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And Glycoarrays DEVELOPMENT AND APPLICATION OF PEPTIDE- AND GLYCOARRAYS A THESIS SUBMITTED TO THE UNIVERSITY OF MANCHESTER FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (PHD) IN THE FACULTY OF ENGINEERING AND PHYSICAL SCIENCES DIPL.-CHEM.MARTIN WEISSENBORN, MSC SCHOOL OF CHEMISTRY 2012 CONTENTS Declaration5 Copyright6 Acknowledgements7 Abstract9 1 Thesis Structure 10 2 Enzymatic Reactions on Solid-Support 11 3 Enzymatic Glycosylations on Arrays 12 4 Glycoarrays on Gold Surfaces 13 5 Objectives of this Thesis 14 5.1 Array Formation.............................. 14 5.2 Analysis of Arrays............................. 15 5.3 Application of Microarrays......................... 16 6 Methodologies applied in this Thesis 17 6.1 Chemical Synthesis............................. 17 6.2 Arrays on Gold............................... 17 6.2.1 Coupling into SAMs........................ 17 6.2.2 MALDI-ToF MS analysis of SAMs................ 19 6.3 Surface Plasmon Resonance (SPR) on SAMs............... 20 6.4 Arrays on Polystyrene............................ 22 7 Preparation of aminoethyl glycosides for glycoconjugation 23 7.1 Supporting Information........................... 24 8 Oxo-ester mediated native chemical ligation 25 8.1 Supporting Information........................... 26 2 CONTENTS 9 MALDI-ToF MS Analysis on Glass and Polystyrene 27 9.1 Supporting Information........................... 28 10 Dual purpose S-trityl-linkers for glycoarray fabrication on both polystyrene and gold 29 10.1 Supporting Information........................... 30 11 High-Throughput Screening of Protein Glycosylation Using Lectin-Binding Biophotonic Microarray Imaging 31 12 Crystal structure of a soluble form of human CD73 with ecto-5’-nucleotidase activity 32 12.1 Supporting Information........................... 33 13 Chemoenzymatic Synthesis of O-Mannosylpeptides 34 13.1 Supporting Information........................... 35 14 Conclusion and Outlook 36 14.1 Conclusion................................. 36 14.2 Outlook................................... 37 15 Bibliography 39 Final word count: 69800 3 LIST OF FIGURES 6.1 Formation of SAMs on gold......................... 18 6.2 Formation and functionalisation of self-assembling monolayers on gold.. 18 6.3 The mechanism of the activation of the linker 2 by EDC to form the acti- vated ester 3................................. 19 6.4 Maleimide functionalised SAMs followed by the formation of thioethers. 19 6.5 Schematic illustration of direct MALDI-ToF MS analysis of SAMs.... 21 6.6 MALDI-ToF MS spectra of unmodified and modified SAMs........ 21 6.7 Illustration of the surface plasmon resonance (SPR) technique....... 22 4 DECLARATION The University of Manchester PhD by published work Candidate Declaration Candidate Name: Martin Weissenborn Faculty: Engineering and Physical Sciences Thesis Title: Development and Application of Peptide- and Glycoarrays Declaration to be completed by the candidate: I declare that no portion of this work referred to in this thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. Signed: Date: December 7, 2012 5 COPYRIGHT The author of this thesis (including any appendices and/or schedules to this thesis) owns any copyright in it (the "Copyright")1and he has given The University of Manchester the right to use such Copyright for any administrative, promotional, educational and/or teaching purposes. Copies of this thesis, either in full or in extracts, may be made only in accordance with the regulations of the John Rylands University Library of Manchester. Details of these regulations may be obtained from the Librarian. This page must form part of any such copies made. The ownership of any patents, designs, trade marks and any and all other intellectual property rights except for the Copyright (the "Intellectual Property Rights") and any re- productions of copyright works, for example graphs and tables ("Reproductions"), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property Rights and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property Rights and/or Reproductions. Further information on the conditions under which disclosure, publication and ex- ploitation of this thesis, the Copyright and any Intellectual Property Rights and/or Repro- ductions described in it may take place is available from the Head of School of Chem- istry(or the Vice-President) and the Dean of the Faculty of Engineering and Physical Sci- ences, for Faculty of Engineering and Physical Sciences candidates. 1This excludes material already printed in academic journals, for which the copyright belongs to said journal and publisher. Pages for which the author does not own the copyright are numbered differently from the rest of the thesis. 