Carbohydrate Microarrays: Methods and Protocols (Methods in Molecular Biology, v808)

Methods

in

Molecular Biology™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK



For further volumes: http://www.springer.com/series/7651

Carbohydrate Microarrays Methods and Protocols Edited by

Yann Chevolot Université de Lyon, Institut des Nanotechnologies de Lyon – INL, UMR CNRS 5270, Site Ecole Centrale de Lyon, Ecully, France

Editor Yann Chevolot Université de Lyon Institut des Nanotechnologies de Lyon – INL – UMR CNRS 5270 Site Ecole Centrale de Lyon Ecully, France [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-372-1 e-ISBN 978-1-61779-373-8 DOI 10.1007/978-1-61779-373-8 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011938687 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or d ­ issimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface Carbohydrate microarrays have emerged in the late 1990s and early twenty-first century as a key technology for the deciphering of the glycospace. A carbohydrate microarray is a multiplex technology, where tens to hundreds of carbohydrate–protein interactions can be probed in parallel. Multiplexing is made possible, thanks to the immobilization of various carbohydrates at a discrete location on the surface of a material or on coded microspheres for example. This book, dedicated to carbohydrate microarray, is aimed to give the reader the theoretical and experimental clues for the fabrication and implementation of carbohydrate microarrays. The fabrication of carbohydrate microarrays requires (1) to obtain the carbohydrate probes (monosaccharides, oligosaccharides, polysaccharides, glycoconjugates, or glycoclusters), (2) to immobilize these probes, (3) to implement the protocols for biological/biochemical interaction with the desired target. Carbohydrate probes can be obtained from natural sources, by synthesis or are commercially available. The synthesis of oligosaccharides is beyond the scope of this book and requires specific skills. Nevertheless, an overview chapter is dedicated to glycochemistry as we felt that notions in the field are essential in particular for the introduction of derivatives (glycosides) for their immobilization. Indeed, immobilization of carbohydrates can be performed without further modifications of the carbohydrate or may require their conjugation with proteins, DNA, lipids, or the introduction of functional groups (thiol, amine, maleimide…). Immobilization can be performed through weak interaction (van der Waals, ionic interactions), covalent bonding or biochemical reactions (streptavidin/biotin, DNA/DNA hybridization). These immobilizations can be performed directly on unmodified surfaces (thiol glycosides on gold surfaces for example) or on chemically prepared surfaces. With the first two chapters, we have given an overview on carbohydrate microarray, carbohydrate chemistry. Chapter 3 is dedicated to sialic acids which display special biological and chemical properties. The following chapters deal with experimental protocols that present immobilization of various carbohydrates (oligosaccharides, glycoproteins, glyco‑ lipids, and polysaccharides) on different substrates (gold, glass, polymers…). Protocols span from the “straight forward” adsorption of unmodified polysaccharides/glycoconjugates from natural sources on commercial substrates to the synthesis of glycosides and their subsequent immobilization on chemically modified surfaces. Some materials used in certain protocols, such as glycosides or chemically modified surfaces are commercially available. Similarly, we have tried to illustrate different detection techniques (Mass Spectrometry, Surface Plasmon Resonance, Dual Polarization Interferometry, Fluorescence…) and ­applications (from lectin/carbohydrates applications to serum antibodies profiling, ­serodiagnosis, enzymes, whole viruses…). Hopefully, the catalogue of protocols will span most of the reader needs/applications. Finally, I gratefully acknowledge Pr. John Walker and all the authors for their contributions which have made the realization of this book possible. Ecully, France

Yann Chevolot

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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  1 Recent Advances and Future Challenges in Glycan Microarray Technology . . . . . . José L. de Paz and Peter H. Seeberger   2 Chemical Synthesis of Carbohydrates and Their Surface Immobilization: A Brief Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniel B. Werz   3 General Consideration on Sialic Acid Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . Hongzhi Cao and Xi Chen   4 Synthesis of Azido-Functionalized Carbohydrates for the Design of Glycoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Samy Cecioni, David Goyard, Jean-Pierre Praly, and Sébastien Vidal   5 Polypyrrole-Oligosaccharide Microarray for the Measurement of Biomolecular Interactions by Surface Plasmon Resonance Imaging . . . . . . . . . . Julia Bartoli, André Roget, and Thierry Livache   6 Glycosylated Self-Assembled Monolayers for Arrays and Surface Analysis . . . . . . . . Fang Cheng and Daniel M. Ratner   7 Carbohydrate Microarrays for Enzymatic Reactions and Quantification of Binding Affinities for Glycan–Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . Myung-Ryul Lee, Sungjin Park, and Injae Shin   8 Neoglycolipid-Based Oligosaccharide Microarray System: Preparation of NGLs and Their Noncovalent Immobilization on Nitrocellulose-Coated Glass Slides for Microarray Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yan Liu, Robert A. Childs, Angelina S. Palma, Maria A. Campanero-Rhodes, Mark S. Stoll, Wengang Chai, and Ten Feizi   9 Preparation of a Mannose-6-Phosphate Glycan Microarray Through Fluorescent Derivatization, Phosphorylation, and Immobilization of Natural High-Mannose N-Glycans and Application in Ligand Identification of P-Type Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xuezheng Song, Jamie Heimburg-Molinaro, Nancy M. Dahms, David F. Smith, and Richard D. Cummings 10 Production of Fluorous-Based Microarrays with Uncharged Carbohydrates . . . . . . Sahana K. Nagappayya and Nicola L.B. Pohl 11 General Procedure for the Synthesis of Neoglycoproteins and Immobilization on Epoxide-Modified Glass Slides . . . . . . . . . . . . . . . . . . . . . . Yalong Zhang and Jeffrey C. Gildersleeve

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12 Immobilization of Polyacrylamide-Based Glycoconjugates on Solid Phase in Immunosorbent Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxana E. Galanina, Alexander A. Chinarev, Nadezhda V. Shilova, Marina A. Sablina, and Nicolai V. Bovin 13 Surface Plasmon Resonance Imaging Analysis of Protein Binding to a Sialoside-Based Carbohydrate Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . Matthew J. Linman, Hai Yu, Xi Chen, and Quan Cheng 14 Glycoarray by DNA-Directed Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . François Morvan, Yann Chevolot, Jing Zhang, Albert Meyer, Sébastien Vidal, Jean-Pierre Praly, Jean-Jacques Vasseur, and Eliane Souteyrand 15 Fabrication of Carbohydrate Surfaces by Using Non-derivatised Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jonathan Popplewell, Marcus Swann, Gavin Brown, and Bob Lauder 16 Polysaccharide Microarrays: Application to the Identification of Heparan Sulphate Mimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Julien Dheur, Nabil Dendane, Rémi Desmet, Fatima Dahmani, Gauthier Goormachtigh, Jérome Vicogne, Véronique Fafeur, and Oleg Melnyk 17 Carbohydrate Antigen Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Denong Wang 18 Probing Virus–Glycan Interactions Using Glycan Microarrays . . . . . . . . . . . . . . . . Jamie Heimburg-Molinaro, Mary Tappert, Xuezheng Song, Yi Lasanajak, Gillian Air, David F. Smith, and Richard D. Cummings 19 MALDI-ToF MS Analysis of Glycosyltransferase Activities on Gold Surface Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicolas Laurent, Rose Haddoub, Josef Voglmeir, and Sabine L. Flitsch 20 Microarray Technology Using Glycans Extracted from Natural Sources for Serum Antibody Fluorescent Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emanuela Lonardi, André M. Deelder, Manfred Wuhrer, and Crina I.A. Balog 21 Studying Modification of Aminoglycoside Antibiotics by Resistance-Causing Enzymes via Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . Matthew D. Disney 22 Microarray Method for the Rapid Detection of Glycosaminoglycan–Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Claude J. Rogers and Linda C. Hsieh-Wilson 23 Neoglycolipid-Based “Designer” Oligosaccharide Microarrays to Define b-Glucan Ligands for Dectin-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angelina S. Palma, Yibing Zhang, Robert A. Childs, Maria A. Campanero-Rhodes, Yan Liu, Ten Feizi, and Wengang Chai 24 Measurement of Antibodies to Pneumococcal Polysaccharides with Luminex xMAP Microsphere-Based Liquid Arrays . . . . . . . . . . . . . . . . . . . . . Jerry W. Pickering and Harry R. Hill

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25 Carbohydrate Microarrays in 96-Well Polystyrene Microtiter Plates . . . . . . . . . . . . Jean-Philippe Ebran, Nabil Dendane, and Oleg Melnyk 26 Photoimmobilization of Saccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gregory T. Carroll and Denong Wang 27 Microwave-Assisted Method for Fabrication of Carbohydrate Cluster Microarrays on 3-Dimensional Hydrazide–Dendrimer Substrate . . . . . . . . . . . . . . Xichun Zhou, Jian Zhang, and Denong Wang 28 Combinatorial Glycoarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Simon Rinaldi, Kathryn M. Brennan, and Hugh J. Willison Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors Gillian Air  •  Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Crina I.A. Balog  •  Department of Parasitology, Leiden University Medical Center, Biomolecular Mass Spectrometry Unit, Center for Infectious Diseases, Leiden, The Netherlands Julia Bartoli  •  Institut de Biologie Structurale, UMR 5075 (CEA, CNRS, UJF), Grenoble, France Nicolaï V. Bovin  •  Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation Kathryn M. Brennan  •  Division of Clinical Neurosciences, University of Glasgow, Glasgow Biomedical Research Centre, Glasgow, UK Gavin Brown  •  Division of Biomedical and Life Sciences, School of Health and Medicine, Lancaster University, Lancaster, UK Maria A. Campanero-Rhodes  •  The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK; Department of Biological Physical Chemistry, Instituto de Química-Física “Rocasolano”, CSIC and CIBERES, Madrid, Spain Hongzhi Cao  •  National Glycoengineering Research Center, Shandong University, Jinan, Shandong, People’s Republic of China Gregory T. Carroll  •  Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands Samy Cecioni  •  Laboratoire de Chimie Organique 2, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Glycochimie, UMR5246, CNRS, Université Lyon 1, Villeurbanne, France Wengang Chai  •  The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK Xi Chen  •  Department of Chemistry, University of California, Davis, CA, USA Fang Cheng  •  Department of Bioengineering, University of Washington, Seattle, WA, USA Quan Cheng  •  Department of Chemistry, University of California, Riverside, CA, USA Yann Chevolot  •  Université de Lyon, Institut des Nanotechnologies de Lyon – INL, UMR CNRS 5270, Site Ecole Centrale de Lyon, Ecully, France Robert A. Childs  •  The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK Alexander A. Chinarev  •  Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation Richard D. Cummings  •  Department of Biochemistry, O. Wayne Rollins Research Center, Emory University School of Medicine, Atlanta, GA, USA

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Fatima Dahmani  •  CNRS UMR 8161, Université Lille Nord de France, Institut Pasteur de Lille, IFR 142 Molecular and Cellular Medicine, Lille, France Nancy M. Dahms  •  Medical College of Wisconsin, Milwaukee, WI, USA Jose L. de Paz  •  Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and US, Sevilla, Spain André M. Deelder  •  Department of Parasitology, Leiden University Medical Center, Biomolecular Mass Spectrometry Unit, Center for Infectious Diseases, Leiden, The Netherlands Nabil Dendane  •  CNRS UMR 8161, Université Lille Nord de France, Institut Pasteur de Lille, IFR 142 Molecular and Cellular Medicine, Lille, France Rémi Desmet  •  CNRS UMR 8161, Université Lille Nord de France, Institut Pasteur de Lille, IFR 142 Molecular and Cellular Medicine, Lille, France Julien Dheur  •  CNRS UMR 8161, Université Lille Nord de France, Institut Pasteur de Lille, IFR 142 Molecular and Cellular Medicine, Lille, France Matthew D. Disney  •  Department of Chemistry, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA Jean-Philippe Ebran  •  CNRS UMR 8161, Université Lille Nord de France, Institut Pasteur de Lille, IFR 142 Molecular and Cellular Medicine, Lille, France Véronique Fafeur  •  CNRS UMR 8161, Université Lille Nord de France, Institut Pasteur de Lille, IFR 142 Molecular and Cellular Medicine, Lille, France Ten Feizi  •  The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK Sabine L. Flitsch  •  Manchester Interdisciplinary Biocentre & School of Chemistry, The University of Manchester, Manchester, UK Oxana E. Galanina  •  Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation Jeffrey C. Gildersleeve  •  Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA Gauthier Goormachtigh  •  CNRS UMR 8161, Université Lille Nord de France, Institut Pasteur de Lille, IFR 142 Molecular and Cellular Medicine, Lille, France David Goyard  •  Laboratoire de Chimie Organique 2, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Glycochimie, UMR5246, CNRS, Université Lyon 1, Villeurbanne, France Rose Haddoub  •  Manchester Interdisciplinary Biocentre & School of Chemistry, The University of Manchester, Manchester, UK Jamie Heimburg-Molinaro  •  Department of Biochemistry, Glycomics Center, Emory University School of Medicine, Atlanta, GA, USA Harry R. Hill  •  ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA Linda C. Hsieh-Wilson  •  Division of Chemistry and Chemical Engineering, California Institute of Technology and the Howard Hughes Medical Institute, Pasadena, CA, USA Yi Lasanajak  •  Department of Biochemistry, Glycomics Center, Emory University School of Medicine, Atlanta, GA, USA Bob Lauder  •  Division of Biomedical and Life Sciences, School of Health and Medicine, Lancaster University, Lancaster, UK

Contributors

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Nicolas Laurent  •  Manchester Interdisciplinary Biocentre & School of Chemistry, The University of Manchester, Manchester, UK Myung-Ryul Lee  •  Department of Chemistry, Center for Biofunctional Molecules, Yonsei University, Seoul, South Korea Matthew J. Linman  •  Department of Chemistry, University of California, Riverside, CA, USA Yan Liu  •  The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK Thierry Livache  •  CREAB, UMR SPRAM 5819 (CEA, CNRS, UJF), INAC CEA Grenoble, Grenoble, France Emanuela Lonardi  •  Department of Parasitology, Leiden University Medical Center, Biomolecular Mass Spectrometry Unit, Center for Infectious Diseases, Leiden, The Netherlands Oleg Melnyk  •  CNRS UMR 8161, Institut Pasteur de Lille, Université Lille Nord de France, IFR 142 Molecular and Cellular Medicine, Lille, France Albert Meyer  •  Institut des Biomolécules Max Mousseron, UMR 5247, CNRS Université Montpellier 1, Université Montpellier 2, Montpellier, France François Morvan  •  Institut des Biomolécules Max Mousseron, UMR 5247, CNRS Université Montpellier 1, Université Montpellier 2, Montpellier, France Sahana K. Nagappayya  •  Department of Chemistry, The Plant Sciences Institute, and the Interdepartmental Program in Microbiology, Iowa State University, Ames, IA, USA Angelina S. Palma  •  The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK; Chemistry and Technology Network Laboratory (REQUIMTE), Department of Chemistry, Faculty of Science and Technology, New University of Lisbon, Caparica, Portugal Sungjin Park  •  Department of Chemistry, Center for Biofunctional Molecules, Yonsei University, Seoul, South Korea Jerry W. Pickering  •  ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA Nicola L.B. Pohl  •  Wilkinson Professor of Interdisciplinary Engineering, Department of Chemistry, Department of Chemical and Biological Engineering, The Plant Sciences Institute, and the Interdepartmental Program in Microbiology, Iowa State University, Ames, IA, USA Jonathan Popplewell  •  Farfield Group Ltd, Voyager West Wing, Level 7, Manchester Airport, Manchester, UK Jean-Pierre Praly  •  Laboratoire de Chimie Organique 2, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Glycochimie, UMR5246, CNRS, Université Lyon 1,Villeurbanne, France Daniel M. Ratner  •  Department of Bioengineering, University of Washington, Seattle, WA, USA Simon Rinaldi  •  SRI International Biosciences Division, Menlo Park, CA, USA

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Contributors

Claude J. Rogers  •  Division of Chemistry and Chemical Engineering, California Institute of Technology and the Howard Hughes Medical Institute, Pasadena, CA, USA André Roget  •  CREAB, UMR SPRAM 5819 (CEA, CNRS, UJF), INAC CEA Grenoble, Grenoble, France Marina A. Sablina  •  Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation Peter H. Seeberger  •  Max-Planck Institute of Colloids and Interfaces, Potsdam, Germany; Freie Universität Berlin, Berlin, Germany Nadezhda V. Shilova  •  Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation Injae Shin  •  Department of Chemistry, Center for Biofunctional Molecules, Yonsei University, Seoul, South Korea David F. Smith  •  Department of Biochemistry, Glycomics Center, Emory University School of Medicine, Atlanta, GA, USA Xuezheng Song  •  Department of Biochemistry, Glycomics Center, Emory University School of Medicine, Atlanta, GA, USA Eliane Souteyrand  •  Université de Lyon, Institut des Nanotechnologies de Lyon – INL, UMR CNRS 5270, Site Ecole Centrale de Lyon, Ecully, France Mark S. Stoll  •  The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK Marcus Swann  •  Farfield Group Ltd, Voyager West Wing, Level 7, Manchester Airport, Manchester, UK Mary Tappert  •  Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Jean-Jacques Vasseur  •  Institut des Biomolécules Max Mousseron, UMR 5247, CNRS Université Montpellier 1, Université Montpellier 2, Montpellier, France Jérome Vicogne  •  CNRS UMR 8161, Université Lille Nord de France, Institut Pasteur de Lille, IFR 142 Molecular and Cellular Medicine, Lille, France Sébastien Vidal  •  Laboratoire de Chimie Organique 2, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Glycochimie, UMR5246, CNRS, Université Lyon 1, Villeurbanne, France Josef Voglmeir  •  Manchester Interdisciplinary Biocentre & School of Chemistry, The University of Manchester, Manchester, UK Denong Wang  •  Tumor Glycomics Laboratory, Center for Cancer Research, SRI International Biosciences Division, Menlo Park, CA, USA Daniel B. Werz  •  Institut für Organische und Biomolekulare Chemie, Georg-August-Universität Göttingen, Göttingen, Germany Hugh J. Willison  •  College of Medicine, Veterinary Medicine and Life Sciences, Glasgow Biomedical Reseach Centre, Glasgow, UK Manfred Wuhrer  •  Department of Parasitology, Center for Infectious Diseases, Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, Leiden, The Netherlands Hai Yu  •  Department of Chemistry, University of California, Davis, CA, USA Jian Zhang  •  ADA Technologies, Inc., Littleton, CO, USA

Contributors

Jing Zhang   •  Université de Lyon, Institut des Nanotechnologies de Lyon – INL, UMR CNRS 5270, site Ecole Centrale de Lyon, Ecully, France Yalong Zhang  •  Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA Yibing Zhang  •  The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK Xichun Zhou  •  Vitan-Biotech LLC, Littleton, CO, USA

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Chapter 1 Recent Advances and Future Challenges in Glycan Microarray Technology José L. de Paz and Peter H. Seeberger Abstract Glycan microarrays, carrying hundreds of different sugars on chip surfaces, have become a standard tool for the study of interactions of biomolecules with carbohydrates. The chip-based format offers important advantages, including the ability to screen in parallel several thousand binding events on a single slide, the minimal amount of sample required for one experiment, and the multivalent display of sugars on the chip that mimics the presentation of carbohydrates in nature. This chapter presents recent advances and future challenges in glycan microarray technology. We describe different immobilization and detection methods as well as applications in glycomics, drug discovery, and biomedicine. Key words: Microarrays, Carbohydrates, Glycomics, Glycobiology, Immobilization chemistry

1. Introduction Carbohydrate microarrays (glycoarrays) (1–6) consist of sugars that are bound, covalently or noncovalently, to a solid surface in a spatially defined and miniaturized fashion. In the past few years, glycoarrays have become a standard tool to screen large number of sugar–biomolecule interactions and investigate the role of carbohydrates in biological systems. The most important advantages of glycoarray technology over conventional approaches, such as enzyme-linked lectin assay, surface plasmon resonance (SPR), or isothermal titration calorimetry, are the ability to screen several thousand binding events on a single slide and the miniscule amounts of both analyte and ligand required for one experiment. Additionally, glycoarrays are ideal platforms to detect interactions that involve carbohydrates because the multivalent display of ligands on a surface overcomes the relative weakness of these interactions by mimicking cell–cell interfaces. Yann Chevolot (ed.), Carbohydrate Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 808, DOI 10.1007/978-1-61779-373-8_1, © Springer Science+Business Media, LLC 2012

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Glycoarrays have been predominantly applied to determine the sugar-binding profiles of proteins, nucleic acids, whole cells, and pathogens. These experiments provide valuable information for the design of carbohydrate-based drugs. Glycoarrays also constitute an attractive platform to test serum samples, opening the way for a wide variety of biomedical applications, such as the identification of novel sugar markers related to early-stage diseases and the detection and diagnosis of pathogenic infections. In this chapter, we focus on recent advances, current limitations, and opportunities for improvement in glycoarray technology. In particular, we first present an overview of the immobilization methods that have been used for glycoarray fabrication. Then, we discuss the different detection methods that have been introduced to analyze binding events on carbohydrate microarrays. Finally, we describe glycoarray applications in glycomics, drug discovery, and biomedical research.

2. Glycoarray Fabrication To prepare glycoarrays, pure carbohydrates have to be attached to appropriate chip surfaces. A variety of different immobilization methods have been developed over the past few years, involving both covalent and noncovalent attachment (7). Glycans used for immobilization can be either chemically synthesized or isolated from natural sources. A standard size microscope glass slide is the most widely used solid surface. The sugars are printed onto the slide using automated arraying robots. The use of robotic equipment designed for DNA microarray preparation is common. A typical microarray spotter generates spots of approximately 200  mm in diameter by delivering 1 nL of sugar solutions. The carbohydrates are usually printed at different concentrations in several replicates. The array can contain up to 10,000 spots. Printing is conducted in a chamber with regulated humidity. After printing, slides are immediately placed in a humidity chamber and usually incubated for several hours. Arrays are then washed to remove the unbound carbohydrates from the surface. A typical printing protocol often includes a quenching step of remaining reactive groups on the slide. Microarrays are finally washed and dried by centrifugation, and can be stored for a long period of time. 2.1. Covalent Immobilization

Chemical synthesis can be designed to afford oligosaccharide probes with an orthogonal functional group that is suitable for a chemical reaction with a reactive solid support (Fig. 1). In this way, sugars are covalently coupled to the microarray surface. The choice of the covalent coupling chemistry mainly depends on the synthetic strategy of the carbohydrate. The coupling reaction should be fast,

1  Recent Advances and Future Challenges in Glycan…

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specific, and high yielding. For instance, thiol-terminated oligosaccharides were prepared and attached to maleimide-derivatized glass slides (8). The selective reaction between thiol and maleimide groups has been widely employed to prepare various bioconjugates (9). Other ligation reactions used to prepare glycoarrays involved amide bond formation between an amine-containing sugar and an N-hydroxysuccinimide surface (10, 11), or a 1,3-dipolar cycloaddition between an azide and an alkyne (12). Epoxide-functionalized solid surfaces can be used to prepare glycan microarrays via immobilization with glycan-conjugated BSA (13). Due to recent advances in carbohydrate chemistry (14–16), including one-pot oligosaccharide synthesis (17) and the development of automated solidphase oligosaccharide synthesizers (18, 19), chemical synthesis has become the most popular sugar source to fabricate glycoarrays (20, 21). However, the synthesis of thousands of oligosaccharides for the construction of microarrays containing, ideally, the entire glycome of an organism is far from trivial. Synthetic libraries are still limited in number and structural diversity. Therefore, further development of more efficient synthetic methods is needed to expand the complexity and utility of glycoarrays containing synthetic probes.

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Fig. 2. Direct attachment of unmodified carbohydrates on hydrazide-containing chip surfaces.

Alternative approaches involving the use of naturally derived oligosaccharides should also be considered. Unprotected glycans can be directly derivatized by taking advantage of the reactivity of the masked aldehyde of the reducing end. Examples of procedures for the functionalization of free sugars include reductive amination (22), oxime bond formation (23), and glycosylamine synthesis (24). The modified sugars are then printed on appropriate slides. An even better method to prepare glycoarrays with isolated sugars is the direct attachment of free carbohydrates on aminooxyor hydrazide-containing surfaces that avoids prior modification (Fig. 2) (25, 26). For example, free reducing end sugars selectively bound to hydrazide groups on the chip surface to initially form the acyclic hydrazone, followed by conversion to cyclic, b-configured adduct. In contrast, acyclic structures were predominantly formed on aminooxy surfaces (25). 2.2. Noncovalent Immobilization

Noncovalent immobilization, mainly through ionic and hydrophobic interactions, has been also employed for the fabrication of glycoarrays. Noncovalent immobilization techniques include nonspecific adsorption of free glycans to underivatized solid surfaces. For example, polysaccharides have been passively adsorbed on nitrocellulose-coated glass slides (27). In these processes, the polysaccharides are site-nonspecifically attached to the chip. Polysaccharides with large contact areas are efficiently adsorbed on the surface, whereas small glycans are removed during washing steps. Therefore, this straightforward method is limited to high molecular weight sugars, leading to a random orientation of carbohydrates on the surface. Other noncovalent methods have been exploited for polysaccharide immobilization. Heparin polysaccharides have been directly attached to poly-l-lysine-coated slides via electrostatic interactions while maintaining their ability to interact with proteins (28). The methods described above are not suitable for the preparation of microarrays containing monosaccharides or low molecular weight oligosaccharides. Conjugation with a carrier molecule, such as a lipid chain, is required for noncovalent immobilization of smaller oligosaccharides onto nitrocellulose via hydrophobic interactions (29).

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Another noncovalent immobilization method involves the attachment of fluorous-tagged glycans to fluoroalkylated slides via fluorousbased interactions (30). The lipid and fluorous tags retain attached glycans even after extensive washing and avoid random sugar orientation on the chip surface that can negatively influence binding assay reproducibility. Noncovalent attachment can also rely on the binding of biotinylated sugars to streptavidin-coated plates (31). A DNA-based glycan microarray has also been reported for the site-specific, noncovalent immobilization of sugars (32). The carbohydrates were conjugated with a DNA strand and immobilization was performed through hybridization using a glass slide coated with a complementary DNA sequence. Relative sugar densities were assessed by the introduction of a fluorescent probe on the DNA-glycoconjugate. This point is of particular interest since despite the impressive progress in the development of carbohydrate immobilization strategies, quantification of immobilized sugars on the array surface remains difficult. To tackle this problem, an alternative solution involved the attachment of a fluorescent linker to the sugar probe by reductive amination (22). This fluorescent linker contained an additional functional group that permitted the immobilization of the conjugate on the appropriate surface. The fluorescent label allowed quantification of immobilized sugar by comparing the fluorescence intensity before and after washing. A cleavable linker was also employed to estimate the yield of the immobilization step by using mass spectrometry (33, 34). However, these methods require more elaborate synthetic strategies to install the cleavable or fluorescent linker, thereby complicating glycoarray fabrication.

3. Detection of CarbohydrateMediated Interactions

Glycoarray technology has been applied most extensively to the high-throughput analysis of carbohydrate–protein interactions. The proteins are incubated on the microarrays to allow for binding to the exposed carbohydrates before unbound proteins are washed away from the surface. In the next step, bound proteins are detected (Fig.  3). Fluorescence-based methods are the most widely used because of high sensitivity and availability of highresolution fluorescence scanner. When fluorescent-labelled proteins are used, microarrays are directly read out and the fluorescence intensities indicate the amount of ligand bound to the chip. It should be noted that protein labelling with fluorescent tags may cause protein denaturation or modification of the sugar-binding domain. Therefore, an alternative approach involves the use of an antibody that specifically recognizes proteins bound to glycoarrays (Fig. 3). The antibody can be directly detected if it contains a fluorescent tag.

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J.L. de Paz and P.H. Seeberger fluorescently labelled antibody

a protein

fluorescence scanner glass surface

b label-free SPR experiment gold chip

Fig. 3. Detection of carbohydrate–protein interactions. (a) Detection of bound protein by using a specific fluorescently labelled antibody; (b) Detection of bound protein by direct SPR experiment.

A typical sandwich procedure involving fluorescently labelled secondary antibodies has also been extensively used. However, specific antibodies can only be applied in some cases due to their limited availability. The introduction of label-free detection methods, such as mass spectrometry or SPR, is convenient since labelling of sugar interaction partners becomes obsolete (Fig. 3) (35). SPR affords kinetic parameters associated with the interaction, measuring association and dissociation constants on the array. For this technique, a gold surface is required as array support (36). Thiol-terminated oligosaccharides can be attached to the gold surface via Au–S linkage formation. The development of multichannel SPR instruments allows for the simultaneous analysis of hundreds of sugar spots (37–39). Another label-free detection method is MALDI-TOF mass spectrometry (40) that is particularly well suited to monitor carbohydrate transformations as a result of enzymatic reactions (41, 42). The specificity of enzymes can be studied by probing a panel of immobilized potential substrates. Moreover, the diversity of sugars on the array can be increased by enzymatic synthesis. Label free MALDI-TOF analysis is crucial for glycoarray applications involving enzymes. Alternatively, monitoring of on-chip enzymatic products with fluorescent lectins is very limited by the availability of such lectins. MALDI-TOF detection has also been employed for the on-chip chemical synthesis of oligosaccharides (43). A hydroxyphenyl-functionalized self-assembled monolayer of alkanethiols on gold was glycosylated using glycosyl trichloroacetimidates. Selective removal of a temporary protecting group unmasked a hydroxyl group of the sugar, allowing their chemical

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glycosylation with another trichloroacetimidate. Thus, an oligosaccharide chain was elongated on the chip surface, observing the synthetic intermediates by mass spectrometry. This technique is however limitated by the inability to determine the configuration of the anomeric linkage. Besides proteins, glycoarrays have been applied to screen the interactions of carbohydrates with nucleic acids (44), whole cells (45, 46) and pathogens such as viruses (10) or bacteria. Binding of RNA to glycoarrays can be measured using either fluorescently labelled RNA or by staining the bound RNA with dyes such as SYBR Green. Cells can be stained with cell permeable nucleic acid dyes prior to incubation. Viruses can be detected by incubating antibodies against proteins present on the virion (10). Most binding experiments with glycoarrays have been performed at one concentration of protein to afford qualitative information about the carbohydrate binding profile. Recently, carbohydrate chips have been used to quantify glycan–protein interactions, determining dissociation constants (Kd) values (47, 48). A series of protein concentrations were incubated on the chip surface, and protein concentration was plotted against fluorescence intensity for each immobilized sugar. These curves were analyzed as Langmuir isotherms to afford Kd values that were similar to those obtained in SPR experiments. IC50 values of soluble inhibitors for protein binding to carbohydrates attached to the chip surface have also been determined (9, 49, 50). For this purpose, glycoarrays were incubated with a series of mixtures containing the protein and different concentrations of the soluble inhibitor. This competition experiment yielded the IC50 values by plotting the inhibitor concentration against the fluorescence intensity.

4. Applications of Carbohydrate Microarrays

Glycoarrays have been predominantly used for the study of the binding properties of a variety of sugar-binding partners such as proteins, antibodies, cells, and viruses. For example, microarrays of heparin oligosaccharides have been employed to establish the binding profiles of several proteins such as growth factors and chemokines (51, 52). Heparin is a highly sulfated, linear polymer that belongs to the glycosaminoglycan (GAG) family and participates in a plethora of biological processes, such as inflammation, angiogenesis, and blood coagulation, by interaction with many proteins. Heparin is formed by disaccharide repeating units of d-glucosamine and either l-iduronic or d-glucuronic acid carrying sulfate groups at different positions of the chain. The sulfate pattern and uronic acid distribution are controlled by a complex biosynthetic pathway that leads to heparin chains with a high level of structural diversity. This heterogeneity

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of heparin, particularly the sulfation patterns, is responsible for its specific interaction with a wide variety of proteins. However, little is known about the exact structural requirements of heparin– protein interactions. Microarrays constitute an ideal platform for the establishment of structure–activity relationships for heparin sequences (53). A series of amine-terminated heparin oligosaccharides of varying length and sulfation pattern were chemically synthesized and immobilized on N-hydroxysuccinimide-activated glass surfaces by amide bond formation (11). These arrays were incubated with several chemokines and growth factors, demonstrating that specific sulfation motifs are needed for binding. In addition, heparin microarrays allowed for the identification of potential low molecular weight inhibitors of GAG–protein interactions such as monosaccharides containing an artificial sulfate distribution that is not found in nature. These arrays give valuable data to understand the biological role of GAG–protein interactions, providing a first step for new strategies to modulate GAG-mediated physiological processes. Incubation of glycoarrays with sera samples opens the way to more medically relevant applications, including identification of carbohydrate cancer markers and specific identification of pathogen infection. The identification of markers in early-stage cancers could lead to improved therapies and survival rates of patients. Cancer cells often display carbohydrates with different structures than those observed in normal cells (54). Therefore, cancer-associated carbohydrates can be considered as cancer markers and glycoarrays constitute an ideal platform for diagnosis. For example, Globo H is a hexasaccharide that is highly expressed on various types of cancers, especially breast, prostate, and ovarian cancers. A microarray containing the Globo H hexasaccharide and several structural analogs (55) was employed to screen sera samples of breast cancer patients and healthy individuals (55, 56). These studies revealed significant differences in antibody levels of cancer and healthy patients. Thus, glycoarrays are valuable tools for cancer diagnosis. Furthermore, the fabrication and use of more complex glycoarrays, with a higher number of carbohydrate structures, will allow the identification of novel carbohydrate markers related to other disease states, increasing the utility of this platform in cancer research. Another important application of glycoarrays is the diagnosis of infection diseases. Most pathogens contain specific polysaccharides on their cell membrane. After infection, protective antibodies are produced against these capsular polysaccharides. Glyco arrays are employed to analyze serum samples, detecting specific antibodies in infected patients to facilitate diagnosis. Moreover, microarray experiments give important information to design carbohydratebased vaccines and discover novel pathogen biomarkers. Salmonella bacteria cause a variety of diseases in humans. The severity of the

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disease depends on the particular Salmonella strain. A glycoarray containing a panel of Salmonella-related carbohydrate antigens was employed to analyze human sera from salmonellosis and healthy patients (57). Specific antibodies against bacterial infection were detected, and different Salmonella subtypes could also be distinguished. Glycosylphosphatidylinositol (GPI) glycolipids are present at the plasma membrane of Plasmodium falciparum, a parasite that causes malaria in humans. GPI has emerged as an important toxin in malaria disease and people living in malariaendemic regions often produce high levels of anti-GPI antibodies. Synthetic GPI glycoarrays (58) served to establish the binding specificities of anti-GPI antibodies, correlate antibody levels and protection from severe malaria and aid the design of efficient carbohydrate-based antitoxin vaccine. A different application of glycoarrays is based on the fact that viruses and bacteria use carbohydrates on the surface of human cells as initial recognition and attachment sites. Hemagglutinin proteins are present on the surface of influenza viruses and mediate the attachment to the host cell by binding to sialylated carbohydrates. The precise structure of these cell surface sialic acid epitopes depends on the host species and anatomical location. For instance, a 2,6-linked sialic acid structures are preferentially found on human epithelial cells in the upper respiratory tract whereas a 2,3-linked sialic acid units are found in the respiratory tract of birds. Hemagglutinins from different influenza variants vary in their sialic acid binding profile. Human influenza hemagglutinins preferentially recognize a 2,6-linked sialic acid residues and avian hemagglutinins are specific for a-2,3 linkages. Glycoarrays bearing many sialylated oligosaccharides were employed to determine the binding preferences of several influenza strains (59), including a highly pathogenic Vietnamese H5N1 avian virus (60) and two pandemic influenza A (H1N1) 2009 viruses (61). Microarray technology can be used to identify influenza virus subtypes from infected serum samples by analyzing their fine sugar binding specificities. Fast glycoarray tests might detect influenza strains in early stages of epidemic infection and, more importantly, identify changes in carbohydrate binding profiles suggesting dangerous virus mutations. Certain Escherichia coli strains cause urinary tract infections. Interestingly, these harmful strains strongly bind to mannosecontaining oligosaccharides on the host cell. In contrast, E. coli mutants exhibiting a reduced affinity to mannose do not cause infections. Glycoarrays were employed to study the binding profiles of E. coli bacteria (46). Different strains were distinguished, paving the way for developing detection tests for pathogens. Moreover, the chip format allowed for the fast screening for anti-adhesion compounds inhibiting carbohydrate–cell interactions that can be considered as therapeutics against pathogen infection.

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Acknowledgments We thank the Spanish Research Council (CSIC) (Grant 200880I041), the Spanish Ministry of Science and Innovation (Grant CTQ200907168), Junta de Andalucía (Grant P07-FQM-02969, “Incentivo a Proyecto Internacional”), and the European Union (FEDER support and Marie Curie Reintegration Grant) for financial support. Generous financial support from the Max-Planck Society is gratefully acknowledged. References 1. Laurent, N., Voglmeir, J., and Flitsch, S. L. (2008) Glycoarrays - tools for determining protein-carbohydrate interactions and glycoenzyme specificity. Chem. Commun., 4400–4412. 2. Horlacher, T., and Seeberger, P. H. (2008) Carbohydrate arrays as tools for research and diagnostics. Chem. Soc. Rev. 37, 1414–1422. 3. Park, S., Lee, M. R., and Shin, I. (2008) Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes. Chem. Commun., 4389–4399. 4. Wu, C. Y., Liang, P. H., and Wong, C. H. (2009) New development of glycan arrays. Org. Biomol. Chem. 7, 2247–2254. 5. de Paz, J. L., Horlacher, T., and Seeberger, P. H. (2006) Oligosaccharide microarrays to map interactions of carbohydrates in biological systems. Methods Enzymol. 415, 269–292. 6. de Paz, J. L., and Seeberger, P. H. (2006) Recent advances in carbohydrate microarrays. QSAR Comb. Sci. 25, 1027–1032. 7. Shin, I., Park, S., and Lee, M. R. (2005) Carbohydrate microarrays: An advanced technology for functional studies of glycans. Chem. Eur. J. 11, 2894–2901. 8. Adams, E. W., Ratner, D. M., Bokesch, H. R., McMahon, J. B., O’Keefe, B. R., and Seeberger, P. H. (2004) Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology: Glycan-dependent gp120/protein interactions. Chem. Biol. 11, 875–881. 9. Park, S., Lee, M. R., Pyo, S. J., and Shin, I. (2004) Carbohydrate chips for studying highthroughput carbohydrate-protein interactions. J. Am. Chem. Soc. 126, 4812–4819. 10. Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M. E., Alvarez, R., Bryan, M. C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D. J., Skehel, J. J., van Die, I., Burton, D. R., Wilson, I. A., Cummings, R., Bovin, N., Wong, C. H., and Paulson, J. C. (2004) Printed

covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U.S.A. 101, 17033–17038. 11. Noti, C., de Paz, J. L., Polito, L., and Seeberger, P. H. (2006) Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions. Chem. Eur. J. 12, 8664–8686. 12. Calarese, D. A., Lee, H. K., Huang, C. Y., Best, M. D., Astronomo, R. D., Stanfield, R. L., Katinger, H., Burton, D. R., Wong, C. H., and Wilson, I. A. (2005) Dissection of the carbohydrate specificity of the broadly neutralizinganti-HIV-1 antibody 2 G12. Proc. Natl. Acad. Sci. U.S.A. 102, 13372–13377. 13. Manimala, J. C., Li, Z., Jain, A., VedBrat, S., and Gildersleeve, J. C. (2005) Carbohydrate array analysis of anti-Tn antibodies and lectins reveals unexpected specificities: implications for diagnostic and vaccine development. ChemBioChem 6, 2229–2241. 14. Seeberger, P. H., and Werz, D. B. (2007) Synthesis and medical applications of oligosaccharides. Nature 446, 1046–1051. 15. Zhu, X. M., and Schmidt, R. R. (2009) New Principles for Glycoside-Bond Formation. Angew. Chem. Int. Ed. 48, 1900–1934. 16. Boltje, T. J., Buskas, T., and Boons, G. J. (2009) Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research. Nature Chem. 1, 611–622. 17. Zhang, Z. Y., Ollmann, I. R., Ye, X. S., Wischnat, R., Baasov, T., and Wong, C. H. (1999) Programmable one-pot oligosaccharide synthesis. J. Am. Chem. Soc. 121, 734–753. 18. Plante, O. J., Palmacci, E. R., and Seeberger, P. H. (2001) Automated solid-phase synthesis of oligosaccharides. Science 291, 1523–1527. 19. Seeberger, P. H. (2008) Automated oligosaccharide synthesis. Chem. Soc. Rev. 37, 19–28.

