Carbohydrate structure can be elucidated or confirmed by using NMR spectroscopy as the prime technique. Prediction of ¹H and ¹³C NMR chemical shifts by computational approaches makes this assignment process more efficient and the program CASPER can perform this task rapidly. It does so by relying on chemical shift data of mono-, di-, and trisaccharides. In order to improve accuracy and quality of these predictions we have assigned ¹H and ¹³C NMR chemical shifts of 30 monosaccharides, 17 disaccharides, 10 trisaccharides and one tetrasaccharide; in total 58 compounds. Due to different rotamers, ring forms, α- and β-anomeric forms and pD conditions this resulted in 74 ¹H and ¹³C NMR chemical shift data sets, all of which were refined using total line-shape analysis for the ¹H resonances in order to obtain accurate chemical shifts. Subsequent NMR chemical shift predictions for three sialic acid-containing oligosaccharides, viz., GD1a, a disialyl-LNnT hexasaccharide and a polysialic acid-lactose decasaccharide, and NMR-based structural elucidations of two O-antigen polysaccharides from E. coli O174 were performed by the CASPER program (http://www.casper.organ.su.se/casper/) resulting in very good to excellent agreement between experimental and predicted data thereby demonstrating its utility for carbohydrate compounds that have been chemically or enzymatically synthesized, structurally modified or isolated from nature.

The highly poisonous botulinum neurotoxins, produced by the bacterium Clostridium botulinum, act on their hosts by a high-affinity association to two receptors on neuronal cell surfaces as the first step of invasion. The glycan motifs of gangliosides serve as initial coreceptors for these protein complexes, whereby a membrane protein receptor is bound. Herein we set out to characterize the carbohydrate minimal binding epitope of the botulinum neurotoxin serotype A. By means of ligand-based NMR spectroscopy, X-ray crystallography, computer simulations, and isothermal titration calorimetry, a screening of ganglioside analogues together with a detailed characterization of various carbohydrate ligand complexes with the toxin were accomplished. We show that the representation of the glycan epitope to the protein affects the details of binding. Notably, both branches of the oligosaccharide GD la can associate to botulinum neurotoxin serotype A when expressed as individual trisaccharides. It is, however, the terminal branch of GD1a as well as this trisaccharide motif alone, corresponding to the sialyl-Thomsen-Friedenreich antigen, that represents the active ligand epitope, and these compounds bind to the neurotoxin with a high degree of predisposition but with low affinities. This finding does not correlate with the oligosaccharide moieties having a strong contribution to the total affinity, which was expected to be the case. We here propose that the glycan part of the ganglioside receptors mainly provides abundance and specificity, whereas the interaction with the membrane itself and protein receptor brings about the strong total binding of the toxin to the neuronal membrane.

To advance the field of glycobiology, efficient synthesis methods of oligosaccharides and glycoconjugates are a requisite. In glycosylation reactions using superarmed donors, both selectivity and reactivity issues must be considered, and we herein investigate these aspects for differently protected beta-linked 2-O-glycosylated glucosyl donors carrying bulky tert-butyldimethylsilyl groups to different extents. The acceptors in reactions being secondary alcohols presents a challenging situation with respect to steric crowding. Conformational pyranose ring equilibria of the superarmed disaccharide donors with axial-rich substituents contained skew and boat conformations, and three-state models were generally assumed. With NIS/TfOH as the promotor, 2,6-di-tert-butyl-4-methylpyridine as the base, and a dichloromethane/toluene solvent mixture, ethyl 1-thio-beta-d-glucosyl disaccharide donors having 6-O-benzyl group(s) besides tert-butyldimethylsilyl groups were efficiently coupled at -40 degrees C to the hydroxyl group at position 3 of glucopyranosyl acceptors to form beta-(1 -> 2),beta-(1 -> 3)-linked trisaccharides, isolated in excellent 95% yield. The more axial-rich donors in skew and boat conformations are thus preorganized closer to the assumed transition state in these glycosylation reactions. The developed methodology was subsequently applied in the synthesis of a multibranched hexasaccharide related to the capsular polysaccharide from Streptococcus pneumoniae type 37, which consists of a beta-(1 -> 3)-linked backbone and a beta-(1 -> 2)-linked side chain of D-glucosyl residues in disaccharide repeating units.

Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system.

Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase beta 4GalT7. Some beta-D-xylosides, such as 2-naphthyl beta-D-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for beta 4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for beta 4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with beta-glycosidic linkages function as good substrates for beta 4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by beta 4GalT7 is dependent on subtle differences in orientation of the xylose moiety.

