In posttranslational modifications of proteins and peptides by glycosylation the two major classes are N-linked and O-linked glycans. The sugar residue proximal to the peptide chain is in N-glycans linked to l-asparagine and in O-linked glycans it is linked to either l-serine, l-threonine or l-tyrosine, although other amino acids may be glycosylated. Identifying and assigning the 1H and 13C NMR chemical shifts of these glycoconjugates are a prerequisite for structural characterization as well as for subsequent conformational and interaction studies thereof. The web-based computer program CASPER (http://www.casper.organ.su.se/casper) is a tool that provides prediction of 1H and 13C NMR chemical shift for glycans, as well as those linked to l-Asn, l-Ser, l-Thr or l-Tyr, for which the predicted NMR chemical shifts of the glycan show good agreement to those from NMR experiments of glycopeptides and glycoproteins. This highlights that an approximation in which a single amino acid is present at the reducing end of the glycan structure is sufficient to predict NMR data well, as shown for different N-linked and O-linked glycans of various complexity.

Carbohydrate structures containing alkyl groups as aglycones are useful for investigating enzyme activity and glycan-protein interactions. Moreover, linker-containing oligosaccharides with a spacer group are commonly used to print glycan microarrays or to prepare protein-conjugates as vaccine candidates. The structural accuracy of these synthesized glycans are essential for interpretation of results from biological experiments in which the compounds have been used and NMR spectroscopy can unravel and confirm their structures. An approach for efficient ¹H and ¹³C NMR chemical shift assignments employed a parallel NOAH-10 measurement followed by NMR spin-simulation to refine the ¹H NMR chemical shifts, as exemplified for a disaccharide with an azidoethyl group as an aglycone, the NMR chemical shifts of which have been used to enhance the quality of CASPER (http://www.casper.organ.su.se/casper/). The CASPER program has been further developed to aid characterization of linker-containing oligo- and polysaccharides, either by chemical shift prediction for comparison to experimental NMR data or as structural investigation of synthesized glycans based on acquired unassigned NMR data. The ability of CASPER to elucidate structures of linker-containing oligosaccharides is demonstrated and comparisons to assigned or unassigned NMR data show the utility of CASPER in supporting a proposed oligosaccharide structure. Prediction of NMR chemical shifts of an oligosaccharide, corresponding to the repeating unit of an O-antigen polysaccharide, having a linker as an aglycone and a non-natural substituent derivative thereof are presented to exemplify the diversity of structures handled. Furthermore, NMR chemical shift predictions of synthesized polysaccharides, corresponding to bacterial polysaccharides, containing a linker are described showing that in addition to oligosaccharide structures also polysaccharide structures having an aglycone spacer group can be analyzed by CASPER.

Glycans are central to information content and regulation in biological systems. These carbohydrate molecules are active either as oligo- or polysaccharides, often in the form of glycoconjugates. The monosaccharide entities are joined by glycosidic linkages and stereochemical arrangements are of utmost importance in determining conformation and flexibility of saccharides. The conformational preferences and population distributions at the glycosidic torsion angles f and y have been investigated for three disaccharides where the substitution takes place at a secondary alcohol, viz., in a-l-Fucp-(1→3)-β-d-Glcp-OMe, a-l-Fucp-(1→3)-a-d-Galp-OMe and a-d-Glcp-(1→4)-a-d-Galp-OMe. Stereochemical differences at or adjacent to the glycosidic linkage were explored by solution state NMR spectroscopy using one‑dimensional 1H,1H-NOESY NMR experiments to obtain transglycosidic proton‑proton distances and one- and two-dimensional heteronuclear NMR experiments to obtain 3JCH transglycosidic coupling constants related to torsion angles f and y. Computed effective proton‑proton distances from molecular dynamics (MD) simulations showed excellent agreement to experimentally derived distances for the a-(1→3)-linked disaccharides and revealed that for the bimodal distribution at the y torsion angle for the a-(1→4)-linked disaccharide experiment and simulation were at variance with each other, calling for further force field developments. The MD simulations disclosed a highly intricate inter‑residue hydrogen bonding pattern for the a-(1→4)-linked disaccharide, including a nonconventional hydrogen bond between H5' in the glucosyl residue and O3 in the galactosyl residue, supported by a large downfield ¹H NMR chemical shift displacement compared to a-d-Glcp-OMe. Comparison of population distributions of the glycosidic torsion angles f and y in the disaccharide entities to those of corresponding crystal structures highlighted the potential importance of solvation on the preferred conformation. 

Methodology development in carbohydrate chemistry entails the stereoselective formation of C–O bonds as a key step in the synthesis of oligo- and polysaccharides. The anomeric selectivity of aglycosylation reaction is affected by a multitude of parameters, such as the nature of the donor andacceptor, activator/promotor system, temperature and solvent. The influence of different solvents onthe stereoselective outcome of glycosylation reactions employing thioglucopyranosides as glycosyldonors with a non-participating protecting group at position 2 has been studied. A large change inselectivity as a function of solvent was observed and a correlation between selectivity and the Kamlet-Taft solvent parameter π* was found. Furthermore, molecular modeling using density functionaltheory methodology was conducted to decipher the role of the solvent and possible reaction pathwayswere investigated.