1  Recent Advances and Future Challenges in Glycan… 20. Seeberger, P. H., and Werz, D. B. (2005) Automated synthesis of oligosaccharides as a basis for drug discovery. Nat. Rev. Drug Discov. 4, 751–763. 21. Werz, D. B., and Seeberger, P. H. (2005) Carbohydrates as the next frontier in pharmaceutical research. Chem. Eur. J. 11, 3194–3206. 22. Xia, B. Y., Kawar, Z. S., Ju, T. Z., Alvarez, R. A., Sachdev, G. P., and Cummings, R. D. (2005) Versatile fluorescent derivatization of glycans for glycomic analysis. Nat. Methods 2, 845–850. 23. Liu, Y., Feizi, T., Carnpanero-Rhodes, M. A., Childs, R. A., Zhang, Y. N., Muiioy, B., Evans, P. G., Osborn, H. M. I., Otto, D., Crocker, P. R., and Chai, W. C. (2007) Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions. Chem. Biol. 14, 847–859. 24. Brun, M. A., Disney, M. D., and Seeberger, P. H. (2006) Miniaturization of microwave-assisted carbohydrate functionalization to create oligosaccharide microarrays. ChemBioChem 7, 421–424. 25. Lee, M., and Shin, I. (2005) Facile preparation of carbohydrate microarrays by site-specific, covalent immobilization of unmodified carbohydrates on hydrazide-coated glass slides. Org. Lett. 7, 4269–4272. 26. Zhi, Z. L., Powell, A. K., and Turnbull, J. E. (2006) Fabrication of carbohydrate microarrays on gold surfaces: Direct attachment of nonderivatized oligosaccharides to hydrazide-modified self-assembled monolayers. Anal. Chem. 78, 4786–4793. 27. Wang, D. N., Liu, S. Y., Trummer, B. J., Deng, C., and Wang, A. L. (2002) Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nat. Biotechnol. 20, 275–281. 28. Shipp, E. L., and Hsieh-Wilson, L. C. (2007) Profiling the sulfation specificities of glycosaminoglycan interactions with growth factors and chemotactic proteins using microarrays. Chem. Biol. 14, 195–208. 29. Fukui, S., Feizi, T., Galustian, C., Lawson, A. M., and Chai, W. G. (2002) Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat. Biotechnol. 20, 1011–1017. 30. Ko, K. S., Jaipuri, F. A., and Pohl, N. L. (2005) Fluorous-based carbohydrate microarrays. J. Am. Chem. Soc. 127, 13162–13163. 31. Alvarez, R. A., and Blixt, O. (2006) Identification of ligand specificities for glycan-binding proteins using glycan arrays. Methods Enzymol. 415, 292–310.

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32. Chevolot, Y., Bouillon, C., Vidal, S., Morvan, F., Meyer, A., Cloarec, J. P., Jochum, A., Praly, J. P., Vasseur, J. J., and Souteyrand, E. (2007) DNA-based carbohydrate biochips: a platform for surface glyco-engineering. Angew. Chem. Int. Ed. 46, 2398–2402. 33. Bryan, M. C., Fazio, F., Lee, H. K., Huang, C. Y., Chang, A., Best, M. D., Calarese, D. A., Blixt, C., Paulson, J. C., Burton, D., Wilson, I. A., and Wong, C. H. (2004) Covalent display of oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126, 8640–8641. 34. Lee, J. C., Wit, C. Y., Apon, J. V., Siuzdak, G., and Wong, C. H. (2006) Reactivity-based onepot synthesis of the tumor-associated antigen N3 minor octasaccharide for the development of a photocleavable DIOS-MS sugar array. Angew. Chem. Int. Ed. 45, 2753–2757. 35. Song, E.-H., and Pohl, N. L. B. (2009) Carbohydrate arrays: recent developments in fabrication and detection methods with applications. Curr. Opin. Chem. Biol. 13, 626–632. 36. Ratner, D. M., Adams, E. W., Su, J., O’Keefe, B. R., Mrksich, M., and Seeberger, P. H. (2004) Probing protein-carbohydrate interactions with microarrays of synthetic oligosaccharides. ChemBioChem 5, 379–382. 37. Smith, E. A., Thomas, W. D., Kiessling, L. L., and Corn, R. M. (2003) Surface plasmon resonance imaging studies of protein-carbohydrate interactions. J. Am. Chem. Soc. 125, 6140–6148. 38. Karamanska, R., Clarke, J., Blixt, O., MacRae, J. I., Zhang, J. Q. Q., Crocker, P. R., Laurent, N., Wright, A., Flitsch, S. L., Russell, D. A., and Field, R. A. (2008) Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays. Glycoconj. J. 25, 69–74. 39. de Boer, A. R., Hokke, C. H., Deelder, A. M., and Wuhrer, M. (2008) Serum antibody screening by surface plasmon resonance using a natural glycan microarray. Glycoconj. J. 25, 75–84. 40. Su, J., and Mrksich, M. (2002) Using mass spectrometry to characterize self-assembled monolayers presenting peptides, proteins, and carbohydrates. Angew. Chem. Int. Ed. 41, 4715–4718. 41. Laurent, N., Voglmeir, J., Wright, A., Blackburn, J., Pham, N. T., Wong, S. C. C., Gaskell, S. J., and Flitsch, S. L. (2008) Enzymatic glycosylation of peptide arrays on gold surfaces. ChemBioChem 9, 883–887. 42. Zhi, Z. L., Laurent, N., Powel, A. K., Karamanska, R., Fais, M., Voglmeir, J., Wright, A., Blackburn, J. M., Crocker, P. R., Russell, D. A., Flitsch, S., Field, R. A., and Turnbull, J. E. (2008) A versatile gold surface approach for

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fabrication and interrogation of glycoarrays. ChemBioChem 9, 1568–1575. 43. Ban, L., and Mrksich, M. (2008) On-chip synthesis and label-free assays of oligosaccharide arrays. Angew. Chem. Int. Ed. 47, 3396–3399. 44. Disney, M. D., and Seeberger, P. H. (2004) Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins. Chem. Eur. J. 10, 3308–3314. 45. Nimrichter, L., Gargir, A., Gortler, M., Altstock, R. T., Shtevi, A., Weisshaus, O., Fire, E., Dotan, N., and Schnaar, R. L. (2004) Intact cell adhesion to glycan microarrays. Glycobiology 14, 197–203. 46. Disney, M. D., and Seeberger, P. H. (2004) The use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens. Chem. Biol. 11, 1701–1707. 47. Liang, P. H., Wang, S. K., and Wong, C. H. (2007) Quantitative analysis of carbohydrateprotein interactions using glycan microarrays: Determination of surface and solution dissociation constants. J. Am. Chem. Soc. 129, 11177–11184. 48. Park, S., and Shin, I. (2007) Carbohydrate microarrays for assaying galactosyltransferase activity. Org. Lett. 9, 1675–1678. 49. de Paz, J. L., Noti, C., Bohm, F., Werner, S., and Seeberger, P. H. (2007) Potentiation of fibroblast growth factor activity by synthetic heparin oligosaccharide glycodendrimers. Chem. Biol. 14, 879–887. 50. Houseman, B. T., and Mrksich, M. (2002) Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. Chem. Biol. 9, 443–454. 51. de Paz, J. L., Noti, C., and Seeberger, P. H. (2006) Microarrays of synthetic heparin oligosaccharides. J. Am. Chem. Soc. 128, 2766–2767. 52. de Paz, L. L., Moseman, E. A., Noti, C., Polito, L., von Andrian, U. H., and Seeberger, P. H. (2007) Profiting heparin-chemokine interactions using synthetic tools. ACS Chem. Biol. 2, 735–744. 53. de Paz, J. L., and Seeberger, P. H. (2008) Deciphering the glycosaminoglycan code with

the help of microarrays. Mol. BioSyst. 4, 707–711. 54. Dube, D. H., and Bertozzi, C. R. (2005) Glycans in cancer and inflammation. Potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4, 477–488. 55. Huang, C. Y., Thayer, D. A., Chang, A. Y., Best, M. D., Hoffmann, J., Head, S., and Wong, C. H. (2006) Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen. Proc. Natl. Acad. Sci. U.S.A. 103, 15–20. 56. Wang, C. C., Huang, Y. L., Ren, C. T., Lin, C. W., Hung, J. T., Yu, J. C., Yu, A. L., Wu, C. Y., and Wong, C. H. (2008) Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer. Proc. Natl. Acad. Sci. U.S.A. 105, 11661–11666. 57. Blixt, O., Hoffmann, J., Svenson, S., and Norberg, T. (2008) Pathogen specific carbohydrate antigen microarrays: a chip for detection of Salmonella O-antigen specific antibodies. Glycoconj. J. 25, 27–36. 58. Kamena, F., Tamborrini, M., Liu, X. Y., Kwon, Y. U., Thompson, F., Pluschke, G., and Seeberger, P. H. (2008) Synthetic GPI array to study antitoxic malaria response. Nat. Chem. Biol. 4, 238–240. 59. Stevens, J., Blixt, O., Glaser, L., Taubenberger, J. K., Palese, P., Paulson, J. C., and Wilson, I. A. (2006) Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol. 355, 1143–1155. 60. Stevens, J., Blixt, O., Tumpey, T. M., Taubenberger, J. K., Paulson, J. C., and Wilson, I. A. (2006) Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312, 404–410. 61. Childs, R. A., Palma, A. S., Wharton, S., Matrosovich, T., Liu, Y., Chai, W. G., Campanero-Rhodes, M. A., Zhang, Y. B., Eickmann, M., Kiso, M., Hay, A., Matrosovich, M., and Feizi, T. (2009) Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray. Nat. Biotechnol. 27, 797–799.

Chapter 2 Chemical Synthesis of Carbohydrates and Their Surface Immobilization: A Brief Introduction Daniel B. Werz Abstract For all carbohydrate microarrays, two important prerequisites are necessary: the carbohydrate of interest has to be obtained either by isolation from natural sources, enzymatic or chemical synthesis; an immobilization of the carbohydrate at the surface of the chip has to be achieved. This chapter provides a very brief overview of the chemical synthesis of carbohydrates (creation of building blocks, assembly, and deprotection) and of immobilization techniques. Numerous methods are known to construct oligosaccharides by chemical methods. A typical monosaccharide building block, used in oligosaccharide assembly, is equipped with different protecting groups that mask the hydroxyl and amine groups. In general, a good leaving group at the anomeric center that can easily be activated is mandatory; especially trichloroacetimidates, phosphates, and thioethers have been widely used for the creation of glycosidic bonds. After the complete assembly of the oligosaccharide, a global deprotection of all permanent protecting groups affords the desired target structure with free hydroxyl groups. Linkers, which were introduced during the synthesis, must often be modified at the end to create appropriate functionalities for surface immobilization. Key words: Glycosylation, Protecting group, Deprotection, Building block, Chemical synthesis, Surface immobilization, Microarrays, Thiols

1. Introduction The chemical synthesis of complex oligosaccharides has been a synthetically difficult and rather time-consuming endeavor (1). In contrast to peptides and oligonucleotides, carbohydrates are often branched and several hydroxyl or amino groups at each building block lead to a dazzling variety of structures, which can be obtained by a small set of subunits. Even more complicated is the creation of a new stereogenic center resulting from the formation of a glycosidic bond during the combination of two monosaccharides.

Yann Chevolot (ed.), Carbohydrate Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 808, DOI 10.1007/978-1-61779-373-8_2, © Springer Science+Business Media, LLC 2012

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Table 1 Number of different oligomers for nucleotides, peptides, and carbohydrates Numbers of different oligomers Oligomer size

Nucleotides

Peptides

Carbohydrates

1

4

20

20

2

16

400

1,360

3

64

8,000

126,080

4

256

160,000

13,495,040

5

1,024

3,200,000

1,569,745,920

6

4,096

64,000,000

192,780,943,360

The numbers for the carbohydrates are based on the “10 mammalian” monosaccharide units. Branching is also taken into account

A recent statistical analysis regarding the diversity of mammalian carbohydrate structures – based on the “10 mammalian monosaccharides” without considering any further attachments – reveals astounding numbers, which dwarf the complexity of both peptides and oligonucleotides (Table  1) (2). Although the number of structural combinations encountered in nature is much smaller than the theoretical ones, it is obvious that synthetic carbohydrate chemists have to face a variety of challenges during the assembly of an oligosaccharide by chemical means.

2. Chemical Synthesis of Carbohydrates

A carefully designed synthetic plan is necessary before one starts with the total synthesis of the desired target structure. Such a plan includes the glycosylation strategy (type of anomeric leaving groups) and some kind of temporary protecting groups (3, 4). After that, mono- or disaccharide building blocks have to be prepared. With these compounds in hand, the assembly process can be started. Besides well-known solution-phase techniques, an automated solid-phase approach has also been developed (5, 6). To gain access to the native structure, a deprotection is required. Before the carbohydrate of interest is attached to the surface of a microarray other required steps include the functionalization of the linker, purification steps (such as dialysis or the use of HPLC), and quality control (mass spectrometry and NMR analyses). A simplified scheme of the different steps from the desired target structure to surface immobilization is depicted in Fig. 1.

2  Chemical Synthesis of Carbohydrates…

15

Fig. 1. Required steps from defining the carbohydrate target structure until the immobilization of the synthesized oligosaccharide.

2.1. Building Blocks

A typical monosaccharide building block used in oligosaccharide assembly is equipped with different protecting groups that mask the hydroxyl and amine groups. Two different kinds of protecting groups, permanent and temporary ones, need to be distinguished. The former mask hydroxyl moieties have to be unveiled at the end of the synthesis whereas the latter mark sites of further glycosylation. Permanent protecting groups are commonly ethers (mostly Bn ethers), which can be cleaved by reduction at the end of the synthesis, but rather stable esters (such as Piv or even Bz esters) are also used. Temporary protecting groups are functional groups that are easy to cleave (but should be stable during the glycosylation reaction) such as trityl ethers, fluorenylmethoxycarbonyl (Fmoc) groups, and several silyl groups, while acetates (Ac) are often employed as temporary protecting groups. However, whether a protecting group is used as permanent or temporary is often depend strongly on the glycosylation strategy (3). Besides controlling regioselectivity by orthogonal protecting groups to account for possible branching of the carbohydrate chain, the stereochemistry

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D.B. Werz

at the anomeric carbon must be controlled during the formation of the glycosidic bond. Placement of participating protective groups at the C-2 hydroxyl or amine groups ensures the formation of trans-glycosidic linkages. In contrast, nonparticipating groups and low temperatures during the glycosylation lead to the preferential installation of cis-glycosides. Furthermore, an anomeric leaving group that can be easily activated to induce the formation of the glycosidic linkage is needed (3, 7). A large variety of anomeric leaving groups are known and several of them are widely utitized in complex oligosaccharide synthesis. Figure  2 depicts a selection of such anomeric leaving groups (1–7) with their respective activation procedures to perform a glycosylation reaction (3). The oldest anomeric leaving groups used for the chemical synthesis of saccharides are glycosyl bromides 1 that are activated by silver salts. Today, the most commonly used anomeric leaving groups are phosphates 2, trichloroacetimidates 3, and thioethers 4 (3). To date, the synthesis of building blocks is the most timeconsuming process of oligosaccharide synthesis. Rather than synthesizing each building block separately integrated synthetic paths that grant access to several building blocks from a common precursor are desirable.

Fig. 2. Various anomeric leaving groups in 1–7 and their respective activation modes.

2  Chemical Synthesis of Carbohydrates…

17

Commonly, the differently protected and functionalized monosaccharides are accessed from naturally occurring sugar starting materials through a series of protection–deprotection maneuvers. Such a process establishes the desired protecting group pattern and typically requires 6–20 steps depending on the sugar, the protecting group ensemble, and the anomeric leaving group. A plethora of different methods to chemically distinguish the different hydroxyls already exists. The reactivity of the anomeric hydroxyl group that is part of a hemiacetal differs significantly from that of the other hydroxyls. The selective removal of acetate esters in the anomeric position and the readily occurring substitution of esters by alcohols such as n-pentenol or p-methoxybenzyl alcohol in the presence of Lewis acids are well established (8, 9). The C-6 hydroxyl also exhibits special reactivity as the only primary hydroxyl group of the sugar moiety. In addition, the C-6 hydroxyl is sterically exposed and increased reactivity often leads to high regioselectivity (9). Another very popular method for the selective protection of four or five hydroxyls relies on rendering two of them silent. cisHydroxyls easily form 5-membered rings, so-called isopropylidenes (9), upon treatment with acetone dimethylacetal in the presence of a Lewis acid (Fig. 3). Benzaldehyde and benzaldehyde dimethylacetal are prone to form 6-membered rings (benzylidene acetals, 10) involving the C-4 and the C-6 hydroxyl. An elegant method to lock the C-1 and C-2 hydroxyls is the formation of an orthoester 11 (Fig.  3). Usually orthoesters are synthesized by nucleophilic

Fig. 3. Methods used in building block synthesis to render two hydroxyls “silent”.

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D.B. Werz

Fig. 4.  Synthesis of glycosyl phosphate 17 starting from d-glucose (13).

attack of an alcohol at the oxocarbenium ion that can be generated by a glycosyl bromide with the neighboring ester group in the presence of a nonnucleophilic base (9). This method commonly yields two stereoisomeric products. The elimination of two hydroxyls to form a 1,2 double bond is yet another method to reduce the number of hydroxyl groups to be distinguished. The remaining hydroxyls of these 1,2-glycals 12 are more easily distinguished. Finally, selective oxidation procedures allow the reinstallation of the eliminated hydroxyls at a later stage (9). For example, Fig.  4 shows the generation of a glucosyl phosphate with Fmoc as a temporary protecting group in position 4. d-Glucose is used as starting material. After peracetylation and anomeric substitution of acetate by bromide, Zn-mediated reduction eliminates two hydroxyl groups of the glucose 13 to afford the glucal 14. Protection with permanent protecting groups (Bn) in positions 4 and 6 mediated by a tin reagent and further protection with FmocCl as temporary protecting group yielded 16. Epoxidation with dimethyl dioxirane (DMDO) is followed by opening of the 1,2-anhydrosugar with dibutylphosphate. Protection of the ensuing C2 hydroxyl group with a participating group (e.g. pivaloyl chloride [PivCl)] produced the desired glucosyl phosphate 17 in excellent yield (10, 11). 2.2. Glycosylation Reaction and Assembly

Definitely it would go beyond the scope of this brief chapter to discuss in detail all the parameters which affect the glycosylation reaction (3, 4, 12). However, a short overview of the mechanistic picture of this important reaction seems to be appropriate. Nevertheless, detailed information about glycosylation mechanisms is mostly fragmented. Commonly, the glycosylation reaction involves nucleophilic displacement at the anomeric carbon. Due to the endocyclic oxygen next to the anomeric center a positive charge

2  Chemical Synthesis of Carbohydrates…

19

Fig. 5. Simplified mechanistic picture of a glycosylation reaction either with non-participating group (Case A) or with participating group (Case B) in position 2.

is strongly stabilized. Thus, after activation of 18 one may consider a glycosyl cation 19 (oxocarbenium ion) as intermediate if no further stabilising substituents are in reach (Fig. 5, Case A). A nucleophile such as a hydroxyl group can attack this species either from the top or from the bottom face; a mixture of trans- and cis-glycosides 20 and 21 results. In the case of a participating group in position 2, such a carbocation might also be stabilized by neighboring group participation leading to an acyloxonium ion as major intermediate (Fig. 5, Case B). Such an acyloxonium ion 23 shields either the top or the bottom face (depending on the stereochemistry at C-2). As a result, the attack of the nucleophilic hydroxyl group can only take place from the nonshielded side leading to transglycoside 24 as major isomer. Using this model, it is easy to understand why participating groups must not be used for the formation of cis-glycosides. For the creation of cis-glycosides, one has to rely, in most cases, on thermodynamic considerations. In the case of glucose, galactose, and fucose, as well as in their corresponding amines and acids, the a product is thermodynamically preferred due to the anomeric effect. Low reaction temperatures favor their formation. However, a complete a selectivity is often difficult to

20

D.B. Werz

achieve. In the case of mannose, the cis-mannosides are the b product. Therefore, the creation of b-mannosides requires other approaches that have been described in the literature (12, 13). In addition to the glycosylation reaction, the second important reaction in oligosaccharide assembly is the highly selective removal of protecting groups (9). For branched oligosaccharides, careful synthetic planning with respect to the temporary protecting group ensemble is required. Permanent protecting groups stay unaffected until the very final steps of the synthesis to procure the completely deprotected carbohydrate. The cleavage of temporary protecting groups after each glycosylation reaction is essential to liberate hydroxyls that are needed as nucleophiles for the next glycosylation step. Silyl protecting groups (e.g. in 25) are cleaved by fluoride sources or strong acids whereas fluorenylmethoxycarbonyl (Fmoc) groups (e.g. in 27) are cleaved by weak bases such as piperidine. Stronger bases such as sodium methoxide are required for the clevage of acetate (Ac) groups (as shown for 29) whereas levulinoate (Lev) esters in 31 need only hydrazine to be cleaved. An overview of four deprotection methods is provided in Fig. 6. Caution has to be taken by employing two temporary protecting groups next to each other. In some instances, a migration of the remaining protecting group might occur when one of the two is removed (especially in the presence of bases) (8). 2.3. Global Deprotection

Once the oligosaccharide is assembled by solution- or solid-phase techniques, the deprotection of all permanent protecting groups (global deprotection) is a challenging endeavour. Two approaches dominate: The first is a single-step procedure with sodium, carried out in liquid ammonia, often referred as Birch reduction (8). Benzyl ether groups as well as esters and carbonates are removed. The fact that olefinic moieties (except allyl) stay unaffected is the major advantage since terminal CC double bonds are often used as a chemical handle, as a future point of attachment to the surface of a microarray or to a carrier protein (8, 14). Thus it should not be attacked during the deprotection sequence. Besides olefinic moieties, the reducing end hemiacetal of an oligosaccharide is also maintainable during Birch debenzylation (15). Sometimes the commonly employed trichloroacetyl (TCA) amino protecting group causes problems resulting in a tremendous reduction of yield. To circumvent these difficulties, the transformation of the TCA group into an acetyl group by a radical initiated reduction with Bu3SnH is advantageous. To facilitate the further purification process, the unprotected sugar, obtained by Birch reduction, is frequently peracetylated using acetic anhydride and pyridine (9, 10). The experimental setup for the Birch reduction using sodium in liquid ammonia is relatively high. Minor impurities in the starting material may cause a serious decrease in yield. A major advantage compared to hydrogenolysis procedures is avoidance of heavy

2  Chemical Synthesis of Carbohydrates…

21

Fig. 6. Four examples of temporary protecting groups (TBS, Fmoc, Ac, and Lev) and methods for their selective removal.

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D.B. Werz

metals. Traces of such metals may cause problems in biological experiments employing the synthetic carbohydrates. The other protocol requires two steps. In a first step, ester groups have to be removed by base, before hydrogenolysis cleaves the benzyl (Bn) ethers (9). In the latter case, palladium on charcoal or Pd(OH)2 on charcoal (Pearlman’s catalyst) are widely used as catalysts. The reaction itself takes place under an atmosphere of hydrogen. Commonly the reaction proceeds well and is finished over night. If problems with the deprotection of several benzyl ethers (e.g. sterically very hindered Bn groups) occur, hydrogen pressure up to 100 bar may be applied. It is evident that the conditions employed for hydrogenolysis also reduce carbon–carbon double bonds. If one needs an alkene as a handle, the Birch reduction is the method of choice for global deprotection. 2.4. Case Study of Blood Group A Antigen

To illustrate the process of carbohydrate assembly, the blood group A antigen 34 with a thiol linker is chosen as an example (16). This glycan is a branched tetrasaccharide consisting of galactosamine, fucose, galactose, and glucosamine (Fig. 7). For the installation of a chemical handle being necessary for surface immobilization, a pentenyl group was attached to the reducing end of the sugar. Such an olefinic moiety can be transformed easily at the end of the total synthesis into a thiol unit. Figure 7 shows the retrosynthetic analysis into five building blocks 36–40. Pivaloyl (Piv) and acetate (Ac) esters are employed as permanent protecting groups for hydroxyls benzyl (Bn) ethers. The amine moieties are protected in the case of galactosamine 36 as azide (N3), in the case of glucosamine 40 as trichloroacetamide (TCANH). In the latter case a participating group, namely TCA, is necessary to achieve b selectivity whereas nonparticipating nitrogen functionality, namely the azide, is necessary to achieve a selectivity. For the branching galactose 37, two orthogonal temporary protecting groups are required. Fmoc in position 2 ensures, with its ability for neighboring groupparticipation, b selectivity and can be cleaved later on by the weak base piperidine without affecting the levulinoate in position 3. In the case of the fucose building block 39, it is again important to have a nonparticipating group in position 2 to afford mainly the a product. For the anomeric leaving groups, a mixed strategy consisting of one trichloroacetimidate and three phosphates was chosen. This selection was based on arguments with respect to the ease of building block synthesis. Figure  8 summarizes the assembly and deprotection process (16). The pentenyl functionalized monosaccharide 41 and galactosyl phosphate 37 were reacted in the presence of the Lewis acid TMSOTf, as activating agent, to produce a disaccharide whose Fmoc group was immediately deprotected to afford 42. Further fucosylation and removal of levulinoate in the presence of hydrazine yielded trisaccharide 43. To achieve high a selectivity,

2  Chemical Synthesis of Carbohydrates…

23

Fig. 7. Retrosynthesis of the A antigen 34 with a thiol linker, building blocks 36–40.

the last glycosylation was carried out at low temperatures. The completely protected tetrasaccharide 35 was subjected to Birch reduction. Sodium in liquid ammonia removed all permanent protecting groups and transformed the azide moiety as well as the

24

D.B. Werz

Fig. 8. Assembly of the tetrasaccharidic A antigen 34 with a thiol linker.

trichloroacetamide into “naked” amines, thus achieving global deprotection. The reaction with acetic anhydride and pyridine converted all hydroxyls to the corresponding acetates and all amines to the respective acetamido groups. This product is much less polar than the compound directly obtained after Birch reduction and can be more easily purified. A radical-initiated addition of thioacetic acid introduced the sulfur moiety (16). In the final step all acetates are cleaved to generate the native structure 34. Dialysis might be a good choice to get rid of salts, which might have accumulated during the last steps of the synthesis. Lyophilisation yields the carbohydrate ready for attachment to microarrays.

2  Chemical Synthesis of Carbohydrates…

25

3. Immobilization Techniques Microarrays in the “chip” format, prepared by attachment of biopolymers to a surface in a spatially discrete pattern, have enabled a low-cost and high-throughput methodology for screening interactions involving these molecules (17–19). The most important advantages compared to classical methods are that microarrays allow for several thousand binding events to be screened in parallel, hence the experiment requires only miniscule amounts of both analyte and ligand. Thus, binding profiles and lead structures can be readily examined. In addition, carbohydrate microarrays are ideal to detect interactions that involve carbohydrates since the multivalent display of ligands on a surface (cluster effect) overcomes the relative weakness of these interactions by mimicking cell–cell interfaces. However, an important question to be tackled is the way in which a carbohydrate becomes immobilized on the surface. This book provides and discusses a large variety of methods in detail. However, in the following paragraphs I would like to provide an overview of some selected techniques from the viewpoint of an organic chemist. Older methods for the preparation of carbohydrate microarrays consist of nitrocellulose-coated slides (in the case of noncovalent immobilization of microbial polysaccharides) (20) or self-assembled monolayers modified by Diels–Alder mediated coupling of cyclopentadiene-derived oligosaccharides (21). Unfortunately, the former method requires large polysaccharides or lipid modified sugars for the noncovalent interaction. The latter requires the preparation of oligosaccharides bearing the sensitive cyclopentadiene moiety. Today noncovalent immobilization is mainly based on fluorine–fluorine interactions (22) or DNA-directed immobilization (23). In the first case, the surface is coated by perfluorinated hydrocarbons and the carbohydrate to be attached bears a perfluorinated alkyl chain. In the latter case, the specific pairing of DNA bases due to the different number of hydrogen bridges is the key to immobilization; one single strand of DNA is attached to the carbohydrate moiety whereas the complementary one is attached to the surface of the microarray. For covalent immobilization (24–26) very good results were obtained by utilizing maleimide functionalization of glass slides and the immobilization of the oligosaccharides with thiol-containing linkers 44. As pointed out in the Subheading 2.4, thiols are obtained in a facile way by reacting double bonds with thiolacetic acid under radical conditions (either initiated by light or radical starters such as AIBN). Sodium methoxide or even potassium carbonate cleaves the thioacetate to generate the free thiol. In a Michaeltype addition the thiol moiety attacks the maleimide functionality. A stable covalent bond is formed to afford modified surfaces of type 45. Care has to be taken when thiol-containing substrates are

26

D.B. Werz

stored for a longer time. Oxygen from the air leads to the formation of disulfide linkages that no longer react with maleimides. Therefore, a weak reducing agent is often added to generate the thiols out of the respective disulfides. Another possible reaction partner is iodoacetyl groups, which are immobilized on a surface. Due to the high nucleophilicity of the sulfur, a simple nucleophilic substitution leads to immobilized saccharides of type 46 (Fig. 9). Also, other moieties such as linkers functionalized with azides are utilized for surface immobilization. In a Huisgen 1,3-dipolar cycloaddition, a five-membered trizole is formed with an alkyne under the catalytic influence of copper. This chemistry is often referred to today as “click-chemistry” (27). Of course, the carbohydrate part can bare an alkyne unit, while the surface bares the respective azide. Also, primary amines, which react under conditions of a reductive amination with aldehydes or vice versa to secondary amines, are utilized. However, the generation of aldehydes – either as part of a linker attached to the carbohydrate moiety or as part of the surface – is difficult, and decomposition may occur readily. If amines are used as reactive group for immobilization, one should use an activated amide as counterpart. Both functionalities are rather stable, and high yields are obtained. If one likes to use thiols instead of amines, but due to synthetic considerations amines have

Fig. 9. Two possibilities to attach thiol-functionalized saccharides to surfaces.

2  Chemical Synthesis of Carbohydrates…

27

Fig. 10. Transformation of an amine-functionalized linker 47 into a thiol-functionalized linker 49 by using Traut’s reagent 48.

been created, Traut’s reagent 48 will be able to convert amines 47 into corresponding thiols 49 (Fig. 10) (28). By this procedure, the length of the linker is increased by five heavy atoms. In general, every simple reaction that is easy to handle and promises a high reaction yield can be used for the immobilization of carbohydrates. The bonds formed have to be stable and should not be cleaved easily by hydrolysis. Of course, the corresponding functional groups have to be installed on both reaction partners and the oligosaccharide as well as the modified surface of the microarray. Further considerations that should be taken into account are the kind of linker, its length, and the point of attachment. Polyethylenglycol (PEG) linkers reveal a higher degree of water solubility than common alkyl chains. Linkers that are too short might cause problems when large proteins interact with the carbohydrate. As point of attachment, in most cases, the anomeric center is chosen to mimic the native structure; however, of course, other functionalization sites are also possible.

4. Conclusion Carbohydrate synthesis is still a rather time-consuming endeavour; however, major efforts have been done during the last decades to fasten this process such as the use of integrated paths to create building blocks, improved glycosylation strategies and even the introduction of automated methods for the assembly of oligosaccharides. The right choice of temporary and permanent protecting groups, participating and nonparticipating ones, is the prerequisite for a successful stereoselective assembly. As anomeric leaving groups especially trichloroacetimidates, phosphates, and thioethers have become prominent. In a final step, global deprotection has to be performed, either by saponification and hydrogenolysis or by Birch reduction. For surface immobilization, handles such as pentenyl chains can be easily converted at the end of the synthesis into thiols.

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Acknowledgments The author would like to thank the Deutsche Forschungsgemeinschaft (Emmy Noether Fellowship) and the Fonds der Chemischen Industrie (Liebig Fellowship) for financial support. References 1. Seeberger, P. H., and Werz, D. B. (2007) Synthesis and medical applications of oligosaccharides. Nature 446, 1046–1051. 2. Werz, D. B., Ranzinger, R., Herget, S., Adibekian, A., von der Lieth, C.-W., and Seeberger, P. H. (2007) Exploring the Structural Diversity of Mammalian Carbohydrates (“Glycospace”) by Statistical Databank Analysis. ACS Chem. Biol. 2, 685–691. 3. Demchenko, A. V. (Ed.) (2008) Handbook of Chemical Glycosylation, Wiley-VCH, Weinheim. 4. Zhu, X., and Schmidt, R. R. (2009) New principles for glycoside-bond formation. Angew. Chem. Int. Ed. 48, 1900–1934. 5. Plante, O. J., Palmacci, E. R., and Seeberger, P. H. (2001) Automated solid-phase synthesis of oligosaccharides. Science 291, 1523–1527. 6. Seeberger, P. H., and Werz, D. B. (2005) Automated synthesis of oligosaccharides as basis for drug discovery. Nature Reviews Drug Discovery 4, 751–763. 7. Schmidt, R. R., Castro-Palomino, J. C., and Retz, O. (1999) New aspects of glycoside bond formation. Pure Appl. Chem. 71, 729–744. 8. Werz, D. B., and Seeberger, P. H. (2005) Total Synthesis of Antigen Bacillus Anthracis Tetrasaccharide – Creation of an Anthrax Vaccine Candidate. Angew. Chem. Int. Ed. 44, 6315–6318. 9. Lindhorst, T. K. (2007) Essentials of Carbohydrate Chemistry and Biochemistry, 3rd edition, Wiley-VCH, Weinheim. 10. Werz, D. B., Castagner, B., and Seeberger, P. H. (2007) Automated Synthesis of the TumorAssociated Carbohydrate Antigens Gb-3 and Globo-H: Incorporation of a-Galactosidic Linkages. J. Am. Chem. Soc. 129, 2770–2771. 11. Love, K. R., and Seeberger, P. H. (2004) Automated solid-phase synthesis of protected tumor-associated antigen and blood group determinant oligosaccharides. Angew. Chem. Int. Ed. 43, 602–605. 12. Crich, D. (2010) Mechanism of a Chemical Glycosylation Reaction. Acc. Chem. Res. 43, 1144–1153.

13. Crich, D. (2007) Stereocontrolled glycosylation: recent advances: b-D-rhamnosides and b-D-mannans. ACS Symposium Series 960, 60–72. 14. Tamborrini, M., Werz, D. B., Frey, J., Pluschke, G., and Seeberger, P. H. (2006) AntiCarbohydrate Antibodies for the Detection of Anthrax Spores. Angew. Chem. Int. Ed. 45, 6581–6582. 15. Dudkin, V. Y., Miller, J. S., and Danishefsky, S. J. (2004) Chemical Synthesis of Normal and Transformed PSA Glycopeptides. J. Am. Chem. Soc. 126, 736–738. 16. Horlacher, T., Oberli, M. A., Werz, D. B., Kröck, L., Bufali, S., Mishra, R., Sobek, J., Simons, K., Hirashima, M., Niki, T., and Seeberger, P. H. (2010) Determination of Carbohydrate-Binding Preferences of Human Galectins with Carbohydrate Microarrays. ChemBioChem 11, 1563–1573. 17. Barnes-Seemann, D., Park, S. B., Koehler, A. N., and Schreiber, S. L. (2003) Expanding the functional group compatibility of small molecule microarrays: Discovery of novel calmodulin ligands. Angew. Chem. Int. Ed. 42, 2376–2379. 18. Fukui, S., Feizi, T., Galustian, C., Lawson, A. M., and Chai, W. (2002) Oligosaccharide microarrays for high-throughput detection and specifity assignments of carbohydrate-protein interactions. Nat. Biotechnol. 20, 1011–1017. 19. Hergenrother, P. J., Depew, K. M., and Schreiber, S. L. (2000) Small Molecule Microarrays: Covalent Attachment and Screening of AlcoholContaining Small Molecules on Glass Slides. J. Am. Chem. Soc. 122, 7849–7850. 20. Wang, D., Liu, S., Trummer, B. J., Deng, C., and Wang, A. (2002) Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells, Nat. Biotechnol. 20, 275–281. 21. Houseman, B. T., and Mrksich, M. (2002) Carbohydrate arrays for the evaluation of protein binding and enzymatic modification, Chem. Biol. 9, 443–454. 22. Ko, K.-S., Jaipuri, F. A., and Pohl, N. L. (2005) Fluorous-Based Carbohydrate Microarrays. J. Am. Chem. Soc. 127, 13162–13163.

2  Chemical Synthesis of Carbohydrates… 2 3. Zhang, J., Pourceau, G., Meyer, A., Vidal, S., Praly, J.-P., Souteyrand, E., Vasseur, J.-J., Morvan, F., and Chevolot, Y. (2009) Specific recognition of lectins by oligonucleotide glycoconjugates and sorting on a DNA microarray. Chem. Commun. 2009, 44, 6795–6797. 24. Love, K. R., and Seeberger, P. H. (2002) Carbohydrate Arrays as Tools for Glycomics. Angew. Chem. Int. Ed. 41, 3583–3586. 25. Kiessling, L. L., and Cairo, C. W. (2002) Hitting the sweet spot, Nat. Biotechnol. 20, 234–235.

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26. Disney, M. D., and Magnet, S., Blanchard, J. S., and Seeberger, P. H. (2004) Aminoglycoside Microarrays to Study Antibiotic Resistance. Angew. Chem. Int. Ed. 43, 1591–1594. 27. Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021. 28. Tada, T., Mano, K., Yoshida, E., Tanaka, N., and Kunugi, S. (2002) SH-group introduction to the N-terminal of subtilisin and preparation of immobilized and dimeric enzymes. Bull. Chem. Soc. Jp. 75, 2247–2251.

Chapter 3 General Consideration on Sialic Acid Chemistry Hongzhi Cao and Xi Chen Abstract Sialic acids, also known as neuraminic acids, are a family of negatively charged a-keto acids with a nine-carbon backbone. These unique sugars have been found at the termini of many glycan chains of vertebrate cell surface, which play pivotal roles in mediating or modulating a variety of physiological and pathological processes. This brief review covers general approaches for synthesizing sialic acid containing structures. Recently developed synthetic methods along with structural diversities and biological functions of sialic acid are discussed. Key words: Sialic acid, Sialoside, Glycosylation, Chemoenzymatic synthesis, Sialyltransferase, Sialylation

1. Sialic Acids: A Unique Family of Monosaccharide 1.1. Structure Diversity of Sialic Acid

Sialic acids constitute a unique family of 2-keto-3-deoxynonulosonic acids. These nine-carbon monosaccharides have a tertiary anomeric center at C-2 position linked to an anomeric carboxylate, a deoxygenated C-3, a glycerol side chain at C-6 and different substituents at C-5. Three basic forms of sialic acids (Fig. 1) are N-acetylneuraminic acid (Neu5Ac, N-acetyl-5-amino-3,5-dideoxy-d-glycero-d-galacto2-nonulosonic acid, 1), N-glycolylneuraminic acid (Neu5Gc, having a hydroxyl group in place of one of the hydrogens at the N-acetyl group of Neu5Ac, 2), and deaminoneuraminic acid (KDN, 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid, 3) (1–3). Further modifications of these three basic sialic acid forms, including O-acetylation, O-methylation, O-lactylation, O-sulfation, and O-phosphorylation at the hydroxyl groups at C-4, C-8, and/ or C-9, changes on the group at C-5, and the formation of

Yann Chevolot (ed.), Carbohydrate Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 808, DOI 10.1007/978-1-61779-373-8_3, © Springer Science+Business Media, LLC 2012

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HO

O

OH 9

8

N H

6

7 5

OH

OH

OH

O 4

CO2-

2

OH

HO

1

3

O

OH

OH

HO

N H HO

CO2-

O

OH

HO

CO2-

O

HO HO

Neu5Gc,2

Neu5Ac,1

OH

OH

KDN,3

Fig. 1. Three basic forms of naturally occurring sialic acids.

