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Exploring the Structure of Oligo- and Polysaccharides Synthesis and NMR Spectroscopy Studies

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Exploring the structure of oligo- and polysaccharides Synthesis and NMR spectroscopy studies Hanna Jonsson ©Hanna Jonsson, Stockholm 2010 Cover picture “Bacteria runs NMR” ISBN 978-91-7447-041-3 Printed in Sweden by US-AB, Stockholm 2010 Distributor: Department of Organic Chemistry, Stockholm University All I ever want is sugar. -Andy Warhol Mina mångåriga akademiska studier har lett mig fram till, den för hela den vetenskapliga forskningen epokgörande slutsatsen, att jag är bäst. -Ur Grallimatik Tage Danielsson Abstract A deeper understanding of the diversity of carbohydrates and the many ap- plications of oligo- and polysaccharides found in nature are of high interest. Many of the processes involving carbohydrates affect our everyday life. This thesis is based on six papers all contributing to an extended perspective of carbohydrate property and functionality. An introduction to carbohydrate chemistry together with a presentation of selected carbohydrate synthesis and analysis methods introduces the reader to the research field. The first paper is an NMR spectroscopy reinvestigation of the structures of the O- antigens from the lipopolysaccharides (LPS) of Escherichia coli O124 and Shigella dysenteriae type 3. The repeating units were concluded to be built of identical branched pentasaccharides now with the correct anomeric con- figurations. Paper II is a structural investigation of the O-antigen from the LPS of E. coli O74 which is built of branched tetrasaccharide repeating units including the uncommon monosaccharide D-Fuc3NAc. Paper III is a con- formational study of a rhamnose derivative, using NMR spectroscopy and X- ray crystallography. The benzoyl ester group positioned at C4 prefers an “eclipsed” conformation in the crystal as well as in solution. The use of site- specifically 13C-labeled compounds in conformational studies is discussed in Papers IV and V. The disaccharide α-L-Rhap-(1→2)-α-L-Rhap-OMe was synthesized together with two 13C-isotopologues and studied with NMR spectroscopy to give five J-couplings related to torsion angles φ and ψ. The trisaccharide α-L-Rhap-(1→2)[α-L-Rhap-(1→3)]-α-L-Rhap-OMe was syn- thesized with 13C-labeling at two positions which presented a solution to a problem of overlapping signals in the 1H NMR spectrum. The site-specific 3 2 labeling also facilitated the measurement of two JCC and two JCH coupling constants. Finally, chapter 6 gives a short introduction to glycosynthase chemistry and discusses the synthesis of α-glycosyl fluorides. A novel cyclic heptasaccharide was synthesized from α-laminariheptaosyl fluoride using a mutant of the enzyme laminarase 16A and subsequently analyzed by NMR spectroscopy. List of publications This thesis is based on the following papers referred to as their Roman nu- merals I-VI. I Structural studies of the O-antigenic polysaccharides from Shigella dysenteriae type 3 and Escherichia coli O124, a rein- vestigation K. Hanna M. Jonsson, Andrej Weintraub and Göran Widmalm Carbohydrate Research 2006, 341, 2986–2989. II Structural determination of the O-antigenic polysaccharide from Escherichia coli O74 K. Hanna M. Jonsson, Andrej Weintraub and Göran Widmalm Carbohydrate Research 2009, 344, 1592–1595. III Methyl 4-O-benzoyl-2,3-O-isopropylidene-α-L-rhamno- pyranoside K. Hanna M. Jonsson, Lars Eriksson and Göran Widmalm Acta Crystallographica C62 2006, o447-o449. IV Studies on the conformational flexibility of α-L-Rhap-(1→2)- α-L-Rhap-OMe using molecular simulation and 13C-site- specific labeling: a model for a commonly occurring disac- charide in bacterial polysaccharides K. Hanna M. Jonsson, Elin Säwén and Göran Widmalm In manuscript n n V NMR analysis of conformationally dependent JC,H and JC,C in the trisaccharide α-L-Rhap-(1→2)[α-L-Rhap-(1→3)]-α-L- Rhap-OMe and a site-specifically labeled isotopologue thereof K. Hanna M. Jonsson, Göran Widmalm In manuscript VI Synthesis of cyclic β-glucan Using Laminarinase 16A Glyco- synthase Mutant from the Basidiomycete Phanerochaete chrysosporium Jonas Vasur, Rie Kawai, K. Hanna M. Jonsson, Göran Widmalm, Åke Engström, Martin Frank, Evalena Andersson, Henrik Hans- son, Zarah Forsberg, Kiyohiko Igarashi, Masahiro Samejima, Mats Sandgren, Jerry Ståhlberg Journal of the American Chemical Society 2010, 132, 1724- 1730. Paper I and II were reprinted with kind permission from Elsevier, paper III from IUCr and Paper VI from ACS. Contents Chapter 1: Introduction to carbohydrate chemistry..........................11 1.1 Carbohydrates in bacteria ................................................................................13 1.2 Short summary of carbohydrate nomenclature...........................................14 Chapter 2: Selected