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University of Cape Town CHEMICAL AND CONFORMATIONAL STUDIES OF BACTERIAL CELL SURFACE POLYSACCHARIDE REPEATING UNITS Zaheer Timol University of Cape Town Supervisors: Neil Ravenscroft, Michelle Kuttel and David Gammon A thesis presented for the degree of Master in Science University of Cape Town March 2017 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Abstract Bacterial cell surface polysaccharides are primarily present as lipopolysaccharides or capsular polysac- charides. They are used by cells for both structure and function and have been shown to be a virulence factor of bacterial pathogens. Cell surface polysaccharides are widely utilised as antigenic components in vaccines and play an important role in the protection against numerous diseases including meningo- coccal disease and shigellosis. This study is composed of two parts: a computational section, which investigates the capsular polysaccharide (CPS) repeating unit (RU) conformations of meningococcal Y and W CPS vaccines and a second experimental component that involves synthetic studies toward the O-specific polysaccharide (O-SP) RU of Shigella sonnei. The CPS RU of MenY [!6)-a-D-Glc(1!4)-a-D-NeuNAc-(2!] and MenW [!6)-a-D-Gal(1!4)-a- D-NeuNAc-(2!] differ only in the orientation of the C-4 hydroxyl: equatorial in MenY and axial in MenW. However, groups Y and W CPS vaccines have different levels of antibody cross-protection. The purpose of the computational study was to determine if these observed differences may be attributed to CPS RU conformation. Potential of mean force calculations were applied to disaccharide RUs of MenY and MenW, and larger three repeating units (3RU) were simulated with molecular dynamics (MD) in solution. The molec- ular modelling showed that differences in RU conformation between the meningococcal groups arise primarily due to the structural differences between glucose and galactose; affecting the behaviour and orientation of the 2!6 dihedral linkage. The 2!6 linkages in the MenY 3RU adopt a single preferred orientation and consequently it has a single dominant molecular conformation. In contrast, the 2!6 linkages in the MenW 3RU move frequently between different rotameric conformations resulting in multiple conformational families. These results indicate significant conformational differences between the MenY and MenW CPS RUs, which may account for the different levels of cross-protection observed. The synthetic component was part of a larger study to develop a novel route towards the O-SP RU of S. sonnei for use in biological testing and physicochemical characterisation for vaccine development. The O-SP RU of S. sonnei is !4-a-L-AltNAc-(1!3)-b-D-FucNAc4N(1!. The multi-step synthesis was performed using known methodology as well as methods developed by the research group. The key 2,3-oxazolidinone protected intermediate was successfully synthesised in good yields and due to time constraints the final product synthesised was two steps away from the protected FucNAc4N residue. Additional studies were performed on the 2,3-oxazolidinone intermediate as part of a divergent syn- thesis strategy toward the AltNAcA residue of S. sonnei. Reactions were conducted whereby b and a derivatives of the 2,3-oxazolidinone intermediate were successfully synthesised in large scale and good yields for further studies to be performed by the group. i Acknowledgments I wish to acknowledge the contribution of the following people without whom this project would not have been possible: First, I would like to thank my supervisors - Associate Professor’s Neil Ravenscroft, Michelle Kuttel and David Gammon for their advice, support and considerable patience throughout the project. To all my friends and family members for their continued encouragement. Students from the Gammon, Hunter and Chibale Research Groups. The University of Cape Town and the National Research Foundation for funding support. The University of Cape Town’s ICTS High Performance Computing team for providing the computation facilities: http://hpc.uct.ac.za. Many thanks to Andrew Lewis for all his assistance. Contents Abstract 1 Introduction . .1 1.1 Cell surface polysaccharides . .1 1.2 Glycobiology and immunology . .3 1.3 Carbohydrate-based vaccines . .5 1.4 Meningococcal disease . .9 1.4.1 Prevalence of meningococcal disease . 10 1.4.2 Capsular polysaccharides of meningococcus . 12 1.4.3 Meningococcal vaccines . 12 1.4.4 Polysaccharide vaccine trials of MenY and MenW . 14 1.5 Shigellosis . 15 1.5.1 Prevalence of shigellosis . 16 1.5.2 O-specific polysaccharides of Shigella ................. 17 1.5.3 Vaccines against Shigella ........................ 17 1.6 Scope of work . 19 2 Molecular modelling and analytical methods . 