Proteoglycans (PGs) consist of a core protein with covalently bound glycosaminoglycan (GAG) chains that are linked via a tetrasaccharide. PGs are important macromolecules that are involved in biological processes such as cell growth and differentiation. A key enzyme in the biosynthesis of PG GAG chains is beta-1,4-galactosyltransferase 7 (beta 4GalT7) that catalyzes the transfer of galactose to a xylose residue in the formation of the linker tetrasaccharide. It is well known that the addition of xylosides containing naphthyl aglycones can initiate the biosynthesis of GAG chains by acting as substrates for beta 4GalT7. Previous studies have shown that its galactosylation ability is increased by using bioisosters, in which the anomeric oxygen is replaced with sulfur or sulfur-containing functional groups. Thus, 2-naphthyl xylosyl sulfoxides were synthesized and characterized by H-1 and C-13 NMR spectroscopy relying on both one- and two-dimensional experiments to differentiate the stereochemistry at the sulfur atom. Notably, the conformationally dependent (3)J(CH) coupling constants between the anomeric proton and the C2 ' atom of the naphthyl group were large and significant, >= 3.3 Hz, for the (S)(S)-configured compound as well as for the O-glycoside and the thio-derivative whereas the corresponding coupling for the (R)(S)-configured compound and the sulfone derivative had (3)J(C2 ',H1) < 0.6 Hz and (3)J(C2 ',H1) < 0.5 Hz, respectively. Quantum mechanical calculations of the (3)J(C2 ',H1) coupling constant corroborated the experimentally observed trends at the phi torsion angle. The galactosylation by beta 4GalT7 of the different acceptor substrates showed the highest affinity for the (R)(S)-configured compound and the sulfone derivative whereas an intermediate affinity was present for the (S)(S)-configured compound and the thio-derivative. The enzyme efficiency exhibited with the latter substrate was more than three times higher than with any other of the thio-derivatives. From molecular docking of the acceptor substrates to the UDP-galactose:beta 4GalT7 complex specific intermolecular interactions were identified. The binding affinity correlates with stacking to a tyrosine residue and a weak C-H & ctdot;O hydrogen bond between the indole group of tryptophan in the enzyme and a proximate oxygen atom of sulfone and sulfinyl derivatives of 2-naphthyl xylosides.

The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D H-1, C-13-HSQC, and H-1,H-1-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that similar to 1/2 of the population was 2,6-di-O-acetylated, similar to 1/4 was 2-O-acetylated, whereas similar to 1/4 did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: -> 2)-beta-D-Manp-(1 -> 3)-beta-D-Manp2Ac6Ac-(1 -> 4)-beta-D-GlcpA-(1 -> 3)-alpha-D-GlcpNAc-(1 ->, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840(T) differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

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.

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.

Enteropathogenic Escherichia coli O125, the cause of infectious diarrheal disease, is comprised of two serogroups, viz., O125ab and O125ac, which display the aggregative adherence pattern with epithelial cells. Herein, the structure of the O-antigen polysaccharide from E. coli O125ac:H6 has been elucidated. Sugar analysis revealed the presence of fucose, mannose, galactose and N-acetyl-galactosamine as major components. Unassigned ¹H and ¹³C NMR data from one- and two-dimensional NMR experiments of the O125ac O-antigen in conjunction with sugar components were used as input to the CASPER program, which can determine polysaccharide structure in a fully automated way, and resulted in the following branched pentasaccharide structure of the repeating unit: →4)[β-D-Galp-(1 → 3)]-β-D-GalpNAc-(1 → 2)-α-D-Manp-(1 → 3)-α-L-Fucp-(1 → 3)-α-D-GalpNAc-(1→, where the side chain is denoted by square brackets. The proposed O-antigen structure was confirmed by ¹H and ¹³C NMR chemical shift assignments and determination of interresidue connectivities. Based on this structure, that of the O125ab O-antigen, which consists of hexasaccharide repeating units with an additional glucosyl group, was possible to establish in a semi-automated fashion by CASPER. The putative existence of gnu and gne in the gene clusters of the O125 serogroups is manifested by N-acetyl-D-galactosamine residues as the initial sugar residue of the biological repeating unit as well as within the repeating unit. The close similarity between O-antigen structures is consistent with the presence of two subgroups in the E. coli O125 serogroup.

Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a(2) is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri. LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a(2)]) with deletions of different Oag modification genes (oacB, oacD, and grrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen.

The structure of the O-antigen from Escherichia coli reference strain O188 (E. coli O188:H10) has been investigated. The lipopolysaccharide shows a typical nonrandom modal chain-length distribution and the sugar and absolute configuration analysis revealed D-Man, D-Glc, D-GlcN and D-GlcA as major components. The structure of the O-specific polysaccharide was determined using one- and two-dimensional H-1 and C-13 NMR spectroscopy experiments, where inter-residue correlations were identified by H-1,C-13-heteronuclear multiple-bond correlation and H-1,H-1-NOESY experiments, which revealed that it consists of pentasaccharide repeating units with the -> 4)-beta-D-GlcpA-(1 -> 2)-beta-D-Manp-(1 -> 4)-beta-D-Manp-(1 -> 3)-beta-D-GlcpNAc-(1 -> following structure: vertical bar alpha-D-Galp-(1 -> 3) Biosynthetic aspects and NMR analysis are consistent with the presented structure as the biological repeating unit. The O-antigen of Shigella boydii type 16 differs only in that it carries O-acetyl groups to similar to 50% at O6 of the branchpoint mannose residues. A molecular model of the E. coli O188 O-antigen containing 20 repeating units extends similar to 100 angstrom, which is similar to the height of the periplasmic portion of polysaccharide co-polymerase Wzz proteins that regulate the O-antigen chain length of lipopolysaccharides in the Wzx/Wzy biosynthetic pathway.

Escherichia coli includes clonal groups of both commensal and pathogenic strains, with some of the latter causing serious infectious diseases. O antigen variation is current standard in defining strains for taxonomy and epidemiology, providing the basis for many serotyping schemes for Gram-negative bacteria. This review covers the diversity in E. coli O antigen structures and gene clusters, and the genetic basis for the structural diversity. Of the 187 formally defined O antigens, six (O31, O47, O67, O72, O94 and O122) have since been removed and three (O34, O89 and O144) strains do not produce any O antigen. Therefore, structures are presented for 176 of the 181 E. coli O antigens, some of which include subgroups. Most (93%) of these O antigens are synthesized via the Wzx/Wzy pathway, 11 via the ABC transporter pathway, with O20, O57 and O60 still uncharacterized due to failure to find their O antigen gene clusters. Biosynthetic pathways are given for 38 of the 49 sugars found in E. coli O antigens, and several pairs or groups of the E. coli antigens that have related structures show close relationships of the O antigen gene clusters within clades, thereby highlighting the genetic basis of the evolution of diversity.