HO HO

OH

CO2-

OH O

AcHN

O

OH O

HO

HO

HO

OH

HO

HO O

O AcHN

HO

CO2-

OH

HO

CO 2-

OH

HO

O

AcHN O

O

(4Siaα2-6Galα1-)n

O

HO

HO

O

O HO

CO 2-

OH O O

OH

OR

CO2-

OH

AcHN

AcHN

n

OH

n

(9Siaα2-9Siaα2-)n HO

O HO

CO2O

HO

OH

O

Siaα2-6GalNAcαOR

OH

n

(8Siaα2-8Siaα2-)n

O

AcHN

OH

HO

HO

CO 2-

OH HO

OR

O AcHN

O

HO

AcHN

O

HO

HO

OH

CO2-

OH

O

HO

OH

Siaα2-6GalβOR

Siaα2-3GalβOR

O AcHN

O

AcHN

OR

CO 2-

OH

O

OH

O HO

(4Siaα2-6Glcα1-)n

OH n

Fig. 2. Common sialic acid containing structures with different sialyl linkages.

intramolecular lactone or lactam leads to the identification of more than 50 natural occurring sialic acids (2, 3). These structurally distinct sialic acids are the key components of many important glycolipids and glycoproteins presented on vertebrate cell surface. In vertebrates, sialic acids are typically presented in (Siaa2-8Sia)n homopolymers or as the outermost carbohydrate units of glycoconjugates, such as in Siaa2-3GalOR, Siaa2-6GalOR, Siaa2-6GalNAcOR structures (Fig. 2). In bacteria, sialic acids have been found in extracellular capsular polysaccharides and lipopolysaccharides (LPS) in homopolymers of sialic acid with a2-8-, a2-9-, or alternating a2-8/9-linkages (4, 5) as terminal residues in the side chains of capsular polysaccharide heteropolymers (2, 3, 6, 7), as terminal residues in the glycans of bacterial lipooligosaccharides (LOS) of various Gram-negative bacteria (8), and as internal residues in heteropolymers of capsular polysaccharides of Neisseria meningitidis (9). 1.2. General Biological Significance of Sialic Acid

As the terminal monosaccharides located at the outermost position of cell surface glycans, sialic acids are the key recognition sites of many biomolecules. Moreover, the structural diversity of sialic acids also help to fine-tune the pivotal roles that sialic acids play in

3  General Consideration on Sialic Acid Chemistry

33

biological and pathological processes, such as cell–cell interaction (10, 11), inflammation (12), fertilization (13), infection (14), differentiation (15), malignancy (16–18), and cell signaling (19, 20). They are also highly regulated during embryonic development (21–23). In bacteria, they are believed to be important virulence factors (24) and used by the bacteria to mimic sialylated host cell surface carbohydrate structures to evade detection and attack by the host’s immune defense mechanisms (3, 8, 25–27). Such examples include a2-8-linked (8Siaa2-8Siaa2-)n capsular polysialic acid (also called colominic acid) expressed by N. meningitidis serogroup B (NmB) (28) and Escherichia coli K1 (29), which mimics the structure of human neural cell adhesion molecules (NCAMs) (30–32). Other examples include sialylated oligosaccharides or polysaccharides produced at surface of pathogenic bacteria such as Campylobacter jejuni, Group B Streptococcus, P. haemolytica A2 (33). The sialic acid-based recognition processes are believed to be closely related to the fine structures of the sialic acids, the underlying glycan structures, and the types of the sialyl linkages. Malignant cells often display surface glycosylation patterns differing from normal cells. The overexpression of tumor-associated carbohydrate antigens (TACAs) is commonly observed on different types of malignant cells (34–36). Sialic acid residue is part of many TACAs and is believed to play essential role in different stages of cancer progress (37). These sialic acid containing TACAs (Fig. 3), such as sialyl Lewis x (sLex), sialy Lewis a (sLea), gangliosides (GM3, GM2, GM1, GD2, GD3, fucosyl GM1), sialyl Tn and sialyl T, and so on, have been the targets of studies for developing carbohydrate-based vaccines (38–40). Several carbohydrate-based

OH OH

HO

AcHN

O

CO2O

OH

OH

HO

O

O OH

O

O

OH O

HO

OR NHAc

OH OH

AcHN

CO 2-

OH NHAc O OR O O O HO OH OH O OH HO OH

O

O

OH

OH

HOOH Sialyl Lewis x (sLex) OH OH

HO

CO2O

AcHN

OH

O

O HO OH

OH

O HO

Sialyl Lewis a (sLea) OH O OH

HO

OR AcHN

OH O

HO

HO O

O O

HO OH

HO OH O HO

AcHN

OH O

OH

O

O OH

OH

O

O HO OH

HO

AcHN

OH -O C 2

CO 2-

HO O

OH O

HO

AcHN OR

O OH O

O OH HO

OH O

OH Fucosyl GM1

HO AcHN OR

CO2-

OH O OH

O AcHN

OH OH

HO

CO 2-

OH

AcHN

O

O

OH

O HO Sialyl Tn (sTn) AcHN OR

Sialyl T (sT)

GM3 HO

OH OH

OH

OH OH

CO2O

OH

O AcHN n

Polysialic acid (PSA)

Fig. 3. Representative sialic acid containing tumor-associated carbohydrate antigens (TACAs).

CO2O

OH

OR

34

H. Cao and X. Chen

vaccines are currently in clinic trails (38). The isolation of carbohydrates from natural source is a tedious process and often yields impurities in the carbohydrate preparations. Therefore, synthetic carbohydrates have been attractive targets for the development of carbohydratebased vaccine. Obtaining structurally defined complex carbohydrates in sufficient amount has been a big challenge (41). Recent advances in chemical, enzymatic, and chemoenzymatic synthesis provide efficient methods to overcome the challenge. Neu5Ac (1), the most abundant form of sialic acid, is synthesized from N-acetylmanosamine (ManNAc, 4) in both eukaryotes and prokaryotes though its biosynthetic pathways differ in bacteria and vertebrates. The synthesis of Neu5Ac from ManNAc in eukaryotes occurs in the cytosol with three consecutive steps as shown in route a in Fig. 4, including the formation of ManNAc-6-phosphate (5) from ManNAc (4) catalyzed by an N-acetylmannosamine kinase (EC2.7.1.60), formation of Neu5Ac-9-phosphate (6) catalyzed by an N-acetylneuraminic acid 9-phosphate synthase (EC2.5.1.57), and dephospholation catalyzed by an N-acetylneuraminic acid 9-phosphate phosphatase (EC3.1.3.29) (42). In bacteria, Neu5Ac is synthesized directly through the reaction of ManNAc with phosphoenolpyruvate (PEP) catalyzed by a Neu5Ac synthase (route b in Fig. 4). Sialic acid aldolase (lyase) catalyzing the reversible cleavage of Neu5Ac for the formation of ManNAc and pyruvate can be used in vitro for the inexpensive production of Neu5Ac from ManNAc and excess amount of pyruvate (28, 43–46). The production of activated sugar nucleotide donor cytidine 5¢-monophosphate sialic acid (CMP-sialic acid) required by sialyltransferases (47, 48) is catalyzed by CMP-sialic acid synthetases (CSSs, EC 2.7.7.43) in both eukaryotes and bacteria. CSSs catalyze the formation of CMP-Neu5Ac and pyrophosphate from Neu5Ac and CTP in the presence of a metal cation such as Mg2+ or Mn2+ (3, 42, 49). The biosynthetic pathway of CMP-KDN in bacteria is believed to follow a similar route as CMP-Neu5Ac though it is not

1.3. Biosynthetic Pathway of Sialic Acid and CMP-Sialic Acid

-2

O3PO NHAc O HO OH HO ManNAc-6-P, 5

ADP

ATP

ManNAc kinase

NHAc HO O HO HO ManNAc, 4

PEP Neu5Ac Pyruvate synt hase or Pi

PEP Neu5Ac-9-P synthase Pi -2O PO 3

OH

AcHN

route (a)

OH

OH O

HO Neu5Ac-9-P, 6

CO2-

route (b)

Neu5Ac-9-P HO phosphatase H2O

Pi

OH

OH

AcHN

OH

OH O

HO Neu5Ac, 1

CO2-

Pyruvate Sialic acid aldolase ( lyase)

CTP CSS

PPi

HO

HO

OCMP

OH

AcHN

O

HO CMP-Neu5Ac, 7

Fig. 4. Biosynthetic routes of Neu5Ac and CMP-Neu5Ac in eukaryotes (route (a)) and bacteria (route (b)).

CO2-

3  General Consideration on Sialic Acid Chemistry

35

as well elucidated (1, 3, 48, 50, 51). CMP-Neu5Gc, which has not been reported in bacterial sources (3), is obtained from CMPNeu5Ac by oxidative conversion of the N-acetyl group of Neu5Ac to the N-glycolyl residue in Neu5Gc catalyzed by a CMP-Neu5Ac hydroxylase (52). In addition to the biosynthesis discussed earlier, Neu5Ac, Neu5Gc, and KDN deliberated from glycoconjugates in catabolic pathways can be recycled via salvage pathways to form the activated sialic acid donors for sialyltransferases (53, 54). Sialyltransferases are the key enzymes that catalyze the transfer of sialic acid residue from CMP-sialic acid to suitable acceptors for the formation of sialyl linkages. Different sialyl linkages are usually formed by different sialyltransferases. Eukaryotic sialyltransferases are usually more specific than their bacterial counterparts in the formation of sialyl linkages and recognizing suitable acceptor substrates, though all are relatively flexible in using CMP-sialic acids containing different sialic acid forms. Due to their high activity, high solubility, and suitability to be expressed in large amount in E. coli system, bacterial sialyltransferases have been cloned from different species and expressed in E. coli for high efficient preparation of complex sialosides (1). Biosynthetically, sialic acid modifications including O-acetylation, O-methylation, O-lactylation, and O-sulfation, usually take place after the formation of sialic bonds. These modifications thus belong to carbohydrate postglycosylational modifications (55). Nevertheless, due to the promiscuity of bacterial sialyltransferases and other bacterial enzymes involved in the CMP-sialic acid synthesis, naturally occurring sialic acid modifications can be introduced to monosaccharide stage by chemical or enzymatic methods, which can be used by multiple enzymes for the synthesis of CMP-sialic acids and sialosides with different sialic acid forms (56–58).

2. Difficulties in Chemical Formation of Sialoside Bond

Synthesis of structurally defined complex carbohydrates poses great challenges due to the inherent structural complexity of the carbohydrates such as the presence of multiple hydroxyl groups of similar reactivity and varied glycosidic linkages in the molecule. Among different chemical glycosylation processes, sialylation is considered as one of the most difficult reactions due to the hindered tertiary anomeric center and the lack of a neighboring participating group in sialic acids (59, 60). Many factors affect the outcome of chemical sialylation, including the protecting group patterns of sialyl donor and glycosyl acceptor, the leaving group of sialyl donor, the promoter, reaction solvent, temperature, and so on. The most significant challenge of chemical sialylation comes from the inherited unique structural feature of sialic acid.

36

H. Cao and X. Chen

Neu5Ac is the most abundant sialic acid form in nature. So far, chemical sialylation has been mainly focused on the synthesis of Neu5Ac-containing structures. General chemical sialylation methods involve an activated sialic acid donor (8) with a proper leaving group (LG) (Fig.  5) and an acceptor (R¢OH) bearing a single free hydroxyl group. The electron-withdrawing carboxylate at the C-2 anomeric carbon destabilizes the formation of the oxocarbenium ion intermediate (9 and 10). Furthermore, the tertiary anomeric center at C-2 of sialic acid donor prevents an easy nucleophilic attacking by the hydroxyl group in the acceptor and makes sialylation a slow process. Therefore, the competing elimination process is usually quite significant to generate a considerable amount of 2,3-dehydro sialic acid byproduct, 12 (Fig. 5). Due to the lack of a hydroxyl group at C-3, the neighboring carbon of C-2 anomeric center of sialic acid, functional protecting group cannot be easily introduced at C-3 to regulate the stereoselectivity of the sialylation process for the formation of desired a-sialyl linkages found in nature. In addition, the desired a-sialyl linked products are thermodynamically less stable than their b-anomers due to anomeric effect (60). As a consequence, many early sialylation methods generally lead to low yields due to poor stereoselectivity and the generation of the elimination byproduct. It is worth to mention that commonly used tricholoracetimidate glycosyl donor (Schmidt donor) is not suitable for sialylation due to the unique structural feature of sialic acid (61). Other than the challenges described earlier for general chemical sialylation, chemical sialylation of a sialic acid to another sialic acid for the formation of a2-8-linked disialyl motif Neu5Aca2-8Neu5 AcaOR has additional complications due to decreased nucleophilic activity of the C-8 hydroxyl group in sialic acid acceptor caused by its intramolecular hydrogen bonding network with C-5 acetamido group, C-1 carboxyl, or C-2 oxygen (Fig. 6) (59).

PO

LG

OP O

AcHN PO

CO2R

Activation

PO

PO

OP

O

AcHN PO

8

CO2R

OP O

AcHN

OR'

PO

PO

OP

O

CO2R

AcHN

El im in at io n

PO

10

+

PO

PO

-H

PO

9

CO2R

R' Gl O yc os H yl at io n

PO

PO

PO

OP

O

AcHN PO

11

Fig. 5. A general chemical sialylation approach and related elimination process.

12

CO2R

3  General Consideration on Sialic Acid Chemistry

PO

O

O

H

OR

OP

PO O

AcHN PO

OR'

O

H

O

R' H O OP PO

OP O

AcHN PO

37

CO2R

AcHN

CO 2R O

OR'

PO

Fig. 6. Intramolecular hydrogen bonding network of C-8 hydroxyl group of Neu5Ac acceptor.

3. The Influence of Different Protecting Groups on Reactivity

The reactivity and stereoselectivity of chemical sialylation can be greatly affected by the protecting groups on sialyl donors. The stereoselective formation of a-sialyl linkages has been improved by introducing different protecting groups at C-1, C-3, and C-5 of sialyl donors.

3.1. C-1 Modification

Several auxiliary protecting groups with potential neighboring participating effect have been introduced to the C-1 carboxyl group in sialyl donors to overcome the low stereoselectivity and low yield of sialylation. For example, several decades ago, Ratcliff and coworkers noticed that benzyl ester protection of carboxyl in Neu5Ac gave better yields and stereoselectivity than commonly used methyl ester protection under Koenigs–Knorr glycosylation conditions (60). Recently, N,N-dimethylglycolamide was developed as a new C-1 auxiliary group in sialyl donors by the Gin group (62). This C-1 auxiliary neighboring participating group can stabilize the oxocarbenium ion intermediate to form a bicyclic intermediate and to improve a-selectivity for the formation of both a2-3- and a2-6-sialyl linkages. In another method, Wong and coworkers converted the C-1 carboxyl into a hydroxymethylene and found this modification can boost the reactivity of sialic acid donor and improve glycosylation yields significantly though the undesired b-sialosides were the predominated products (63).

3.2. C-3 Modification

Using a C-3 directing auxiliary group (Y in sialyl donor 13, Fig. 7) was one of the most common approaches for chemical sialylation a decade ago, and it was usually classified to “indirect glycosylation” methods (59, 60). The C-3 auxiliary group was introduced as a neighboring participating group to stabilize the positive charge of oxocarbenium ion intermediate by forming a bicyclic intermediate 14 and to prevent competing 2,3-elimination reaction. Several directing groups have been introduced and successfully employed to the synthesis of a broad library of sialosids (59). The C-3 hydroxyl group was initially introduced by Goto and coworkers for the synthesis of a2-9- and a2-8-linked Neu5Ac dimers under Koenigs–Knorr glycosylation conditions (64). They found that an O-acetyl-protected hydroxyl group or bromine at C-3 can provide

38

H. Cao and X. Chen

AcO

OAc OAc

Y O

AcHN

AcO 14

CO 2Me

Sialylation AcO

OAc OAc

AcO

AcO 15

R'OH

AcO

AcO

LG Y

OAc OAc AcO

Ph

O S

CO2Me O

AcHN

13

Y = OAc, Br, SPh, SePh,

OR' Y

Removal of Y

CO2Me O

AcHN

O

AcHN

Activation OAc OAc

CO2Me

OR'

16

S or

Fig. 7. Schematic illustration of using C-3 auxiliary groups to improve sialylation stereoselectivity.

better stereoselectivity compared to the same types of sialyl donors with no C-3 directing group. Ogawa et al. introduced C-3 phenylthiol and phenylselenyl groups as the neighboring participating groups (65). The phenylthiol group was shown to be a superior directing group due to its increased size and polarizability compared to oxygenbased C-3 auxiliaries. Furthermore, it can be easily removed after glycosylation by one-step treatment with Bu3SnH and AIBN under radical reaction conditions (65). Other C-3 auxiliary groups have also been employed to the chemical synthesis, such as 2,4-dimethylbenzenesulfenyl (66) and phenoxythiocarbonyloxy (67) groups, which have similar properties as the phenylthiol directing group. The auxiliary participating group strategy can dramatically increase the desired a-sialoside products and minimize the elimination byproduct. However, the disadvantage is obvious due to the required additional steps for adding the C-3 auxiliary group during the synthesis of sialyl donor and removal of the group after glycosylation. Currently, this “indirect strategy” is rarely used in chemical sialylation reactions. 3.3. C-5 Modification

In the process of looking for other positions for adding auxiliary groups to help chemical sialylation, Boons and Demchenko found that adding an additional acetyl group at the nitrogen in the N-acetyl group at C-5 in Neu5Ac (17) can significantly improve the reactivity of both sialyl donor and acceptor compared to the corresponding mono-N-acetylated sialyl donor and acceptor (68). This second N-acetyl group can be easily introduced by acetylation to mono-N-acetylated sialyl donor or acceptor and can be conveniently removed under Zemplén conditions after the glycosylation. Encouraged by these results, many other functional groups, such as azido (18) (69), N-trifluoroacetyl (NHTFA, 19) (70), N-trichloroethoxycarbonyl (NHTroc, 20) (71), N-t-butoxycarbonylacetamido (NAcBoc, 21)

3  General Consideration on Sialic Acid Chemistry

AcO

OAc OAc

Ac2N

AcO

CO2Me O

AcO

LG

CO2Me O

AcO

OAc OAc N3

17

OAc OAc

AcBocN

AcO

21

LG

AcO

CO2Me O

AcO

LG

AcO

22

LG

CO2Me O

TFAHN

CO2Me AcO O

HO

OAc OAc

18

OAc OAc

PhthN

AcO

O

23

CO 2Me O

TrocHN AcO

CO2Me O

HN

OAc OAc

19

OAc OAc

O

LG

AcO

LG

O

LG

20 CO2Me

O

O

HN OP

39

OP OP

LG

24

Fig. 8. Examples of C-5 modified sialyl donors.

(72), phthalimido (NPhth, 22) (73), 5-N,4-O-oxazolidinone (23) (74), and 5-N,7-O-oxazinone (24) (75) groups (Fig. 8) have been introduced to the C-5 position to increase the reactivity of sialyl donor or acceptor. The C-5 modification strategies along with the novel sialyl donor developed in the last decades have been demonstrated to be applicable as general approaches for chemical sialylation (76).

4. Different Chemical Sialylation Strategies

4.1. 2-Halo Sialyl Donors: The Koenigs– Knorr Protocol

Different sialyl donors have been developed. Several common and effective ones are discussed later with the corresponding suitable protecting groups. The first example of chemical sialylation was reported by Meindl et al. in 1965 (77). The Koenigs–Knorr glycosylation method was employed using 2-halo sialic acid derivatives as the donors and a silver salt such as Ag2CO3 (classical Koenigs–Knorr) or Hg(CN)2/ HgBr2 (Helferich modification) as the promoter (Fig.  9) (77). The 2-chloro derivative of Neu5Ac 25 has reasonable stability and is suitable for glycosylation with a number of highly reactive alcohols, such as primary alcohols, phenols, and primary hydroxyl groups of sugars in good yields and reasonable stereoselectivity (78, 79). This easily obtainable sialyl donor is still one of the popular sialyl donors for the synthesis of simple sialosides under Koenigs– Knorr conditions. In comparison, the 2-bromide and 2-fluoride derivatives of Neu5Ac have only limited applications due to the low stability and the tendency in forming nonnatural b-linkage, respectively (59). Nevertheless, the method is not ideal for sialylation of secondary or hindered hydroxyl groups on sugar acceptors, which favor the elimination pathway to give the 2,3-dehydro-Neu5Ac elimination byproduct in significant amount. Moreover, the application of this method is limited by the cost and the toxicity concern of using stoichiometric amount of heavy metal salts as the promoters.

40

H. Cao and X. Chen

AcO

OAc OAc

Cl O

AcHN AcO

CO2Me

+

O

25

O

Ag2CO3

OO

67%, α only

O

26

OAc OAc AcO AcHN AcO

OAc OAc CO2Me O O AcHN O AcO

AcO

OH

O

Cl O

CO 2Me

27

BnO OH Hg(CN)2/HgBr2 O + OBn BnO 84%, α/β = 3/4 OBn

25

O OO

OAc OAc CO2Me O O AcHN BnO AcO

AcO

28

29

BnO

O

OBn OBn

Fig. 9. Sialyl chloride as a donor for sialylation under Koenigs–Knorr glycosylation conditions.

AcO

OAc OAc

SMe O

RHN AcO

30

CO 2Me

AcO

OAc OAc

SEt O

RHN AcO

31

CO 2Me

AcO

OAc OAc

SPh O

RHN AcO

32

AcO CO 2Me

OAc OAc RHN

CO 2Me O

AcO 33 S

S OEt

Fig. 10. Representative 2-thio sialyl donors.

4.2. 2-Thio Sialyl Donors

Using 2-thio derivatives of sialic acid as sialyl donors is a great improvement in chemical sialylation during the last decade. The 2-thio derivatives of Neu5Ac are usually thio-alkyl (methyl 30, ethyl 31) or thio-aryl (phenyl 32, p-methylphenyl, etc.) sialosides, and sialyl xanthates (ethoxydithiocarbonate 33) (Fig. 10) (59, 80). These 2-thio derivatives of Neu5Ac can be readily prepared and are compatible with many protecting group manipulation conditions. The thioglycoside donors can be activated under mild conditions in the presence of stoichiometric amount of a thiophilic promoter such as N-iodosuccinimide/trifluoromethanesulfonic acid (NIS/TfOH). It can also be easily transformed to other glycosyl donors. Due to these advantages, 2-thio derived sialyl donors provide a great platform to accommodate many other protecting group modification strategies for the synthesis of a broad spectrum of sialyloligosaccharides and sialylglycoconjugates. Some representative examples are shown in the following section. As shown in Fig. 11, Boons and Demchenko used N-acetylacetamido (di-N-acetyl) group in a 2-thiomethyl sialyl donor to greatly shorten the reaction time from more than 2 h with monoN-acetyl 2-thiomethyl sialyl donor to a few minutes with increased yield (68). Solvent effect plays an important role in the stereoselective formation of desired natural a-sialosides using thioglycoside donors (59, 81). As shown in Fig. 12, thioglycoside donor (34) forms the oxocarbenium ion intermediate (39) upon activation by a suitable promoter. The acetonitrile as a solvent can attack this oxocarbenium ion intermediate to generate both a- (40) and

3  General Consideration on Sialic Acid Chemistry OAc OAc

AcO

AcO

34

OAc OAc

AcO

Ac2N

CO 2Me + HO

O

AcHN

35 HO

SMe O

AcO

HO

SMe

CO2Me + HO

37

35

OBz O

NIS, TfOH, MS 3A, CH3CN, -40oC

OTE OH

OBz O

AcO

OAc MeO 2C OAc O

AcHN

2-6 h, 61%

AcO NIS, TfOH, MS 3A, CH3CN, -40oC

OTE

5 min, 72%

OH

HO O

41 OBz O

OTE

OH

36

OAc MeO 2C HO OAc O O Ac2N AcO 38

AcO

OBz O

OTE OH

Fig. 11. Sialylation using di-N-acetyl thiosialoside donor.

AcO

OAc OAc

O

AcHN

Solvent CO 2Me participation

AcO

OAc OAc O

AcHN AcO

39

N

C CH 3

CO2Me N

OAc OAc

AcO

AcO

Activation AcO

OAc OAc

SMe O

AcHN AcO

AcO

CO2Me

O

AcHN

AcO 40

N CO2Me

41

ROH

Sialylation OAc CO2Me OAc

AcHN

O

OR

AcO 42

34

Fig. 12. a-Stereoselective sialylation via solvent participation.

b-nitrilium (41) intermediates. The predominance of the kinetically controlled b-nitrilium ion intermediate leads to a-selective sialylation via an SN2 mechanism. Many protecting group modification strategies have been developed in recent years for thiosialoside donors, such as N-trifluoroacetyl (NHTFA) (70), azido (69), N-trichloroethoxycarbonyl (NHTroc) (71), N-t-butoxycarbonylacetamido (NAcBoc) (72), and phthalimido (NPhth) (73) groups at C-5 position of sialyl donors. A higher a-stereoselectivity was observed by Wong and coworkers with a C-5 azido derivative of thiosialoside donor (45) compared to its C-5 N-acetyl derivative (34) (69). As shown in Fig. 13, the desired a-stereoselectivity was dramatically improved from 1:1.25 to 10:1 (a/b) by changing the C-5 acetamido group to C-5 azido under same glycosylation conditions (69). Unfortunately, the higher stereoselectivity controlled by the C-5 azido group of sialyl donor was associated with its deactivating effect leading to low yields. Ando et  al. developed novel 1,5-lactam sialic acid acceptors (47, 51, Fig.  14) with a thiophenylsialoside donor (48) for the synthesis of the carbohydrate components of gangliosides HLG-2 (50) (82) and Hp-s6 (53) (83) oligosaccharides (82, 83). These lactam acceptors render high reactivity at C4- and C8-hydroxyl groups to high yields production of a2-4- and a2-8-linked disialyl intermediates, respectively. Tanaka et al. developed a 5-N,4-O-carbonyl-protecting group for Neu5Ac and found this protecting group is superior for both

42

H. Cao and X. Chen

OAc OAc SMe AcO CO2Me O AcHN

O +

O

AcO

N3 AcO

o

NIS, TfOH, MS 3A, CH3CN, -40 C OTBDPS

45%, α/β = 1:1.25

OAc OAc

AcHN AcO

CO2Me O

N3

34 OAc OAc AcO

OH O

AcO

STol O

CO2Me

45

+

O

44 AcO

OH O N3

O

O

43 O

O O

OTBDPS

NIS, TfOH, MS 3A, CH 3CN, -40o C 53%, α/β = 10:1

43

OTBDPS

N3

OAc MeO C 2 OAc O N3 O AcO O 46

O

O

OTBDPS

N3

Fig. 13. Sialylation using C-5 N-acetyl versus C-5 azido thiosialoside donors.

Fig. 14. 1,5-Lactam sialyl acceptors/donors for the synthesis of gangliosides HLG-2 and Hp-s6.

Fig. 15. 5-N,4-O-Carbonyl protected sialyl donor and acceptor for the synthesis of oligosialic acids.

sialyl donor (54) and acceptor (55) (Fig. 15) in selective formation of a2-8-disialyl sequence in good yields (74). The efficiency of the approach was demonstrated in the synthesis of a2-8-linked oligosialic acid (57) (74), a2-9-linked oligosialic acid (84), and complex ganglioside GP1c (85). 4.3. 2-Phosphite Sialyl Donors

Sialyl phosphite donors (Fig. 16) were independently developed by Wong (58) (86) and Schmidt (61) (87) in 1992. These 2-phosphites derivatives of sialic acid can be synthesized from readily available 2-hydroxyl sialic acid derivatives. They can be activated at low temperature by catalytic amount of many common Lewis acid promoters, such as trimethylsilyl trifluoromethanesulfonate (TMSOTf). Since the sialyl phosphite donors are synthesized

3  General Consideration on Sialic Acid Chemistry

OAc OAc AcO AcHN

OP(OBn)2 O CO2Me

+

BnO BnO

AcO

OAc OAc

OP(OEt) 2 O

AcHN

+

CO2Me

TMSOTf, CH3CN, -42oC

BnO OMe 59

58 (Wong's Donor) AcO

AcO

OH O

BnO BnO

AcO

61 (Schmidt's Donor)

OH O BnO

59

OAc OAc

O BnO BnO

AcO

80%, α/β = 6/1

60 OAc OAc AcO

TMSOTf, CH3CN, -40oC

OMe

CO2Me O

AcHN

70%, α/β = 4/1

AcHN

43

O BnO

OMe

CO2Me O

AcO 60

O BnO BnO

O BnO

OMe

Fig. 16. Sialylation using 2-phosphite sialyl donors.

OAc MeO2C O OAc O O P OBu AcN OBu O O 62

AcO

63, CH2Cl 2, 4A MS, TMSOTf -78oC to -40oC

64, NIS

OAc MeO2C OAc O O AcN HO O O O BzO O 65 BzO BnO (80%, single isomer)

AcO

-40oC

OBn O

O(CH2)5N3 NPhth

Ph OAc MeO2C O OAc O O P OBu AcN OBu O O 62

66, CH2Cl 2, 4A MS, TMSOTf

AcO

HO BzO

-78oC to -40oC

64, NIS

O OAc MeO 2C O OAc O O O O AcN BzO BnO O 67 O (79%, single isomer )

AcO

-40o C Ph

OH O

STol

BzO 63

HO BnO

OBn O

O(CH2)5N 3 NPhth 64

O O HO

O

OBn O

O(CH2)5N3 NPhth

STol

BzO 66

Fig. 17. One-pot glycosylation using a 5-N-acetyl-5-N,4-O-carbonyl 2-phosphate sialyl donor.

predominately as b-anomers and activated at low temperature, the glycosylation usually leads to predominate a-sialoside either through direct SN2 mechanism or solvent participating pathway (Fig. 16) (86, 87). Most recently, Wong and coworkers demonstrated the 2-phosphate derivative of Neu5Ac containing a 5-N-acetyl-5-N,4O-carbonyl protecting group (62) is an ideal sialyl donor for the synthesis of complex sialosides (88). As shown in Fig.  17, the 2-phosphate sialyl donor (62) can be used in a programmable one-pot glycosylation protocol developed in the Wong group to stereoselectively produce a-sialyl products in good yields (88). 4.4. 2-Trifluoroace timidate Sialyl Donors

It was well known that the Schmidt type sialyl donors with a tricholoroacetimidate as the leaving group are not suitable for sialylation (61). Quite interestingly, a trifluoroacetimidate sialyl donor developed by Yu and coworkers (68 in Fig. 18) by substituting the trichloroacetimidate in the Schmidt donor with trifluoroacetimidate has been successfully applied to direct sialylation with impressive yields and a-stereoselectivity (Fig.  18a) (89, 90). Fukase and coworkers

44

H. Cao and X. Chen OAc MeO2C O OAc O O P OBu AcN OBu O O 62

AcO

63, CH2Cl2, 4A MS, TMSOTf -78oC to -40oC

64, NIS

OAc MeO2C OAc O O AcN HO O O O BzO O 65 BzO BnO (80%, single isomer)

AcO

-40oC

OBn O

O(CH2)5N3 NPhth

Ph OAc MeO2C O OAc AcO O O P OBu AcN OBu O O 62 HO BzO

66, CH2Cl2, 4A MS, TMSOTf

64, NIS

-78oC to -40oC

O OAc MeO 2C O OAc O O O O AcN BzO BnO O 67 O (79%, single isomer)

-40oC Ph

OH O

STol

BzO

HO BnO

O O

OBn O

O(CH2)5N 3 NPhth

63

OBn O

AcO

HO

O

O(CH2)5N3 NPhth

STol

BzO 66

64

Fig. 18. Sialylation using trifuloroacetimidate sialyl donors.

a AcO

OAc OAc

OH O

AcHN AcO

CO 2Me

+

BnO BnO

72

b AcO

OAc OAc

AcHN AcO

OH O

O 74

O

NMe2 O

+

OH O

BnOOMe 59

AcO

OH O

O

AcHN AcO

74

O

NMe2 O

66%, α/β = 2:1

OAc OAc

CO2Me O

AcHN AcO 73

26

Ar 2SO, Tf 2O

O

BnO BnO

OAc O OAc AcO O OH O AcHN O (p-NO C H )(Ph)SO, Tf O, CH Cl 2 6 4 2 2 2 O AcO OO 70%, α/β = 4:1

c OAc OAc

(p-NO 2C6H4)(Ph)SO, Tf2O, CH2Cl2

AcO

O BnO OMe

Ar S OTf O Ar AcO O O NMe2 AcHN O AcO O 76

AcO

OAc OAc AcO

O

O

OO

TfO O O

AcHN

NMe2 O O

75

OAc OAc

O

O

77

NHMe2 O O

Fig. 19. Dehydrative sialylation approaches using C-2 hemiketal Neu5Ac derivative as a sialyl donor.

found the “fixed dipole effect” of a C-5 N-phthaloyl protecting group on trifluoroacetimidate donor (69) provided predominant a-sialosides under the similar glycosylation conditions (Fig. 18b) (91). 4.5. C-2 Hydroxyl Derivatives of Neu5Ac as Donor for Dehydrative Sialylation

Recently, Gin and coworkers expanded their dehydrative glycosylation protocol to chemical sialylation (92, 93). As shown in Fig.  19, anomeric hemiketal Neu5Ac derivative with C-1 N,Ndimethylglycolamide ester auxiliary group (74) (Fig. 19b) produced higher a-selectivity compared to its counterpart without the C-1 participating group (72) (Fig.  19a). The higher a-stereoselecitive was contributed by the C-1 N,N-dimethylglycolamide ester auxiliary group as it can attack the anomeric center to form b-oriented bicyclic intermediate (77) due to anomeric effect (Fig. 19c).

3  General Consideration on Sialic Acid Chemistry

5. Enzymatic Sialylation With or Without CMPSia Regeneration

As described earlier, chemical sialylation is a time-consuming process, which involves tedious protecting group manipulation. So far, only a small library of sialosides has been prepared through chemical synthesis. Furthermore, the sialic acid form in most of these sialosides is restricted to Neu5Ac, the most abundant sialic acid form. Sialosides can be effectively synthesized by sialyltransferasecatalyzed enzymatic reactions. In these processes, CMP-sialic acid is used by sialyltransferases as an activated nucleotide donor. Galactose (Gal) or N-acetylgalactosamine (GalNAc)-terminating structures are common sialyltransferase acceptors for producing monosialylated products. Using different sialyltransferases, a2-3-, a2-6-, and a2-8-linked sialosides (Siaa2-3GalbOR, Siaa26GalbOR, and Siaa2-6GalNAcaOR) can be synthesized (Fig. 20a–c). For the enzymatic synthesis of Siaa2-8Sia-containing structures, an a2-3-linked monosialylated compound serves as an acceptor for a2-8-sialyltransferase for the synthesis of a2-8-disialyl

OH HO

OH O

GalβOR

OR

OH

a

HO

b HO

O

AcHN

OH

CO2-

HO

HO

c

CMP-Neu5Ac, 7

OR

OH

d HO

HO

O

O

OH O

HO

Siaα2-6GalβOR HO OR

HO

CO 2-

OH O

AcHN

O

HO

Siaα2-6GalNAcαOR

OH

HO

O

HO

AcHN

OR

OR

α-linked sialoside α2-8SiaT

OR

OH

CO2O

OR

OH CO 2-

HO

OH

AcHN

OH OH O

Siaα2-3GalβOR

OH

AcHN

GalNAcαOR α2-6SiaT OH

HO

OH O

AcHN

O

O

AcHN

α2-6SiaT

OCMP

OH

CO2-

OH HO

OH OH O

GalβOR

HO

HO

α2-3SiaT

OH

45

HO

CO 2-

OH HO AcHN

O HO

O AcHN

OH

Siaα2-8Siaα HO

Fig. 20. Sialyltransferase-catalyzed formation of four common sialyl linkages.

CO 2-

OH O

OR

46

H. Cao and X. Chen OPO32O

CO 2-

(PEP)

CO 2-

PK HO

HO

CO2- OH

OH O

AcHN HO

ATP

O

OH

O

HO

OH NHAc

79 HO HO

CMP

O O HO

78 2Pi

OH O

OPO32-

CDP

α2-6SiaT

OH

OH

NMK

O

O OH HO

ADP

CO 2-

PK

CMP-Neu5Ac, 7

CTP

O CO 2-

OH NHAc

PPase

PPi

CMP-Neu5Ac HO synthetase HO OH

OH O

AcHN HO

CO2-

1

Fig. 21. Sialyltransferase-catalyzed sialylation with CMP-sialic acid regeneration.

products (Fig.  20d). Polysialic acids can also be synthesized by polysialyltransferases using CMP-Neu5Ac as a donor substrate and sialic acid containing acceptors (94). To avoid the use of stoichiometric amount of expensive CMPsialic acid and to minimize product inhibition by CMP formed in the sialyltransferase reaction, Wong and coworkers developed an in situ CMP-Neu5Ac regeneration system (Fig.  21) for sialyltransferase-catalyzed synthesis of sialosides (95). In this system, CMPNeu5Ac can be regenerated from sialyltransferase byproduct CMP via a nucleoside monophophate kinase (NMK) in the presence of ATP, a pyruvate kinase (PK), phosphoenolpyruvate (PEP), a CMP-Neu5Ac synthetase and Neu5Ac. An a2-6-sialyltransferase (a2-6SiaT)-catalyzed reaction successfully produced a2-6-linked sialoside product (79). The pyrophosphate byproduct of the CMP-Neu5Ac synthetase reaction was decomposed to inorganic phosphate by pyrophosphatase (PPase) and the ADP byproduct of the nucleoside monophophate kinase (NMK) was recycled back to ATP by the pyruvate kinase (PK) (Fig. 21) (95). In this system, only Neu5Ac, sialyltransferase acceptor LacNAc (78) and phosphoenolpyruvate (PEP) (2 equivalent), needed to be used in stoichiometric amounts, thus decreased the cost for the synthesis of sialosides. However, the number (five) of required enzymes in the system and the limited availability of mammalian a2-6-sialyltransferase prevented this method for common application in largescale synthesis of sialosides.

3  General Consideration on Sialic Acid Chemistry

6. Chemoenzymatic Sialylation in Generating Diverse Sialosides

47

Recently, Chen and coworkers developed a highly efficient one-pot three-enzyme approach for the synthesis of diverse sialosides with different naturally occurring and nonnatural sialosides, various sialyl linkages, and diverse underlying glycans (56–58, 96). Many of the sialosides produced have a propyl azide aglycone at the reducing end of the glycans, which can be conveniently converted to an amino group for direct coupling to N-hydroxysuccinamide (NHS)or epoxy-activated slides for glycan microarray studies (97–99). Alternatively, the amino group can also be used to conjugate to biotin or other biomolecules (100–103) for bioassays. As shown in Fig.  22, chemical or enzymatic modification of N-acetylmannosamine (ManNAc) or mannose (Man) produced diverse sialic acid precursors, which can be coupled with pyruvate catalyzed by an E. coli (104) or a Pasteurella multocida (105) sialic acid aldolase for the formation of diverse naturally occurring and nonnatural sialic acid forms. These sialic acid forms can then be activated by a bacterial CMP-sialic acid synthetase (104) and transferred by different sialyltransferases to diverse glycans or glycoconjugates terminated with a Gal, GalNAc, or a sialic acid for the formation of a2-3-, a2-6-, or a2-8-linked sialosides (56–58, 106, 107). Efficient large-scale synthesis of diverse sialoside libraries has been successfully achieved due to the availability of highly active promiscuous bacterial sialoside biosynthetic enzymes in large amounts. The chemoenzymatic methods have been successfully

Fig. 22. One-pot three-enzyme system for chemoenzymatic synthesis of sialoside libraries.

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H. Cao and X. Chen

applied for the synthesis of sialosides with naturally occurring labile sialic acid modification such as O-acetylation and O-lactylation in addition to O-methylation and nonnatural modification at different positions of sialic acid, most of which have not been achieved by chemical or pure enzymatic synthesis. Using the one-pot three-enzyme chemoenzymatic method described earlier, a library of a2-3-linked sialosides containing naturally occurring or nonnatural sialic acid modifications were prepared in preparative scale. Three enzymes used were E. coli sialic acid aldolase, N. meningitidis CMP-sialic acid synthetase (NmCSS), and a multifunctional a2-3-sialyltransferase from P. multocida (PmST1) (56). Some representative a2-3-linked sialosides synthesized are listed in Fig. 23 (56, 106–108). Following a similar approach, a library of a2-6-linked sialosides were also prepared in preparative scales by substituting the sialyltransferase from PmST1 to a multifunctional Photobacterium damsela a2-6-sialyltransfearse (Pd2,6ST) (57, 109, 110). Some representative a2-6-linked sialosides synthesized are listed in Fig. 24 (57, 106–109).

Fig. 23. Representative a2-3-linked sialosides prepared using the one-pot multienzyme chemoenzymatic approach described in Fig. 22.

3  General Consideration on Sialic Acid Chemistry

49

Fig. 24. Representative a2-6-linked sialosides prepared using the one-pot multienzyme chemoenzymatic approach described in Fig. 22.