6 ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor, Prof Sabine Flitsch, for giving me the opportunity to work on this great project in a fascinating and highly interdisci- plinary network. Thanks also goes to my former supervisor Prof Thisbe Lindhorst for recommending me to Prof. Flitsch and for the fruitful collaboration. I am furthermore grateful for the generous support of the European Commission for the Marie Curie research fellowship. This made many collaborations possible. Thanks to these collaborators for hosting me in their labs and giving rise to many inter- esting and successful projects. Mein Dank an meine Familie ist nicht in einem Satz zusammenzufassen. Ich danke euch für eure Unterstützung in allen Lebenslagen, die Idee Chemie zu studieren und das Studium zu finanzieren. Und noch so vieles mehr. I am also very proud of being a part of the Turner/Flitsch group. It was great to work in such a nice atmosphere for three years. Special thanks goes to my colleagues Dr Robert Sardzik, Dr Josef Voglmeir, Dr Damien Debecker, Dr Mark Corbett, Paul Kelly, Roberto Castangia, Dominique Richardson and Christopher Gray. Dank an an meine Freunde aus Kiel, für eine tolle gemeinsame Zeit in der ich viel Hilfe von euch erfahren habe, and to my friends in Manchester for a great time. Lastly, I would like to thank Dr Ivan Vilotijevic, Dr Mark Corbett and Paul Kelly for their fantastic and numerous proof readings — including this section. 7 Ich widme diese Arbeit in Liebe und Dankbarkeit meiner Familie. Abstract Microarrays enable high throughput analysis with minute amounts of analyte. They are widely used in the ’omics’ fields both as diagnostic and analytical tools. Their ability to dramatically impact an entire field of research has focused our attention on the de- velopment of novel methods for the formation, analysis and applications of microarrays to study carbohydrate-protein interactions and the analysis of glycosylation patterns of biomolecules. Availability of appropriately modified ligands is often a limiting factor in the prepara- tion of microarrays. To address this issue robust routes for the synthesis of nine aminoethyl glycosides were developed that can be employed for microarray formation. The syntheses of more complex ligands typically deliver small quantities of mate- rial despite the requirements for special skills, equipment and long preparation times. Considering the number of complex oligosaccharides that are necessary for systematic microarray studies, the problem of availability of these complex structures is difficult to address solely with synthetic ligands. A modified native chemical ligation (NCL) strategy, in which a surface bound oxo-ester is used instead of a thioester, was optimised and used for efficient chemoselective immobilisation of sugars and peptides carrying N-terminal cysteines. The reaction proceeds under physiological conditions and has the potential to become a valuable tool for immobilisation of N-terminal cysteine-containing molecules from biological samples. The new NCL coupling methodology was developed on gold surfaces and analysed by MALDI-ToF MS. The majority of array systems, however, rely on secondary pro- tein interactions on glass or polystyrene surfaces. A direct, more accurate analytical tool could ease the analysis and significantly improve the quality of data read-out from glass microarrays. MALDI-ToF MS that is applicable to gold microarrays cannot be used on surfaces that do not provide the necessary electrical conductivity. The undertaken exper- iments indicated that application of conductive tape to the back of glass or polystyrene slides made MALDI-ToF analysis on poorly conducting surfaces possible. Furthermore, the triphenylmethyl (trityl) groups attached to the surface-molecules were shown to act as ’internal-matrix’ and enable the direct MALDI analysis. Once the new array formation and analysis techniques were developed, we turned our attention towards the application of microarrays to analyse carbohydrate-protein interac- tions. The tools for analysis of glycosylation of biomolecules are laborious and can only be used in specialised labs. As glycosylated biomolecules gain prominence in research, clinical and industrial settings, high throughput analysis of glycosylation patterns is be- coming a requirement for quality control. A technique for screening of glycosylation patterns in glycopeptides on microarrays was developed based on biophotonic scattering. This technique enables the detection of glycosylation patterns by screening immobilised glycoproteins with
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