Carbohydrates is one of the three classes of biomolecules found in nature. It is the most common one in comparison to the other two classes, lipids and proteins. However, this simple categorization does not reflect the reality since carbohydrates often are covalently linked to e.g., proteins, so-called glycoproteins where, for example, N-glycans are used as markers of quality control during the process of protein folding. Another example is lipopolysaccharides, which cover the cell surfaces of gram-negative bacteria and which contain both a lipid moiety (Lipid A) and a carbohydrate chain. The outer part of the carbohydrate chain is a polysaccharide, also called O-antigen, as it interacts with the immune system of the host. The polysaccharide has, like a polymer, a repeating unit consisting of 2-7 monosaccharides. The repeating unit varies between different bacteria. Determining the structure of these polysaccharides is important in order to be able to categorize the various strains that exist, but also to be able to develop future glycoconjugate vaccines. This is important as the WHO estimates that antibiotic resistance is expected to be more lethal than cancer by 2050, and therefore a vaccine is needed to slow down this development.

Nuclear Magnetic Resonance Spectroscopy (NMR) is a useful analytical tool to analyze these carbohydrates at the atomic level in order to determine their structures.

The first part (Paper I-III) of this thesis will summarize the structural determination of three Escherichia coli serogroups with hitherto unknown lipopolysaccharides.

The second part (Paper IV) will discuss the structure determination, using NMR spectroscopy, for various mono-, di-, and tri-saccharides that have recently been implemented in the structure-determination program, CASPER. The chapter will also present examples of predictions of complex carbohydrates that CASPER can perform.

The third part (Paper V) of the thesis will investigate conformational aspects of the polysaccharides from Shigella flexneri serotypes 7a and 7b using NMR spectroscopy and molecular dynamics simulations.

Carbohydrates are ubiquitous components in nature involved in a range of tasks. They cover every cell and contribute both structural stability as well as identity. Lipopolysaccharides are the outermost exposed part of the bacterial cell wall and the primary target for host-pathogen recognition. Understanding the structure and biosynthesis of these polysaccharides is crucial to combat disease and develop new medicine. Structural determinations can be carried out using NMR spectroscopy, a powerful tool giving information on an atomistic scale. This thesis is focused on method development to study polysaccharide structures as well as application on bacterial lipopolysaccharides. The focus has been to incorporate a bioinformatics approach prior to analysis by NMR spectroscopy, and then computer assisted methods to aid in the subsequent analysis of the spectra.

The third chapter deals with the recent developments of ECODAB, a tool that can help predict structural fragments in Escherichia coli O-antigens. It was migrated to a relational database and the aforementioned predictions can now be made automatically by ECODAB. The fourth chapter gives insight into the program CASPER, a computer program that helps with structure determination of oligo- and polysaccharides. An approach to determine substituent positions in polysaccharides was investigated. The underlying database was also expanded and the improved capabilities were demonstrated by determining O-antigenic structures that could not previously be solved. The fifth chapter is an application to O‑antigen structures of E. coli strains. This is done by a combination of NMR spectroscopy and bioinformatics to predict components as well as linkages prior to spectra analysis. In the first case, a full structure elucidation was performed on E. coli serogroup O63, and in the second case a demonstration of the bioinformatics approach is done to E. coli serogroup O93. In the sixth chapter, a new version of the CarbBuilder software is presented. This includes a more robust building algorithm that helps build sterically crowded polysaccharide structures, as well as a general expansion of possible components. 

The outer surface of bacteria is composed of around 75% carbohydrates, which are vital for the bacteria to survive and communicate with the host biological system. The thesis discusses different properties of carbohydrates that are essential for understanding the bacterial behavior in biological systems. The first three chapters give an overview of carbohydrates.

The fourth chapter discusses the synthesis of four amide-substituted 3,6-dideoxy-α-D-galactopyranosides, namely, methyl α-3,6-dideoxy-3-formamido-, acetamido-, (R)-3-hydroxybutyramido-, and (4-hydroxybutyramido)-D-galactopyranoside. These sugars were found as components of some bacterial O-antigens; the study is a step toward the synthesis of oligosaccharides that contain them. The fifth chapter describes the exchange kinetics of the formyl and acetyl derivatives that were synthesized. Both of them have two conformational states for the amide side-chain. ¹³C-NMR saturation transfer experiments are utilized for these measurements to reveal more about their properties in solution.

In chaptr six, NMR and conformational analysis of oligosaccharides related to the O-antigen of Yersinia enterocolitica O:3 bacteria were carried out to obtain more information regarding their 3D structure.

Chapter seven is focusing on the development of CASPER, a program for rapid assignment of ¹H- and ¹³C-NMR chemical shifts of bacterial lipopolysaccharides, by adding more sugars into its database and testing it for naturally occurring LPS as well as extending the scope for synthetic carbohydrates, which is planned to be developed further in the future.