In this thesis, within the topic of bioorganic chemistry, the molecular structure of carbohydrates has been studied. Carbohydrates, or glycans, are ubiquitous biomolecules exhibiting a wide range of biological roles. The specific functions of these molecules are largely determined by their interactions with proteins and molecular structure ultimately governs such specialized recognition events.

Glycan-binding proteins, such as lectins or enzymes, often interact with their sweet ligands in a transient fashion and nuclear magnetic resonance spectroscopy (NMR) is a viable technique to probe these complexes. In particular, ligand-based NMR techniques have been employed, typically in combination with other biophysical as well as biochemical and computational methods. The aim of this work has been to gain new insights about specific biological systems, to develop methods and to devise protocols for their studies.

The first two papers cover NMR-interaction studies of native ligands as well as inhibitor glycans with the enzyme hen egg-white lysozyme and the lectin botulinum neurotoxin type A. Screening experiments were performed to investigate ligand affinities and selectivities. Solution models in combination with X-ray crystal structures were compared in order to evaluate their agreement and the details of interactions.

A method for application in carbohydrate ligand NMR-screening was developed in paper three. The heteronucleus selenium was exploited as a reporter of selenoglycosides binding to lectins. ⁷⁷Se NMR spectroscopy proved sensitive to binding events and the presented approach should be useful in large screenings of glycomimetic inhibitors.  In order to obtain sufficient amounts of glycans for bioorganic studies their production often relies on chemical synthesis. In the last paper, the structure of some conformationally highly activated glycosyl donors was thoroughly investigated and related to their reactivity in synthetic glycosylation reactions.  

We report a method for the screening of interactions between proteins and selenium-labeled carbohydrate ligands. SEAL by NMR is demonstrated with selenoglycosides binding to lectins where the selenium nucleus serves as an NMR-active handle and reports on binding through Se-77 NMR spectroscopy. In terms of overall sensitivity, this nucleus is comparable to C-13 NMR, while the NMR spectral width is ten times larger, yielding little overlap in Se-77 NMR spectroscopy, even for similar compounds. The studied ligands are singly selenated bioisosteres of methyl glycosides for which straightforward preparation methods are at hand and libraries can readily be generated. The strength of the approach lies in its simplicity, sensitivity to binding events, the tolerance to additives and the possibility of having several ligands in the assay. This study extends the increasing potential of selenium in structure biology and medicinal chemistry. We anticipate that SEAL by NMR will be a beneficial tool for the development of selenium-based bioactive compounds, such as glycomimetic drug candidates.

H-1 and C-13 NMR chemical shift data are used by the computer program CASPER to predict chemical shifts of oligo- and polysaccharides. Three types of data are used, namely, those from monosaccharides, disaccharides, and trisaccharides. To improve the accuracy of these predictions we have assigned the H-1 and C-13 NMR chemical shifts of eleven monosaccharides, eleven disaccharides, twenty trisaccharides, and one tetrasaccharide; in total 43 compounds. Five of the oligosaccharides gave two distinct sets of NMR resonances due to the alpha- and beta-anomeric forms resulting in 48 H-1 and C-13 NMR chemical shift data sets. In addition, the pyranose ring forms of Neu5Ac were assigned at two temperatures, due to chemical shift displacements as a function of temperature. The H-1 NMR chemical shifts were refined using total line-shape analysis with the PERCH NMR software. H-1 and C-13 NMR chemical shift predictions were subsequently carried out by the CASPER program (http://www.casper.organ.su.se/casper/) for three branched oligosaccharides having different functional groups at their reducing ends, namely, a mannose-containing pentasaccharide, and two fucose-containing heptasaccharides having N-acetyllactosamine residues in the backbone of their structures. Good to excellent agreement was observed between predicted and experimental H-1 and C-13 NMR chemical shifts showing the utility of the method for structural determination or confirmation of synthesized oligosaccharides.

By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using H-1 saturation transfer difference (STD) effects and transferred H-1,H-1-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides beta-D-Galp-(1 -> 4)-beta-D-GlcpNAc-OMe and beta-D-GlcpNAc-(1 -> 4)-beta-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/pi-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on H-1 NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.

In the title compound, C₃₀H₃₁NO₆S, the plane of the N-phthalimido group is nearly orthogonal to the least-squares plane of the sugar ring (defined by atoms C2, C3, C5 and O5 using standard glucose nomenclature), making a dihedral angle of 72.8 (1)°. The thioethyl group has the exo-anomeric conformation. The hydroxy group forms an intermolecular hydrogen bond to the O atom in the sugar ring, generating [100] chains. There are four close - contacts with centroid-centroid distances less than 4.0 Å, all with dihedral angles between the interacting systems of only 8°, supporting energetically favourable stacking interactions