., -GlcNS3S6S-). Importantly, the 3-O-sulfated Y3/C3 fragment was generated predominantly from UVPD of the [Fonda-2H+]2- precursor, increased with activation time, and was observable using data-dependent HILIC-MS/MS UVPD analysis of fondaparinux spiked into a semi-complex HSGAG mixture. The discovery of this antithrombin-like 3-O-sulfated fragment provides a potential strategy for screening complex HSGAG mixtures in a data-dependent or data-independent acquisition mode to determine the presence of this therapeutic and biologically significant HSGAG modification. Graphical abstract.

NMR solution geometry of saccharides containing the 6-O-(α-D-glucopyranosyl)-α/ß-D-glucopyranose (isomaltose) or 6-O-(α-D-galactopyranosyl)-α/ß-D-glucopyranose (melibiose) core.

The solution geometries of D-Glcp, Me-D-Glcp, 6-O-Me-D-Glcp, Me-6-O-Me-D-Glcp, D-Glcp-(α-1,6)-D-Glcp (isomaltose), D-Glcp-(α-1,6)-D-Glcp-(α-1,6)-D-Glcp (isomaltotriose), D-Galp-(α-1,6)-D-Glcp (melibiose), D-Galp-(α-1,6)-D-Glcp-(α-1,2)-D-Fruf (raffinose), and D-Galp-(α-1,6)-D-Galp-(α-1,6)-D-Glcp-(α-1,2)-D-Fruf (stachyose) in water are described by NMR spectroscopy, molecular dynamic simulations and quantum mechanical calculations. Overall, a change in anomeric configuration at the reducing end and/or anomeric substitution (methylation) changed the conformational space of the terminal CH2OH group significantly. Conformational analysis of the free monosaccharides matched literature results very well. Dihedral angle histograms weighted against published Karplus equations yielded excellent matches of experimental J-values in some cases but significant deviations in other. The anomeric hemiacetal configuration appeared to have a significant remote influence on the conformational space of the α-1,6-glycosidic linkage. Rigid glycosidic φ-conformations (g+) combined with mostly st-conformations for glycosidic ψ-angles from computations matched experimental nuclear Overhauser enhancements in all cases. While the investigated Glcp-α-1,6-Glcp linkages were nearly identical in φ/ψ-conformation, differences were apparent in the Galp-α-1,6-Galp linkage of stachyose. Of twenty-one crystal structures, a total of fourteen had ligand conformations corresponding to the most abundant or second-most abundant solution geometry determined in this study.

Synthesis and conformational analysis of d-gluco-pyranosyl-(6,6')-d-gluco-pyranuronate, a model compound for the inter-glycan 6,6'-ester linkage.

The synthesis of a 6,6'-ester linked disaccharide analog model compound was achieved in five steps from d-glucose and featured a key oxidative esterification transformation. The synthesized d-gluco-pyranosyl-(6,6')-d-gluco-pyranuronate was characterized in D2O using NMR spectroscopy. Using the experimental data together with molecular dynamics simulations (TIP3P, water), a model of the compound's conformational behavior was established. The effect of the 6,6'-ester linkage on the solution phase structure was compared to that of the previously reported 6,6'-ether linkage in a disaccharide analog. Based on the established models, the ester linkage was found to have a profound effect on the overall shape of the molecule.