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«DISS. ETH NO. 19662 I. Synthetic Bacterial Lipopolysaccharide Core Structures as Vaccine Candidates against Clamydia trachomatis and Yersinia pestis ...»

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DISS. ETH NO. 19662

I. Synthetic Bacterial Lipopolysaccharide Core Structures

as Vaccine Candidates against

Clamydia trachomatis and Yersinia pestis

II. Synthesis of a Fungal Galectin Epitope Trisaccharide

and the HNK-1 Epitope Trisaccharide

A dissertation submitted to


for the degree of

Doctor of Sciences

presented by

Xiaoqiang Guo

M.Sc., Tsinghua University

Born October 27, 1981

citizen of P.R.China

accepted on the recommendation of

Prof. Dr. Karl-Heinz Altmann

Prof. Dr. Peter H. Seeberger Prof. Dr. Markus Aebi Acknowledgements First of all, I would like to thank Professor Seeberger for having given me the opportunity of working in this very ‘international’ group on these challenging projects.

I am also grateful to Professor Altmann for generously agreeing to supervise this thesis and Professor Aebi for agreeing to co-chair my doctoral examination.

Four and a half years of being in this diverse working environment with colleagues from all over the world has expanded my vision not only in chemistry but also in many other areas in my life. This experience has been invaluable to me and I will always cherish it. In particular, I have enjoyed the pleasant atmosphere in the lab together with Lenz Kröck, Laila Hossain, Reiko Wada, Xiao-Yin Mak, Matthias Oberli, Cullen Klein and Christopher Martin and will treasure the friendship between us very much. Going jogging with Arjan Odedra, Paola Laurino, Karolin Geyer, Faustin Kamena, Dominea Rathwell every week was another enjoyable part of my experience in this group these past few years.

I want to express my appreciation to Dr. Rajan Pragani, Dr. Dominea Rathwell, Dr.

Chris Rathwell and Mr. Christopher Martin for their help proof-reading parts of this thesis. Very special thanks to Dr. Xiao-Yin Mak, for proof-reading all the chapters and for valuable comments. I would also like to thank Mr. Christopher Martin for the German translation of the abstract.

Thanks to Mrs. Eva Settels for her help in MALDI-TOF measurements and Mrs.

Annette Wahlbrink for the immunological studies with mice and ELISA assays.

I also thank Dr. Yin Jian and his wife Dr. Hu Jing, Mr. Yu-Hsuan Tsai and his wife Mrs. An-Ting Chang for their friendship and support. Our cheerful dinner parties together made my life abroad much more enjoyable.

Foremost, I would like thank my parents for their support during the years. Talking with them every weekend was always my happiest time.

Parts of this thesis have been published and communicated:

Publication Butschi, A.; Titz, A.; Wälti, M.A.; Olieric, V.; Paschinger, K.; Nöbauer, K., Guo, X.;

Seeberger, P.H.; Wilson, lain B.H.; Aebi, M.; Hengartner, M.O.; Künzler, M.

‘Caenorhabditis elegans N-glycan Core β-galactoside Confers Sensitivity towards Nematotoxic Fungal Galectin CGL2’ PLOS Pathog. 2010, 6, e1000717.

Posters ‘Solution and Solid Phase Synthesis of HNK-1 Carbohydrate’ Xiaoqiang Guo, Lenz Kröck, Peter H. Seeberger, at the Competence Center for Materials Science & Technology (CCMX) Annual Conference, Fribourg, Switzerland, March 20, 2007 ‘The Approach to the Synthesis of HNK-1 Trisaccharide’ Xiaoqiang Guo, Peter H. Seeberger, at the Annual Meeting of the Competence Center for Materials Science & Technology (CCMX), Bern, Switzerland, April 9, 2008.


–  –  –

Abstract Lipopolysaccharide (LPS) is present on the outer membrane of Gram-negative bacteria and plays a critical role in cell adhesion and bacterial infection processes. The O-antigen and core oligosaccharide fragments of LPS can be used as potential antigens for anti-bacterial vaccine development. Carbohydrates alone are T-cell independent antigens, however when conjugated with carrier protein they are able to trigger the participation of T-helper cells and induce long-term protection.

Chlamydia trachomatis is a gram-negative bacterium that causes genital tract infection and trachoma, which can lead to blindness. To date, no vaccine against Chlamydia has been marketed. In Chapter 2, the chlamydial LPS core structure, a potential antigen consisting of a 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) trisaccharide α-Kdo-(2→8)-α-Kdo-(2→4)-Kdo, was chemically synthesized. The trisaccharide was assembled using three Kdo monosaccharide building blocks from the non-reducing end. Different Kdo glycosylating agents were investigated to optimize the glycosylation yields and stereoselectivities. Although Kdo glycosyl phosphates generally afforded better yields than the related thioglycosides, the α/βselectivities on the newly generated glycosidic bonds were poor and β-elimination to the 2,3-glycal remained a major side reaction. These findings suggest that those traditional glycosylating agents may not be ideal for Kdo glycosylations and new methods have to be investigated.