The one-pot three-enzyme approach was also used in sequential for a two-step one-pot multienzyme synthesis of complex GD3 oligosaccharides. In the first step, a2-3-linked sialosides were synthesized using a one-pot three-enzyme system containing PmST1 as the sialyltransferase. In the second step, a2-8-linked sialosides were synthesized from a2-3-linked sialosides as acceptors using a one-pot three-enzyme system containing a multifunctional C. jejuni a2-8-sialyltransferase (CstII). These disialyl glycans are key components for developing ganglioside-based carbohydrate vaccines (58). A modified multiple enzyme approach was also developed by the Chen group for the synthesis of CMP-sialic acids and sialosides containing a 3-fluoro-sialic acid residue (111). These fluorinated derivatives are invaluable mechanistic probes for protein crystal structural studies of sialic acid processing enzymes (Fig. 25).

50

H. Cao and X. Chen

Fig. 25. Enzymatic synthesis of fluorinated sialic acid derivatives as mechanistic probes for sialic acid processing enzymes.

7. Conditions for Preventing De-O-Acetylation or O-Acetyl Migration

O-Acetylation is the most common postglycosylational sialic acid modification (3, 55). This modification has great impact on sialic acid-dependent recognition processes. For example, 9-O-acetylation of the sialic acids on host cell surface is necessary for influenza C virus binding and subsequent invasion (112, 113) but prevents the attachment of malaria parasites (114) and influenza A and B viruses (115, 116). In the biosynthetic pathway, the acetyl groups are usually introduced to the C-7 or C-9 hydroxyl groups of sialic acid moiety after the formation of sialic linkages. Using the chemoenzymatic method described earlier, 9-O-acetylated sialic acids can be either synthesized from sialic acid by chemical selective acetylation (117) or from 6-O-acetylated sialic acid precursors (6-O-acetylated ManNAc, ManNGc, or Man) followed by enzymatic aldol-addition reaction (96, 118). Other O-acetylated sialic acids are generally obtained from the hydrolysis products of naturally occurring O-acetylated sialic acid containing structures by treated with sialidases (119).

3  General Consideration on Sialic Acid Chemistry

51

The acetyl groups are labile under both acidic and basic conditions. For example, the C-7 O-acetyl groups can spontaneously migrate to the C-8 and C-9 hydroxyl groups under physiological conditions (3, 119). The deacetylated sialic acids or its containing structures are the common byproducts generated during the synthesis or purification processes. To minimize de-O-acetylation or O-acetyl migration during the enzymatic synthesis, a suitable buffer is generally employed to maintain the reaction mediate at near neutral conditions (pH 7.5), and excess amount of enzymes are added to shorten the reaction time (57, 58, 96). A buffer in a pH range of 3.0–6.0 is frequently used to prevent de-O-acetylation or O-acetyl migration during the process of purification and analysis (120). To minimize de-O-acetylation or O-acetyl migration during storage, purified samples should be dried and kept at −20°C or −80°C.

8. Prospective and Conclusion The emerging glycan microarray platform provides a high-throughput approach for studying structure–activity relationship (SAR) of sialic acid containing structures. The synthesis of structurally defined diverse sialosides is a bottleneck step for these studies. Recently developed chemical and enzymatic synthetic approaches allow the access to some sialosides. Taking advantages of high expression level in E. coli expression system, high activity, and substrate promiscuity of bacterial sialoside biosynthetic enzymes, onepot multi-enzyme chemoenzymatic approach opens up a new avenue to quickly access many natural occurring and nonnatural sialosides in sufficient amount for diverse glycan microarray-based SAR studies.

Acknowledgments We are grateful for financial supports from Shandong University (to H.C.), the National Science Foundation of China (No. 20902087 to H.C.), the Natural Science Foundation of Shandong Province (SDNSF, No. ZR2010BM018 to H.C.), the University of CaliforniaDavis (to X.C.), the National Institutes of Health (R01GM076360 and U01CA128442 to X.C.), the National Science Foundation (CAREER Award 0548235 to X.C.), Alfred P. Sloan Foundation (to X.C.), and the Camille & Henry Dreyfus Foundation (to X.C.). X.C. is an Alfred P. Sloan Research Fellow, a Camille Dreyfus Teacher-Scholar, and a UC-Davis Chancellor’s Fellow.

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of KDN (2-keto-3-deoxy-D-glycero-Dgalacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis. J. Biol. Chem. 274, 22949–22956. 49. Kean, E. L. (1991) Sialic acid activation. Glycobiology 1, 441–447. 50. Angata, T., Kitazume, S., Terada, T., Kitajima, K., Inoue, S., Troy, F. A., 2nd, and Inoue, Y. (1994) Identification, characterization, and developmental expression of a novel alpha 2 → 8-KDN-transferase which terminates elongation of alpha 2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins. Glycoconj. J. 11, 493–499. 51. Terada, T., Kitazume, S., Kitajima, K., Inoue, S., Ito, F., Troy, F. A., and Inoue, Y. (1993) Synthesis of CMP-deaminoneuraminic acid (CMP-KDN) using the CTP:CMP-3deoxynonulosonate cytidylyltransferase from rainbow trout testis. Identification and characterization of a CMP-KDN synthetase. J. Biol. Chem. 268, 2640–2648. 52. Kawano, T., Koyama, S., Takematsu, H., Kozutsumi, Y., Kawasaki, H., Kawashima, S., Kawasaki, T., and Suzuki, A. (1995) Molecular cloning of cytidine monophospho-N-acetylneuraminic acid hydroxylase. Regulation of species- and tissue-specific expression of N-glycolylneuraminic acid. J. Biol. Chem. 270, 16458–16463. 53. Hirschberg, C. B., Goodman, S. R., and Green, C. (1976) Sialic acid uptake by fibroblasts. Biochemistry 15, 3591–3599. 54. Oetke, C., Hinderlich, S., Brossmer, R., Reutter, W., Pawlita, M., and Keppler, O. T. (2001) Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells. Eur. J. Biochem. 268, 4553–4561. 55. Yu, H., and Chen, X. (2007) Carbohydrate post-glycosylational modifications. Org. Biomol. Chem. 5, 865–872. 56. Yu, H., Chokhawala, H., Karpel, R., Wu, B. Y., Zhang, J. B., Zhang, Y. X., Jia, Q., and Chen, X. (2005) A multifunctional Pasteurella multocida sialyltransferase: A powerful tool for the synthesis of sialoside libraries. J. Am. Chem. Soc. 127, 17618–17619. 57. Yu, H., Huang, S. S., Chokhawala, H., Sun, M. C., Zheng, H. J., and Chen, X. (2006) Highly efficient chemoenzymatic synthesis of naturally occurring and non-natural alpha2,6-linked sialosides: A P. damsela alpha-2,6sialyltransferase with extremely flexible donor-substrate specificity. Angew. Chem. Int. Ed. 45, 3938–3944. 58. Yu, H., Cheng, J. S., Ding, L., Khedri, Z., Chen, Y., Chin, S., Lau, K., Tiwari, V. K., and

Chen, X. (2009) Chemoenzymatic synthesis of GD3 oligosaccharides and other disialyl glycans containing natural and non-natural sialic acids. J. Am. Chem. Soc. 131, 18467–18477. 59. Boons, G. J., and Demchenko, A. V. (2000) Recent advances in O-sialylation. Chem. Rev. 100, 4539–4565. 60. Halcomb, R. L., and Chappell, M. D. (2002) Recent developments in technology for glycosylation with sialic acid, J. Carbohydr. Chem. 21, 723–768. 61. Schmidt, R. R., and Kinzy, W. (1994) Anomeric-oxygen activation for glycoside synthesis-the trichloroacetimidate method. Adv. Carbohydr. Chem. Biochem. 50, 21–123. 62. Haberman, J. M., and Gin, D. Y. (2001) A new C(1)-auxiliary for anomeric stereocontrol in the synthesis of alpha-sialyl glycosides. Org. Lett. 3, 1665–1668. 63. Ye, X. S., Huang, X. F., and Wong, C. H. (2001) Conversion of the carboxy group of sialic acid donors to a protected hydroxymethyl group yields an efficient reagent for the synthesis of the unnatural beta-linkage. Chem. Commun., 974–975. 64. Okamoto, K., Kondo, T., and Goto, T. (1986) An effective synthesis of alpha-glycosides of N-acetylneuraminic acid by use of 2-betahalo-3-beta-hydroxyl-4,7,8,9-tetra-O-acetylneuraminic acid methyl-ester. Tetrahedron Lett. 27, 5233–5236. 65. Ito, Y., and Ogawa, T. (1987) An efficient approach to stereoselective glycosylation of N-acetylneuraminic acid-Use of phenylselenyl group as a stereocontrolling auxilliary. Tetrahedron Lett. 28, 6221–6224. 66. Martichonok, V., and Whitesides, G. M. (1996) A practical method for the synthesis of sialyl alpha-glycosides. J. Am. Chem. Soc. 118, 8187–8191. 67. CastroPalomino, J. C., Tsvetkov, Y. E., Schneider, R., and Schmidt, R. R. (1997) 8-O-Sialylation of neuraminic acid acceptor reactivity and anomeric stereocontrol. Tetrahedron Lett. 38, 6837–6840. 68. Demchenko, A. V., and Boons, G. J. (1999) A novel direct glycosylation approach for the synthesis of dimers of N-acetylneuraminic acid. Chemistry-Eur. J. 5, 1278–1283. 69. Yu, C. S., Niikura, K., Lin, C. C., and Wong, C. H. (2001) The thioglycoside and glycosyl phosphite of 5-azido sialic acid: Excellent donors for the alpha-glycosylation of primary hydroxy groups. Angew. Chem. Int. Ed. 40, 2900–2903. 70. De Meo, C., Demchenko, A. V., and Boons, G. J. (2001) A stereoselective approach for the synthesis of alpha-sialosides. J. Org. Chem. 66, 5490–5497.

3  General Consideration on Sialic Acid Chemistry 71. Ren, C. T., Chen, C. S., and Wu, S. H. (2002) Synthesis of a sialic acid dimer derivative, 2¢alpha-O-benzyl Neu5Ac-alpha-(2 → 5)Ne u5Gc. J. Org. Chem. 67, 1376–1379. 72. Sherman, A. A., Yudina, O. N., Shashkov, A. S., Menshov, V. M., and Nifant’ev, N. E. (2001) Synthesis of Neu5Ac- and Neu5Gcalpha-(2 → 6’)-lactosamine 3-aminopropyl glycosides. Carbohydr. Res. 330, 445–458. 73. Fujita, S., Numata, M., Sugimoto, M., Tomita, K., and Ogawa, T. (1994) Total synthesis of a modified ganglioside, de-N-acetyl GM(2). Carbohydr. Res. 263, 181–196. 74. Tanaka, H., Nishiura, Y., and Takahashi, T. (2006) Stereoselective synthesis of oligo-alpha-(2,8)-sialic acids. J. Am. Chem. Soc. 128, 7124–7125. 75. Crich, D., and Wu, B. L. (2008) Imposing the trans/gauche conformation on a sialic acid donor with a 5-N,7-O-oxazinanone group: effect on glycosylation stereo selectivity. Tetrahedron 64, 2042–2047. 76. De Meo, C., and Priyadarshani, U. (2008) C-5 modifications in N-acetyl-neuraminic acid: scope and limitations. Carbohydr. Res. 343, 1540–1552. 77. Meindl, P., and Tuppy, H. (1965) Uber synthetische ketoside der N-acetyl-Dneuraminsaure. 1. Darstellung einer reihe durch neuraminidase spaltbarer ketoside. Monatsh. Chem. 96, 802–815. 78. Paulsen, H., and Tietz, H. (1982) Oligosaccharide building-blocks. 43. Synthesis oftrisaccharidemoietiesfrom N-acetylneuraminic acid and N-acetyllactosamine. Angew. Chem. Int. Ed. 21, 927–928. 79. Ogawa, T., and Sugimoto, M. (1985) Synthetic studies on cell-surface glycans. 31. Synthesis of alpha-Neu5Acp-(2-)3)-D-Gal and alpha-Neu5Acp-(2-)3)-beta-D-Galp-(1-) 4)-D-Glc, Carbohydr. Res. 135, C5–C9. 80. Ress, D. K., and Linhardt, R. J. (2004) Sialic acid donors: Chemical synthesis and glycosylation. Curr. Org. Syn. 1, 31–46. 81. Schmidt, R. R., and Rucker, E. (1980) Stereoselective glycosidations of uronic-acids. Tetrahedron Lett. 21, 1421–1424. 82. Iwayama, Y., Ando, H., Ishida, H., and Kiso, M. (2009) A first total synthesis of ganglioside HLG-2. Chem-Eur. J. 15, 4637–4648. 83. Ando, H., Koike, Y., Koizumi, S., Ishida, H., and Kiso, M. (2005) 1,5-Lactamized sialyl acceptors for various disialoside syntheses: Novel method for the synthesis of glycan portions of Hp-S6 and HLG-2 gangliosides. Angew. Chem. Int. Ed. 44, 6759–6763. 84. Tanaka, H., Nishiura, Y., and Takahashi, T. (2009) Stereoselective synthesis of alpha(2,9)

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di- to tetrasialic acids, using a 5,4-N,Ocarbonyl protected thiosialoside. J. Org. Chem. 74, 4383–4386. 85. Tanaka, H., Nishiura, Y., and Takahashi, T. (2008) An efficient convergent synthesis of GP1c ganglioside epitope. J. Am. Chem. Soc. 130, 1724–7125. 86. Kondo, H., Ichikawa, Y., and Wong, C. H. (1992) Beta-sialyl phosphite and phosphoramidite-synthesis of CMP-sialic acid and sialyl oligosaccharides. J. Am. Chem. Soc. 114, 8748–8750. 87. Martin, T. J., and Schmidt, R. R. (1992) Eficient sialylation with phophite as leaving group. Tetrahedron Lett. 33, 6123–6126. 88. Hsu, C. H., Chu, K. C., Lin, Y. S., Han, J. L., Peng, Y. S., Ren, C. T., Wu, C. Y., and Wong, C. H. Highly alpha-selective sialyl phosphate donors for efficient preparation of natural sialosides. Chemistry-Eur. J. 16, 1754–1760. 89. Cai, S. T., and Yu, B. (2003) Efficient sialylation with phenyltrifluoroacetimidates as leaving groups. Org. Lett. 5, 3827–3830. 90. Yu, B. A., and Sun, J. S. (2010) Glycosylation with glycosyl N-phenyltrifluoroacetimidates (PTFAI) and a perspective of the future development of new glycosylation methods. Chem. Commun. 46, 4668–4679. 91. Tanaka, K., Goi, T., and Fukase, K. (2005) Highly efficient sialylation towards alpha(2– 3)- and alpha(2–6)-Neu5Ac-Gal synthesis: Significant ‘fixed dipole effect’ of N-phthalyl group on alpha-selectivity. Synlett, 2958–2962. 92. Garcia, B. A., Poole, J. L., and Gin, D. Y. (1997) Direct glycosylations with 1-hydroxy glycosyl donors using trifluoromethanesulfonic anhydride and diphenyl sulfoxide. J. Am. Chem. Soc. 119, 7597–7598. 93. Haberman, J. M., and Gin, D. Y. (2003) Dehydrative sialylation with C2-hemiketal sialyl donors. Org. Lett. 5, 2539–2541. 94. Ferrero, M. A., and Aparicio, L. R. Biosynthesis and production of polysialic acids in bacteria. Appl. Microbiol. Biotechnol. 86, 1621–1635. 95. Ichikawa, Y., Liu, J. L. C., Shen, G. J., and Wong, C. H. (1991) A highly efficient multienzyme system for the one-step synthesis of a sialyl trisaccharide-In situ generation of sialic acid and N-acetyllactosamine coupled with regeneration of UDP-glucose, UDP-galactose, and CMP-sialic acid. J. Am. Chem. Soc. 113, 6300–6302. 96. Yu, H., Chokhawala, H. A., Huang, S. S., and Chen, X. (2006) One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities. Nat. Protoc. 1, 2485–2492.

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C5-diversified sialic acid. Bioorg. Med. Chem. Lett. 19, 5869–5871. 109. Cheng, J. S., Huang, S. S., Yu, H., Li, Y. H., Lau, K., and Chen, X. Trans-sialidase activity of Photobacterium damsela alpha2,6-sialyltransferase and its application in the synthesis of sialosides. Glycobiology 20, 260–268. 110. Sun, M. C., Li, Y. H., Chokhawala, H. A., Henning, R., and Chen, X. (2008) N-Terminal 112 amino acid residues are not required for the sialyltransferase activity of Photobacterium damsela alpha2,6-sialyltransferase. Biotechnol. Lett. 30, 671–676. 111. Chokhawala, H. A., Cao, H. Z., Yu, H., and Chen, X. (2007) Enzymatic synthesis of fluorinated mechanistic probes for sialidases and sialyltransferases. J. Am. Chem. Soc. 129, 10630–10631. 112. Rogers, G. N., Herrler, G., Paulson, J. C., and Klenk, H. D. (1986) Influenza C virus uses 9-O-acetyl-N-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells. J. Biol. Chem. 261, 5947–5951. 113. Herrler, G., Rott, R., Klenk, H. D., Muller, H. P., Shukla, A. K., and Schauer, R. (1985) The receptor-destroying enzyme of influenza C virus is neuraminate-O-acetylesterase. Embo J. 4, 1503–1506. 114. Schauer R, S. H., Pommerencke J, Iwersen M, Kohla G. (2001) Metaolism and role of O-acetylated sialic acids. In Molecular immunology of complex carbohydrates 2, edited by Wu AM, Plenum, New York, 2001., 325–342. 115. Gottschalk, A. (1957) Neuraminidase: the specific enzyme of influenza virus and Vibrio cholerae. Biochim. Biophys. Acta 23, 645–646. 116. Klenk, E., Faillard, H., and Lempfrid, H. (1955) Enzymatic effect of the influenza virus. Hoppe Seylers Z Physiol. Chem. 301, 235–246. 117. Ogura, H., Furuhata, K., Sato, S., Anazawa, K., Itoh, M., and Shitori, Y. (1987) Studies on sialic acids. 6. Synthesis of 9-O-acetyl-sialic acid and 4-O-acetyl-sialic acid. Carbohydr. Res. 167, 77–86. 118. Liu, J. L. C., Shen, G. J., Ichikawa, Y., Rutan, J. F., Zapata, G., Vann, W. F., and Wong, C. H. (1992) Overexproduction of CMO-sialic acid synthesis. J. Am. Chem. Soc. 114, 3901–3910. 119. Lewis, A. L., Nizet, V., and Varki, A. (2004) Discovery and characterization of sialic acid O-acetylation in group B Streptococcus. Proc. Natl. Acad. Sci. USA 101, 11123–11128. 120. Varki, A., and Diaz, S. (1984) The release and purification of sialic acids from glycoconjugates: methods to minimize the loss and migration of O-acetyl groups. Anal. Biochem. 137, 236–247.

Chapter 4 Synthesis of Azido-Functionalized Carbohydrates for the Design of Glycoconjugates Samy Cecioni, David Goyard, Jean-Pierre Praly, and Sébastien Vidal Abstract As carbohydrates play a major role in numerous biological processes through their interactions with lectins and also appear as one of the most crucial post-translational modifications of proteins, chemists have developed several approaches for the design of glycoconjugates based on a series of conjugation methodologies. The recent development of copper(I)-catalyzed azide-alkyne cycloaddition (CuACC) paved the way to a novel conjugation strategy in which azido-functionalized carbohydrate derivatives can be readily connected to alkyne-functionalized (bio)molecules. This so-called “click chemistry” methodology has now found numerous applications both in chemistry and biology. The azido moiety can be introduced either directly at the anomeric carbon of the carbohydrate derivative, or attached to a spacer arm. We describe here the syntheses of 2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl azide as well as 1-azido-3,6-dioxaoct-8-yl 2,3,4, 6-tetra-O-acetyl-b-d-galactopyranoside and 1-azido-3,6-dioxaoct-8-yl 2,3,6,2¢,3¢,4¢,6¢-hepta-O-acetyl-bd-lactoside. These molecules can then be used in the construction of glycoconjugates to find applications in chemical biology. Key words: Carbohydrates, Click chemistry, 1,3-Dipolar cycloaddition, Azide, Alkyne, Glycoconju­ gates, Glycosylation, Triazole

1. Introduction Biological processes occur mainly through receptor–ligand interactions or chemical reactions catalyzed by enzymes. Among these processes, lectin–carbohydrate interactions are involved in both normal and pathological phenomena such as cell–cell communication, viral infection, cancer metastasis or even fecundation (1, 2). The design of high affinity ligands of lectins is therefore a promising strategy for the treatment of some pathological disorders involving

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such receptor–ligand interactions. In this respect, chemists have designed several types of multivalent glycoconjugates (3–7) mimicking the natural multivalency associated with such interactions, a phenomenon usually termed the “cluster effect” (8, 9). Multivalent glycoconjugates are macromolecules displaying multiple copies of a carbohydrate moiety connected to a central core scaffold. Chemists have studied the possible strategies to connect carbohydrate derivatives to its scaffold. The chemical reaction selected as the conjugation technique must provide the desired glycoconjugates in high yields and short reaction times, with high chemo- and regioselectivities and must be usually compatible with aqueous media due to the properties of most biomolecules. The recent development of Cu(I)-catalyzed azide-alkyne cycloaddition (CuACC) (10–12) provides a very reliable and powerful technique for the conjugation of azides and alkynes, which are highly stable in biological media and also orthogonal to most functional groups present in biomolecules. This approach has found applications for chemical ligation of carbohydrate probes to synthetic multivalent scaffolds (13). The synthesis of glycoconjugates through CuAAC can be achieved from azido-functionalized carbohydrates bearing the azido moiety attached either directly at the anomeric center or connected through a spacer arm (Fig. 1).

Fig. 1. Schematic representation for the conjugation of azido-functionalized carbohydrates to alkynylated scaffolds (grey ball). “Click” refers to the conjugation through copper(I)-catalyzed azide-alkyne cycloaddition (CuACC).

4  Synthesis of Azido-Functionalized Carbohydrates for the Design…

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2. Materials 2.1. General methods

Compounds 1 to 16 were from commercial sources and used without further purification. 1. N,N-Dimethylformamide (DMF, >99%, Sigma-Aldrich). 2. Ethyl acetate (EtOAc, technical, SDS). 3. Petroleum ether (PE, technical, SDS). 4. Dimethylsulfoxide (DMSO, 99.7%, Acros Organics). 5. 2-[2-(2-chloroethoxy)ethoxy]ethanol (97%, Acros Organics). 6. CH2Cl2 (Acros Organics). 7. Tin(IV) chloride: 1 M SnCl4 solution in CH2Cl2. 8. Silver trifluoroacetate (98%, Acros Organics). 9. Sodium azide (99%, Sigma-Aldrich). 10. Tetra-n-butyl ammonium iodide (98%, Sigma-Aldrich). 11. 2,3,4,6-tetra-O-acetyl-a-d-glucopyranosyl bromide 1 (99%, Sigma-Aldrich). 12. 1,2,3,4,6-penta-O-acetyl-b-d-galactopyranose 3 (99%, Carbo­ synth) (14). 13. Diethyl ether (technical grade, Sigma-Aldrich). 14. Magnesium sulfate (MgSO4, technical grade, VWR). 15. Sodium sulfate (Na2SO4, technical grade, Laurylab). 16. Sodium hydrogenocarbonate (NaHCO3, technical grade, Laurylab). 17. Sodium chloride (NaCl, technical grade, Laurylab). 18. 1,2,3,6,2¢,3¢,4¢,6¢-Octa-O-acetyl-d-lactose 5 was prepared from previously reported procedures (15). 19. Thin-layer chromatography plates: (TLC) was carried out on aluminum sheets coated with silica gel 60F254 (Merck). 20. TLC charring (9:1 v/v).

solution:

10%

H2SO4

in

EtOH/H2O

21. Geduran® silica gel Si 60 (40–63 mm) purchased from Merck (Darmstadt, Germany). 2.2. Analytical Techniques

1. Saturated aqueous NaHCO3: The solution was prepared using an excess of solid (NaHCO3, >100 g/L) in water in order to obtain a saturated aqueous solution meaning that some solid was still present at the bottom of the bottle even after vigorous shaking. 2. Saturated aqueous NaCl (Brine): The solution was prepared using an excess of solid (NaCl, >360 g/L) in water in order

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to obtain a saturated aqueous solution meaning that some solid was still present at the bottom of the bottle even after vigorous shaking. 3. Calcium hydride: CaH2. 4. Dry Dichloromethane (CH2Cl2, Carlo Erba): Dichloromethane (CH2Cl2, Carlo Erba) was distilled over CaH2 under argon atmosphere. 5. Proton (1H) and carbon (13C) NMR spectra were recorded at 298  K using a Bruker Avance DRX300 spectrometer at 300  MHz with the residual solvent as the internal standard (CHCl3 at 7.26 ppm for 1H and 77.16 for 13C). 6. ESI mass spectra were recorded in the positive mode using a Thermo Finnigan LCQ spectrometer. 7. Optical rotations were measured using a Perkin Elmer polari­ meter and values are given in 10−1 deg/cm2/g.

3. Methods The azido-functionalized carbohydrates can be conjugated to the desired (bio)molecules as their acetylated precursors, which can then be unmasked to the corresponding hydroxylated derivatives under standard conditions (e.g. NaOH/H2O, MeOH/Et3N/ H2O, NaOMe/MeOH). This final deprotection step can be avoided by using the hydroxylated azido-functionalized carbohydrates but the purification of the intermediates is sometimes troublesome. Acetylated precursors are usually preferred for most of the synthetic organic multivalent glycoconjugates since their purification can be readily achieved by silica gel column chromatography, while bioorganic glycoconjugates (e.g. proteins, peptides, DNA) would be conjugated with hydroxylated carbohydrate probes. Water used for liquid/liquid extractions was de-ionized and bacteria-free by treatment with a EASYpureRoDI purification system. All reactions were performed under argon. Solutions in organic solvents were dried with anhydrous Na2SO4 and concentrated under reduced pressure at 30–35°C. Compounds were visualized by UV light (l = 254 nm) and/or by charring with 10% H2SO4 in EtOH/H2O (9:1 v/v). Purification was performed by flash-chromatography with Geduran® silica gel Si 60 (40–63 mm) purchased from Merck (Darmstadt, Germany). 3.1. Synthesis of Glycosyl Azides

When subjected to CuAAC reaction, glycosyl azides displaying an azide moiety directly at the anomeric center of carbohydrates will afford N-heterocyclic derivatives, which could present interesting properties and possible resistance to carbohydrate processing

4  Synthesis of Azido-Functionalized Carbohydrates for the Design… OAc O

AcO AcO 1

AcO

Br

a 81 %

AcO AcO

OAc O 2

61

N3

OAc

Scheme 1. Synthesis of the azido-functionalized glucose derivative 2. Reagents and conditions: (a) NaN3, DMSO, r.t., 3 h.

enzymes (e.g. glycosidases, glycosyltransferases). This strategy could therefore prove beneficial for the design of enzyme-resistant glycoconjugates, which might not be eliminated too quickly from living systems (e.g. cells, bacteria, viruses, animals). The synthesis is usually achieved from the peracetylated brominated carbohydrates available from commercial sources. Never­ theless, glycosyl bromides are water and temperature sensitive and may decompose to the corresponding hemiacetal. The reactions must therefore be performed under strictly anhydrous conditions. The anomeric bromine atom is displaced by the azide anion via a SN2 mechanism (i.e. with inversion of configuration at the anomeric carbon) to obtain the desired azido-functionalized carbohydrates (Scheme 1). In addition, 1,1-diazides can be readily prepared from the bis-halogenated carbohydrate precursor (16). 3.1.1. Synthesis of the Glucose Derivative 2

1. Introduce sodium azide (Caution: see Note 1) (1.58  g, 24.2 mmol) in a 100-mL round-bottom flask. Close the flask with a rubber septum and inert with argon. 2. Add DMSO (40 mL) and stir the suspension at room temperature until complete dissolution of the solid. 3. Open the flask and introduce quickly peracetylated glucosyl bromide 1 (5 g, 12.2 mmol) and close the flask again with the rubber stopper then inert with argon. 4. Stir the reaction at room temperature for 3 h when TLC (eluent: Et2O/PE 1:1) shows complete conversion of the starting material 1 (Rf = 0.66) and the formation of the desired azide 2 as a more polar product (Rf = 0.49). 5. Dilute the solution with Et2O (150 mL) and wash with H2O (100 mL). Extract the aqueous layer with Et2O (2 × 50 mL). Combine organic layers and wash with H2O (4 × 150 mL), dry over MgSO4, filter the solid, wash with Et2O (2 × 15 mL) and concentrate the filtrate under reduced pressure on a rotatory evaporator (Buchi, 30°C, 20 mmHg). 6. Dissolve the crude material (beige solid) in a minimum of Et2O and precipitate with petroleum ether. Filter the solid to obtain a first crop. Concentrate the filtrate and precipitate again more solid off the solution as previously from Et2O and petroleum ether.

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Fig. 2. Proton NMR spectrum (1H, 300 MHz) of compound 2 in CDCl3 recorded at 25°C.

7. Both precipitations afford the product 2 as an analytically pure white powder (3.12 g, 9.83 mmol, 81%). Analytical data for 2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl azide (2, see Ref. 17 and Figs. 2 and 3): Rf = 0.49 eluent: Et2O/PE 1:1; M.p. = 125.6–126.5°C; [a]D = −29° (c = 2, CHCl3); 1H NMR (CDCl3, 300 MHz) d 5.22 (pdd, 1H, J = 9.4 Hz, H-3), 5.10 (pdd, 1H, J = 9.7 Hz, H-4), 4.96 (pdd, 1H, J = 9.2 Hz, H-2), 4.65 (d, 1H, J = 8.8 Hz, H-1), 4.28 (dd, 1H, J = 12.5, 4.8 Hz, H-6), 4.17 (dd, 1H, J = 12.5, 2.3 Hz, H-6¢), 3.79 (ddd, 1H, J = 9.9, 4.7, 2.4 Hz, H-5), 2.10, 2.08, 2.03, 2.01 (4s, 4 × 3 H, 4 × CH3CO); 13C NMR (CDCl3, 75 MHz) d 170.5, 170.0, 169.3, 169.1 (4s, 4 × CH3CO), 87.8 (C-1), 73.9 (C-5), 72.5 (C-3), 70.6 (C-2), 67.8 (C-4), 61.6 (C-6), 20.6, 20.4, 20.4, 20.4 (4s, 4 × CH3CO). 3.2. Synthesis of w-AzidoTriethyleneglycol Glycosides

Glycosylation reactions (18) usually require the activation of the anomeric carbon atom with a proper leaving group but the synthesis of such activated derivatives is adding up to three synthetic steps and the corresponding products are not always stable enough to be stored under simple conditions. Peracetylated carbohydrates are easily prepared from the corresponding native carbohydrates or can be also purchased from most chemical suppliers. They are very

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Fig. 3. Carbon NMR spectrum (13C, 75 MHz) of compound 2 in CDCl3 recorded at 25°C. AcO AcO

OAc O 3

AcO AcO

OAc O

O OAc AcO 5

OAc OAc OAc O

a,b 60%

AcO AcO

a,b OAc 62% OAc

OAc O O OAc

AcO AcO

OAc O

O

O

N3

4

O OAc AcO 6

OAc O OAc

O

O

O

N3

Scheme 2. Syntheses of the azido-functionalized galactose and lactose derivatives. Reagents and conditions: (a) SnCl4, CF3CO2Ag, H(OCH2CH2)3Cl, CH2Cl2, r.t., 2–3 h, (b) NaN3, nBu4NI, DMF, 70°C, 16 h. Yields are indicated for the complete two-step process.

stable and can be stored for longer periods of time. The glycosylation from peracetylated carbohydrates usually requires a Lewis acid as a promoter of the reaction (e.g. boron trifluoride etherate, titanium(IV) chloride). We have recently revisited a glycosylation protocol (19) involving tin(IV) chloride (SnCl4) and silver trifluoroacetate (CF3CO2Ag) for the large-scale synthesis (typically 5  g and up to 20  g) of w-azido-triethyleneglycol glycosides and obtained reproducibly the desired 1,2-trans glycosides in short reaction times (typically 2–3 h) and high yields (Scheme 2). When a mixture of anomeric acetates is used, the b-derivative will be consumed in a few hours (~2–3 h) while the a-anomer will remain

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unchanged in the reaction mixture over a long period of time (>24 h). The b-configured sugar peracetates will therefore always be preferred to their a-isomers due to the greater reactivity of the b-anomers. 3.2.1. Synthesis of the Galactose Derivative 4

1. Introduce the peracetylated galactose 3 (5 g, 12.8 mmol) and silver trifluoroacetate (4.24 g, 19.2 mmol) in a 250-mL roundbottom flask. Close the flask with a rubber septum and inert with argon. The reaction flask will be kept away from light by wrapping into aluminum foil in order to prevent the silver salts from decomposing slowly. 2. Add freshly distilled dichloromethane (120  mL) and 2-[2-(2-chloroethoxy)ethoxy]ethanol (2.80 mL, 19.2 mmol) dropwise to obtain a solution. Keep an argon atmosphere over the reaction. 3. Add SnCl4 (1 M in CH2Cl2, 38.4 mL, 38.4 mmol) dropwise within ~30 min at 0°C (ice bath) to the stirred solution. Stir the solution under argon at room temperature. 4. Monitor the reaction completion by TLC (eluent: PE-EtOAc 1:1, Rf = 0.60 for 3 and 0.34 for 4). Disappearance of the starting material was observed within 1–3 h, occasionally with the mixture turning to a pale pink color. 5. Add a solution of saturated aqueous NaHCO3 (Solution 1, 100 mL) in order to adjust pH above 8 (pH paper) and stir the solution vigorously for 20 min (see Note 2). 6. Dilute the resulting biphasic system with 500 mL of water (see Note 3) and extract the aqueous layer with CH2Cl2 (3 × 150 mL). 7. Combine the organic layers and wash (see Note 4) with saturated aqueous NaHCO3 (Solution 1, 150  mL), water (3 × 150  mL), brine (Solution 2, 2 × 150  mL) and dry over sodium sulfate (Na2SO4). 8. Filter the solid and wash with CH2Cl2 (2 × 50 mL). Evaporate the solvent on a rotatory evaporator (Buchi, 30°C, 20 mmHg) to obtain a crude pale yellow gum and transfer the gum into a 250-mL round bottom flask by dissolving in CH2Cl2 and ­re-evaporating the solvent (see Note 5). 9. Add sodium azide (Caution: see Note 1) (4.16 g, 64.0 mmol) and tetra-n-butyl ammonium iodide (4.73  g, 12.8  mmol). Close the flask with a rubber septum and inert with argon. 10. Add DMF (150 mL) and stir the mixture at 70°C under argon for 16 h. 11. Cool the reaction mixture to room temperature, filter the solids off and wash with EtOAc (3 × 100 mL).

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12. Dilute the filtrate with EtOAc to obtain a total volume of 1 L. Wash the organic layer (see Note 6) with saturated aqueous NaHCO3 (Solution 1, 3 × 300 mL), water (3 × 500 mL), brine (Solution 2, 400 mL) and dry over sodium sulfate (Na2SO4). 13. Filter the solid and wash with EtOAc (2 × 50 mL). Evaporate the solvent on a rotatory evaporator (Buchi, 30°C, 20 mmHg) to obtain a crude yellow to orange gum. 14. Purify the product by silica gel column chromatography (eluent: PE-Et2O 1:4, internal diameter = 45  mm; length = 250– 300 mm) to obtain 4 (3.88 g, 60% yield) as a pale yellow gum (see Note 7). Analytical data for 1-azido-3,6-dioxaoct-8-yl 2,3,4,6-tetra-Oacetyl-b-d-galactopyranoside (4, see Refs. 20–22 and Fig.  4): Rf = 0.34 (1:1 PE-EtOAc). 1H NMR (CDCl3) d 1.99, 2.04, 2.07, 2.15 (4s, 4 × 3H, 4 × CH3CO), 3.40 (t, 2H, J = 5.0 Hz, CH2N3), 3.60– 3.82 (m, 9H, OCH2), 3.88–4.02 (m, 2H, OCH2, H-5), 4.08–4.20 (m, 1H, H-6, H-6¢), 4.59 (d, 1H, J1,2 = 7.9 Hz, H-1), 5.02 (dd, 1H, J3,4 = 3.4 Hz, J3,2 = 10.5 Hz, H-3), 5.21 (dd, 1H, J2,1 = 7.9 Hz, J2,3 = 10.5 Hz, H-2), 5.39 (dd, 1H, J4,5 = 0.7 Hz, J4,3 = 3.4 Hz, H-4). 13 C NMR (CDCl3) d 20.3, 20.4, 20.4, 20.5 (4s, 4 × CH3CO), 50.4 (CH2N3), 61.0 (C-6), 66.8 (C-4), 68.5 (C-2), 68.8, 69.8, 70.1 (3s, 3 × CH2O), 70.4 (C-3), 70.4, 70.5 (2  s, 2 × CH2O), 70.6 (C-5), 101.1 (C-1), 169.2, 169.9, 170.0, 170.1 (4s, 4 × CH3CO).

2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 .0

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 −0.5 f1 (ppm)

Fig. 4. Proton NMR spectrum (1H, 300 MHz) of compound 4 in CDCl3 recorded at 25°C. Traces of EtOAc were observed in the spectrum (triplet at 1.25 ppm).

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Fig. 5. Proton NMR spectrum (1H, 300 MHz) of compound 6 in CDCl3 recorded at 25°C. Traces of EtOAc were observed in the spectrum (triplet at 1.25 ppm).

3.2.2. Synthesis of the Lactose Derivative 6

The synthesis of the lactose derivative 6 can be performed under the same conditions as presented for compound 4. The only difference is in the purification over silica gel where a gradient was used from 7:3 to 2:3 PE-EtOAc with 10% increase every 400  mL. Compound 6 was obtained as a pale yellow gum in 62% yield from 5 g of peracetylated lactose 7. Analytical data for 1-azido-3,6-dioxaoct-8-yl 2,3,6,2¢,3¢,4¢, 6¢-hepta-O-acetyl-b-d-lactoside (6, see Ref. 23 and Fig. 5): Rf = 0.26 (1:1 PE–EtOAc). 1H NMR (CDCl3) d 1.94, 2.02, 2.04, 2.10, 2.13 (5 s, 21H, CH3CO), 3.37 (t, 2H, J = 6.0 Hz, CH2N3), 3.57–4.15 (m, 16H), 4.42–4.48 (m, 2H), 4.59 (d, 1H, J = 7.9  Hz), 4.87 (dd, 1H, J = 8.7  Hz, J = 8.9  Hz), 4.93 (dd, 1H, J = 2.8  Hz, J = 8.7 Hz), 5.09 (dd, 1H, J = 8.0 Hz, J = 11.5 Hz), 5.17 (t, 1H, J = 11.5 Hz), 5.31–5.33 (m, 1H).

4. Notes 1. CAUTION: Sodium azide, when inhaled, is highly toxic and cases of death have been reported (MSDS J.T. Baker). Precautions must be taken when weighing the material such as using a powder mask and a teflon spatula (metallic spatula may cause explosion). The azidation reaction was performed behind

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a plastic shield due to the potential explosion. DMF is used as a polar solvent favoring the reaction but also to maintain a basic pH (>8) of the solution. Hydrazoic acid (HN3) may be formed in acidic pH, which may explode and/or, when inhaled, may cause intoxication, damage of the central nervous system and blood pressure effects. DMSO can be used for azidation procedures but halogenated solvents (CHCl3 and CH2Cl2) must be avoided as they may lead to unstable azide derivatives prone to explosive decomposition. 2. When performing the reaction on a larger scale (typically > 10 g), it is better to transfer the solution in a 1 L erlenmeyer containing 500  mL of saturated aqueous NaHCO3 (Solution 1). Special care should be taken with the formation of foam during the neutralization of tin(IV) chloride with NaHCO3. 3. The solid present in the suspension may be filtered through a bed of Celite for small scale syntheses (>1 g). 4. The water layers should be disposed with proper care considering the presence of tin and silver salt. 5. The removal of trace amounts of CH2Cl2 should be considered seriously to avoid the formation explosive species with sodium azide. 6. The aqueous layers must not be in contact with acidic solutions to avoid the formation of hydrazoic acid (see Note 1). 7. TLC analyses did not show a significant difference between the polarities of the chlorinated precursors and the azido compounds.