20 2.1 Molecular mechanics . 20 2.2 Force fields . 20 2.3 Molecular dynamics . 21 2.3.1 The integrator . 21 2.3.2 The water model . 22 2.3.3 Periodic boundary conditions . 22 2.4 Metadynamics . 22 2.5 File formats . 24 2.6 Analytical methods . 24 2.6.1 Root mean square deviation . 24 2.6.2 Radius of gyration . 24 2.6.3 Distance . 25 2.6.4 Cluster analysis . 25 2.6.5 Solvent accessible surface area . 25 3 Conformations of meningococcal Y and W capsular polysaccharide vaccines . 27 3.1 Introduction . 27 3.2 Methods . 31 3.3 Results and discussion . 33 3.3.1 Potential of mean force of calculations of Glc14N and Gal14N . 33 3.3.2 Potential of mean force calculations for N26Glc and N26Gal . 33 3.3.3 Polymer conformations . 36 iii 3.3.4 Capsular polysaccharide conformation and immune response . 47 3.4 Conclusions . 48 4 Synthesis of the S. sonnei O-SP FucNAc4N residue . 50 4.1 Overview . 50 4.2 Synthetic routes toward the repeating unit of S. sonnei .............. 50 4.2.1 Previous routes . 50 4.2.2 Novel route towards FucNAc4N employing a 2, 3 oxazolidinone pro- tected intermediate . 52 4.3 Results and discussion . 53 4.3.1 Synthesis of the b thioglycoside (33).................. 53 4.3.2 Synthesis of the 2, 3-oxazolidinone protected glucosamine intermedi- ate (37).................................. 54 4.3.3 De-oxygenation at C-6 for the synthesis of Phenyl 2-amino-2-N,3-O- carbonyl-2,6-dideoxy-1-thio-b-D-glucopyranoside (40)........ 56 4.3.4 Synthesis of the protected form of FucNAc4N, 42 ........... 56 4.4 Conclusions . 57 5 Experimental . 59 5.1 General procedures . 59 5.2 Synthesis . 59 6 Conclusions . 64 Bibliography Appendices List of Figures 1.1 Cross-sectional view of a gram-negative cell envelope . .1 1.2 Proposed structure of the conserved reducing terminal glycolipid in E. coli and N. menin- gitidis CPS .........................................2 1.3 TI-1 B-cell activation and antibody formation and carbohydrate antigen pathways . .5 1.4 Notable history in the development of carbohydrate-based vaccines . .6 1.5 B-cell immune response to polysaccharide vaccines . .7 1.6 Immune response to conjugate vaccines . .8 1.7 Structure of the meningococcal outer membrane . .9 1.8 Worldwide distribution of major meningococcal groups . 11 1.9 Distribution of major meningococcal groups in Africa . 12 1.10 Capsular polysaccharide repeating unit structures of the six most prevalent meningococ- cal groups . 13 1.11 Attributable incidence of pathogen-specific dysentery per 100 child-years by age . 15 1.12 Global distribution of predominant shigella species . 17 1.13 O-Specific polysaccharide repeating unit of S. sonnei.................... 18 2.1 MD simulations with and without periodic boundary conditions . 23 3.1 De-O-acetylated structures CPS dimer of MenY and MenW . 27 3.2 Space filling model of 10 RUs of MenY and MenW . 28 3.3 dOA MenY and MenW trisaccharide . 29 3.4 Hydroxymethyl conformations of glucose and galactose . 30 3.5 Glc14N and Gal14N dihedral PMF surfaces and representative structures . 34 3.6 N26Glc and N26Gal dihedral PMF surfaces and representative structures . 35 3.7 Overlay of MD 3RU dihedral angles of MenY and MenW on the metadynamics PMF surfaces . 38 3.8 Probability density functions of the N26Glc and N26Gal linkage f dihedrals . 39 3.9 Probability density functions of the N26Glc and N26Gal linkage w dihedrals . 39 3.10 Time series of end-to-end distance and w dihedral of MenY and MenW 3RU . 41 3.11 Time series of H-bond length and angles of MenY and MenW 3RU simulation . 42 3.12 Change in solvent accessible surface area of MenY and MenW 3RU structures over 200 ns 43 3.13 SASA potential map on the surface of MenY and MenW 3RU structures . 44 3.14 Mean representative structures of the MenY and MenW 3RU clusters . 46 3.15 Proposed immune response to MenY and MenW vaccine antigens . 48 4.1 O-Specific polysaccharide repeating unit of S. sonnei ................... 50 4.2 Proposed synthesis of the S. sonnei O-SP repeating unit . 52 4.3 Mechanism for b-phenylthioglycoside formation . 54 4.4 Mechanism for oxazolidinone intermediate formation . 55 v List of Tables 1.1 Functions of bacterial extracellular polysaccharides . .3 1.2 Estimated annual number of cases and deaths from selected pathogens . .6 1.3 Repeating unit structures of selected important bacterial cell surface polysaccharides involved in vaccine development . .7 1.4 Immunological responses (bactericidal antibody) of volunteers vaccinated with group Y, group W, or a combined Y-W CPS vaccine . 14 1.5 O-SP repeating unit structures of the four major serotypes of Shigella .......... 17 3.1 Immunogenicity of meningococcal group Y and W CPS vaccines .
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