methods of carbohydrate synthesis.................17 2.1 Protecting group chemistry ..............................................................................17 2.2 O-Glycosylation methods..................................................................................18 Chapter 3: Methods of carbohydrate analysis ....................................21 3.1 Sugar analysis.....................................................................................................21 3.2 NMR spectroscopy analysis ..............................................................................22 3.2.1 Methods of structural investigation .......................................................23 3.2.2 Methods of conformational analysis ......................................................25 3.2.3 Selected NMR techniques for J-coupling measurements..................26 Chapter 4: Structural studies of bacterial polysaccharides (Papers I & II)..............................................................................................................29 4.1 Introduction .........................................................................................................29 4.2 Structural investigation of the O-antigenic polysaccharides from E. coli O124 and S. dysenteriae type 3 (Paper I)...........................................................30 4.3 Structural investigation of the O-antigen of E. coli O74 (Paper II) ........33 Chapter 5: Conformational analysis of rhamnose-containing saccharides (Paper III, IV & V) ..............................................................37 13 5.1 Synthesis of C-labeled rhamnopyranoside oligosaccharides .................38 5.2 Conformational study of a rhamnose derivative (Paper III).....................40 5.3 Studies on the conformational flexibility of α-L-Rhap(1→2)-α-L-Rhap- OMe (Paper IV)...........................................................................................................41 5.4 NMR studies on of the trisaccharide α-L-Rhap-(1→2)[α-L-Rhap-(1→3)]- α-L-Rhap-OMe (Paper V)..........................................................................................44 Chapter 6: Enzymatic synthesis using glycosynthases (Paper VI).47 6.1 A short introduction to glycosynthase chemistry ........................................47 6.2 Synthesis of α-glycosyl fluoride donors.........................................................49 6.3 Investigating a cyclic heptaglucan synthesized by mutant laminarase 16A (Paper VI) ...........................................................................................................51 Chapter 7: Concluding remarks and future prospects ......................54 Acknowledgements ...................................................................................56 References ..................................................................................................58 Abbreviations 1DLR One dimensional long-range CPS Capsular polysaccharide DAST Diethylaminosulfur trifluoride DOSY Diffusion-ordered spectroscopy DQF-COSY Double-quantum filtered correlated spectroscopy EIEC Enteroinvasive Escherichia coli Fuc Fucose GalNAc N-acetyl galactosamine GLC Gas-liquid chromatography GlcNAc N-acetyl glucosamine HECADE (Heteronuclear Couplings from ASSCI-domain experiments with E.COSY-type crosspeaks) HETCOR Heteronuclear correlation HMBC Heteronuclear multiple bond correlation HSQC Heteronuclear single quantum correlation LPS Lipopolysaccharide MD Molecular dynamics Me Methyl NIS N-iodosuccinimide NOESY Nuclear Overhauser effect spectroscopy OAc O-acetyl OBz O-benzoyl p pyranose Rha Rhamnose TOCSY Total correlation spectroscopy 2 TSP Sodium 3-methylsilyl-(2,2,3,3- H4)-propanoate Chapter 1: Introduction to carbohydrate chemistry Today when carbohydrates are mentioned one immediately thinks of the main source of energy obtained from our food. People in general also distin- guish between sugar and carbohydrates, the former being the dangerous white food-sweetener and the latter being healthy slow-digesting “good” energy. For a carbohydrate chemist, the difference only lies in the use of the words; sugar is the common word and carbohydrate is the scientifically cor- rect term. Of course, carbohydrates are not only found in the food we eat, they are also the major constituent of wood and the shells of insects and shellfish, and they are present in almost all cell walls of living tissue. They contribute as signaling substances recognized by many proteins and define our blood group antigens. Carbohydrates are the most abundant of the bio- logical substances, produced in thousands of tons per year by photosynthesis in plants and microorganisms. Carbohydrate chemistry was founded by the German researcher Hermann Emil Fischer (1852-1919)1 who already in 1890 established the stereochemi- cal nature and isomerism of the common
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