Acknowledgments The authors thank the University Claude Bernard Lyon 1 and the CNRS for financial support. S.C. thanks the Région Rhône-Alpes (Cluster de Recherche Chimie) for a PhD stipend and D.G. thanks the Agence Nationale de la Recherche (ANR) for a PhD stipend. References 1. Dwek, R. A. (1996) Glycobiology: Toward Understanding the Function of Sugars. Chem. Rev. 96, 683–720. 2. Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G. W., and Marth, J. Essentials of glycobiology, Cold Spring Harbor Laboratory Press: New York, 1999. 3. Flitsch, S. L. (2000) Chemical and enzymatic synthesis of glycopolymers. Curr. Opin. Chem. Biol. 4, 619–625.

4. Cloninger, M. J. (2002) Biological applications of dendrimers. Curr. Opin. Chem. Biol. 6, 742–748. 5. de la Fuente, J. M., and Penadés, S. (2006) Glyconanoparticles: Types, synthesis and applications in glycoscience, biomedicine and material science. Biochim. Biophys. Acta. 1760, 636–651. 6. Imberty, A., Chabre, Y. M., and Roy, R. (2008) Glycomimetics and Glycodendrimers as High

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Affinity Microbial Anti-adhesins. Chem. Eur. J. 14, 7490–7499. 7. Chabre, Y. M., and Roy, R. R. (2010) Design and Creativity in Synthesis of Multivalent Neoglycoconjugates. Adv. Carbohydr. Chem. Biochem. 63, 165–393. 8. Mammen, M., Choi, S.-K., and Whitesides, G. M. (1998) Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew. Chem. Int. Ed. 37, 2754–2794. 9. Lundquist, J. J., and Toone, E. J. (2002) The Cluster Glycoside Effect. Chem. Rev. 102, 555–578. 10. Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B. (2002) A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. 41, 2596–2599. 11. Tornøe, C.W., Christensen, C. and Meldal, M. (2002) Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. J. Org. Chem. 67, 3057–3064. 12. Meldal, M., and Tornøe, C. W. (2008) Cu-Catalyzed Azide-Alkyne Cycloaddition. Chem. Rev. 108, 2952–3015. 13. Dondoni, A. (2007) Triazole: the Keystone in Glycosylated Molecular Architectures Cons­ tructed by a Click Reaction. Chem. Asian J. 2, 700–708. 14. Wolfrom, M. L., and Thompson, A. (1963) in Methods in Carbohydrate Chemistry, Academic Press: New York, vol. 2, pp. 211–215. 15. Westerlind, U., Hagback, P., Tidbäck, B., Wiik, L., Blixt, O., Razib, N., and Norberg, T. (2005) Synthesis of deoxy and acylamino derivatives of lactose and use of these for probing the active site of Neisseria meningitidis

N-acetylglucosaminyltransferase. Carbohydr. Res. 340, 221–233. 16. Praly, J.-P., Péquery, F., Di Stefano, C., and Descotes, G. (1996) Synthesis of protected glycopyranosylidene 1,1-diazides. Synthesis, 577–579. 17. Tropper, F. D., Anderson, F. O., Braun, S., and Roy, R. R. (1992) Phase Transfer Catalysis as a General and Stereoselective Entry into Glycosyl Azides from Glycosyl Halides. Synthesis, 618–620. 18. Fugedi, P. Glycosylation methods In The organic chemistry of carbohydrates. Levy, D. E., and Fugedi, P. Eds, CRC Taylor and Francis: New York, 2006, Chapter 4, pp. 89–151. 19. Xue, J. L., Cecioni, S., He, L., Vidal, S., and Praly, J.-P. (2009) Variations on the SnCl4 and CF3CO2Ag-promoted glycosidation of sugar acetates: a direct, versatile and apparently simple method with either a or b stereocontrol. Carbohydr. Res. 344, 1646–1653. 20. Szurmai, Z., Szabo, L., and Liptak, A. (1989) Diethylene and triethylene glycol spacers for the preparation of neoglycoproteins. Acta Chim. Hung. 126, 259–269. 21. Chevolot, Y., Bouillon, C., Vidal, S., Morvan, F., Meyer, A., Cloarec, J.-P., et  al. (2007) DNA-Based Carbohydrate Biochips: A Platform for Surface Glyco-Engineering. Angew. Chem. Int. Ed. 46, 2398–2402. 22. Cecioni, S., Lalor, R., Blanchard, B., Praly, J.-P., Imberty, A., Matthews, S.E., and Vidal, S. (2009) Achieving High Affinity towards a Bacterial Lectin through Multivalent Topological Isomers of Calix[4]arene Glycoconjugates. Chem. Eur. J. 15, 13232–13240. 23. Li, J., Zacharek, S., Chen, X., Wang, J., Zhang, W., Janczuk, A., and Wang, P. G. (1999) Bacteria targeted by human natural antibodies using a-gal conjugated receptor-specific glycopolymers. Bioorg. Med. Chem. 7, 1549–1558.

Chapter 5 Polypyrrole-Oligosaccharide Microarray for the Measurement of Biomolecular Interactions by Surface Plasmon Resonance Imaging Julia Bartoli, André Roget, and Thierry Livache Abstract The polypyrrole approach initially developed for the construction of DNA chips, has been extended to other biochemical compounds such as proteins and more recently oligosaccharides. The copolymerization of a pyrrole monomer with a biomolecule bearing a pyrrole group by an electrochemical process allows a very fast coupling of the biomolecule (probe) to a gold layer used as a working electrode. Fluorescencebased detection is the reference method to detect interactions on biochips; however an alternative label free method, could be more convenient for rapid screening of biointeractions. Surface Plasmon Resonance (SPRi) is a typical label-free method for real time detection of the binding of biological molecules onto functionalized surfaces. This surface sensitive optical method is based upon evanescent wave sensing on a thin metal layer. The SPR approach described herein is performed in an imaging geometry that allows simultaneous monitoring of biorecognition reactions occurring on an array of immobilized probes (chip). In a SPR imaging experiment, local changes in the reflectivity are recorded with a CCD camera and are exploited to monitor up to 100 different biological reactions occurring onto the molecules linked to the polypyrrole matrix. This method will be applied to oligosaccharide recognition. Key words: Polypyrrole, Oligosaccharide, Array, Biochip, Surface plasmon resonance

1. Introduction Recently, there has been an increasing interest for the development of microarray devices adapted to the high-throughput analysis of biological events. The presentation of a large set of probes (DNA, proteins …) in the format of an array provides a mean to simultaneously monitor multiple binding events, with a compatible detection method. A number of techniques have been developed to

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immobilize these probes on a high variety of supports. More recently, in the wake of DNA and proteins, the study of oligosaccharides structures and protein-oligosaccharide interactions became of special interest, and the development of carbohydrate chips is an emerging field. The understanding of the different roles of oligosaccharides is in an early stage as compared with our knowledge of DNA and proteins. If DNA and protein syntheses are well developed, oligosaccharide synthesis is much more difficult to perform and remains challenging. The development of DNA and protein chips, including on chip synthesis (1, 2) has been particularly important, but only a few strategies are described to prepare oligosaccharides chips. Although these approaches are of general interest for these biomolecules, the problem is not so easy to solve. A few approaches of in situ synthesis are described but suffer from the extreme complexity of the chemistry (3). A number of methods involve either the chemical synthesis in solution (4, 5) the enzymatic synthesis (6) or the extraction of the biomolecule and the conjugation to a chemical moiety allowing the immobilization on a great variety of support (glass, plastic, gold…). A comparison between different supports was done by Angenendt showing high signal uniformity and reproducibility of most plain glass and plastic slides (7). An efficient method, namely electrospotting, has been developed to covalently immobilize DNA sequences or proteins on gold surfaces using a simplified electrochemical process (8). This process allows the grafting of biomolecules on a nonstructured conducting layer (homogenous gold film). Initially developed for DNA chips, this approach has been adapted for oligosaccharides but necessitates a functionalization step. A number of functions are available on the oligosaccharide backbone and are a potential source of anchoring sites but as for DNA and proteins it is necessary that the chemical coupling doesn’t involve a functional domain of the molecule. For this reason it is more convenient to modify the oligosaccharide reducing extremity, although there is a lack of reactivity in this position (see Note 1). We describe in this chapter an approach combining the functionalization of oligosaccharides in two steps, the fast grafting of these oligosaccharides on a microarray, via an electrodeposition process and a polypyrrole film, and the detection of oligosaccharide–protein interactions by Surface Plasmon Resonance imaging (SPRi). SPRi is a label-free, surface sensitive technique, which allows the real-time measurement of biological binding events occurring onto a gold surface bearing covalently attached probes. An application example is given, using glycosaminoglycans (GAG) or GAG fragments as models of oligosaccharide. For that purpose, heparin and heparin derived octasaccharide were modified by a pyrrole moiety on their reducing extremity and immobilized on a gold surface through electrocopolymerization. The interactions with proteins (SDF-1alpha and IFN-gamma) on the surface are monitored without labelling in real time by SPRi.

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2. Materials 2.1. Synthesis of the NHS Activated Pyrrole: N-Hydro­xy succinimidyl-6(Pyrrole-yl)-Caproate (NHS-Pyrrole)

1. 2,5-Dimethoxytetrahydrofuran, mixture of cis- and trans-isomers 99% (Acros organics). 2. 6-Aminocaproic acid 99 + % (Acros organics). 3. 1,4-Dioxan, synthesis grade. 4. Acetic acid, purex analytical grade. 5. N-hydroxysuccinimide 97% (Aldrich). 6. N,N¢-Dicyclohexylcarbodiimide 99% (Acros organics). 7. N,N-Dimethylformamide (DMF) for analysis. 8. Dimethyl sulfoxide, analytical reagent. 9. Dichloromethane (CH2Cl2) purex for analysis. 10. Ethyl Alcohol anhydrous (EtOH), for analysis. 11. Silica gel PF254 containing CaSO4 for preparative layer chromatography (MERCK). 12. Chloroform-d, 99.9 atoms % D, stabilized with 0.5% wt silver foil.

2.2. Two Steps Preparation of Pyrrolylated Oligosaccharides

1. 15 kDa Heparin (Sigma-Adrich). 2. Heparin fragment octasaccharide (dp8) obtained by heparinase I depolymerization (9). 3. Adipic dihydrazide (Merck). 4. First coupling buffer: 100 mM CH3COONa, adjusted at pH 5 with acetic acid. Store at 4°C. 5. NHS-Pyrrole, synthesized in the laboratory. 6. Second coupling buffer: 50% Phosphate buffer saline (PBS, Euromedex)/50% Dimethyl sulfoxide (DMSO, Sigma-Aldrich) v/v. 7. Dialysis membrane MWCO = 1,000 Da (Standard RC Dialysis Tubing, Pre-treated, Spectra Por 7, Spectrum labs). 8. SpeedVac Concentrator system. 9. Freeze-dryer.

2.3. Quantification of the Coupling Reaction: Tnbsa Method

1. TNBSA solution: 5% (w/v) Picrylsulfonic acid in H2O (P229710 mL, Sigma-Aldrich). The solution has to be stored at 4°C, and to be diluted at 1:10 in buffer 1, just before use. 2. Buffer 1 (reacting buffer): 100  mM Na2CO3/NaHCO3, pH 9.6 (the solution can be stored several months at 4°C). 3. Buffer 2 (stopping buffer): 98.5% 100  mM NaH2PO4/1,5% 100 mM Na2SO4 (each solution has to be stored at –20°C, and mixed before use).

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4. 10  mM stock solution of adipic dihydrazide in buffer 1, for calibration, prepared extemporaneously. 5. 96-well plate (Nunc). 6. Victor 1420 Multilabel Counter. 2.4. Oligosaccharides Immobilization on SPR Prisms: Electrospotting

1. 1  M Pyrrole (Tokyo Kasei, Japan) in anhydrous acetronitrile 99.8% (Sigma-Aldrich). Store at −20°C in a dark bottle to ­minimize pyrrole oxidation. 2. Pyrrole-oligosaccharide conjugates (prepared as in Sub­ heading 3.2), at different concentrations. 3. Electrocopolymerization buffer: 50  mM NaH2PO4, 50  mM NaCl, 10% (p/v) glycerol adjusted at pH 6.8 with a NaOH solution. Store at 4°C (−20°C for a conservation longer than 2 weeks). 4. Glass prisms (n = 1.717 at l = 633 nm) covered by 50 nm-thick gold layer (Genoptics, Orsay, France). 5. Pre-treatment solution: mixture of 70% H2SO4 and 30% H2O2 (v/v) (prepared just before use). Care must be taken as this reaction is highly exothermic and reacts strongly with organic compounds. 6. 96-well plate (Nunc, conical, #249946). 7. Microarrayer for the immobilization the oligosaccharides on the chip (Genomic Solutions), with an acquisition card U12 (LabJack), and a software developed from Labview. 8. Ceramic needle with an inox extremity (X-Tend Pin, 350 mm external diameter/150 mm internal diameter, Genoptics, Orsay, France), containing a platinum wire (diameter 200 mm).

2.5. SPRi Interactions Monitoring

1. “SPRi lab”: SPR imaging system (Genoptics, Orsay, France) and Software Genovision (Genoptics) and Labview. The apparatus is placed in an air oven, under controlled temperature (25°C). 2. 7.2 mL PEEK flow cell. 3. Syringe pump (Cavro/Tecan XL3000) and software developed from LabView. 4. Degasser (Alltech). 5. Injection valve with 500  mL injection loop and tubing in PEEK. 6. Injection and Washing buffer: 10 mM HEPES, 150 mM NaCl, 0,005% Tween 20, pH 7.4. 7. Saturation buffer: Bovine Serum Albumin (BSA, SigmaAldrich) 1% (w/v) in washing buffer.

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8. Regeneration buffers: (a) 1 M NaCl (Sigma-Aldrich) in washing buffer. (b) 1% (w/v) Sodium Dodecyl Sulphate (SDS) (Euromedex) in water. (c) 2 M Guanidine Hydrochloride (Sigma-Aldrich) in washing buffer. 9. All the buffers are filtrated on a 0.22 mm membrane, degassed, aliquoted (50  mL, 15  mL, or 1  mL) and stored at –20°C, except for the SDS buffer, stored at room temperature (RT) (see Note 2). 10. Stromal cell-derived factor-1a (SDF-1a) and interferon-g (IFNg) obtained from H. Lortat-Jacob, prepared as previously described (9, 10).

3. Methods 3.1. Synthesis of the NHS Activated Pyrrole: N-Hydro­xysuccinimidyl-6(Pyrrol-yl)-Caproate (NHS-Pyrrole)

1. Heat under reflux for 4  h a mixture of 2,5-Dimetho­ xytetrahydrofuran (490  mmol, 64.85  mL), 6-Aminocaproic acid (430 mmol, 56.33 g), acetic acid (430 mL) and 1,4-Dioxan (570 mL) and stir it at room temperature overnight (11). 2. Remove the volatiles under reduced pressure in a rotavapor; dissolve the residue in ethanol and coevaporate it (2 × 100 mL) to eliminate acetic acid. 3. The product 1, 6-pyrrolyl caproic acid, is obtained with a yield of 86% after chromatographic purification on silica gel (500 g) column; CH2Cl2/EtOH as eluents. Start the elution by 500 mL CH2Cl2, then 300 mL 98% CH2Cl2/2% EtOH, then 400 mL 95%/5% EtOH, product 1 goes out the column for a gradient of 90%/10% EtOH. This product 1 looks like a brownish oil. 4. M.S. product 1 (m/z) = 182.1 (M+) 5. 1H-RMN product 1 (200 MHz; CDCl3/TMS) d (ppm): 1.72 (m, 6H, –CH2–(CH2)3– CH2–); 2.34 (t, 2H, –CH2–CO2H); 3.87 (t, 2H, –CH2–N); 6.13 (dd, 2H, 3-H and 4-H pyrrole); 6.64 (dd, 2H, 2-H and 5-H pyrrole). 6. Mix and stir overnight, at room temperature, product 1 (144  mmol, 26.05  g), N-Hydroxysuccinimide (144  mmol, 16.56 g), N,N  ¢-Dicyclohexylcarbodiimide (159 mmol, 32.75 g) and DMF (1,500 ml). 7. Filter the mixture to eliminate N,N¢-Dicyclohexylurea. 8. Remove the volatiles under reduced pressure. Product 2, N-Hydroxysuccinimidyl-6-(pyrrole-yl)-caproate, is used without any other purification.

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9. 1H-RMN product 2 (200 MHz; CDCl3/TMS) d (ppm): 1.72 (m, 6H, –CH2–(CH2)3– CH2–); 2.34 (t, 2H, –CH2–CO); 2.88 (tt, 4H, –CH2–CH2–: NHS); 3.87 (t, 2H, –CH2–N); 6.13 (dd, 2H, 3-H and 4-H pyrrole); 6.64 (dd, 2H, 2-H and 5-H: pyrrole). 3.2. Two Steps Preparation of Pyrrolylated Oligosaccharides

In a first step, the oligosaccharide is coupled with adipic dihydrazide, and after purification, the glycosylhydrazide is finally functionalized with NHS-Pyrrole (Fig. 1).

3.2.1. Functionalization of Oligosaccharides with Adipic Dihydrazide

The reaction between the aldehyde function of the oligosaccharide reducing end with the hydrazide function of adipic dihydrazide, in an acidic medium, is the limiting step. Hydrazides are attractive for coupling reactions with oligosaccharides as they retain their nucleophilicity in acidic aqueous media, and the acylhydrazone which is formed is comparatively stable. Equilibration to the tautomeric ringclosed glycosylhydrazide is favoured, which is an advantage since the native form of the reducing end monosaccharide is preserved (12). 1. Prepare a 500  mM adipic dihydrazide stock solution, in the CH3COONa coupling buffer. 2. Prepare the reacting solution by mixing 10 mL of 100 mg/mL oligosaccharide (solubilised in water) with 50 mL of 500 mM adipic dihydrazide and complete to 500 mL final volume with

Fig. 1. Preparation of modified oligosaccharide bearing a pyrrole group and a chemical linker, in two steps.

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coupling buffer. These conditions lead to a 2  mg/mL final concentration of oligosaccharide and a 50 mM final concentration of adipic dihydrazide. 3. Prepare negative controls by omitting adipic dihydrazide. 4. Incubate 72 h, at 56°C, after which the samples can be stored at 4°C before purification. 5. Purify the samples by dialysis against water (the membranes have to be rinsed in water before use), in order to eliminate remaining adipic dihydrazide and salts. For a 500 mL sample, this is usually performed against 600 mL of water. For an optimum purification, the dialysis is done for 24 h and includes five changes of water. After that, samples (that can be stored at 4°C) are recovered in pre-weighed microtubes, concentrated (SpeedVac) and then freeze-dried. 6. Weigh the dry sample accurately to determine the total quantity of recovered oligosaccharide. 7. Solubilise the samples in PBS at 20 mg/mL. It can be stored at 4°C. 8. The determination of the linkage yield is performed as described in Subheading 3.3. 3.2.2. Functionalization with N-Hydroxysuccinimidyl-6(Pyrrole-yl)-Caproate (NHS-Pyrrole)

In a second step, the glycosylhydrazides react with NHS-Pyrrole, by an addition-elimination reaction between the activated ester of NHS-Pyrrole and the free NH2 function of the adipic dihydrazide arm. During this reaction, NHS is regenerated. 1. The reaction has to be performed in a 50% PBS/50% DMSO v/v buffer, in order to favour the solubility of NHS-Pyrrole without precipitating the oligosaccharide. 2. Prepare a 50  mM NHS-Pyrrole stock solution in DMSO. It can be stored at –20°C for several weeks. 3. Prepare the reacting solution by mixing 50 mL of 20 mg/mL oligosaccharide functionalized with adipic dihydrazide (solubilised in PBS), 30 mL of DMSO and 20 mL of 50 mM NHSPyrrole. The final concentrations are 10  mg/mL for the oligosaccharide and 10  mM for the NHS-Pyrrole, in a final volume of 100 mL. 4. Prepare negative controls by performing the reaction with oligosaccharide which has not been coupled with adipic dihydrazide. 5. Incubate 2 h at RT, in the dark to minimize pyrrole oxidation. 6. Complete to a 500 mL final volume with reacting buffer. 7. Purify the samples by dialysis against the 50% PBS/50% DMSO v/v buffer, during 4 h, in order to eliminate remaining NHS-Pyrrole. For a 500 mL sample, we use 125 mL of buffer.

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Then, dialyse against water, to eliminate PBS and DMSO. Use 600 mL of water for a 500 mL sample. For an optimum purification, the dialysis is performed during 24 h and the water is replaced five times. After that, samples are recovered in preweighed microtubes (it can be stored at 4°C from now), concentrated (Speed-Vac) and then freeze-dried. 8. Weigh the dry sample accurately to determine the total quantity of recovered oligosaccharide. 9. Solubilize the samples in water at 20 mg/mL. It can be stored at 4°C. The determination of the linkage yield is performed as described in Subheading 3.3. 3.3. Determination of the Linkage Yield

The TNBSA method is a colorimetric method allowing the quantification of primary amines and hydrazines. In a first step, it enables to determine the quantity of oligosaccharide coupled with adipic dihydrazide, which presents a free hydrazine function. In a second step, the method allows the estimation of pyrrolylated oligosaccharides content, by measuring the diminution of the hydrazine concentration (see Note 3). 1. Prepare a series of adipic dihydrazide (di-NH2 molecule) standards in the buffer 1: the solutions at 0–7.8–15.6–31.25– 62.5–125–250–500  mM are prepared by serial dilutions of a 10 mM adipic dihydrazide stock solution in water. The standard solutions have to be prepared just before use. 2. In a 96-well plate, dispense 10 mL of standard or sample (either oligosaccharide coupled with adipic dihydrazide, or coupled with pyrrole, or negative controls), with 40  mL of buffer 1 (dilution: 5). 3. Add in each well 10 mL of the TNBSA solution prepared by dilution at 1:10 of the stock solution in buffer 1, and incubate for 4 min. 4. Stop the colorimetric reaction by adding 100 mL of buffer 2 in each well. 5. Read the optical density at l = 450 nm with the Victor 1420 Multilabel Counter.

3.4. Oligosaccharides Immobilization on SPR Prisms: Electrospotting

All solutions are prepared extemporaneously. 1. Prepare a 20  mM pyrrole solution by diluting 10  mL of the 1  M stock solution with 490  mL of electrocopolymerization buffer. 2. Prepare the pyrrolylated oligosaccharide solutions at 3 different concentrations (for example, 100, 20, and 2 mM of pyrrolylated oligosaccharide, or 500, 100, and 10  mM of total oligosaccharide) by diluting the 20  mg/mL oligosaccharide

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samples in the 20 mM pyrrole solution (see Note 4). The molar concentration of the pyrrolylated oligosaccharide solutions has been estimated as described in Subheading 3.3. The final concentration of free pyrrole must be 20 mM (see Note 5), in a 30 mL final volume (see Note 6). 3. Transfer the spotting solution in a 96-well plate. Be careful to ensure a good homogenization of the solution without introducing air bubbles. 4. The prism has to be pre-treated with a mixture of 70% H2SO4 and 30% H2O2 (v/v): incubate the prism in 20 mL of the solution, under ventilation, during 10 min. Care must be taken as this reaction is highly exothermic and reacts strongly with organic compounds. Rinse it thoroughly with water and dry it with an argon jet, before utilization. 5. Insert the plate in the microarrayer to carry out the spotting on the chip. It mainly consists in a ceramic needle (150  mm internal diameter) containing a platinum wire which is filled with the solution (6 nL) containing the pyrrolylated oligosaccharide to be grafted and which can move to a precise location on the chip. A 2.4 V electrical pulse for 0.1 s between the needle (counter electrode) and the gold surface of the chip (working electrode) induces the synthesis of the polypyrrole film and its deposition on the gold surface (Fig.  2). In-between each sample deposition and at the end of the cycle, the program includes a procedure where the needle is rinsed with water and subsequently dried.

Fig. 2. General scheme of oligosaccharides addressing on a glass prism coated with gold. The different pyrrolylated oligosaccharides and pyrrole monomer solutions are in the 96-well plate. The electrospotting is carried out on the gold surface via the needle containing the solution to be copolymerized.

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Kinetic Curves

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Fig. 3. Scheme of the Surface Plasmon Resonance Imaging (SPRi) system. The imaging system enables the visualisation in real time of molecular interactions on each plot. A typical reflectivity curve versus incidence angle is represented. The choice of the measurement angle (maximum of the slope) allows kinetic monitoring with optimal amplitude.

6. When all the spots are synthesized, disconnect the prism from the microarrayer, rinse with water and dry it perfectly with argon. In these conditions, the prism can be stored up to 6 months at 4°C. 3.5. SPRi Interactions Monitoring

1. The optical setup is described elsewhere (13) and constructed by Genoptics (Fig.  3). Briefly, the prism is inserted in the apparatus and connected to a flow cell inside the instrument which is hermetically sealed over the prism. A light source (650  nm) illuminates the prism and a 12-bit CCD camera monitors the changes of the reflectivity caused by the ligand– probe interactions, as grey-level contrasts. During interaction experiments, images are recorded at fixed intervals of time (0.2 s) and are analyzed on a PC computer with Genovision Software (Genoptics). The SPRi technique allows the obtention of sensorgrams, corresponding to the association and dissociation phases of the oligosaccharide-protein complexes (Fig.  4). Finally, the raw data are treated with an Excel program.

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5.0

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Fig. 4. Detection of ligand binding by SPRi: sensorgram obtained on five spots (Heparin at three concentrations of total oligosaccharide, and controls) upon injection of SDF-1 at 400 nM.

2. Insert the chip into the SPRi reader and start the apparatus. Work with freshly defrosted buffers. Be sure that they are at room temperature before starting. The flow rate of running solutions within the cell is 70 mL/min. Before starting kinetic measurements, one has to determine the working incidence angle, by drawing plasmon curves: the optimum angle is located at the inflexion point of a plasmon curve (see Fig. 3 and Note 9). The standard procedure for a SPRi experiment includes the following steps: Prism saturation (at the start) 1. Saturate the prism by injecting saturation buffer containing 1% BSA during 7 min. 2. Rinse the prism with washing buffer during 10 min. 3. Inject regeneration buffer containing 1 M NaCl during 7 min to remove remaining BSA. 4. Rinse with washing buffer and wait until the base line is stable. The chip is ready for sample injections. Sample injections 1. Inject the sample diluted in washing buffer during 7 min. 2. Rinse with washing buffer for at least 10  min to remove unbound molecules and observe the oligosaccharide-protein complex dissociation.

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3. Regenerate with 1 M NaCl during 7 min. If spots are properly regenerated, one can inject another sample, after a washing step. If proteins remain on some spots, one can use a denaturing buffer containing 1% SDS or 2 M Guanidine Hydrochloride. In this case, it is recommended to repeat the saturation step before the following injection. 4. Wash during 10 min. Ending procedure 1. At the end of the experiment, stop the running buffer, remove the prism and wash the system (tubing, flow cell, injection loop) with 1% SDS in water and then rinse with water. Rinse the prism with water and dry it perfectly with argon, store it at 4°C. 3.6. Application to Heparin–Cytokine Interactions

The biological application is carried out using glycosaminoglycans (GAG) as a model of oligosaccharides. GAGs are negatively charged polysaccharides that have been shown to bind and directly regulate the bioactivity of a variety of proteins, such as enzymes, growth factors and cytokines (14). Among them, Heparin (Hp) and Heparan Sulfate (HS) are complex and highly sulphated polymers. In order to illustrate the approach described above, we studied interactions between Heparin and a Heparin fragment of 8 monosaccharides (dp8) with stromal cell-derived factor-1a (SDF1-a) and interferon-g (IFN-g) (for another illustration, see ref. 15). The chemokine SDF1-1a is the natural ligand for CXC chemokine receptor 4 (CXCR4). This chemokine inhibits cells infection by human immunodeficiency virus (HIV) and interacts specifically with Hp and HS (16). IFN-g is a T cell secreted cytokine, centrally involved in the immune response, which is regulated upon binding to GAGs (17, 18). The binding domain of HS for the cytokine, contains two N-sulfated regions (8 monosaccharides) linked to each other by an internal N-acetylated domain (around 30 monosaccharide units) (19). Three parallel functionalizations were performed for each sample (A, B, C) in order to determine the reproducibility of the coupling reaction. Thus, the yields of the oligosaccharides pyrrolylation, determined as described in Subheading 3.3, were in the range of 15–25%, and were very similar for the same species. After functionalization, the pyrrolylated oligosaccharides were grafted at four different concentrations onto the prism by electropolymerization, and the chip was used to measure oligosaccharide–cytokine interactions by SPR imaging. The concentrations referred in this example are expressed as concentrations of total oligosaccharide. Free heparin or dp8, which have not been modified, were also grafted (see Fig. 5).

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Fig. 5. Design of the 70 spots chip: copolymerizations were performed with a mix of 20 mM free pyrrole and various concentrations of oligosaccharide probes. Each condition was realized in duplicate. Six polypyrrole (PPy) spots were also immobilized, as negative controls spots.

The two proteins were successively injected at different concentrations in the cell. Each run was followed by a NaCl regeneration step. Representative association and dissociation curves obtained during cytokine injection are shown in Fig.  6. As anticipated, grafted oligosaccharide spots recognize the protein proportionally to their grafting concentration, and the interactions are oligosaccharide specific, with regard to the weak binding of the proteins upon gold surface and polypyrrole spots. As shown in Fig. 7 with an IFN-g injection, the signals obtained for pyrrolylated oligosaccharides resulting from different coupling reactions (A, B or C) are quite similar, which underlines the relative reproducibility of the functionalization reactions. Moreover, the non modified Heparin does not properly bind the cytokine, as the oligosaccharide cannot be grafted on the support. The signal intensity is also directly linked to the protein concentration injected, as shown in Fig. 8. By increasing the number of concentrations tested, equilibrium dissociation constant (Kd) and kinetics parameters (rate constants) can be obtained for each probe analyzed, allowing the direct comparison of molecular interactions for an important number of compounds. This approach can be applied for multiple studies of synthetic and natural oligosaccharides properties, making it a powerful tool in the glycobiology field.

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Fig. 6. Injection of the cytokines on Heparin or dp8 spots immobilized at different concentrations: association and dissociation curves obtained for each oligosaccharide spot, and controls. At the top: differential images of the prism, at the end of the protein injection. The biological interactions cause reflectivity increment for the concerned spots.

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Injection of IFN-γ at 100 nM on different Heparin spots

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Fig. 7. Association and dissociation curves of IFNg injected at 100 nM with different heparin spots, grafted at the higher concentration (100 mM).

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Fig. 8. Association and dissociation curves of IFNg and SDF-1a injected at several concentrations with heparin spots, grafted at the higher concentration (100 mM).

4. Notes 1. The reaction rate is limited by the cycle opening in acidic medium (one can assume a 1% opened form presenting the aldehyde reactive function). It is important to notice that some oligosaccharides are not very stable in acidic medium. That’s why, different changes in the buffer composition, pH and reaction length should be performed to determine optimum conditions,

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leading to an efficient coupling and avoiding oligosaccharide degradation. 2. It is necessary to work with the same stock of buffers from an experiment to another, in order to minimize results variations due to the buffer composition. That is why it is recommended to prepare an important stock of buffers, to aliquote in 50-mL sterile tubes (for washing and regeneration buffer) and in 1.5  ml-microtubes (for saturation buffer), and to store it at –20°C. Thus, one can defrost the needed quantity of buffer for an experiment and store it temporarily at 4°C. The saturation buffer containing BSA cannot be stored more than one day at 4°C, otherwise bacteria contamination can occur. 3. The TNBSA method allows one to determine the molar ratio of oligosaccharides functionalized against the total amount of oligosaccharides. The latter is determined by weighing the dry sample after the freeze-dryer step. The molar concentration of oligosaccharide coupled with adipic dihydrazide is calculated as follows: (a) The neat optical density (OD) of the oligosaccharide bearing a primary amine (NH2) group is obtained by subtraction of the control OD (unmodified oligosaccharide) to the modified sample. (b) The OD of the adipic dihydrazide standards enable to draw a calibration curve and to establish a mathematical relation between the OD and the NH2 group concentration, taking into consideration that adipic dihydrazide standards contain two NH2 groups. This curve allows the determination of the functionalized oligosaccharide concentration from its neat OD. After the second coupling of oligosaccharides with NHS-Pyrrole, the method enables with the same approach, to determine the remaining oligosaccharides bearing a NH2 group. By subtracting the number of mole of oligosaccharide functionalized with adipic dihydrazide, introduced for the NHS-Pyrrole coupling reaction, one can calculate the number of mole of pyrrolylated oligosaccharides. 4. Different final pyrrolylated oligosaccharide concentrations (200 mM to 200 nM) have to be prepared in order to determine the concentration leading to the best reactivity with the tested ligand. Moreover, the coupling yield (between 10 and 50% usually) can differ from one sample to another, so it is recommended to work with a large range of concentrations. 5. Final 20mM free pyrrole concentration is essential for electropolymerisation processus. Depending on the wanted oligosaccharides concentration, corresponding volume needed may impose to prepare different free pyrrole solutions higher than 20mM.

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6. The volume taken by the needle is in the nL range, so the 30  mL final volume is not crucial. However, we recommend preparing 15–30 mL solutions, since a significant evaporation can occur if the samples remain for a long time in the plate before being electrodeposited. 7. It is recommended to realize the spots at least in duplicates, to ensure the reliability of the results. These spots have to be spotted perpendicularly to the flow direction, to avoid potential ligand depletion between the first and second spot. 8. Control spots of polypyrrole and gold, as negative samples have to be performed, to quantify the background level. 9. Because of the considerable number of spots created on the gold surface and the possible variability of the used oligosaccharides, one have to realize a compromise by choosing the most suitable working angle for a majority of spots. 10. In order to verify the stability of the chip upon the several protein injections and SDS regenerations, it is better to inject a ligand of one grafted oligosaccharide, at the beginning of an experiment and then at regular intervals. The interaction intensities have to be very similar. The injection of the ligand at the beginning and at the end can be a sufficient control.

Acknowledgments We thanks H. Lortat-Jacob’s group at the “Institut de Biologie Structurale” – Grenoble for the preparation of recombinant IFNg and SDF-1a, and the “CREAB” group for their technical support with the microarrayer and SRPi apparatus. This work has been partially granted by the Carbinfec project from FUI and Lyonbiopôle. References 1. Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D (1991) Light-directed, spatially addressable parallel chemical synthesis. Science 251(4995): 767–73 2. R. Frank (1992) Spot synthesis of peptides on membrane supports. Tetrahedron 48:9217–32 3. Ban L, Mrksich M (2008) On-chip synthesis and label-free assays of oligosaccharide arrays. Angew Chem Int Ed Engl 47(18):3396–9 4. Kiessling LL, Splain RA (2010) Chemical approaches to glycobiology. Annu Rev Biochem 79:619–53 5. Lepenies B, Yin J, Seeberger PH (2010) Applications of synthetic carbohydrates to

chemical biology. Curr Opin Chem Biol 14(3):404–11 6. Weijers CA, Franssen MC, Visser GM (2008) Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides. Biotechnol Adv 26(5):436–56 7. Angenendt P, Glökler J, Murphy D, Lehrach H, Cahill DJ (2002) Toward optimized antibody microarrays: a comparison of current microarray support materials. Anal Biochem 309(2):253–60 8. Livache T, Guedon P, Brakha C, Roget A, Levy Y and Bidan G (2001) Polypyrrole electrospotting for the construction of oligonucleotide arrays compatible with a surface plasmon

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r­ esonance hybridization detection. Synth. Met 121 (2–3):1443–1444 9. Sarrazin S, Bonnaffé D, Lubineau A, LortatJacob H (2005) Heparan sulfate mimicry: a synthetic glycoconjugate that recognizes the heparin binding domain of interferon-gamma inhibits the cytokine activity. J Biol. Chem 280:37558–64 10. Laguri C, Sadir R, Rueda P, Baleux F, Gans P, Arenzana-Seisdedos F and Lortat-Jacob H (2007) The novel CXCL12g isoform encodes an unstructured cationic domain which regulates bioactivity and interaction with both glycosaminoglycans and CXCR4. PLoS One 2, e1110 11. Jirkowski I. and Baudy R (1981) A facile large scale preparation of 1H-pyrrole-1-ethanamine and syntheses of substituted pyrrolo[1,2-a] pyrazines and hydro derivatives thereof. Synthesis 481–483 12. Emiliano Gemma, Odile Meyer, Dušan Uhrín and Alison N. Hulme (2008) Enabling methodology for the end functionalisation of glycosaminoglycan oligosaccharides. Mol. BioSyst 4:481–95 13. Guedon P, Livache T, Martin F, Lesbre F, Roget A, Bidan G, Levy Y (2000) Characterization and optimization of a realtime, parallel, label-free, polypyrrole-based

DNA sensor by surface plasmon resonance imaging. Anal Chem 72:6003–9 14. Capila I, Linhardt RJ (2002) Heparin-protein interactions. Angew Chem Int Ed Engl 41:391–412 15. Mercey E, Sadir R, Maillart E, Roget A, Baleux F, Lortat-Jacob H, Livache T (2008) Polypyrrole Oligosaccharide Array and Surface Plasmon Resonance Imaging for the Measurement of Glycosaminoglycan Binding Interactions. Anal Chem 80:3476–82 16. Sadir R, Baleux F, Grosdidier A, Imberty A, Lortat-Jacob H (2001) Characterization of the stromal cell-derived factor-1alpha-heparin complex. J Biol Chem 276:8288–96 17. Sadir R, Forest E, Lortat-Jacob H (1998) The heparan sulfate binding sequence of interferongamma increased the on rate of the interferongamma-interferon-gamma receptor complex formation. J Biol Chem 273:10919–25 18. Lortat-Jacob H, Brisson C, Guerret S, Morel G (1996) Non-receptor-mediated tissue localization of human interferon-gamma: role of heparan sulfate/heparin-like molecules. Cytokine 8:557–66 19. Lortat-Jacob H, Turnbull JE, Grimaud JA (1995) Molecular organization of the interferon-gamma-binding domain in heparan sulfate. Biochem J 310:497–505

Chapter 6 Glycosylated Self-Assembled Monolayers for Arrays and Surface Analysis Fang Cheng and Daniel M. Ratner Abstract Over the past few decades, carbohydrates (glycans) have received growing attention for their many roles in biological systems, including pathogenesis, receptor-ligand interactions, and cell signaling. To unravel the biology of this important category of biomolecules, a host of new tools have been developed for glycomics investigation. At the forefront is the carbohydrate microarray, developed to immobilize functional glycans on a solid substrate to rapidly screen a variety of potential binding partners (carbohydrates, proteins, nucleic acids, cells, and viruses). The essential role played by surface modification on glycan microarray performance requires new methods to rigorously characterize glycan surface chemistries. Due to their highly reproducible nature and well-studied properties, self-assembled monolayers (SAMs) on gold are powerful models for presenting glycans on a solid substrate, engineering biomimetic microenvironments and exploring the bioactivity of immobilized carbohydrates via surface plasmon resonance (SPR). However, it can be challenging to prepare high quality glycosylated SAMs (glyco-SAMs) that retain their biological function following surface immobilization. Herein, a selection of versatile methods for the preparation of glyco-SAMs using natural and chemically modified glycans is described. This chapter will highlight the following three immobilization techniques: (1) direct self assembly using thiolated glycosides onto gold, (2) tethering aminated glycosides onto ­amine-reactive SAMs, and (3) conjugating natural glycan onto divinyl sulfone-activated SAMs. Key words: Surface plasmon resonance, Self-assembled monolayer, Glyco-SAM, Thiolated glycoside, Divinyl sulfone

1. Introduction Surface-immobilized carbohydrates can be used to explore glycanmediated recognition and binding events, e.g. pathogen and toxin detection, (1, 2) cell adhesion (3), and characterizing the carbohydrate-specificity of novel carbohydrate-binding proteins (lectins) (4). However, displaying bioactive mono- and oligosaccharides on

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a surface often requires significant chemical derivation prior to immobilization. Functional groups, such as thiols (5), amines (6), fluorocarbons (7), or azides (8), must typically be introduced into a chemically modified glycan to be paired with surfaces bearing compatible chemistries (maleimide, amine-reactive activated ester, hydrophobic, or alkyne). These linking strategies represent a highly rational and engineered approach to surface modification, but can also become a significant synthetic burden to the user. Carbohydrate synthesis frequently requires multiple synthetic steps, complex protecting group manipulations, and deprotection strategies; (9) introducing linkers for surface immobilization only increases the difficulty of said synthesis. Due to the lack of a universal synthetic strategy for producing linker-modified glycans, the following describes the preparation of glyco-SAMs based upon three complementary immobilization methods: thiolated glycosides on gold, aminated glycans on amine reactive SAMs, and free reducing sugar on vinyl sulfone-activated SAMs (Scheme 1). Whether the glycans are prepared synthetically to include a thiolated linker, functionalized via simple amination chemistries, or isolated from natural sources, these techniques permit the facile preparation of bioactive monolayers presenting ­glycans for biological assays, biosensing or surface analysis applications.