Yersinia pestis is a gram-negative bacterium that causes plague. Due to its potential use as an agent of biological warfare and terrorism, Y. pestis is listed in the ‘Category A’ of ‘Bioterrorism Agents/Disease’ by the U.S. Centers for Disease Control and Prevention (CDC). The development of vaccines against Y. pestis has been the subject of investigation for more than a hundred years. Unfortunately, so far none of them has been licensed. The Y. pestis LPS core, consisting of a Kdo disaccharide, α-Kdo4)-Kdo, and an L-glycero-α-D-manno-heptose (LD-Hep) trisaccharide, α-LDHep-(1→7)-α-LD-Hep-(1→3)-LD-Hep, is a potential antigen for Y. pestis and studies on its synthesis and immunological properties are described in Chapter 3. The heptose trisaccharide thioglycoside and the Kdo disaccharide were successfully

i Abstract

synthesized. However, due to the poor reactivity of the Kdo disaccharide, the [3+2] glycosylation of those two fragments did not achieve the assembly of the target pentasaccharide. Instead, a heptose trisaccharide equipped with an aminopentyl linker was synthesized and subsequently conjugated to the carrier protein CRM197 via a squarate method. The glycoconjugate was injected into two female mice. Preliminary results from ELISA studies showed significant anti-trisaccharide IgG responses in the post-immunization sera. This result reveals that the synthetic trisaccharide is immunogenetic and the anti-trisaccharide IgG antibodies were successfully generated.

Lectins are sugar-binding proteins without enzymatic activity. Galectin is a type of lectin that binds β-galactoside. The physiological role of fungal galectins has remained elusive. Studies of isogalectin CGL2, found in fungus Coprinopsis cinerea, and the soil nematode Caenorhabditis elegans suggested that CGL2-mediated nematotoxicity depends on the interaction between the isogalectin and a fucosecontaining glycoconjugate. As described in Chapter 4, in order to provide evidence for the interaction in detail, the trisaccharide β-Gal-(1→4)-α-Fuc-(1→6)-GalNAc was synthesized. The crystal structure of CGL2 and the synthetic trisaccharide at 1.5 Å resolution revealed the biophysical basis of their interaction. This result suggests that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms. In addition, the synthetic trisaccharide was conjugated to the carrier protein KLH via a squarate method and monoclonal antibodies were generated. Future studies will focus on the specificity of these antibodies.

The human natural killer-1 (HNK-1) carbohydrate epitope plays important roles in cell-cell and cell-substrate interactions, cell migration and neurite outgrowth. Its structure has been elucidated as the sulfated trisaccharide HSO3→3-β-GlcA-(1→3)Gal-β-(1→4)-GlcNAc. Chapter 5 describes the synthesis of this trisaccharide. Two different glucuronate building blocks were synthesized. The glycosylation with the reducing disaccharide moiety Gal-β-(1→4)-GlcNAc resulted however in low yields.

The glucuronate glycoside proved to be a poor glycosylating agent and unreactive.

The synthetic HNK-1 trisaccharide serves as an important tool for studying its biological properties.

–  –  –

Zusammenfassung Lipopolysaccharide (LPS) kommen in der äußeren Membran von gramnegativen Bakterien vor und spielen eine entscheidende Rolle in den Vorgängen der Zelladhäsion und bakteriellen Infektion. Das und KernO-Antigen Oligosaccharidfragment des LPS können als mögliche Antigene für die Entwicklung antibakterieller Impfstoffe verwendet werden. Kohlenhydrate selbst sind T-Zell unabhängige Antigene, werden diese jedoch mit einem Trägerprotein konjugiert, können sie T-Helferzellen aktivieren und so zu einem lang anhaltenden Impfschutz führen.

Chlamydia trachomatis ist ein gramnegatives Bakterium, das Infektionen im Genitalbereich und Trachomen verursacht, welche zur Erblindung führen können.

Bisher gibt es noch keinen kommerziell erhältlichen Impfstoff gegen Chlamydien. In Kapitel 2 wird die chemische Synthese der LPS Kernstruktur von Chlamydien beschrieben. Dieses mögliche Antigen besteht aus einem 3-Desoxy-α-D-manno-oct-2ulosonsäure (Kdo) Trisaccharide mit der Struktur α-Kdo-(2→8)-α-Kdo-(2→4)-Kdo.

Das Trisaccharid wurde unter Verwendung von drei Kdo Monosaccharidbausteinen vom nicht-reduzierenden Ende her zusammengesetzt. Dabei wurden verschiedene Glykosylierungsmittel verwendet um die Ausbeute und Stereoselektivität der Glykosylierungsreaktionen zu optimieren. Obwohl die Verwendung von KdoGlykosylphosphaten bessere Ausbeuten als die entsprechenden Thioglykoside ergab, war die α/β-Selektivität der neugeformten glykosidischen Bindung gering und die βEliminierung zum 2,3-Glycal blieb eine häufig beobachtete Nebenreaktion. Diese Ergebnisse legen nahe, dass traditionelle Glykosylierungsmethoden für KdoBausteine nicht optimal geeignet sind und daher neue Methoden untersucht werden müssten.

