The research is focused on understanding structure-dynamics-interaction relationships of carbohydrates in general and glycan-protein interactions in particular. Three different avenues are chosen to this end, viz., (i) organic synthesis; (ii) experimental techniques, especially NMR spectroscopy; and (iii) molecular simulations, in particular molecular dynamics simulations. We carry out investigations on the following subjects:

• Development of rapid and robust methodology for the determination of carbohydrate structure, i.e., towards fully automatic structure determination by NMR
• Chemical synthesis of oligosaccharides, glycoconjugates, and of protein inhibitors
• In depth studies of conformational dynamics of glycoconjugates found in biologically and medically relevant systems
• Interaction and docking studies of glycoconjugates and of small organic molecules with proteins in general and enzymes in particular with the aim of improving antibiotic uptake in gram-negative bacteria

The research spans from Glycobiology to Biophysis

Carbohydrates play an essential part in a great deal of biological and biochemical processes. These molecules are commonly known as glycans in biochemical systems where they make up the carbohydrate chains of glycoproteins, proteoglycans and glycolipids. Glycosylation, i.e., substitution of these carbohydrate chains onto other molecules, most commonly proteins and glycolipids but also steroid skeletons, is a very common post-translational modification. About half of all known proteins in eukaryots are glycosylated, indicating their importance as modifiers of different biological processes. Changes in oligo- or polysaccharide structures are associated with pathological and physiological events. These span a wide variety of processes including intercellular signaling, cell growth and cell trafficking, migration, growth regulation, differentiation, tumor invasion, host-pathogen interaction and trans-membrane signaling. The bioactive signaling is decoded by lectins, i.e., carbohydrate binding proteins but carbohydrate-carbohydrate interactions are also present as part of cell-cell interactions. The task of deciphering the information encoded by the glycome, which is defined as the repertoire of complex sugar structures expressed by cells and tissues, will require a huge effort but promises large scientific benefits once significant parts of unknown processes and relationships have been unraveled. A class of galactose-recognizing lectins known as galectins has diverse roles in immune related processes in cells including pathogen recognition, the development progress of adaptive immune responses and adjusting inflammatory responses. The presence or absence of a single sugar residue in the core region of N-linked glycoproteins is essential to whether cell growth can be regulated or not, i.e., when fucosylation is present on the membrane-associated receptor the growth factor (protein) may bind and signaling is properly modulated whereas in its absence dysfunction occurs for EGF signaling and uncontrolled cell growth may occur.

A large number of diseases related to glycan structures may be addressed by inhibiting or perturbing the proteins involved in the biosynthesis or regulation of these processes. In order to intervene in the biochemical machinery antagonists and inhibitors is a current line of investigation and is a fount for drug discovery. The antagonists are small molecule enzyme inhibitors with or without a carbohydrate structure as part to the scaffold. The targeted glycan structures are e.g. related to O-linked glycans, proteoglycans and glycosaminoglycans, glycosphingolipids and GPI-anchors. As part of the present approaches to understand the biochemical processes, the research area of chemical biology is rapidly evolving in which chemical techniques coupled with biological systems are used to gain knowledge in particular by applying non-natural modifications to biomolecules, that being tagged subsequently can be visualized by a biophysical detection technique.

In organic synthesis of carbohydrates the formation of the glycosidic linkage is the key reaction being carried out. Since long the 1,2-trans-relationship at the anomeric positions is readily handled by the synthesis protocols hitherto developed. The 1,2-cis-relationship still presents problems in stereoselectivity and improvements of these glycosylation reactions are still needed. It is today possible to synthesize essentially any oligosaccharide or even polysaccharide of interest. However, it may be very laborious and highly time consuming to attain the target molecule. Chemo-enzymatic procedures for synthesis of carbohydrate molecules are therefore attractive alternatives to the pure organic synthesis. It is anticipated that during the next decade chemo-enzymatic synthesis in conjunction with automated solid-phase synthesis will facilitate rapid and straightforward production of glycans for research in the glycobiology field.

Techniques for the analytical challenges of glycan structure and interactions include more recently microarry platforms but mass spectrometry has a continued strong position in the field, in particular due to the small amounts of material that are needed for the analysis. There are continued developments in which chemical modifications are used together with mass spectrometry to unravel structures of complex biological materials. However, recent observations using various mass spectrometry techniques have shown that rearrangements of the glycan structure do occur in the mass spectrometer, resulting in artifact structures. This may be very problematic when materials of unknown structure are investigated and a caution in use of the technique is warranted. Linkage substitution patterns of oligosaccharides may be differentiated by collision-induced dissociation thereby resolving whether an oligosaccharide is e.g. 4- or 6-substituted. However, the accuracy in the determinations is on the order of 90%, good enough in a screening process but not if a structure should be firmly established. Recent developments for quantitative glycomics have used stable ¹³C-isotopic labeling in tagging procedures of mixtures of glycans.

NMR spectroscopy is a very versatile technique for studying molecules in solution, whether it will be structure determination or molecular interactions. Multidimensional NMR experiments make use of sophisticated pulse-sequences using several channels including pulsed-field-gradients as an important part. These experiments are aimed at obtaining e.g. better resolved spectra and one recent development results in HSQC spectra that are decoupled in all frequency dimensions. In the late 1990s the use of residual dipolar couplings in high-resolution NMR studies of biomolecules was initiated and has emerged as a very powerful approach to obtain structural and dynamics data. Novel alignment-media are proposed and developed and various approaches are applied to the study of protein structure as well as to carbohydrate molecules. In addition, automatic NMR assignment protocols are being developed.

Computational chemistry is an essential tool in analyzing experimental data, to make predictions that may be tested experimentally and to unravel and explain chemical processes at the atomic level. Molecular dynamics simulations utilize molecular mechanics force fields and these are continuously being developed and refined for proteins and for carbohydrates with the aim of obtaining a generalized force field that can be used for the investigation of biologically important systems. Recent developments in quantum chemical computations of experimental parameters make these tools accessible in the analysis of oligosaccharides as well as protein structures. Density functional theory calculations of chemical processes, such as interconversions between anomeric configurations, and experimental observables, e.g. IR frequencies of compounds, have been described. The methodology also shows great promise for calculation of ¹H and ¹³C NMR chemical shifts and of coupling constants, both for isolated structures as well as for ensembles of molecules.

To target carbohydrate-protein interactions from a drug perspective, therapeutic opportunities and new angles to finding lead compounds for glycomimetic drugs, small organic molecules in library screening facilitate possible avenues in future developments. Fragment-based methodologies and the relationships between the ‘fragments’ and the drug compounds have been investigated during the last few years as an effort to understand how to obtain drug-like compounds from library screening using e.g. NMR spectroscopy. Furthermore, there are now automated approaches to address data from NMR screening experiments using small molecule libraries. In addition, in the screening process the small molecules can be chemically modified to enhance detection and to form inhibitors linked via ‘fragments’.