2. Materials 2.1. Preparation of Au Substrates

Titanium (2  nm) and gold (45  nm) films sequentially deposited onto cleaned silicon wafers (Silicon Valley Microelectronics, San Jose, CA) and SF-10 glass (SCHOTT Glass Technology, Duryea, PA) for surface analysis and SPRi, respectively. Metal films prepared via electron beam evaporation at the Washington Technology Center (Seattle, WA) (see Note 1).

2.2. Preparation of Solutions

1. Thiolated mannoside: 4–10  mM thiolated mannoside (5) in ultrapure water. Store in single-use aliquots at −20°C and dilute at 0.1–1 mM prior to daily use (see Note 2). 2. 11-Mercaptoundecanol: 10  mM 11-Mercaptoundecanol powder (Sigma-Aldrich, St. Louis, MO) in ethanol (200 proof, USP) (Decon Labs, King of Prussia, PA). Store in single-use aliquots at −20°C and dilute at 1 mM prior to daily use (see Note 3). 3. HEPES buffer (10×): 0.1  M HEPES, 1.5  M NaCl, 10  mM Ca2+ and Mn2+, pH 7.4. Store at room temperature. 4. PBS buffer (10×): 27  mM KCl, 1.37  M NaCl, 0.1  M phosphate, pH 7.4. Store at room temperature.

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Scheme 1. Glycan immobilization on to gold or modified gold substrates. (a) Direct self-assembly using thiolated glycosides on to a gold surface. (b) Tethering aminated glycans onto amine-reactive surfaces. (c) Conjugating natural or ­modified glycans onto hydroxyl terminated SAMs via divinyl sulfone chemistry.

5. Carbonate buffer: 0.5 M Na2CO3, pH 10 or pH 11. Store at room temperature. 6. Surface plasmon resonance imaging (SPRi) running buffers: Filter and degass via lab vacuum (4 torr) for at least 30 min prior to daily use (see Note 4). 7. BSA-T buffer (10×): 10  mg/mL Bovine serum albumin ­powder (Sigma-Aldrich, St. Louis, MO), 0.05% Tween-20

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(Sigma-Aldrich, St. Louis, MO), pH 7.4. Filter and store at −20°C and dilute in 1× PBS or 1× HEPES for daily use. 8. Con A-HRP (10×): 2,500 ng/mL Con A horseradish peroxidase conjugate (EY Laboratories, San Mateo, CA), 10× BSA-T HEPES, pH 7.4. Store at −20°C, and dilute in 1× HEPES for daily use. 9. Con A (10×): 10 mg/mL Concanavalin A (MP Biomedicals, Solon, OH), 10× HEPES, pH 7.4. Store at −20°C, and dilute in 1× HEPES for daily use. 10. RCA120-HRP (10×): 2,500 ng/mL Ricin horseradish peroxidase conjugate (EY Laboratories, San Mateo, CA), 1× BSA-T PBS, pH 7.4. Store at −20°C, dialyze overnight against 1× PBS, and dilute in 1× PBS for daily use. 11. RNase A: 10  mg RNase A (New England Biolabs, Ipswich, MA), 10× PBS, pH 7.4. Store at −20°C. 12. RNase B: 10  mg RNase B (New England Biolabs, Ipswich, MA), 10× PBS, pH 7.4. Store at −20°C. 13. Divinyl sulfone (DVS): 10% v/v divinyl sulfone (Sigma-Aldrich, St. Louis, MO), 0.5 M sodium carbonate buffer, pH 11. Mix vigorously prior to daily use. 14. Urea: 8 M urea (Sigma-Aldrich, St. Louis, MO). Sonicate and store at room temperature. 15. Glycine: 10 mM glycine (Sigma-Aldrich, St. Louis, MO), pH 2. Filter and store at room temperature. 2.3. XPS Analysis

Sample X-ray photoelectron spectroscopy (XPS) composition data was acquired on a Kratos AXIS Ultra DLD instrument equipped with a monochromatic Al-Ka X-ray source (hn = 1,486.6 eV). All the samples were grounded and data were obtained at 0° take-off angle in the hybrid mode. The take-off angle is defined as the angle between the sample surface normal and the axis of the XPS analyzer lens. Compositional survey and detailed scans (N 1s, O 1s, and S 2p) were acquired using a pass energy of 80  eV. Highresolution scans (C 1s) were acquired using a pass energy of 20 eV. Data analysis was performed with the CasaXPS software (Casa Software Ltd.).

2.4. ToF-SIMS Analysis

Sample time-of-flight secondary ion mass spectrometry (ToFSIMS) spectra was acquired using an ION-TOF TOF.SIMS 5–100 system (ION-TOF GmbH, Münster, Germany). Positive spectra were recorded by rastering a pulsed 25 keV Bi3+ primary ion source over a (100 × 100) mm2 area, while maintaining the total ion dose below the static limit (i.e. 1012  ions/cm2). The mass resolution (m/Dm) for the positive ion spectra was typically 6,000 at m/z = 27 for all spectra, which were mass calibrated using the CH3+, C2H3+, C3H7+, and AuC2H4+ peaks.

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Sample carbohydrate-mediated protein binding was performed on a surface plasmon resonance imaging system (SPRi), SPRimagerII (GWC Technologies). The SPRimagerII was operated at room temperature using a standard flow cell and a peristaltic pump (BioRad-EconoPump) at 100  mL/min. All surfaces were passivated with BSA-T for 30 min and equilibrated in an appropriate running buffer prior to protein binding. Data acquisition consisted of the averaging of 30 images over a short duration to create an average image. The SPR signal is measured via the average pixel intensity, which can be converted to normalized percentages change in reflectivity according to GWC protocol. Urea (8 M) or glycine (10  mM, pH 2.0) was used to strip bound protein and regenerate the array surfaces. For sensorgram acquisition, a 500 mm × 500 mm region-of-interest (ROI) was selected. For visual clarity, the contrast and brightness of SPR difference images were adjusted by ImageJ software (U.S. National Institutes of Health, Bethesda, MD).

3. Methods Preparing high quality self-assembled monolayers (SAMs) bearing carbohydrates is a prerequisite for exploring the bioactivity and surface chemistry of surface resident glycans. Ensuring the quality of glyco-SAMs is complicated by the cleanness of the substrate, purity of reagents, intrinsic instability of SAMs, and adventitious contamination originating from self-assembly and storage. Therefore, to produce reliable and reproducible glyco-SAMs for analysis, it is necessary to verify surface quality, track surface contaminants and their orgins, establish SAM stability, and examine the compositional change following surface treatment. XPS (10) and ToF-SIMS (11) are routinely utilized to obtain surface information at a molecular level, e.g. atomic composition, the chemical environment of elements at the interface, and surface species relevant to the immobilized biomolecules (Figs. 1 and 2) (12, 13). Ultimately, it is necessary to determine whether glycan presented on the surface retain their bioactivity. This is best accomplished via a biological assay using a natural binding partner, such as a lectin. Exploiting the sensitivity of the enzyme-linked lectin assay, ELLA (14) is a common and low cost method that derives its origins from the enzyme-linked immunosorbent assay (ELISA). As a label-free method, SPR is routinely employed to directly probe carbohydrate–protein interactions at the interface, providing accurate structure/function information on binding specificity and kinetics (Fig. 3) (15).

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a

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Fig. 1. XPS characterization of a glyco-SAMs formed using direct self-assembly of thiolated mannosides: (a) XPS survey scan, (b) detail sulfur 2p scan, and (c) high resolution carbon 1 s scan, indicating the high quality of SAMs bearing mannose on Au. (Note: sulfur 2p3/2 at 162.0 eV and C 1 s at 288.0 eV are assigned to bound thiol and mannose acetal species, respectively).

3.1. Preparing Glyco-SAMs 3.1.1. Direct Self-Assembly Using Thiolated Glycosides

Molecular self-assembly of thiols onto gold is arguably one of the most well studied methods for surface modification (16). Thiolated biomolecules, including carbohydrates, can be used to produce highly uniform and reproducible SAMs and are ideal as model surfaces for surface analysis and bioassays. In addition to the ease with which thiol-based SAMs can be assembled onto gold surfaces, their composition can be controlled through mixed self-assembly (10, 15). While no single chemistry has been described for the generalized

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synthesis of thiolated glycosides for surface modification, a wide variety of published methods describe the synthesis of thiol-bearing linkers for the assembly of modified carbohydrates onto gold surfaces or maleimide-activated substrates (5, 15, 17, 18). 1. Self-assembly of thiolated glycosides to form a uniform glycoSAM is achieved by immersing fresh gold substrate in a 0.1–1 mM solution of the thiolated glycan for 2–12 h at ambient temperature (incubations longer than 2 h do not significantly affect the quality of the glyco-SAM). Assembly may be performed from thiol solutions in water, ethanol or an ethanolwater mix, in accordance with the solubility of the thiolated glycoside. During self-assembly, the sample should be stored in the dark, and kept under an inert atmosphere, if possible (see Notes 2 and 3). 2. Following self-assembly, the modified gold substrate must be rinsed thoroughly. This can be achieved by dipping the ­sample into a vigorously stirring bath of fresh ultrapure water ­(50–100 ml) for 1 min (see Note 5). 3. The rinsed glyco-SAMs should be gently dried by a stream of argon and stored in the dark under an inert atmosphere (see Note 6). 3.1.2. Tethering Aminated Glycans onto Sams Bearing Activated Esters

While thiol-modified glycosides are ideal for direct self-assembly onto gold, the synthetic complexity of their preparation often precludes their use by all but the most highly specialized synthetic laboratories. As a less synthetically burdensome alternative,

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a

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Fig. 3. SPRi reflectivity image and the corresponding sensorgrams on a spotted glyco-SAM array composed of thiolated mannose, galactose and the glycoprotein RNase B. The bioactivity of immobilized glycans were verified via lectin binding with Con A (500 nM). The array includes (a) thiolated mannose, (b) the mannosylated glycoprotein RNase B and (c) thiolated galactose.

a­ minated glycans can easily be prepared by a variety of simple methods from natural isolated reducing sugars (4, 19–21). Glycans bearing a nucleophilic amine are ideal partners for amine reactive chemistries. Utilizing modified surfaces bearing activated ester functionality, it is possible to covalently couple aminated glycans directly to a surface via an amide bond. This provides a facile route for the synthesis of stable glycosylated surfaces, while minimizing the synthetic complexity of the precursors. 1. Amine-reactive surfaces are prepared by assembling SAMs bearing activated esters, e.g. hydroxysuccinimide or tetrafluorophenyl (11, 22). To prepare the activated surface, fresh Au

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substrates should be immersed in 1 mM ethanolic solution of the amine reactive disulfide for 2  h at room temperature to form the precursor SAMs for bioconjugation. 2. After SAM formation, rinse the surfaces in a stirring ethanol bath (50 ml) for 1 min (see Note 5). 3. Gently dry the cleaned surfaces by a stream of argon and store in the dark under an inert atmosphere (see Note 6). 4. The amine-reactive surfaces can be modified by printing or covering the surface with 0.1–1 mM aminated glycan (23) (pH 7.4) for hydroxysuccinimide ester or pH 10 carbonate for tetrafluorophenyl ester for 24 h at room temperature. 5. To quench unreacted active ester functionality, immerse the glycosylated substrate in BSA-T for 30 min, thoroughly rinse in a stirring water bath for 1 min (see Note 5), and dry by a stream of argon prior to storage in the dark under an inert atmosphere. 3.1.3. Conjugating Isolated Glycans onto HydroxylTerminated Sams via Divinyl Sulfone Chemistries

One of the ultimate goals for carbohydrate surface modification is a universal method for the covalent conjugation of glycans that does not require chemical synthesis of the precursor sugar species. In certain cases, the difficultly of carbohydrate synthesis, or lack of structural identification, prohibits synthetic access to the prerequisite glycans. In other instances, it is desirable to avoid repeated synthesis of glycosylated targets that have been prepared previously for alternative array surface chemistries (e.g. thiolated vs. aminated glycans). As an alternative, divinyl sulfone (DVS) represents a unique method for the covalent conjugation of thiolated-, aminated-, and unmodified sugars and proteins through the modification of a hydroxyl-terminated SAM or polymer. Upon DVS activation, hydroxyl-bearing surfaces display the highly electrophilic vinyl sulfone group, which will react with glycans bearing amines, thiols and hydroxyls under slightly basic conditions via a Michael addition (1,4-addition) (24–26). 1. Hydroxyl-terminated SAMs can be prepared by immersing fresh Au substrates in 1 mM 11-mercaptoundecanol for 2 h at room temperature to form a uniform SAM. 2. Dip surfaces in a stirring ethanol bath (50 ml) for 1 min and dry under a stream of argon for 1 min (see Note 5). 3. Immerse the cleaned hydroxyl-bearing surface in a solution of DVS (10% v/v, 0.5 M carbonate buffer, pH 11) with vigorous shaking at room temperature for 1 h. 4. The DVS-treated surface should be thoroughly rinsed with a stream of water, dried under a stream of argon, and stored in the dark at 4°C.

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5. Functionalize the DVS activated surface by printing or covering with a solution of free reducing sugar (0.5–20% w/v, 0.5 M carbonate buffer, pH 10) at ambient temperature for 16–24 h. Thiolated and aminated glycosides should be conjugated at 0.1–1 mM using pH 7.4 PBS. 6. Quench the printed/functionalized surface for 30  m with BSA-T, thoroughly rinse with water, dry under a stream of argon for 1 min and store in the dark at 4°C. 3.2. CarbohydrateMediated Protein Binding 3.2.1. Enzyme-Linked Lectin Assay

ELLA is a popular qualitative probe for interrogating carbohydratemodified surfaces based on a modified enzyme-linked immunosorbent assay (ELISA). Utilizing commercial peroxidase or alkaline phosphatase-conjugated lectins, ELLAs provide users with an accurate method to compare the bioactivity of carbohydrates on various glycosylated substrates. This method is particularly attractive for general use, as it requires little to no specialized equipment for array fabrication or analysis. The following ELLA-based assay for the bioavailability of glyco-SAMs is based upon the quantification of HRP-modified lectins via a commercial fluorogenic peroxidase substrate. 1. The QuantaBlu fluorogenic peroxidase substrate kit (Thermo Fisher Scientific, Rockford, IL) must be equilibrated to room temperature for 30 min and the QuantaBlu working solution prepared in accordance with the manufacturer’s specifications. 2. Prior to ELLA, glyco-SAMs must be immersed in the appropriate working buffer (PBS or HEPES, depending on the lectin) and incubated for 30 min at room temperature to rehydrate the glycosylated surface. Small gold-coated chips can be placed at an angle in a 24-well plate and fully immersed by 1.5  ml buffer. Following incubation, the buffer can be removed by aspiration (see Note 7). 3. To passivate the glyco-SAM substrates against nonspecific protein interactions, gently add BSA-T PBS or HEPES (1.5 ml) to each well and incubate for 1 h at room temperature. Remove the BSA-T buffer and rinse both sides of the glyco-SAM ­sample three times with BSA-T (see Note 7). 4. Carefully remove the glycosylated substrate from the 24-well plate using forceps and clean by flushing three times with PBS or HEPES working buffer on both sides of the chip. Place cleaned chips in a fresh 24-well plate, resting flat on the surface. 5. Add the lectin-HRP solution (250 ng/mL, 1 ml) to each well, and incubate on a shaking platform at room temperature for 1 h (see Note 8). 6. Following incubation, remove the lectin-HRP solution by aspiration and carefully rinse the chip three times with BSA-T

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on both the gold-coated and unfunctionalized side to remove any unbound lectin-HRP. 7. Carefully remove the glyco-SAM substrates from the 24-well plate using clean forceps and place flat on the surface of a fresh 24-well plate. 8. Add the QuantaBlu working solution (1.0  mL) to each well and incubate on shaking platform at room temperature, in the dark, for 30 min. 9. Add the QuantaBlu stop solution (100 mL) to each well and mix briefly. 10. Transfer the fully developed QuantBlu solution (50 mL) to a 96-well plate and measure the Relative Fluorescence Units (RFU) (325 nm ex, and 420 nm em), in accordance with the manufacturer’s specifications. 3.2.2. SPRi

Surface plasmon resonance imaging (SPRi) is a powerful method for interrogating complex arrays of immobilized glycans in a highthroughput and label-free fashion. The versatility of SPRi makes it ideal for validating glyco-SAM bioactivity and optimizing surface modification based on the response to protein binding. Glycan arrays for SPRi can be easily prepared using any of the aforementioned glyco-SAM chemistries, including direct assembly of thiolated glycans on a gold surface, coupling of aminated glycans to an activated ester SAM or conjugation of glycan (regardless of source) to a DVS-activated substrate on gold. Arrays can be printed using microcontact printing, (27) pin-based printing, (28) inkjet printing, (4) or prepared by hand (10). The following protocol is based upon a silicone gasket cut to form individual wells and does not require specialized array printing instrumentation. 1. A 1 cm × 1 cm sheet of culture-well silicone (Grace Bio-Labs, Bend, OR) can be punched using a flat needle (φ 2 mm) to create an array of wells that can be overlaid on a gold surface or activated SAM substrate by gently pressing the pre-punched sheet onto the SPRi chip. 2. Spot ~2  mL of modified or unmodified glycan solution (in accordance with the specific immobilization chemistries) into the silicone well on the SPRi chip and incubate in a 100% relative humidity chamber overnight (see Note 9). 3. Following overnight incubation, remove the SPR chip from the humidity chamber and flush the array spots with water in a bottom-to-top sequence and gently remove the silicone mask. Following removal of the mask, the surface should be briefly rinsed with water (see Note 10). 4. Dry the SPR chip in a stream of argon and immerse the chip in BSA-T on a shaking platform at room temperature for 1 h to

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passivate unfunctionalized areas on the array surface. The BSA blocking step will make it easier to identify glycan-modified spots on the array by increasing the SPRi contrast between the printed and unprinted regions on the chip. This will subsequently facilitate identification of printed spots during regionof-interest (ROI) selection on the SPR instrument. 5. Prior to installation in the SPRi instrument, the functionalized SPR must be rinsed in a stirring water bath, dried in a stream of argon, and positioned in the instrument according to the manufacturer’s recommendations (see Note 11). 6. Running buffer, protein dilutions, and protein stripping solutions (8.0 M urea or 10 mM glycine, pH 2.0) can be applied to the SPR chip to measure SPR response according to a preestablished binding recipe. Care must be taken to avoid caustic solutions or oxidative damage to glyco-SAMs. However, if treated gently, chips can be used in repeated binding and regeneration cycles (see Notes 12 and 13). 7. Following an SPR experiment, the regenerated, rinsed and dried glyco-SAMs can be saved and stored at 4°C for additional SPR experiments or surface analysis (see Note 14).

4. Notes 1. High quality surfaces require clean gold substrates. To ensure the purity of the gold surface, storage conditions must be carefully controlled – in particular, fresh gold substrates must be stored in a polydimethylsiloxane (PDMS)-free environment. Gold can be easily contaminated by PDMS, sulfur, and iodine, even following only brief exposure to the environment in a chemical hood or contaminated lab bench. Gold substrates should be packed/unpacked in a contaminant-free laminar flow hood and stored under an inert atmosphere in the dark. 2. Unless stated otherwise, all aqueous solutions need to be prepared in ultrapure water (18.2  MΩ  cm). In the event where disulfide formation needs to be avoided, 0.5 stoichiometric equivalents of tris(2-carboxyethyl)phosphine (TCEP) can be added to the aqueous thiol solution. This amount of TCEP has not been observed to foul SAMs, according to XPS analysis. 3. Unless otherwise stated, all ethanolic solutions are prepared using 200 proof ethanol (USP test) established to be free from copper contamination. The existence of copper in ethanol affects SAM formation and packing density. To quickly ascertain the purity of the ethanol in question, elemental composition of a hydrophilic SAM prepared from the ethanol can be performed by XPS to identify the presence of copper; for this application SAMs bearing oligo(ethylene) glycol on Au can be used.

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4. Glass vials with occasional magnetic stirring are recommended for degassing. Glassware should be checked for scratches and cracks to mitigate the implosion hazard. 5. It is essential to thoroughly rinse the surface of unbound molecular species, while minimizing exposure to heat, light, and oxygen. A stirring water bath, rather than sonication, should be used to remove free thiol/disulfide from the surface. The water bath avoids unnecessary heating of the sample and potential damage to the glyco-SAM. 6. Unless stated otherwise, all glyco-SAMs must be stored at room temperature in a dark and oxygen-free environment. Petri dishes in glass jars backfilled with argon and sealed with parafilm can reduce airborne contamination and oxidation of the thiol-based SAM. Alternatively, samples can be stored in a vacuum desiccator. However, care must be exercised to avoid the introduction of contaminating species when venting the desiccator. 7. It is essential that the entire gold-coated chip be exposed to the incubation and blocking buffers. By positioning the chips at an angle, propped against the side-wall of the 24-well plate, it is possible to ensure that both the coated and uncoated side are blocked by BSA-T to reduce nonspecific adsorption lectin HRP conjugates to the substrate (bottom side of the chip) and the gold-coated top. Blocking both sides of the chip has been observed to significantly reduce background lectin-binding to the substrate. 8. Lectin-HRP stored at −20°C gradually loses its peroxidase activity, particularly after a number of freeze-thaw cycles. To verify the HRP activity, a small amount of lectin-HRP should be added to the QuantaBlu working solution and mixed briefly. In the event that the fluorescent product is not observed, the lectin-HRP stock should be discarded in favor of a fresh solution. 9. The specific concentration of glycans used for surface modification (array fabrication) is a function of the immobilization chemistry. On DVS-activated surfaces, Unmodified reducing sugars are employed as 0.5–20% w/v solutions, pH 10, and aminated and thiolated glycosides are used at 0.1–1 mM, pH 10 and 0.1–1 mM, pH 7.4, respectively. On bare Au and amine-reactive surfaces thiolated glycosides and aminated glycosides are used at 0.1–1 mM. 10. In the event that glycoprotein or protein spots (e.g. RNase B and RNase A) are used in the SPRi array, particular care must be exercised to avoid contamination of the surface during rinsing. Protein spots should be placed on the edge of the array, and the surface rinsed so that unbound protein flows off the chip, and not across the patterned array.

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11. This rinsing and drying step is essential to ensure the optical quality of the chip by removing excess BSA-T blocking solution and buffer salts. If the chip is not sufficiently rinsed, remaining protein or salts will interfere with refractive index matching to the SPR prism. 12. All buffers and solutions should be degassed prior to introduction to the SPR instrument to reduce the appearance of bubbles during binding experiments. 13. During SPR binding experiments, it is critical to avoid the introduction of air bubbles prior to rinsing the surface with water. Air bubbles have been found empirically to damage glyco-SAMs, as observed by an altered SPRi response. In addition, if bubbles are passed over a surface following the specific capture of proteins, surface-bound proteins can become resistant to stripping by urea or glycine, and the glyco-SAM cannot be completely regenerated. 14. To preserve the quality of the glyco-SAMs, the SPR chip should be thoroughly stripped of bound protein by 8.0  M urea or 10 mM glycine (pH 2) and rinsed with water to remove buffering salts. Following removal from the SPR instrument, the SPR chip (glyco-SAM) must be rinsed with water, dried in a stream of argon, and stored in the dark at 4°C under an inert atmosphere.

Acknowledgements The authors gratefully acknowledge support from the Washington Research Foundation, the University of Washington Royalty Research Fund, and the Department of Bioengineering, and NESAC/BIO (NIH Grant P41 EB002027). References 1. Shi, J., Yang, T., Kataoka, S., Zhang, Y., Diaz, A. J., and Cremer, P. S. (2007) GM1 clustering inhibits cholera toxin binding in supported phospholipid membranes. J. Am. Chem. Soc. 129, 5954–5961. 2. Disney, M., and Seeberger, P. (2004) The use of carbohydrate microarrays to study carbohydrate­cell interactions and to detect pathogens. Chemistry & Biology 11, 1701–1707. 3. Esaki, K., Yokota, S., Egusa, S., Okutani, Y., Ogawa, Y., Kitaoka, T., Goto, M., and Wariishi, H. (2009) Preparation of lactose-modified cellulose films by a nonaqueous enzymatic reac-

tion and their biofunctional characteristics as a scaffold for cell culture. Biomacromolecules 10, 1265–1269. 4. Song, X., Lasanajak, Y., Xia, B., Smith, D., and Cummings, R. (2009) Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays. ACS Chem. Biol. 4, 741–750. 5. Ratner, D., Adams, E., Su, J., O’Keefe, B., Mrksich, M., and Seeberger, P. (2004) Probing protein-carbohydrate interactions with microarrays of synthetic oligosaccharides. Chembiochem 5, 379–383.

6  Glycosylated Self-Assembled Monolayers for Arrays and Surface Analysis 6. Song, X., Xia, B., Stowell, S. R., Lasanajak, Y., Smith, D. F., and Cummings, R. D. (2009) Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chemistry & Biology 16, 36–47. 7. Ko, K., Jaipuri, F., and Pohl, N. (2005) Fluorous-based carbohydrate microarrays. J. Am. Chem. Soc. 127, 13162–13163. 8. Zhang, Y., Luo, S., Tang, Y., Yu, L., Hou, K.-Y., Cheng, J.-P., Zeng, X., and Wang, P. G. (2006) Carbohydrate-protein interactions by “clicked” carbohydrate self-assembled monolayers. Anal. Chem. 78, 2001–2008. 9. Boons, G., and Lindhorst, T. (1998) Carbohydrate chemistry, Blackie Academic & Professional. 10. Dhayal, M., and Ratner, D. A. (2009) XPS and SPR Analysis of Glycoarray Surface Density. Langmuir 25, 2181–2187. 11. Cheng, F., Gamble, L. J., Grainger, D. W., and Castner, D. G. (2007) X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and principal component analysis of the hydrolysis, regeneration, and reactivity of N-hydroxysuccinimide-containing organic thin films. Anal. Chem. 79, 8781–8788. 12. Castner, D., Hinds, K., and Grainger, D. (1996) X-ray photoelectron spectroscopy sulfur 2p study of organic thiol and disulfide binding interactions with gold surfaces. Langmuir 12, 5083–5086. 13. Lee, C. Y., Canavan, H. E., Gamble, L. J., and Castner, D. G. (2005) Evidence of impurities in thiolated single-stranded DNA oligomers and their effect on DNA self-assembly on gold. Langmuir 21, 5134–5141. 14. Shankar, S. P., Chen, I. I., Keselowsky, B. G., García, A. J., and Babensee, J. E. (2010) Profiles of carbohydrate ligands associated with adsorbed proteins on self-assembled monolayers of defined chemistries. J. Biomed. Mater. Res. A 92, 1329–1342. 15. Smith, E., Thomas, W., Kiessling, L., and Corn, R. M. (2003) Surface plasmon resonance imaging studies of protein-carbohydrate interactions. J. Am. Chem. Soc. 125, 6140–6148. 16. Ulman, A. (1996) Formation and structure of self-assembled monolayers. Chem. Rev. 96, 1533–1554. 17. Ratner, D. M., Adams, E. W., Disney, M. D., and Seeberger, P. H. (2004) Tools for glycomics: mapping interactions of carbohydrates in biological systems. Chembiochem 5, 1375–83.

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18. Zhi, Z., Laurent, N., Powell, A., Karamanska, R., Fais, M., Voglmeir, J., Wright, A., Blackburn, J., Crocker, P., and Russell, D. (2008) A versatile gold surface approach for fabrication and interrogation of glycoarrays. Chembiochem 9, 1568–1575. 19. Gildersleeve, J., Oyelaran, O., Simpson, J., and Allred, B. (2008) Improved procedure for direct coupling of carbohydrates to proteins via reductive amination. Bioconjugate Chem. 19, 1485–1490. 20. Lubineau, A., AugÈ, J., and Drouillat, B. (1995) Improved synthesis of glycosylamines and a straightforward preparation of N-acylglycosylamines as carbohydrate-based detergents. Carbohyd. res. 266, 211–219. 21. Rodriguez, E., Marcaurelle, L., and Bertozzi, C. (1998) Aminooxy-, hydrazide-, and thiosemicarbazide-functionalized saccharides: versatile reagents for glycoconjugate synthesis. J. Org. Chem 63, 7134–7135. 22. Lockett, M., Phillips, M., Jarecki, J., Peelen, D., and Smith, L. (2008) A tetrafluorophenyl activated ester self-assembled monolayer for the immobilization of amine-modified oligonucleotides. Langmuir 24, 69–75. 23. Martinez-Avila, O., Hijazi, K., Marradi, M., Clavel, C., Campion, C., Kelly, C., and PenadÈs, S. (2009) Gold manno-glyconanoparticles: multivalent systems to block HIV-1 gp120 binding to the Lectin DC-SIGN. Chem-Eur. J. 15, 9874–9888. 24. Fornstedt, N., and Porath, J. (1975) Characterization studies on a new lectin found in seeds of Vicia ervilia. FEBS letters 57, 187. 25. Cheng, F., Shang, J., and Ratner, D. M. (2011) A versatile method for functionalizing surfaces with bioactive glycans. Bioconjugate Chem. 22, 50–57. 26. Ding, Z., Long, C., Hayashi, Y., Bulmus, E., Hoffman, A., and Stayton, P. (1999) Temperature control of biotin binding and  release with A streptavidin-poly (N-isopropylacrylamide) site-specific conjugate. Bioconjugate Chem. 10, 395–400. 27. Michel, O., and Ravoo, B. (2008) Carbohydrate microarrays by microcontact click chemistry. Langmuir 24, 12116–12118. 28. Bolles, K., Cheng, F., Burk-Rafel, J., Dubey, M., and Ratner, D. M. (2010) Imaging analysis of carbohydrate-modified surfaces using ToF-SIMS and SPRi. Materials 3, 3948–3964.

Chapter 7 Carbohydrate Microarrays for Enzymatic Reactions and Quantification of Binding Affinities for Glycan–Protein Interactions Myung-Ryul Lee, Sungjin Park, and Injae Shin Abstract Glycans are involved in a variety of physiological and pathological processes through interactions with proteins. Thus, the molecular basis of glycan–protein interactions provides valuable information on understanding biological phenomena and exploiting more effective carbohydrate-based therapeutic agents and diagnostic tools. Carbohydrate microarray technology has become a powerful tool for evaluating glycanmediated biological events in a high-throughput manner. This technology is mostly applied for rapid analysis of glycans–protein interactions in the field of functional glycomics. In order to expand application areas of glycan microarrays, we have used carbohydrate microarrays for measurement of binding affinities between glycans and proteins and profiling of glycosyltransferase activities. The glycan microarrays used for these studies are constructed by immobilizing maleimide or hydrazide-conjugated glycans on the thiol or hydrazide-derivatized glass slides, respectively. This protocol describes the fabrication of carbohydrate microarrays and their applications to enzymatic reactions and determination of quantitative binding affinities. Key words: Binding affinity, Carbohydrates, Chemoselectivity, Enzymatic activity, Enzymatic glycosylation, High-throughput analysis, Immobilization, Lectins, Microarrays

1. Introduction Cell surface is highly decorated with glycans which are present in the form of glycoconjugates such as glycolipids, glycoproteins, and proteoglycans. These glycans serve as important recognition markers for a wide variety of physiological and pathological processes through interactions with proteins. Glycan–protein interactions

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are involved in cell communication, cell adhesion, and immune responses (1–4). Interestingly, these biomolecular interactions also play a role in deleterious disease processes including tumor metastasis, inflammation, and bacterial and viral infection (5–8). Therefore, the molecular basis of glycan–protein interactions sheds light on understanding of biological systems as well as development of potent therapeutic agents. Owing to the significance of glycan–protein interactions for biological research and biomedical applications, a number of analytic methods have been exploited to probe these interactions. For example, hemagglutination inhibition assay (9), enzyme-linked lectin assay (10), surface plasmon resonance (11) and isothemal titration calorimetry (12) have been widely utilized to evaluate glycan–protein interactions and to determine their binding affinities. Although these traditional approaches have been successfully employed for understanding of the details of glycan–protein recognition events, they are not suitable for high-throughput analysis of glycan–protein interactions since they are labor-intensive and require large amounts of samples. To overcome this shortcoming, carbohydrate microarray-based technology has been developed for rapid and quantitative analysis of glycan–protein interactions. Since the seminal technology of carbohydrate microarrays was developed by us and others in 2002, this microarray technology has become a powerful tool for studies of glycan-mediated biological events (13–25). Carbohydrate microarrays facilitate fast, quantitative, and simultaneous analysis of a large number of biomolecular interactions using a tiny amount of samples. To prepare glycan microarrays, various immobilization strategies of glycans on the solid surface, which rely on chemical ligation reactions, have been reported (26–33). The constructed carbohydrate microarrays have been mostly applied for rapid analysis of carbohydrate-protein interactions. Using this microarray technology, the glycan binding properties of various proteins have been investigated. In addition, glycan microarrays have also been utilized to detect pathogens for diagnosis of diseases. Furthermore, the microarrays have been applied to determine quantitative binding affinities between glycans and proteins (19, 33) as well as to investigate enzymatic glycosylations and glycosyltransferase activities (17, 19). In this chapter, we provide protocols for applications of carbohydrate microarrays, that are constructed by immobilization of maleimide or hydrazide-conjugated carbohydrates on thiol or epoxide-functionalized glass slides, respectively, to measuring quantitative binding affinities (IC50 and Kd values) (see Fig. 1). We also describe their applications to synthesis of sialyl Lex and profiling of glycosyltransferase activities.

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Fig. 1. Construction of glycan microarrays by respective immobilization of maleimide or hydrazide-conjugated glycans on the thiol or epoxide derivatized glass slide and their applications to (a) determination of binding affinities between glycans and proteins and (b) profiling of glycosyltransferase activities.

2. Materials 2.1. Fabrication of Carbohydrate Microarrays

1. Maleimide (13, 17) and hydrazide-linked carbohydrates (18, 19, 25) are not commercially available and thus should be prepared according to the known procedure. 2. Buffers for dissolving maleimide-linked glycans; phosphate buffered saline (PBS, pH 6.8) containing 40–50% (v/v) glycerol (see Note 1). 3. Buffer for dissolving hydrazide-linked glycans; 100 mM sodium phosphate buffer (pH 5.0) containing 40–50% (v/v) glycerol. 4. Thiol and epoxide-derivatized glass slides are purchased from commercial suppliers such as TeleChem International, Inc. and Schott Nexterion or prepared according to the known procedure (see Note 2) (13, 17–19). 5. 1% N-Ethylmaleimide (Sigma-Aldrich) in water to quench the unreacted thiol groups after immobilization of maleimidelinked glycans on the thiol-coated glass slide. 6. 1–3% 2-Aminoethanol in 10 mM NaHCO3 (pH 8.3) to quench the unreacted epoxide groups after immobilization of hydrazide-linked glycans on the epoxide-coated glass slide. 7. PBS (pH 7.4) containing 0.1% Tween 20 (Sigma-Aldrich). 8. A plastic film (thickness: 0.1–0.2 mm, B.S. Inc.) that is coated by adhesive at one side. 9. MicroSys 5100 microarrayer (Cartisian Technologies) fitted with Stealth Micro Spotting pins (TeleChem International, Inc.).

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2.2. Applications of Carbohydrate Microarrays

1. PBS (pH 7.4) containing 0.1% Tween 20 and 1% bovine serum albumin (BSA). BSA should be added to the buffer prior to use.

2.2.1. Enzymatic Reactions on Glycan Microarrays

2. For enzymatic galactosylation: a solution of β-1,4-galactosyltransferase (1 or 23 mU) (Calbiochem), 10 mM MnCl2 and UDP-Gal (0.1 or 1 mM) in 50 mM HEPES buffer (pH 7.5). 3. For enzymatic sialylation: a solution of α-2,3-sialyltransferase (1 mU) (Calbiochem), 5 mM MnCl2, alkaline phosphatase (20 μU) and 0.1 mM CMP-NeuNAc in 100 mM HEPES buffer (pH 7.0). 4. For enzymatic fucosylation: a solution of α-1,3-fucosyltransferase (1 mU) (Calbiochem), 15 mM MnCl2, alkaline phosphatase (20 μU), and 0.1 mM GDP-Fuc in 50 mM MES buffer (pH 6.0). 5. Mouse anti-sialyl Lex antibody (5–10 μg/ml) (Calbiochem) in 10 mM sodium phosphate (pH 7.2) containing 500 mM NaCl and 0.02% Tween 20. Tween 20 should be added to the solution prior to use. 6. 10 mM sodium phosphate (pH 7.2) containing 500 mM NaCl and 0.02% Tween 20 for washing unbound anti-sialyl Lex antibody. 7. Goat anti-antibody (10 μg/ml) (Calbiochem) in 10 mM sodium phosphate (pH 7.2) containing 500 mM NaCl and 0.02% Tween 20. Tween 20 should be added to the solution prior to use. 8. Cy3-labeled Ricinus communis agglutinin I (Cy3-RCA120, 10–20 μg/ml) in PBS (pH 7.4). 9. 35 mM SDS in PBS buffer (pH 7.4) for wash of the slide after treatment of β-1,4-galactosyltransferase for profiling β-1,4galactosyltransferase activity. 10. Cartisian AxSys software (Cartisian Technologies). 11. Temperature and humanity-controlled incubator (Daihan Scientific, Korea). 12. ArrayWorx™ microarray scanner (Applied Precisions, USA). 13. ImaGene 6.1 software and origin Pro7.0 software.

2.2.2. Determination of Binding Affinities Between Carbohydrates and Proteins

1. PBS (pH 7.4) containing 0.1% Tween 20 and 1% BSA. 2. A series of mixture of a fluorophore-labeled protein and a soluble inhibitor in PBS (pH 7.4) containing 0.1% Tween 20. 3. A solution of 0.1 nM–1 μM concentrations for Cy3-RCA120 (10–20 μg/ml) in PBS (pH 7.4) containing 0.1% Tween 20. 4. Cartisian AxSys software (Cartisian Technologies).

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5. Temperature and humanity-controlled incubator (Daihan Scientific, Korea). 6. ArrayWorx™ microarray scanner (Applied Precisions, USA). 7. ImaGene 6.1 software and origin Pro7.0 software.

3. Methods Glycan microarrays are constructed by printing very small quantities (usually 1 nl) of maleimide or hydrazide-conjugated glycans on the thiol or epoxide-derivatized glass slides using a microarrayer. Buffer solutions used for glycan immobilization should contain 40–50% glycerol to prevent undesired evaporation of nanodroplets during spotting and immobilization steps. For applications of glycan microarrays to enzymatic reactions, they are incubated with glycosyltransferases followed by treatment with fluorescent dyelabeled proteins to detect the transferred glycans on the microarrays. IC50 values of soluble inhibitors are measured by incubating glycan microarrays with the fluorescent dye-labeled protein in the presence of various concentrations of an inhibitor and then quantitating fluorescence intensity of bound protein with a fluorescence scanner. Dissociation constants (Kd values) for glycan–protein interactions are determined by incubating glycan microarrays with various concentrations of fluorescent dye-labeled protein and then quantitating fluorescence intensity of the bound protein with a fluorescence scanner. 3.1. Fabrication of Glycan Microarrays 3.1.1. Immobilization of Maleimide-Conjugated Carbohydrates on the Thiol-Derivatized Glass Slide

1. Dissolve maleimide-conjugated glycans used for immobilization at 0.1–1.0 mM in PBS (pH 6.8) containing 40–50% glycerol. 2. Transfer 5–10 μl of the solution into a 384-well microplate. The V-shaped 384-well microplate is recommended because a small amount of solutions can be loaded into the V-shaped well. The microplate containing the solutions can be stored at −70°C for a month (see Note 3). 3. Cut parts of a plastic film (thickness: 0.1–0.2 mm) that is coated by adhesive at one side with a knife (see Fig. 2). Attach the compartmentalized plastic film to the thiol-derivatized glass slide. 4. Print 1 nl of maleimide-conjugated glycan solutions from a 384-well microplate in predetermined places on the thiolcoated glass slide by using a robotic printing microarrayer (see Note 4).

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Derivatized glass slide Attach a plastic film to a glass slide Print glycans

Fig. 2. Scheme for the attachment of a plastic film to the glass slide.