Yersinia pestis ist ein gramnegatives Bakterium und Verursacher der Pest. Aufgrund der möglichen Verwendung als Biowaffe und terroristische Zwecke, ist Y. pestis in der „Kategorie A“ für „Bioterroristische Erreger/Krankheiten“ des U.S. Centers for Disease Control and Prevention (CDC) aufgeführt. Schon seit mehr als hundert Jahren wird an der Entwicklung eines Impfstoffs gegen Y. pestis geforscht, bisher gibt es jedoch noch keinen lizenzierten Impfstoff. Die Y. pestis LPS Kernstruktur besteht aus

iii Zusammenfassung

einem Kdo Disaccharid, α-Kdo-(2→4)-Kdo und einem L-glycero-α-D-manno-heptose (LD-Hep) Trisaccharid α-LD-Hep-(1→7)-α-LD-Hep-(1→3)-LD-Hep. Die Synthese und immunologische Eigenschaft beider möglichen Y. pestis Antigene ist in Kapitel 3 beschrieben. Das Heptose Trisaccharid Thioglykosid und das Kdo Disaccharid wurden erfolgreich synthetisiert. Jedoch gelang die Verknüpfung der beiden Strukturen in einer [3+2] Glykosylierung zum Pentasaccharid aufgrund der Unreaktivität des Kdo Disaccharides nicht. Stattdessen wurde das Heptose Trisaccharid mit einem Aminopentyl Linker ausgestattet und anschließend an das Trägerprotein CRM197 unter Verwendung der Quadratsäuremethode konjugiert. Diese Glykokonjugat wurde weiblichen Mäusen injiziert. Vorläufige Ergebnisse von ELISA Untersuchungen zeigten eine signifikante anti-Trisaccharid IgG Antwort im Blutserum nach Immunisierung. Diese Ergebnisse zeigen, dass das synthetisierte Trisaccharid immunogen ist und die IgG Antikörper gegen dieses Trisaccharid erfolgreich generiert wurden.

Lektine sind zuckerbindende Proteine, die keine enzymatische Aktivität aufweisen.

Galektin ist ein Lektin, welches β-Galaktoside bindet. Die physiologische Rolle von Galektinen in Pilzen ist nach wie vor ungeklärt. Isogalektin CGL2 kommt im Pilz Coprinopsis cinerea und in dem Fadenwurm Caenorhabditis elegans vor.

Untersuchungen an CGL2 haben ergeben, dass die CGL2 vermittelte Toxizität der Fadenwürmer von den Wechselwirkungen zwischen dem Isogalektin und einem fukosehaltigen Glykokonjugat beruht. In Kapitel 4 wird die Synthese des Trisaccharids β-Gal-(1→4)-α-Fuc-(1→6)-GalNAc beschrieben, welches zur genauen Untersuchung der Wechselwirkungen verwendet wurde. Die Kristallstruktur von CGL2 zusammen mit dem synthetischen Trisaccharid mit einer Auflösung von1.5 Å zeigte die biophysikalische Grundlage der Wechselwirkungen. Die Ergebnisse legen nahe, dass Pilze Galektine zum Schutz vor Feinden einsetzen, indem sie an bestimme Glykokonjugate im Feindorganismus binden. Ferner wurde das synthetisierte Trisaccharid unter Verwendung der Quadratsäuremethode an das Trägerprotein KLH konjugiert und monoklonale Antikörper erzeugt. Die Spezifität der Antikörper wird Gegenstand weiterer Untersuchungen sein.

Das menschliche natürlichen Killer-1 (HNK-1) Kohlenhydrat Epitop spielt eine wichtige Rolle in Zell-Zell und Zell-Substrat Interaktionen, sowie der Zellwanderung

–  –  –

und dem Neurit-Auswuchs. Die Struktur besteht aus dem sulfatiertem Trisaccharid HSO3→3-β-GlcA-(1→3)-Gal-β-(1→4)-GlcNAc. Kapitel 5 beschreibt die Synthese des Trisaccharides. Die Glykosylierung mit dem reduzierenden Disaccharid Epitopfragment Gal-β-(1→4)-GlcNAc ergab jedoch geringe Ausbeuten. Die Glucuronsäure Bausteine erwiesen sich als unreaktives Glykosylierungsmittel. Das synthetische HNK-1 Epitop dient als wichtiges Werkzeug für die Untersuchung der biologischen Eigenschaften.

–  –  –

1.1 Bacterial Lipopolysaccharide (Endotoxin) Richard Pfeiffer, a collaborator of Robert Koch, discovered at the end of 19th century a toxin that was localized within the bacterial cell of Vibrio cholerae and thus named it ‘endotoxin’, in order to distinguish it from the previously known exotoxins [1].

Exotoxins are formed and secreted by the bacterial cell, and found free in the surrounding medium.

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