5. After completion of printing, leave the slide in the print chamber (60% humidity) for 3 h at room temperature. The slide can be left overnight at room temperature in the print chamber. 6. After washing the slide with 30 ml of deionized water (5 min × 2), dry the slide by purging with Ar gas. 7. Drop 20–30 μl of a solution of 1% N-ethylmaleimide in water on each block of the slide by using a micropipette and incubate in the print chamber (60% humidity) for 1 h at room temperature. Unreacted thiols on the slide are removed at this step (see Note 5). 8. After washing the slide with 30 ml of deionized water (5 min × 2), dry the slide by purging with Ar gas. The slide can be stored at room temperature in a desiccator for several weeks. However, for best results, prepare the slide freshly prior to use. 3.1.2. Immobilization of Hydrazide-Conjugated Glycans on the EpoxideDerivatized Glass Slide

1. Prepare 100 μl of 0.1–1.0 mM hydrazide-conjugated glycan solutions in 100 mM sodium phosphate buffer (pH 5.0) containing 40–50% glycerol (see Note 1). 2. Transfer 5–10 μl of the solution into a 384-well microplate. 3. Cut parts of a plastic film (thickness: 0.1–0.2 mm) that is coated by adhesive at one side with a knife (see Fig. 2). Attach the compartmentalized plastic film to the epoxide-derivatized glass slide. 4. Print 1 nl of hydrazide-conjugated glycan solutions from a 384-well microplate in predetermined places on the epoxidecoated glass slide (see Note 4). 5. After completion of printing, leave the slide in the print chamber (60% humidity) for 3 h at room temperature. The slide can be left overnight at room temperature in the print chamber. 6. After washing the slide with 30 ml of deionized water (5 min × 2), dry the slide by purging with Ar gas.

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7. Drop 20–30 μl of a solution of 1–3% 2-aminoethanol or glycine in 10 mM sodium bicarbonate (pH 8.3) on each block of the slide by using a micropipette and incubate in the print chamber (60% humidity) for 1 h at room temperature. Unreacted epoxides on the slide are removed at this step. 8. After washing the slide with 30 ml of deionized water (5 min × 2), dry the slide by purging with Ar gas. The slide can be stored at room temperature in a desiccator for several weeks. However, for best results, prepare the slide freshly prior to use. 3.2. Applications of Carbohydrate Microarrays for Enzymatic Reactions 3.2.1. Enzymatic Glycosylations to Prepare Sialyl Le x from GlcnacImmobilized Glass Slides

1. Prepare the carbohydrate microarray immobilized by GlcNAc according to the procedure described in Subheading 3.1. 2. Drop 20–30 μl of a solution of PBS (pH 7.4) containing 1% BSA and 0.1% Tween 20 on each block of the slide by using a micropipette and incubate in the print chamber (60% humidity) for 0.5–1 h at room temperature (see Note 6). 3. Rinse the slide with PBS (pH 7.4) containing 0.1% Tween 20 to remove protein solution and wash with 30 ml of the same buffer with gentle shaking (10 min × 3). Rinse the slide with deionized water. 4. For galactosylation of GlcNAc, drop 15 μl of a solution of β-1,4-galactosyltransferase (GalT, 23 mU), MnCl2 (10 mM) and UDP-Gal (0.1 mM) in HEPES buffer (50 mM, pH 7.5) on each block of the BSA-pretreated slide by using a micropipette. Incubate the slide in a temperature-controlled humidity chamber (80% humidity) for 15 h at 37°C. 5. Wash the slide with PBS (pH 7.4) containing 0.1% Tween 20 with gentle shaking (15 min × 3) and rinse with water. Dry the slide by purging with Ar gas. 6. Incubate the GalT-treated slide with 15 μl of a solution of α-2,3-sialyltransferase (SialT, 1 mU), MnCl2 (5 mM), alkaline phosphatase (20 μU) and CMP-NeuNAc (0.1 mM) in HEPES buffer (100 mM, pH 7.0) in a temperature-controlled humidity chamber (80%) for 15 h at 37°C for sialylation of LacNAc. 7. Wash the slide with PBS (pH 7.4) containing 0.1% Tween 20 with gentle shaking (15 min × 3) and rinse with water. Dry the slide by purging with Ar gas. 8. Incubate the GalT and SialT-treated slide with 15 μl of a solution of α-1,3-fucosyltransferase (FucT, 1 mU), MnCl2 (15 mM), alkaline phosphatase (20 μU) and GDP-Fuc (0.1 mM) in MES buffer (50 mM, pH 6.0) in a temperaturecontrolled humidity chamber (80%) for 15 h at 37°C for fucosylation of NeuNAcα2,6LacNAc. 9. Wash the slide with PBS (pH 7.4) containing 0.1% Tween 20 with gentle shaking (15 min × 3) and rinse with water. Dry the slide by purging with Ar gas.

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10. To examine successful glycosylation, incubate the slide with PBS (pH 7.4) containing 0.1% Tween 20 and 1% BSA for 30 min at room temperature and then treat with mouse antisialyl Lex antibody (5–10 μg/ml) in 10 mM sodium phosphate (pH 7.2) containing 500 mM NaCl and 0.02% Tween 20 for 1 h at room temperature. 11. Wash the slide with 10 mM sodium phosphate (pH 7.2) containing 500 mM NaCl and 0.02% Tween 20 (10 min × 3) and rinse with water. Dry the slide by purging with Ar gas. 12. Incubate the antibody-treated slide with Cy5-labeled goat anti-antibody (10 μg/ml) in 10 mM sodium phosphate (pH 7.2) containing 500 mM NaCl and 0.02% Tween 20 for 1.5 h at room temperature (see Note 7). 13. Remove the compartmentalized plastic film from the glass slide and wash the slide with 10 mM sodium phosphate (pH 7.2) containing 500 mM NaCl and 0.02% Tween 20 (10 min × 3). Rinse the slide with water and dry by purging with Ar gas. 14. Scan the slide using a microarray scanner fitted with the appropriate filters. Process fluorescence data using ImaGene 6.1 software and analyze data using Origin Pro 7.0 or other software (see Fig. 3). 1. Prepare the carbohydrate microarray containing various carbohydrates according to the procedure described in Subheading 3.1.

3.2.2. Profiling of b-1,4Galactosyltransferase Activity

2. Drop 20–30 μl of a solution of PBS (pH 7.4) containing 1% BSA and 0.1% Tween 20 on each block of the slide by using a OH OH HO O O HO OH

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Fig. 3. Enzymatic synthesis of sialyl Lex from GlcNAc immobilized on the glass surface by using three glycosyltransferases. Treatment of microarrays, which are incubated with three glycosyltransferases, with anti-sialyl Lex antibody and then Cy5-anti-antibody shows the successful preparation of the tetrasaccharide from GlcNAc.

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micropipette and incubate in the print chamber (60% humidity) for 0.5–1 h at room temperature (see Note 6). 3. Rinse the slide with PBS (pH 7.4) containing 0.1% Tween 20 to remove protein solution and wash with the same buffer with gentle shaking (10 min × 3). Rinse the slide with deionized water. 4. Drop 20–30 μl of a solution of β-1,4-galactosyltransferase (1 mU), MnCl2 (10 mM), and UDP-Gal (1 mM) in HEPES buffer (50 mM, pH 7.5) on each block and incubate the slide in a temperature-controlled humidity chamber (80% humidity) at 37°C for 3 h. 5. Rinse the slide with HEPES buffer to remove enzyme solutions and wash with PBS buffer (pH 7.4) containing 0.1% Tween 20 with gentle shaking (10 min × 3). 6. Further wash the slide with 35 mM SDS in PBS (pH 7.4) under sonication at 60°C–65°C for 5 min (see Note 8) and then rinse with deionized water. Dry the slide by purging with Ar gas. 7. To detect galactose transferred by GalT, drop 20–30 μl of a solution of Cy3-RCA120 in PBS (pH 7.4) containing 0.1% Tween 20 on each block of the slide by using a micropipette. Incubate the slide in the print chamber (60% humidity) for 0.5–1 h at room temperature (see Note 7). 8. Rinse the slide with PBS (pH 7.4) to remove protein solutions. Remove the compartmentalized plastic film from the glass slide and wash with PBS (pH 7.4) containing 0.1% Tween 20 with gentle shaking (10 min × 3). Rinse the slide with deionized water. 9. Dry the slide by purging with Ar gas. The slide can be stored in a desiccator at room temperature for several weeks. 10. Scan the slide using a microarray scanner fitted with the appropriate filters. Process fluorescence data using ImaGene 6.1 software and analyze data using Origin Pro 7.0 or other software (see Fig. 4). 3.3. Measurements of Quantitative Binding Affinities 3.3.1. Determination of IC50 Values

1. Prepare the carbohydrate microarray containing α-GlcNAc and α-GalNAc according to the procedure described in Subheading 3.1. 2. Drop 10 μl of a solution of PBS (pH 7.4) containing 1% BSA and 0.1% Tween 20 on each block of the slide by using a micropipette and incubate in the print chamber (60% humidity) for 0.5–1 h at room temperature (see Note 6). 3. Wash the slide with PBS (pH 7.4) containing 0.1% Tween 20 with gentle shaking (15 min × 3) and rinse with water. Dry the slide by purging with Ar gas.

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Fig. 4. Applications of carbohydrate microarrays for profiling of β-1,4-galactosyltranserase activity (1: Fuc-α, 2: Fuc-β, 3: Gal-α, 4: Gal-β, 5: GalNAc-α, 6: Glc-α, 7: Glc-β, 8: GlcNAc-α, 9: GlcNAc-β, 10: Man-α, 11: Xyl-α, 12: Xyl-β, 13: Glcβ1,4Glc-β, 14: Galβ1,4Glc-β, 15: Glcα1,4Glc-β, 16: Galβ1,4GlcNAc-β, 17: Galβ1,6Man-α, 18: Glcβ1,6Man-α, 19: Manα1,6Man-α, 20: Manα1,6Manα1,6Man-α). Carbohydrate microarrays were incubated with 1 mU β-1,4-GalT in the presence of 10 mM MnCl2 and 1 mM UDP-Gal for 3 h at 37°C. To detect transferred Gal, the microarrays were probed with Cy3-RCA120. Fluorescence images of RCA120-treated slides before (left ) and after (right ) treatment with enzyme. α- and β-GlcNAc were converted to the corresponding LacNAc by this enzyme. Data are the average ± S.D. of triplicate determinants.

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2.5 3.0 3.5 4.0 4.5. 5.0 5.5 Log[α-GlcNAc-OMe] (mM)

Fig. 5. Determination of IC50 values of methyl N-acetyl-α-glucosaminide (α-GlcNAc-OMe) to inhibit 50% of WGA (wheat germ agglutinin) binding to (a) α-GlcNAc and (b) α-GalNAc on the carbohydrate microarray.

4. Incubate the carbohydrate microarray with a series of mixtures of a dye-labeled protein and a soluble inhibitor in PBS (pH 7.4) containing 0.1% Tween 20 for 1 h in the print chamber (60% humidity) at room temperature (see Note 7). 5. Wash the slide with PBS (pH 7.4) containing 0.1% Tween 20 with gentle shaking for 15 min three times and rinse with water. 6. Dry the slide by purging with Ar gas and measure fluorescence intensities using a fluorescence scanner. 7. Determine IC50 values of inhibitors from the obtained graph by finding points where concavity changes (see Fig. 5). In other

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words, IC50 values are obtained by calculating concentrations where d2f(x)/dx2 = 0 (f(x); fluorescence intensity and x; concentration of an inhibitor). 1. Prepare the carbohydrate microarray containing α- and β-LacNAc according to the procedure described in Subheading 3.1 and then each block is treated with BSA solution.

3.3.2. Determination of Dissociation Constants (Kd Values) for Protein– Carbohydrate Interactions

2. Drop 20–30 μl of a solution of 0.1 nM–1 μM concentrations of Cy3-labeled RCA120 (20 μg/ml) in PBS (pH 7.4) containing 0.1% Tween 20 on each block of the slide by using a micropipette. 3. Incubate the slide in the print chamber (60% humidity) for 1 h at room temperature. 4. Rinse the slide with PBS (pH 7.4) containing 0.1% Tween 20 to remove protein solution and wash with wash buffer with gentle shaking (3 min × 3). Rinse the slides with deionized water.

[P]0/FI (10-12)

5. Scan the slide using a microarray scanner fitted with the appropriate filters. Process fluorescence data using Imagene 6.1 software and analyze data using Origin Pro 7.0. Kd values are determined by using equation [P]o/FI = Kd/Flmax + [P]o/Flmax (Fl; fluorescence intensity, Flmax; maximum fluorescence intensity and [P]0; concentration of protein) (Fig. 6).

14 12 10 8 6 4 2 0

Kd = 3.4 x 10-8 (LacNAc-α)

[P]o

(Kd + [P]o) = FI Flmax Fl: fluorescence intensity Flmax: maximum fluorescence intensity [P]0: concentration of protein

Kd = 3.3 x 10-8 (LacNAc-β) 0

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Fig. 6. Applications of carbohydrate microarrays to determination of dissociation constants (Kd) for RCA120-surface-linked LacNAc interactions (filled square : LacNAc-β, filled circle: LacNAc-α, FI: fluorescence intensity). Kd values are determined by using equation [P]o/FI = Kd/Flmax + [P]o/Flmax (19).

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4. Notes 1. Glycerol was added into sample solutions to prevent the unwanted evaporation of nanodroplets during spotting and immobilization. Evaporation of solutions causes uneven, inefficient immobilization of glycans. 2. Quality of the modified glass slides is critical for reproducible results. Therefore, it is highly recommended that those with little experience should purchase the modified glass slides from commercial suppliers. 3. Do not repeat thawing and freezing of the sample solutions many times. Repetitive thawing and freezing will result in decomposition of modified glycans in solutions. 4. To prevent contamination of slides, the print chamber and printing pins should be cleaned. 5. Capping of unreacted thiol groups with N-ethylmaleimide prevents oxidative disulfide-bond formation between thiols on the surface and cysteine residues of proteins during incubation with labeled proteins. N-Methylmaleimide can be used instead of N-ethylmaleimide without any problem. 6. One of the most serious problems for microarray experiments is the nonspecific interaction of the probing proteins with derivatized surfaces, leading to high background fluorescence. BSA treatment of microarrays considerably attenuates the nonspecific interaction. Hydrophilic surfaces modified with poly(ethylene glycol) greatly suppress the nonspecific interaction even without treatment with BSA. 7. Tween 20 should be added to protein solutions prior to use since Tween 20 results in the decrease of protein activity during storage. 8. Omission of sonication of the enzyme-treated slide in a hot SDS solution results in a high background fluorescence.

Acknowledgments This work was supported by grants of the National Creative Research Initiative and WCU programs.

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References 1. Bertozzi, C. R., and Kiessling, L. L. (2001) Chemical glycobiology. Science 291, 2357–2364. 2. Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130. 3. Roth, J. (2002) Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control, and cell interactions. Chem. Rev. 102, 285–303. 4. Park, S., Lee, M.-R., and Shin, I. (2008) Chemical tools for functional studies of glycans. Chem. Soc. Rev. 37, 1579–1591. 5. Smith, A. E., and Helenius, A. (2004) How viruses enter animal cells. Science 304, 237–242. 6. Fuster, M. M., and Esko, J. D. (2005) The sweet and sour of cancer: glycans as novel therapeutic targets. Nat. Rev. Cancer 5, 526–542. 7. Lasky, L. A. (1992) Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science 258, 964–969. 8. Scanlan, C.N., Offer, J., Zitzmann, N., and Dwek, R.A. (2007) Exploiting the defensive sugars of HIV-1 for drug and vaccine design. Nat. Insight 446, 1038–1045. 9. Lis, H., and Sharon, N. (1972) Lectins: cellagglutinating and sugar-specific proteins. Science 177, 949–959. 10. McCoy Jr, J. P., Varani, J., and Goldstein, I. J. (1983) Enzyme-linked lectin assay (ELLA): use of alkaline phosphatase-conjugated Griffonia simplicifolia B4 isolectin for the detection of alpha-D-galactopyranosyl end groups. Anal. Biochem. 130, 437–444. 11. Duverger, E., Frison, N., Roche, A. C., and Monsingny, M. (2003) Carbohydrate-lectin interactions assessed by surface plasmon resonance. Biocheimie 85, 167–179. 12. Dam, T. K., and Brewer, C. F. (2002) Thermodynamic studies of lectin-carbohydrate interactions by isothermal titration calorimetry. Chem. Rev. 102, 387–430. 13. Park, S., and Shin, I. (2002) Fabrication of carbohydrate chips for studying proteincarbohydrate interactions. Angew. Chem. Int. Ed. 41, 3180–3182. 14. Wang, D., Liu, S., Trummer, B. J., Deng, C., and Wang, A. (2002) Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nat. Biotechnol. 20, 275–281. 15. Fukui, S., Feizi, T., Galustian, C., Lawson, A. M., and Chai, W. (2002) Oligosaccharide

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microarrays for high-throughput detection and specificity assignments of carbohydrateprotein interactions. Nat. Biotechnol. 20, 1011–1017. Houseman, B. T., and Mrksich, M. (2002) Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. Chem. Biol. 9, 443–454. Park, S., Lee, M.-R., Pyo, S. J., and Shin, I. (2004) Carbohydrate chips for studying highthroughput carbohydrate-protein interactions. J. Am. Chem. Soc. 126, 4812–4819. Lee, M.-R., and Shin, I. (2005) Fabrication of chemical microarrays by efficient immobilization of hydrazide-linked substances on epoxide-coated glass surfaces. Angew. Chem. Int. Ed. 44, 2881–2884. Park, S., and Shin, I. (2007) Carbohydrate microarrays for assaying galactosyltransferase activity. Org. Lett. 9, 1675–1678. Lee, M.-R., and Shin, I. (2005) Facile Preparation of Carbohydrate Microarrays by Site-Specific, Covalent Immobilization of Unmodified Carbohydrates on HydrazideCoated Glass Slides. Org. Lett. 7, 4269–4272. Blixt, O. et al. (2004) Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. USA 101, 17033–17038. de Paz, J. L., Noti, C., and Seeberger, P. H. (2006) Microarrays of synthetic heparin oligosaccharides. J. Am. Chem. Soc. 128, 2766–2767. Disney, M. D., and Seeberger, P. H. (2004) The use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens. Chem. Biol. 11, 1701–1707. Park, S., Lee, M.-R., and Shin, I. (2009) Construction of carbohydrate microarrays by using one-step, direct immobilizations of diverse unmodified glycans on solid surfaces. Bioconjugate Chem. 20, 155–162. Park, S., Lee, M.-R., and Shin, I. (2007) Fabrication of carbohydrate chips and their use to probe protein-carbohydrate interactions. Nat. Protoc. 2, 2747–2758. Shin, I., Cho, J. W., and Boo, D. W. (2004) Carbohydrate arrays for functional studies of carbohydrates. Comb. Chem. High Throughput Screening 7, 565–574. Shin, I., Park, S., and Lee, M.-R. (2005) Carbohydrate microarrays: an advanced technology for functional studies of glycans. Chem. Eur. J. 11, 2894–2901.

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28. Shin, I., Tae, J., and Park, S. (2007) Carbohydrate microarray technology for functional glycomics. Curr. Chem. Biol. 1, 187–199. 29. Shin, I. (2006) Carbohydrate microarrays for high-throughput analysis of carbohydrate-protein interactions. In Protein-Carbohydrate Interactions in Infectious Diseases. (Ed. Carole A. Bewley) 221–46 (RSC Publishing, Cambridge, UK). 30. Park, S., Lee, M.-R., and Shin, I. (2008) Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes. Chem. Commun. 4389–4399.

31. Horlacher, T., and Seeberger, P. H. (2008) Carbohydrate arrays as tools for research and diagnostics. Chem. Soc. Rev. 37, 1414–1422. 32. Laurent, N., Voglmeir, J., and Flitsch, S. L. (2008) Glycoarrays-tools for determining protein-carbohydrate interactions and glycoenzyme specificity. Chem. Commun. 4400–4412. 33. Liang, P.-H., Wang, S.-K., and Wong, C.-H. (2007) Quantitative analysis of carbohydrateprotein interactions using glycan microarrays: determination of surface and solution dissociation constants. J. Am. Chem. Soc. 128, 11177–11184.

Chapter 8 Neoglycolipid-Based Oligosaccharide Microarray System: Preparation of NGLs and Their Noncovalent Immobilization on Nitrocellulose-Coated Glass Slides for Microarray Analyses Yan Liu, Robert A. Childs, Angelina S. Palma, Maria A. Campanero-Rhodes, Mark S. Stoll, Wengang Chai, and Ten Feizi Abstract Carbohydrate microarrays, since their advent in 2002, are revolutionizing studies of the molecular basis of protein–carbohydrate interactions both in endogenous recognition systems and pathogen–host interactions. We have developed a unique carbohydrate microarray system based on the neoglycolipid (NGL) technology, a well-validated microscale approach for generating lipid-tagged oligosaccharide probes for use in carbohydrate recognition studies. This chapter provides an overview of the principles and key features of the NGL-based oligosaccharide microarrays, and describes in detail the basic techniques – from the preparation of NGL probes to the generation of microarrays using robotic arraying hardware, as well as a general protocol for probing the microarrays with carbohydrate-binding proteins. Key  words: Neoglycolipids, Microarrays, Nitrocellulose, Reductive amination, Noncovalent immobilization

1. Introduction 1.1. Neoglycolipid Technology: A Unique and Well-Validated Microscale Approach for Carbohydrate Ligand Discovery

The neoglycolipid (NGL) technology was originally introduced by Feizi and colleagues in 1985 as a novel approach to the study of the antigenicities and receptor functions of carbohydrate chains of glycoproteins (1). It was designed to address the need for microscale analysis and presentation of oligosaccharides in a multivalent form for studying carbohydrate–protein interactions which are generally weak. The technology involves conjugating oligosaccharides by reductive amination to an aminolipid, 1,2-dihexadecyl-sn-glycero3-phosphoethanolamine (DHPE). A library of NGLs from diverse

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oligosaccharide sequences has thus been synthesized: O-glycans or parts thereof (1, 2), N-glycans (3), oligosaccharide fragments of glycosaminoglycans (4, 5) and polysaccharides (5–9), as well as synthetic oligosaccharides (10, 11). The lipid tag confers amphipathic properties and enables efficient immobilization of NGLs in an oriented and clustered display on solid matrices, such as plastic wells, silica-gel thin layer chromatography (TLC) plates or nitrocellulose membranes, for probing with various carbohydraterecognizing systems. Moreover, there is the special advantage that highly heterogeneous mixtures of oligosaccharides when converted into NGLs can be probed for binding after being resolved on highperformance TLC (HPTLC) plates, and the components bound on the plate can be sequenced in situ by mass spectrometry (Fig. 1).

Fig. 1. Schematic presentation of the NGL-based oligosaccharide microarray platform.

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This approach has proven to be a powerful means of identifying hitherto unsuspected oligosaccharides; metaphorically speaking, finding the needle in a haystack (12–14). Highlights during the early stages of application of the technology include the finding of sulfated rather than the familiar sialylated ligands for the selectins among highly heterogeneous oligosaccharides in an epithelial mucin (15) and the discovery of yeast type O-mannosyl glycans in the brain O-glycome (2), amounting to 30% of O-glycans in the brain glycome (16), and now known to be affected in congenital neuromuscular diseases. Other than ring-opening of the monosaccharide residues at the reducing ends, oligosaccharides remain intact in DHPE-derived NGLs (Fig. 2). Among the further developments of methodology are (1) the generation of fluorescent NGLs by conjugating oligosaccharides, via reductive amination, to a fluorescence-labeled lipid, the anthracene-modified DHPE (ADHP) (Fig.  2); these can be detected by UV or fluorescence after TLC and during HPLC with subpicomole detection sensitivity (17); and (2) the creation of a new generation of NGL probes prepared by oxime ligation (18, 19) (Fig.  2), of which a significant proportion of the core monosaccharides are ring-closed so that diverse short oligosaccharides are efficiently presented for direct binding assays. In addition, oligosaccharides that are hard to derivatize by reductive amination, e.g., fragments of glycosaminoglycans and fungal/bacterial

Fig. 2. Principles of the preparation of NGL probes from reducing oligosaccharides by reductive amination and oxime ligation. Lipid reagents ADHP (14, 16) and AOPE (17, 18) are prepared from DHPE.

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polysaccharides, are efficiently derivatized to oxime-linked NGLs ((19) and unpublished results). The scope of the NGL-based oligosaccharide library has thus been further broadened. 1.2. NGL-Based Oligosaccharide Microarrays: Current State of the Art

The NGL technology was adapted to generate the first microarray system for sequence-defined oligosaccharides in 2002 (5). With the use of high precision robotic arraying hardware and imaging, the technology is now the basis of an advanced carbohydrate microarray system currently encompassing more than 700 lipidlinked oligosaccharide probes that are printed onto nitrocellulosecoated glass slides at low femtomole levels (Fig. 1). The repertoire of probes include NGLs derived from diverse oligosaccharide sequences of mammalian-type glycoproteins (N- and O-linked glycans), glycolipids, glycosaminoglycans, and polysaccharides of bacterial, fungal, and plant origins. Natural and synthetic glycolipids are also arrayed in parallel. The repertoire is continually expanding in number and structural diversity. A special feature of the NGLbased oligosaccharide microarray system is that the lipid-linked oligosaccharide probes are noncovalently immobilized on solid matrices and thus have potential lateral mobility; this is an advantage compared with most carbohydrate microarray systems in which oligosaccharides are covalently immobilized (by chemical reactions) and thus “fixed” on the array surfaces; this noncovalent presentation mimics to some extent the arrangement of clustered oligosaccharide structures at the cell surface. The NGL-based microarrays are providing crucial information on specificities of a variety of carbohydrate recognition systems operating endogenously and in pathogen–host interactions (6, 20). Among recent contributions are (1) the assignment of the ligands for a key receptor of the innate immune system against fungal pathogens, Dectin-1 (9); (2) the demonstration of sulfation as a modulator of carbohydrate recognition by sialic acid-binding receptors of the immune system, known as siglecs (21); (3) the discovery of the N-glycolyl analog of ganglioside GM1 (which humans do not synthesize) as the preferred host-cell receptor for Simian virus 40 (SV40) (22), which may explain the high susceptibility of simians to this pathogen; (4) the clinching of the ligand for a novel protein in the endoplasmic reticulum, malectin (23, 24), a candidate new player in the early steps of protein N-glycosylation, folding and quality control; (5) the elucidation of carbohydrate-binding specificities of several key surface-adhesive proteins of Toxoplasma gondii and other apicomplexan parasites (25–28), thus providing clues to host/tissue tropisms and pathobiology of infections by these parasites; and (6) the demonstration of a distinctive receptor-binding profile of the pandemic, swineorigin, influenza A (H1N1) 2009 (H1N1pdm) compared with that of seasonal human H1N1 influenza virus (29) which becomes more pronounced with hemagglutinin D222G mutants of

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H1N1pdm viruses isolated from cases of fatal infection (30), thus uncovering a potential mechanism linking the receptor-binding specificity to severity of disease. 1.3. Purpose and Scope of This Chapter

In this chapter, we provide guidance for biochemists and molecular biologists who wish to generate and apply NGL-based oligosaccharide microarrays for studies of protein–carbohydrate interactions. We focus on the basic techniques for preparing, purifying, and quantifying DHPE-derived NGLs from reducing oligosaccharides by reductive amination (DH-NGLs) for the purpose of generating microarrays. The preparation of NGLs from reduced oligosaccharides and the preparation of fluorescent and oxime-linked NGLs (Fig. 2), including the step-by-step chemical syntheses of the lipid reagents (ADHP and AOPE in Fig.  2) from DHPE, have been described in detail elsewhere (14, 19) and can be readily adapted for the generation of NGL microarrays described in this chapter. Here, we provide protocols for preparing NGLs for arraying onto nitrocellulose-coated glass slides. Procedures for microarray analyses of plant lectins and antibodies are also given as a guide to carry out binding analyses on nitrocellulose-based surface. Procedures of analyses of recombinant proteins (Fc-tagged and His-tagged) are described in Chapter 23 (by Palma et al.) of this book series, which describes the application of the NGL-based microarray technology in oligosaccharide ligand discovery for Dectin-1.

2. Materials 2.1. Preparation of NGLs from Reducing Oligosaccharides

1. A wide range of free reducing oligosaccharides can be ­purchased from companies, including Dextra Laboratories (Reading, UK), GlycoTech (Gaithersburg, USA), Megazyme (Wicklow, Ireland), and Elicityl (Crolles, France). Stock solutions of oligosaccharides can be prepared in water, e.g., at 1 mg/ml, and can be stored in well-sealed sample vials at −20°C for several years. 2. Screw-top glass vials (1.1 ml tapered, 2 and 4 ml) with screw caps and silicone/PTFE seals are available from Chromacol (Herts, UK). 3. All organic solvents are of HPLC grade. Unless otherwise stated, deionised water is used. Compositions of mixed solvents are by volume. 4. DHPE stock solution: 8 nmol/ml of DHPE (1,2-dihexadecylsn-glycero-3-phosphoethanolamine, available from SigmaAldrich) in chloroform/methanol 1:1. This solution is made in a 4-ml screw-cap glass vial and is stable at −20°C (well sealed) at least for 1 year.

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5. Sodium cyanoborohydride solution: 10  mg/ml sodium cyanoborohydride (Sigma-Aldrich) in methanol. This solution should be prepared freshly just before the conjugation reaction. 6. For incubation of reaction vials, a heating block (Dri-Block® heater) from Techne (Staffordshire, UK) is used. For evaporation of samples under a nitrogen stream, a Reacti-Therm Heating Module equipped with a Reacti-Vap Evaporator from Thermo Scientific (Rockford, USA) is used. 7. Aluminum-backed silica gel HPTLC plates (Merck, Darmstadt, Germany), semiautomatic TLC sample applicator Linomat 4 (Camag, Muttenz, Switzerland), glass TLC developing tank and sprayer (available from Sigma-Aldrich or Camag) are used for HPTLC analysis. 8. Primulin stock solution: 1 mg/ml of primulin (Sigma-Aldrich; referred to as primuline or Direct Yellow 59 in the catalogue) in acetone/water 1:9. This solution can be stored at 4°C (in dark) at least for 1 year. 9. Primulin spraying solution: 1/100 dilution of the primulin stock solution (above) in acetone/water 4:1. This solution can be stored at ambient temperature (in dark) for 2 weeks. 10. Orcinol spraying solution is prepared as follows: Dissolve 900 mg of orcinol monohydrate (Sigma-Aldrich) in 25 ml of water, and add 375 ml of ethanol. Cool on ice. Gradually add 50  ml of concentrated sulfuric acid (95–98%) with stirring, maintaining the temperature below 10°C. This solution can be stored at 4°C (in dark) and is stable for at least 1 year. 2.2. Purification of NGL Products

1. C18 cartridges (Sep-Pak Vac 3  cm3, 200  mg) and silica ­cartridges (Sep-Pak Vac 3 cm3, 500 mg) from Waters (Milford, USA). 2. Ammonium acetate solution: 0.2 M in water. 3. Materials and equipment used for semipreparative HPTLC purification are as described above in Subheading 2.1. 4. Empty solid-phase extraction (SPE) tubes fitted with frits can be purchased from Sigma-Aldrich and Agela Technologies (Newark, USA).

2.3. Quantification of NGLs

1. Many of the materials and equipment used for NGL quantitation on TLC plates are as described above in Subheading 2.1, but the following are required in addition. 2. Phosphate-buffered saline (PBS) solution: 10 mM phosphate buffer, pH 7.4, 150 mM sodium chloride (prepare freshly from concentrated PBS buffer or PBS tablet).

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3. A flying-spot scanning densitometer, e.g., Shimadzu CS-9000 dual wavelength densitometer or Camag Scanner (Muttenz, Switzerland). 2.4. Arraying of NGLs on NitrocelluloseCoated Glass Slides 2.4.1. Preparation of Sample Solutions of NGL (or Glycolipid) Probes for Robotically Arraying

1. Snap-cap polypropylene microcentrifuge tubes (0.5 ml) can be purchased from Alpha Laboratories (Hampshire, UK). 2. Phosphatidylcholine (from egg yolk, ³99% pure) and cholesterol (³99%) can be purchased from Sigma-Aldrich as lyophilized powder. These are made into 200  pmol/ml stock solutions in methanol in 4-ml glass screw-cap glass vials (Chromacol), which can be stored at −20°C (well sealed) over 6 months. 3. The 5 pmol/ml working solution of NGLs and natural glycolipids are prepared as described below in Subheading 3.1–3.3. 4. Cy3 stock solution: 10  mg/ml Cy3 mono NHS ester (GE Healthcare) in HPLC grade water. This solution can be stored at 4°C in the dark for at least 2 years. 5. Ultrasonic water bath (Branson 2510, Danbury, USA).

2.4.2. Generating Microarrays Using Robotic Arrayer and Slide Handling

1. Plastic 384-well plates (Bio-Rad Microseal PCR plates 384well CLR, v2.0); polyolefin sealing film StarSeal for 384-well plates (Starlab, Milton Keynes, UK); Sigma benchtop centrifuge with swing out rotor for microtiter plates (DJB Labcare Ltd, Newport Pagnell, UK). 2. Noncontact microarraying system: We are using a Piezorray instrument (PerkinElmer LAS, Beaconsfield, UK) housed in an environmental enclosure located within a clean laboratory environment. 3. Nitrocellulose-coated single or multiple pad slides are available from commercial sources, e.g., FAST slide from Whatman (Kent, UK) and Nexterion® Slide NC from Schott (Elmsford, USA). 4. Microarray scanner: We are using ProScanArray microarray scanner equipped with red (633 nm) and green (543 nm) lasers and ScanArray Express software (PerkinElmer LAS, Beaconsfield, UK).

2.5. Microarray Analyses of CarbohydrateBinding Proteins

1. FAST slide 16-well incubation chamber with silicon gasket and FAST frame multislide plate are available from Whatman (Kent, UK). 2. Hepes-buffered saline (HBS): 5 mM Hepes, pH 7.4, 150 mM NaCl, 2  mM CaCl2. Other buffers (detergent free) may be used as appropriate for different proteins. 3. Blocking solutions: 3% (w/v) bovine serum albumin (BSA; protease free, Sigma-Aldrich) in HBS.

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4. Carbohydrate-binding proteins and detection antibodies: A  wide range of biotinylated plant lectins can be purchased from Vector Laboratories (Peterborough, UK) and EY Laboratories (San Mateo, USA). Biotinylated secondary antibodies (e.g., against human, mouse, rat, rabbit) are available from Sigma-Aldrich, Vector Laboratories and many other suppliers. 5. Alexa Fluor 647-labeled streptavidin is from Molecular Probes (Invitrogen, Carlsbad, USA) available as 1  mg/ml solution. This can be stored as 20 ml aliquots at −20°C (in dark) and is stable for several years.

3. Methods 3.1. Procedure for Preparation of NGLs from Reducing Oligosaccharides

1. Dry down 100 nmol of oligosaccharides in a 1.1-ml tapered screw-cap glass vial either by lyophilization or under a stream of nitrogen gas. Add 1 ml of water to dissolve the oligosaccharide (see Note 1). 2. Add 100 ml of DHPE stock solution and 20 ml of freshly prepared sodium cyanoborohydride (10  mg/ml in methanol) to the vial. Seal the vial and heat at 60°C for 16 h. 3. Dilute the reaction mixture with chloroform/methanol/water 25:25:8 to give a concentration of 1  nmol starting oligosaccharide/5 ml, for HPTLC analysis and for storage. 4. Add a suitable solvent (see Note 2) to the TLC tank and allow vapor to equilibrate (>30 min, at ambient temperature). Cut a TLC plate along the aluminum-backed side to the desired size (e.g., 10 cm long and 5–10 cm wide). 5. Apply NGL solution (equivalent to 1 nmol of starting oligosaccharide) to the HPTLC plate using a nitrogen-assisted TLC applicator, at 15 mm from the bottom edge, as a 4 mm band, and allow 15 mm free at both edges. Carefully place the plate into the tank, with minimum disturbance to the equilibrated vapor layer and develop the chromatogram to 5 mm below the top edge. Remove the plate from the tank and air-dry the plate. 6. NGLs and excess lipids can be viewed and image recorded under the UV lamp after staining with the primulin reagent (see Note 3). The staining can be carried out by spraying the HPTLC plate with the primulin reagent until the surface appears slightly wet, and then allowing the surface to dry before viewing under longwave UV light (365 nm). 7. The same HPTLC plate (after primulin staining) can be stained with orcinol reagent for visualization of hexoses (in both

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Fig. 3. HPTLC analyses of conjugation mixtures of DHPE-NGLs derived from lactose, 3¢ and 6¢ sialyllactose and their quantitation on an HPTLC plate. (a) Images of the chromatogram of the NGL conjugation mixtures of lactose (lane 1), 3¢ sialyllactose (lane 2 ) and 6¢ sialyllactose (lane 3 ). The NGL products can be visualized with both primulin (left ) and orcinol (right ) staining. Judging from the results in orcinol staining, the conjugation yields are almost 100%, as no reducing oligosaccharides are detected near the origin of the HPTLC plate. Asterisk, NGL derived from a minor component (lactose) present in the starting material, a synthetic compound of 3¢ sialyllactose. (b) Chromatography of the purified NGL products (lane 5, lactose; lane 6, 3¢ sialyllactose; lane 7, 6¢ sialyllactose) on an HPTLC plate and primulin staining for quantitation by densitometry. Malto5-DH (in a reaction mixture, see Note 8) is used as a standard: 500 pmol (lane 1), 250 pmol (lane 2 ); 100 pmol (lane 3 ); 50 pmol (lane 4 ). TLC development solvent system is chloroform/methanol/water 60:35:8 (for both panels).

unconjugated oligosaccharides and the NGL products). The staining can be carried out by spraying with orcinol reagent until the plate appears slightly wet. The plate is then heated in a vented oven at 105°C for 2–5 min until the violet color given by hexoses is maximal (see Note 4). An HPTLC chromatogram of conjugation mixtures of lactose, 3¢- and 6¢-sialyllactose is shown in Fig. 3a. 3.2. Purification of NGL Reaction Products

The NGL conjugation mixtures contain an excess of aminolipid DHPE and salts, such as the reducing agent cyanoborohydride, and possibly some unconjugated oligosaccharides. A two-step procedure, namely, desalting and further purification, is described here for obtaining purified NGL products. Depending on the results of HPTLC analyses of an NGL conjugation mixture, one of the two methods (Option A or B, as described below in Subheadings 3.2.2 and 3.2.3) can be used for the further purification step. In general, if the NGL is derived from an oligosaccharide longer than trisaccharide and appears to be a single band in HPTLC analysis (e.g., lane 3 in Fig. 3a), Option A (see Subheading 3.2.2) can be used; whereas for NGLs of mono- and disaccharides and those giving multiple primulin and orcinol positive bands in HPTLC (e.g., lane 2 in Fig.  3a), Option B (see Subheading  3.2.3) should be used.

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Mass spectrometry analysis (see Note 5) should be performed to corroborate the molecular masses of the purified NGLs before quantitation and arraying. 3.2.1. Step 1: Desalting by C18 Cartridge

1. Prepare a C18 cartridge by sequential washing with 2  ml of chloroform/methanol 1:1, 2  ml of methanol and 2  ml of water. 2. Dry the NGL conjugation mixture under a nitrogen stream. Dissolve the dried mixture in a minimal amount of chloroform/methanol/water 15:70:30 (e.g., 100 ml), and carefully apply the sample solution to the prewashed C18 cartridge. Run the column to take up the applied solution. 3. Wash the column sequentially with 2 × 1 ml water followed by 1 ml of methanol, and collect the washings as fractions 1–3. 4. Elute the NGLs and excess lipid with 4 × 500  ml of chloroform/methanol/water 60:35:8. Combine the eluents and evaporate to dryness under a nitrogen stream (see Note 6).

3.2.2. Step 2 – Option A: Further Purification of NGL by Silica Cartridge

1. Wash the cartridge sequentially with 4 ml of methanol, 4 ml of water, 6 ml of 0.2 M ammonium acetate, 12 ml of water, 4 ml of methanol, and 6 ml of chloroform. 2. Dissolve the dried NGL mixture (<100 nmol of starting sugar) with up to 200 ml of chloroform/methanol/water 130:50:9. Apply the solution onto the prewashed cartridge and collect the fall through as fraction 1. 3. Wash the column sequentially with 4 × 300 ml of chloroform/ methanol/water 130:50:9 and collect the washings as fractions 2–5. 4. Elute the cartridge with 4 × 300 ml of chloroform/methanol/ water 60:35:8 followed by 4 × 300  ml of chloroform/methanol/water 25:25:8 and collect these as fractions 6–13. 5. Apply aliquots (1–2 ml) of each fraction for HPTLC analysis to identify, by primulin and orcinol staining, fractions containing the NGLs. 6. Pool the NGL fractions in a glass vial (1.1 or 2 ml with screw cap) and dry under a nitrogen stream.

3.2.3. Step 2 – Option B: Further Purification of NGL by Semipreparative HPTLC

1. Apply NGL reaction mixture as a long band to an aluminumbacked HPTLC plate using the nitrogen-assisted TLC applicator. Try not to overload the plate, e.g., as a guide do not apply more than 15 nmol NGL per cm. 2. Develop the plate using a suitable solvent (see Note 2), and air-dry the plate. 3. To locate the positions of migrating NGL bands, carefully cut out both vertical edges of the plate (see Note 7). The band

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position can be identified and lightly pencil marked on the ­silica surface after staining the cut edges with primulin and orcinol. 4. After localizing the NGL bands, cut out the required strips of plate with a scalpel (on the aluminum-backed side of the plate). Carefully loosen and scrape off the silica gel from the TLC strips and collect the gel in a glass vial. 5. Pack the scraped silica gel as a mini-column in an empty SPE tube fitted with frits and elute the NGLs with 3 × 500  ml of chloroform/methanol/water 25:25:8. Evaporate the solution under a stream of nitrogen gas to obtain the purified NGLs (see Note 8). 3.3. Quantitation of NGL Probes Based on the Lipid Tag

Quantitation of NGLs can be carried out on HPTLC plates using a densitometer after staining with primulin reagent by spraying as described above (see Subheading  3.1, step 6). To obtain better quantitation results, some precautions need to be taken and these are described below. 1. As a standard, DH-NGL of maltopentaose is prepared (see Note 9) and applied as 4  mm bands on an HPTLC plate at four levels (500  pmol, 250  pmol, 100  pmol, and 50  pmol) using a nitrogen-assisted TLC applicator. 2. Dissolve the dried purified NGL, to be quantified, in chloroform/methanol/water 25:25:8 to have a stock solution with an estimated concentration in the range of 50–100 pmol/ml. Up to six NGLs can be applied and quantified on the same plate (10 cm long and 10 cm wide); an example of quantifying three NGLs on a plate is shown in Fig. 3b. 3. Carefully place the plate into a TLC tank equilibrated (for at least 1  h) with solvent chloroform/methanol/water 60:35:8 and develop the chromatogram to 5 mm below the top edge. The plate is removed from the tank and air-dried. 4. Slowly lower the plate into a PBS solution, being careful to avoid trapping air bubbles. Leave to soak for 10 min. 5. Transfer the plate without drying to the primulin working solution (stock primulin solution/PBS 1:100, v/v). Leave to soak for 20 min in the dark. 6. Transfer the plate to a fresh PBS solution without drying, and soak for 10 min in the dark. Drain and air-dry the plate. 7. NGLs can be quantified on TLC plates by a TLC densitometer. The densitometer is used in linear reflectance fluorescence mode with excitation at 370 nm and detection through a filter with a cut-off at 460 nm. 8. Dilute the quantified NGLs in chloroform/methanol/water 25:25:8–5  pmol/ml and these are kept in glass sample vials

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with screw cap as working solutions. The vials should be well sealed and stored at −20°C (see Subheading 3.4.1 below and Note 10). 3.4. Arraying of NGLs on NitrocelluloseCoated Glass Slides

3.4.1. Preparation of Sample Solutions of NGL (or Glycolipid) Probes for Robotically Arraying

Purified and quantified NGLs in organic solvents (chloroform/ methanol/water, 25:25:8) can be directly arrayed, using a nitrogenassisted TLC applicator, onto nitrocellulose membranes to produce macroarrays for binding assays as described (5). An example is given in Chapter 23 of this book series. We describe below, the procedures in current use in our laboratory for preparing robotically generated microarrays of NGL and glycolipid probes in the screening format (each probe spotted at two levels, 2 and 5 fmol/spot in duplicate), using a noncontact robotic arrayer (see Note 11). 1. Add to a 0.5 ml microtube the two carrier lipids, phosphatidylcholine and cholesterol (5  ml each of the stock solutions at 200 pmol/ml), followed by 10 ml (for the 2 fmol level) or 30 ml (for the 5  fmol level) of the 5  pmol/ml working solution of NGL (or glycolipid) (see Note 12). 2. Many samples can be prepared at one time; the tubes are left uncapped and samples are allowed to evaporate to complete dryness at 37°C in an incubator for ~16 h. 3. Prepare Cy3 water (20 ng/ml, 26 fmol/ml) from the 10 mg/ ml Cy3 stock solution by 1:500 dilution in water (HPLC grade). 4. Reconstitute each dried mixture in 10 ml of Cy3 water, vortex (approximately 5 s), and centrifuge briefly (see Note 13). 5. Sonicate the tubes for 15 min in a sonic water bath at 30°C, and centrifuge at 8,000´ g (approximately 30 s) in a bench microcentrifuge.      

6. The sample solutions are now ready to be transferred, according to the planned microarray layout, into the “source plate” (e.g., a 384-well plate) for arraying. Place a piece of sealing film on top of the plate and centrifuge the plate at 300 × g for 3  min. The samples in the source plate are now ready for arraying. 3.4.2. Generating Microarray Slides Using “Piezorray” and Slide Handling

1. Piezorray is housed in an environmental enclosure located within a clean laboratory environment. Air supply to the laboratory is passed through an HEPA filter and the room has a slight positive pressure relative to the exterior. The environmental temperature is controlled in the range 19°C–20°C and the humidity within the enclosure is maintained in the range 40–50% using, if necessary, pumped humidified air.

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2. The Piezorray source plate holder is temperature-controlled by circulating water chilled to a temperature just above the dew point to avoid condensation on the metal plate holder. Operation of the Piezorray is through manufacturer’s supplied software run from a dedicated PC. 3. Carefully remove the sealing film on the source plate and fit the plate on the chilled metal source plate holder. Start the preconstructed array program (see Note 14). 4. After arraying, carefully remove the microarray slides in order and store in the original plastic slide container. Slides can be stored at 19°C–20°C with no discernible loss of performance over a period of 1 year or more. 5. The quality of microarrays can be monitored by evaluation of the spot image obtained from scanning each slide for Cy3 dye (543 nm laser) using a microarray scanner. Scan image files are stored and used later in conjunction with Alexa Fluor 647 scan files (see Subheading 3.5) to locate spots at the time of quantitation of carbohydrate binding (Fig. 4).

Fig. 4. The 16-pad nitrocellulose-coated glass slides in current use for generating NGL-based microarrays and their scan images. Sixty four lipid-linked oligosaccharide probes were printed in duplicate at two levels (256 arrayed spots) in each pad with Cy3 dye included in the diluent as a marker. Binding is detected with Alexa Fluor 647-labeled streptavidin.

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3.5. Protocols for Microarray Analyses of Carbohydrate-Binding Proteins

The carbohydrate-binding assays are most commonly performed with soluble proteins, such as plant lectins (19), anti-carbohydrate antibodies (31–33) and recombinant proteins with suitable tags (e.g., IgG-Fc) (9, 21) or hexa-His (23, 25). Whole viruses (29, 30) or virus-like particles (22) may also be analyzed, either after suitable labeling (e.g., biotinylation) (22) or using nonblocking antibodies for detection of binding (29, 30). It is generally necessary to have the carbohydrate-binding domains of the soluble proteins in oligomeric or multivalent state (see Note 15). Here, we elaborate on protocols for microarray analyses of biotinylated plant lectins and anti-carbohydrate antibodies (these do not require oligomerization) as a general guide giving the steps involved in a binding assay using microarrays of the lipid-linked probes on the 16-pad nitrocellulose-coated glass slides. Analyses of IgG-Fc- and hexa-His-tagged are described Chapter 23 of this book series. 1. Place a 16 pad silicon gasket over the slide and insert the assembly into an FAST Frame (see Note 16). 2. Wet the pads by pipetting onto the surface HBS buffer. Leave for 1 min and flick to remove solution; briefly blot the inverted slide/frame. 3. Add 140 ml of blocking solution (see Note 17) using an 8-channel multipipette to each pad and place the slide on a horizontal platform. Incubate at ambient temperature for 60 min. 4. Remove the blocker solution by flicking the frame and wash the wells by pipetting HBS buffer (flick to remove solution). Excess fluid is removed by briefly blotting the inverted frame. 5. Add the solutions of biotinylated plant lectins or anti-carbohydrate antibodies (diluted in blocking solution to a desired concentration as recommended by the provider or determined by performing optimization experiments) to the pads (50–100 ml of each), and incubate at ambient temperature for 90  min. Remove overlay materials by flicking or by careful introduction of pipette tips into the well to aspirate solution (see Note 18). 6. Wash pads by pipetting 140 ml HBS buffer into each well and flick or aspirate (see Note 19); three wash steps over 5  min should be performed. 7. For anti-carbohydrate antibodies, add appropriate biotinylated secondary antibody (diluted in blocking solution to the desired concentration recommended by the provider). Incubate at ambient temperature for 60  min and wash as in step 6. For biotinylated plant lectins, omit this step. 8. Add 100 ml of Alexa Fluor 647-labeled streptavidin (1 mg/ml in blocking solution). Incubate at ambient temperature (in dark) for 40 min. Remove by flicking or aspirating.

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9. Wash pads by pipetting HBS buffer as in step 6. 10. Carefully remove the slide/gasket from the frame and wash the slide with HBS buffer, followed by purified water (approximately 5 s). Drain the slide and dry in dark at ambient temperature or at 37°C. 11. Scan the slide for Alexa Fluor 647 dye (633 nm laser) using the microarray scanner. Image files of the scanned slides are stored and used in conjunction with Cy3 scan files (to locate spots) for quantitation with the scanner software (see Note 20).

4. Notes 1. The water is added to assist the dissolution of the oligosaccharides in the chloroform/methanol-based reaction mixture. However, as an inhibitory effect of water has been found for reductive amination (34), the amount of water should be strictly kept at less than 5%. For pentasaccharides or shorter oligosaccharides, 1  ml of water (or no water) is preferred; whereas for longer chain oligosaccharides (longer than hexasaccharides) or highly acidic oligosaccharides, 5 ml of water in maximum can be added to 100 nmol of oligosaccharide. Brief sonication (10–15 s) is advised to ensure the dissolution. 2. Different TLC development solvent systems should be used for oligosaccharides of different sizes: Chloroform/methanol/ water 130:50:9, for NGLs of mono- to trisaccharides; 60:35:8, for NGLs of tri- to pentasaccharides; 55:45:10, for NGLs of penta- to octasaccharides; 105:100:28, for NGLs of longer oligosaccharides. 3. Primulin staining is required for visualization of DH-NGLs and the oxime-linked AO-NGLs under longwave UV light; whereas the fluorescent NGLs derived from ADHP can be viewed directly under a longwave UV light (365 nm) without primulin staining, and shortwave UV light (254  nm) gives more sensitive detection (14, 17). 4. As the orcinol reagent contains concentrated sulfuric acid, the spraying process should be carried out in a ventilated TLC spray hood (available from CAMAG) or an acid-resistant fume hood. For color development of the HPTLC plate, a TLC heating plate (CAMAG) set up at 105°C in a fume hood can be used instead of the oven. It should be noted that orcinol staining is for visualization of hexose-containing oligosaccharides (e.g., those having glucose, galactose, mannose, or fucose residues). It is not applicable to the visualization of nonhexose oligosaccharides, e.g., oligosaccharides derived from chitin,

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polysialic acid, and glycosaminoglycans. Resorcinol reagent (14) can be used for specific staining of sialic acid containing oligosaccharides on TLC plates. 5. Molecular masses of the NGLs can be determined by matrixassisted laser desorption/ionization mass spectrometry as ­previously described (14). 6. Careful elution, namely collection of fractions of smaller volume (e.g., 200 ml) or using shallower gradient of polar solvent compositions (e.g., by including intermediate eluting solvents, such as chloroform/methanol/water 50:70:30 and 25:25:8 before 60:35:8), may achieve separation of the NGL products from both excess lipid and salt. HPTLC analysis of the fractions would be required to identify the fractions of NGLs and lipids. 7. Cutting should be carefully performed on the aluminumbacked side of the plate on a clean surface using a scalpel. For a 100 × 100 mm HPTLC plate with a 70 mm wide band applied in the middle, a 17 mm wide strip can be cut out on each vertical side for subsequent TLC staining procedures. For the preparative TLC purification of ADHP-derived fluorescent NGLs, the NGL band can be visualized directly under UV and thus there is no need to cut out the edges of the plate. 8. It may be necessary to remove the residual silica gel contained in the NGL solution when the preparative TLC method is used for purification: A small amount of chloroform/methanol/ water (e.g., 100 ml) is added for dissolution of the dried NGL in the 1.1 tapered glass vial. The undissolved silica residue can thus be separated after centrifugation and transfer of the NGL solution to a new sample vial. This reextraction process is normally repeated at least three times to minimize the loss of NGL. 9. The maltopentaose-DH standard can be prepared essentially as described in Subheading 3.1 using anhydrous maltopentaose (>95% pure, Sigma-Aldrich) as the starting material. To ensure a complete conversion of maltopentaose to its NGL, the reaction is finished by evaporation to dryness of the reaction mixture at 60°C (over approximately 1 h) by loosening the screw cap of the reaction vial. The standard maltopentaose-DH solution is made by adding 2 ml of chloroform/methanol/water 25:25:8 to the dried mixture without purification. HPTLC analysis (as in Subheading 3.1) can be carried out to corroborate the full conversion of the maltopentaose to NGL. 10. NGLs prepared by oxime-ligation, and natural and synthetic glycolipids are quantified in the same way using maltopentaoseDH as the standard. ADHP-derived fluorescent NGLs may be quantified by densitometry on HPTLC plates and by spectrophotometry in solution (14).

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11. The use of contact arrayers has not proven ideal for generating microarrays of lipid-linked oligosaccharides into nitrocellulosecoated glass slides since damage may occur to the delicate nitrocellulose membrane of the slide. 12. Probes should be prepared under clean laboratory conditions; workers should wear disposable powder free gloves and a face mask. NGL working solutions stored at −20°C need to be warmed up to ambient temperature before pipetting. Seal the vials immediately after the pipetting is finished to minimize the evaporation of the organic solvents. 13. Dried lipid/NGL mixture should be reconstituted on the day of arraying. 14. As different laboratories may have different arraying operation systems, we do not go into technical and programming details of using Piezorray. The arraying time for one source plate is set to be no longer than 4 h in order to minimize any evaporation of the samples in the plate. Multiple source plates can be used, if required, to complete the arraying process. 15. Whereas proteins that are multivalent or have sufficiently high avidities for carbohydrate ligands are used without further treatment, those comprising single or double carbohydratebinding domains often require “artificial” oligomerization, e.g., by precomplexing with nonblocking antibodies in order to elicit detectable binding signals. As an example, a recombinant IgG Fc chimera is usually precomplexed with biotinylated anti-IgG in a 1:3 (w/w) ratio, and the complex is allowed to stand at ambient temperature for approximately 45 min before dilution for the binding experiments. 16. The gasket/slide combination should be a tight fit in the FAST frame (not to allow leaking from one pad to another); be careful that the slide does not break when assembling. 17. Optimization of blocking conditions is often required when analyzing a new protein. Frequently used blocking solutions include (a) 3% BSA in HBS and (b) blocker Casein in TBS, 1% (w/v) (Pierce, Rockford, USA) with addition of 1% (w/v) BSA. It should be noted that different batches (lots) of the same product (e.g., the Casein blocker) have been found to have differing blocking strengths (supplier may not provide an explanation). A variety of commercial blocking solutions may also be used, but the solutions containing detergents, which are likely to interfere the immobilization of lipid-linked oligosaccharide probes, should be avoided. 18. Introduction of pipette tips into the well to aspirate solution must be done very carefully (try to position the tips at a corner), as it may cause irreparable damage/scratching to the nitrocellulose surface. Aspiration is obviously necessary when

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analyzing infectious agents. Care should be taken when handling infectious agents to ensure all safety precautions are in place and a risk assessment has been undertaken. If necessary, the binding assay should be performed inside a Class 1 microbiological safety cabinet and all liquid and solid waste disposed of appropriately according to local rules. It may be necessary to “fix” (using, for example, 4% paraformaldehyde) the slide before it can be handled in the open laboratory and put through the slide scanner; we typically include a fixation step after the incubation of influenza viruses. 19. It is important not to let the nitrocellulose dry once the protein being investigated has been added. 20. We use PerkinElmer ScanArray Express software for quantifying the bound fluorescence in the array spots, and a dedicated in-house-designed software suite for storing, retrieving, and displaying carbohydrate microarray data (35).

Acknowledgments We gratefully acknowledge collaborations of other members (past and present) in the Glycosciences Laboratory, in particular Alex Lawson, Yibing Zhang, and Colin Herbert. The Glycosciences Laboratory acknowledges with gratitude collaborators over the years with whom our microarray probes were studied. We also acknowledge UK Research Councils’ Basic Technology Initiative “Glycoarrays” (GRS/79268) and follow on EPSRC Translational Grant (EP/G037604/1), MRC and Wellcome Trust and NCI Alliance of Glycobiologists for Detection of Cancer and Cancer Risk (U01 CA128416) for financial support. A.S.P. is a fellow of the Fundação para a Ciência e Tecnologia (SFRH/ BPD/26515/2006, Portugal) and M.A.C. of the Ministerio de Ciencia e Innovación (JCI-2007-123-160, Spain). References 1. Tang PW, Gooi HC, Hardy M, Lee YC, Feizi T (1985) Novel approach to the study of the antigenicities and receptor functions of carbohydrate chains of glycoproteins. Biochem Biophys Res Commun 132:474–480 2. Yuen C-T, Chai W, Loveless RW, Lawson AM, Margolis RU, Feizi T (1997) Brain contains HNK-1 immunoreactive O-glycans of the sulfoglucuronyl lactosamine series that terminate in 2-linked or 2,6-linked hexose (mannose). J Biol Chem 272:8924–8931 3. Mizuochi T, Loveless RW, Lawson AM, Chai W, Lachmann PJ, Childs RA, Thiel S, Feizi T

(1989) A library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins reveals that conglutinin binds to certain complex type as well as high-mannose type oligosaccharide chains. J Biol Chem 264: 13834–13839 4. Leteux C, Chai W, Nagai K, Herbert CG, Lawson AM, and Feizi T (2001) 10E4 Antigen of scrapie lesions contains an unusual non­ sulfated heparan motif. J Biol Chem 276:12539–12545 5. Fukui S, Feizi T, Galustian C, Lawson AM, Chai W (2002) Oligosaccharide microarrays

8  Neoglycolipid-Based Oligosaccharide Microarray System… for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat Biotechnol 20:1011–1017 6. Feizi T, Chai W (2004) Oligosaccharide microarrays to decipher the glyco code. Nat Rev Mol Cell Biol 5:582–588 7. Galustian C, Park CG, Chai W, Kiso M, Bruening SA, Kang YS, Steinman RM, Feizi T (2004) High and low affinity carbohydrate ligands revealed for murine SIGN-R1 by carbohydrate array and cell binding approaches, and differing specificities for SIGN-R3 and langerin. Int Immunol 16:853–866 8. Reddy ST, Chai W, Childs RA, Page JD, Feizi T, Dahms NM (2004) Identification of a Low Affinity Mannose 6-Phosphate-binding Site in Domain 5 of the Cation-independent Mannose 6-Phosphate Receptor. J Biol Chem 279:38658–38667 9. Palma AS, Feizi T, Zhang Y, Stoll MS, Lawson AM, Diaz-Rodríguez E, CampaneroRhodes AS, Costa J, Brown GD, Chai W (2006) Ligands for the beta-glucan receptor, Dectin-1, assigned using ‘designer’ microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides. J Biol Chem 281:5771–5779 10. Komba S, Galustian C, Ishida H, Feizi T, Kannagi R, Kiso M (1999) The first total synthesis of 6-sulfo-de-N-acetylsialyl Lewisx ganglioside: A superior ligand for human L-selectin. Angew Chem Int Ed Engl 38: 1131–1133 11. Green PJ, Tamatani T, Watanabe T, Miyasaka M, Hasegawa A, Kiso M, Stoll MS, Feizi T (1992) High affinity binding of the leucocyte adhesion molecule L-selectin to 3¢-sulphatedLea and -Lex oligosaccharides and the predominance of sulphate in this interaction demonstrated by binding studies with a series of lipid-linked oligosaccharides. Biochem Biophys Res Commun 188:244–251 12. Feizi T, Stoll MS, Yuen C-T, Chai W, Lawson AM (1994) Neoglycolipids: probes of oligosaccharide structure, antigenicity and function. Methods Enzymol 230:484–519 13. Feizi T, Childs RA (1994) Neoglycolipids: probes in structure/function assignments to oligosaccharides. Methods Enzymol 242: 205–217 14. Chai W, Stoll MS, Galustian C, Lawson AM, Feizi T (2003) Neoglycolipid technology deciphering information content of glycome. Methods Enzymol 362:160–195 15. Yuen C-T, Lawson AM, Chai W, Larkin M, Stoll MS, Stuart AC, Sullivan FX, Ahern TJ, Feizi T (1992) Novel sulfated ligands for the cell adhesion molecule E-selectin revealed by the neoglycolipid technology among O-linked

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oligosaccharides on an ovarian cystadenoma glycoprotein. Biochemistry 31:9126–9131 16. Chai W, Yuen CT, Kogelberg H, Carruthers RA, Margolis RU, Feizi T, Lawson AM (1999) High prevalence of 2-mono- and 2,6-di-substituted Manol-terminating sequences among O-glycans released from brain glycopeptides by reductive alkaline hydrolysis. Eur J Biochem 263:879–888 17. Stoll MS, Feizi T, Loveless RW, Chai W, Lawson AM, Yuen CT (2000) Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery. Eur J Biochem 267:1795–1804 18. Liu Y, Chai W, Childs RA, Feizi T (2006) Preparation of Neoglycolipids with Ring-Closed Cores via Chemoselective Oxime-Ligation for Microarray Analysis of Carbohydrate-Protein Interactions. Methods Enzymol 415C:326–340 19. Liu Y, Feizi T, Campanero-Rhodes MA, Childs RA, Zhang Y, Mulloy B, Evans PG, Osborn HM, Otto D, Crocker PR, Chai W (2007) Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharideprotein interactions. Chem Biol 14:847–859 20. Liu Y, Palma AS, Feizi T (2009) Carbohydrate microarrays: key developments in glycobiology. Biol Chem 390:647–656 21. Campanero-Rhodes MA, Childs RA, Kiso M, Komba S, Le Narvor C, Warren J, Otto D, Crocker PR, Feizi T (2006) Carbohydrate microarrays reveal sulphation as a modulator of siglec binding. Biochem Biophys Res Comm 344:1141–1146 22. Campanero-Rhodes MA, Smith A, Chai W, Sonnino S, Mauri L, Childs RA, Zhang Y, Ewers H, Helenius A, Imberty A, Feizi T (2007) N-glycolyl GM1 ganglioside as a receptor for simian virus 40. J Virol 81: 12846–12858 23. Schallus T, Jaeckh C, Feher K, Palma AS, Liu Y, Simpson JC, Mackeen M, Stier G, Gibson TJ, Feizi T, Pieler T, Muhle-Goll C (2008) Malectin - A Novel Carbohydrate-binding Protein of the Endoplasmic Reticulum and a Candidate Player in the Early Steps of Protein N-glycosylation. Mol Biol Cell 19:3404–3414 24. Palma AS, Liu Y, Muhle-Goll C, Butters TD, Zhang Y, Childs R, Chai W, Feizi T (2010) Multifaceted approaches including neoglycolipid oligosaccharide microarrays to ligand discovery for malectin. Methods Enzymol 478:265–286 25. Blumenschein TMA, Friedrich N, Childs RA, Saouros S, Carpenter EP, Campanero-Rhodes MA, Simpson P, Chai W, Koutroukides T, Blackman MJ, Feizi T, Soldati-Favre D, Matthews S (2007) Atomic resolution insight into host cell recognition by Toxoplasma gondii. EMBO J 26:2808–2820

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26. Garnett JA, Liu Y, Leon E, Allman SA, Friedrich N, Saouros S, Curry S, Soldati-Favre D, Davis BG, Feizi T, Matthews S (2009) Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii. Protein Sci 18:1935–1947 27. Allman SA, Jensen HH, Vijayakrishnan B, Garnett JA, Leon E, Liu Y, Anthony DC, Sibson NR, Feizi T, Matthews S, Davis BG (2009) Potent Fluoro-oligosaccharide Probes of Adhesion in Toxoplasmosis. Chembiochem 10:2522–2529 28. Friedrich N, Santos JM, Liu Y, Palma AS, Leon E, Saouros S, Kiso M, Blackman MJ, Matthews S, Feizi T, Soldati-Favre D (2010) Members of a novel protein family containing MAR domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites. J Biol Chem 285:2064–2076 29. Childs RA, Palma AS, Wharton S, Matrosovich T, Liu Y, Chai W, Campanero-Rhodes MA, Zhang Y, Eickmann M, Kiso M, Hay A, Matrosovich M, Feizi T (2009) Receptorbinding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray. Nat Biotechnol 27:797–799 30. Liu Y, Childs RA, Matrosovich T, Wharton S, Palma AS, Chai W, Daniels R, Gregory V, Uhlendorff J, Kiso M, Klenk HD, Hay A, Feizi T, Matrosovich M (2010) Altered receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus. J Virol 84:12069–12074

31. Capodicasa C, Chiani P, Bromuro C, De BF, Catellani M, Palma AS, Liu Y, Feizi T, Cassone A, Benvenuto E, Torosantucci A (2011) Plant production of anti-beta-glucan antibodies for immunotherapy of fungal infections in humans. Plant Biotechnol J. 9:776–787 32. Torosantucci A, Chiani P, Bromuro C, De Bernardis F, Palma AS, Liu Y, Mignogna G, Maras B, Colone M, Stringaro A, Zamboni S, Feizi T, Cassone A (2009) Protection by antibeta-glucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence. PLoS ONE 4:e5392 33. Dunlop C, Bonomelli C, Mansab F, Vasiljevic S, Doores KJ, Wormald MR, Palma AS, Feizi T, Harvey DJ, Dwek RA, Crispin M, Scanlan CN (2010) Polysaccharide mimicry of the epitope of the broadly neutralising anti-HIV antibody, 2G12, induces enhanced antibody responses to self oligomannose glycans. Glycobiology 20:812–823 34. Stoll MS, Mizuochi T, Childs RA, Feizi T (1988) Improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities from reducing oligosaccharides. Biochem J 256:661–664 35. Stoll, Feizi T (2009) Software tools for storing, processing and displaying carbohydrate microarray data. In: Kettner C (ed) Proceeding of the Beilstein Symposium on Glyco-Bioinformatics, 4–8 October, 2009, Potsdam, Germany. pp 123–140 (available online at http://www.beilsteininstitut.de/en/symposia/overview/procee dings/2009-glycobioin­formatics/)

Chapter 9 Preparation of a Mannose-6-Phosphate Glycan Microarray Through Fluorescent Derivatization, Phosphorylation, and Immobilization of Natural High-Mannose N-Glycans and Application in Ligand Identification of P-Type Lectins Xuezheng Song, Jamie Heimburg-Molinaro, Nancy M. Dahms, David F. Smith, and Richard D. Cummings Abstract Glycan microarrays prepared by immobilization of amino-functionalized glycans on NHS-activated glass slides have been successfully used to study protein–glycan interactions. Fluorescently tagged glycans with an amino functional group can be prepared from natural glycans released from glycoproteins. These tagged glycans can be enzymatically modified with various glycosyltransferases, phosphotransferases, sulfotransferases, etc., to quickly expand the size and diversity of the tagged glycan libraries (TGLs). The TGLs, presented in the format of microarrays, provide a convenient platform for identifying the glycan ligands of glycan-binding proteins (GBPs). The chapter provides the background to prepare a defined glycan microarray and uses as an example glycans generated as phosphodiesters and phosphomonoesters of high-mannose type N-glycans. The method describes the preparation of high-mannose type glycan-AEAB conjugates (GAEABs), the purification of their phosphodiesters, and the subsequent mild acid hydrolysis to obtain corresponding phosphomonoesters. These GAEABs are covalently printed as a phosphorylated glycan microarray and used for analysis of the glycan ligand specificities of P-type lectins, such as the mannose-6phosphate receptors (Man-6-P receptors or MPRs). Key words: Glycan microarray, Glycan derivatization, Phosphorylation, Mannose-6-phosphate, Man-6-P receptor, P-type lectin

1. Introduction Historically, the identification and characterization of protein–glycan interactions were determined using hapten inhibition of agglutination assays or hapten inhibition of binding detected in ELISA-like formats.

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These assays were sequential (one glycan hapten at a time), labor-intensive, and required large amounts of samples, which were difficult to obtain. The development of glycan microarrays (1–4), which is an extension of gene microarray technologies for genomics and protein microarray technologies of proteomics, has transformed the study of protein–glycan interactions into a microscale assay that can analyze hundreds of glycans simultaneously for their ability to bind proteins. The repertoire of glycans encompasses compounds that are either chemo/enzymatically synthesized (5, 6) or isolated from natural sources (7–9) and are able to be covalently (5, 10, 11) or non-covalently (12, 13) immobilized to an activated glass surface. This method can be used to test the binding specificity of any potential glycan binding protein (GBP) that can be detected fluorescently, including lectins, antibodies, viruses, and receptors. The high-mannose type phosphorylated N-glycan array (14) is an example of a defined glycan array generated from naturally occurring glycans. In this example, glycans released by PNGase F digestion of bovine ribonuclease B (RNaseB) and soybean agglutinin (SBA) are purified and labeled with a bifunctional fluorescent linker; subsequently, the isomeric structures are separated and phosphorylated using recombinant form of the UDPGlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) (15). The resulting phosphodiesters are separated by HPLC, purified, structurally identified, and subsequently converted to their corresponding phosphomonoesters by mild acid hydrolysis as described previously (14). Thus, a library of fluorescently labeled N-linked high-mannose type N-glycans and their phosphodiesters and phosphomonoesters was available for printing a phosphorylated N-glycan array. Prior to this array, there were no formats available for studying large panels of phosphomonoesters and phosphodiesters of high-mannose N-glycans, due to the lack of methods for their chemical synthesis. The availability of a large panel of phosphomonoesters and phosphodiesters of high-mannose N-glycans permitted the investigation of the detailed specificity of P-type lectins, notably the cation-dependent and cation-independent mannose-6-phosphate receptors (Man-6-P receptors or MPRs), which are critical for the intracellular delivery of hydrolytic enzymes to the lysosome (16–19). Although the specificity of MPRs for phosphomonoesters and phosphodiesters had been observed (20), the array described here provides a platform for studying these important interactions (14, 21) and is available as a general platform for studies on P-type lectins.

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2. Materials 2.1. Preparation of Functional Fluorescent High-Mannose Glycan Conjugates

1. Ribonuclease B (RNaseB) (Sigma-Aldrich). 2. SBA (prepared as described previously) (22). 3. PNGase F (New England Biolabs), including glycoprotein denaturing buffer (10×), G7 reaction buffer (10×), and NP-40 (10%). 4. 2-(N-aminoethyl)amino benzamide (AEAB) (9). 5. C18 Sep-Pak column (Millipore, 500 mg and 2 g). 6. Carbograph SPE columns (Alltech, 150  mg, 300  mg and 1 g). 7. Conjugation solvent: 7/3 (v/v) dimethyl sulfoxide (DMSO)/ acetic acid (AcOH). 8. Methanol. 9. Sodium 95%).

cyanoborohydride

(NaCNBH3)

(Sigma-Aldrich,

10. Trifluoroacetic acid (TFA) (Fisher Scientific, HPLC grade). 11. MilliQ water (dH2O). 12. Analytical HPLC system with UV and Fluorescence detection (Shimadzu). 13. Semipreparative normal-phase (250 × 9.2 mm, Agilent).

(NP)

HPLC

column

14. Hypercarb HPLC column (PGC-HPLC column, 150 × 4.6 mm, Thermo Scientific). 15. Hypercarb guard column (20 × 4 mm, Thermo Scientific). 16. Centri-vap (Labconco) at room temperature. 17. Sodium azide (NaN3). 18. Centrifuge filter (Nylon 0.2 mm, Costar). 19. MALDI-TOF/TOF (Bruker UltraflexII). 20. Matrix solution: 5 mg/mL 2,5-dihydroxybenzoic acid in 50% acetonitrile with 0.1% TFA. 21. Solution A: 50% acetonitrile in 0.1% TFA. 22. HPLC solvent A: Acetonitrile (Fisher Scientific, HPLC grade). 23. HPLC solvent B: 0.25 M ammonium acetate, pH 4.5. 24. HPLC solvent C: water. 25. HPLC solvent D: 1% TFA in water. 26. Normal phase (NP) HPLC linear gradient: 0 min: 80% A, 4% B, 16% C; 90 min: 10% A, 50% B, 40% C.

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2.2. Synthesis of GlcNAc-6-PhosphoGAEABs and 6-Phospho-GAEABs

1. Fluorescent conjugates of purified isomers of high-mannose glycans were converted to their corresponding phosphodiesters using a recombinant GlcNAc-phosphotransferase (20) and kindly provided by Stuart Kornfeld and Marielle Boonen (Washington University, St. Louis, MO). 2. Hydrochloric acid (HCl). 3. Acetonitrile. 4. Trifluoroacetic acid. 5. LNnT-AEAB (prepared as described previously from LNnT) (9). 6. Hypercarb HPLC column (PGC-HPLC column, 150 × 4.6 mm, Thermo Scientific). 7. Hypercarb guard column (20 × 4 mm, Thermo Scientific). 8. Phosphorylation buffer: 0.1 M Tris-Hcl, pH 7.5, 0.1 M MgCl2, and 0.1 M MnCl2. 9. Hypercarb (PGC) HPLC linear gradient: 0 min: 15% A, 75% C, 10% D; 30 min: 45% A, 45% C, 10% D.

2.3. Printing Microarrays

1. MALDI-TOF/TOF (Bruker UltraflexII). 2. Piezorray noncontact printer (Perkin-Elmer). 3. ProScanArray Scanner (Perkin-Elmer). 4. Printing buffer (2×): 600 mM sodium phosphate buffer, pH 8.5. 5. NHS-activated America).

microarray

glass

slides

(Schott

North

6. Biotin-hydrazine (Sigma-Aldrich). 7. Blocking buffer: 50  mM ethanolamine in 50 mm sodium borate So Tris buffer, pH 9.0. 8. 384-well plate, V-shape (Biorad). 2.4. Interrogation of Phosphorylated Glycan Microarray with CD-MPR and CI-MPRs

1. CI-MPR and CD-MPR and antibodies for their detection were kindly provided by Nancy Dahms (Medical College of Wisconsin, Milwaukee, WI). 2. Assay buffer: 50  mM imidazole, pH 6.5, 150  mM NaCl, 10 mM MnCl2. 3. Binding buffer: Assay buffer containing 1% bovine serum albumin (BSA) and 0.05% Tween-20. 4. 16-Chamber adapter (Grace Biolabs). 5. Coplin jars. 6. Biotinylated lectins Concanavalin A (ConA) and Ricinus communis agglutinin-I (RCA-I) (Vector labs). 7. Cyanine5 (Cy5)-streptavidin (Zymed).

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8. Rabbit polyclonal antibodies specific for CD-MPR (B3.5) and CI-MPR (B14.5) (Generated in rabbits immunized with corresponding MPRs purified from bovine liver). 9. Alexa Fluor 488-Goat anti-rabbit IgG (Invitrogen).

3. Methods 3.1. Preparation of Functional Fluorescent High-Mannose Glycan Conjugates from RNaseB

1. Dissolve RNaseB (100 mg) in 5 mL 1× glycoprotein denaturing buffer and boil for 10 min at 100°C. To the cooled solution, add 1  mL of 10% NP40, 1  mL of 10× G7 buffer and 1  mL of 0.2% sodium azide solution. Then add PNGase F (30 mL, 500,000 units/mL) and 2 mL water. Digest the glycoprotein at 37°C for 3  days in a stoppered tube to prevent loss of water (see Note 1). 2. Precondition C18 Sep-pak cartridges by washing with 1 column volume (cv) of methanol followed by 2 cv of dH2O; Precondition carbograph cartridges by washing with 1 column volume (cv) Solution A followed by 3 cv dH2O. 3. The PNGase F digested solution is boiled for 5  min, passed through a 2  g C18 Sep-Pak cartridge and washed with dH2O (see Note 2). The flow-through and 2 cv water wash are pooled and applied on a 1 g carbograph cartridge, which is subsequently washed with 6 cv dH2O. Free glycans are then eluted from the carbograph cartridge with 3 cv of Solution A. The eluent is dried by Centri-vap prior to fluorescent derivatization. 4. For fluorescent derivatization, prepare a solution of AEAB by adding 88 mg AEAB to 1 mL conjugation solvent; vortex the mixture for 10 min. This solution can be stored at −20°C for future use. Prepare fresh sodium cyanoborohydride solution by adding 64  mg NaCNBH3 to 1  mL conjugation solvent; vortex for 2 min. 5. To the dried free glycans, add 500 mL AEAB solution and an equal volume of NaCNBH3 solution. Vortex the mixture for 1 min and incubate at 65°C for 2 h. Add acetonitrile (10 mL) to precipitate the glycans. Cool the mixture at −20°C for 30 min. 6. Centrifuge the mixture at 5,000 × g for 5  min. Discard the supernatant. Dry the pellet in a Centri-vap for 5  min and reconstitute it in 500 mL dH2O. The solution is then centrifuge-filtered and ready for normal phase HPLC purification. 7. A linear gradient (0 min: 80% A, 4% B, 16% C; 90 min: 10% A, 50% B, 40% C) is applied for the semipreparative NP-HPLC separation of GAEABs from RNaseB. Five well-separated peaks are collected, which corresponds to Man5-AEAB, Man6-AEAB, Man7-AEAB (isomers), Man8-AEAB (isomers), and Man9AEAB, respectively. Fractions are dried in a Centri-vap.

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8. Resuspend the dried fractions in dH2O and pass them individually through a preconditioned carbograph (use 1 g carbograph for ~10 mg GAEABs) for desalting as described in step 3. The eluent is lyophilized. Quantify these fractions based on their UV 330 nm absorption and fluorescence (Ex 330 nm/Em 420 nm); they are then ready for enzymatic phosphorylation. 9. Characterize the desalted fractions in step 8 using MALDITOF. Spot 0.5  mL matrix solution and then 0.5  mL sample. Run MALDI-TOF using reflective positive mode. 3.2. Preparation of GlcNAc-6-PhosphoGAEABs and 6-Phospho-GAEABs

1. Incubate the purified GAEABs (0.25–0.5 mmol) separately at 37°C with 0.5 mg of purified GlcNAc-phosphotransferase (20), and 1 mmol of UDP-GlcNAc in 20 mL of phosphorylation buffer. The reactions are stopped by freezing and lyophilizing after 48 h incubation. 2. The crude products are reconstituted into 100 mL dH2O and centrifuged. The clear supernatant is injected into porous graphitized carbon (PGC)-HPLC for separation (see Note 3). Apply a linear gradient (0 min: 15% A, 75% C, 10% D; 30 min: 45% A, 45% C, 10% D) (Fig. 1). The mono- and diphosphodiesters elute later than the starting GAEABs. Collect individual fluorescent peaks, which are then analyzed by MALDI-TOF and dried in Centri-vap. Structures are assigned according to the molecular weights and the HPLC behavior. 3. A portion of each fraction of phosphodiester is converted to its corresponding phosphomonoester by mild acid hydrolysis (0.01  M HCl, 1  h, 100°C). After heating in 10  mM HCl (100 mL) at 100°C for 1 h, the solution is cooled down and injected into PGC-HPLC for purification (see Note 4). 4. The AEAB derivatives of the phosphomonoesters, as well as the starting nonphosphorylated and phosphodiesters are quantified based on their UV absorbance relative to LNnT-AEAB standards and reconstituted to 200  mM in dH2O and stored frozen until use (see Note 5).

3.3. Printing Microarray of High-Mannose Glycan AEAB Derivatives and Corresponding Phosphomonoesters and Phosphodiesters

1. We use a noncontact Piezorray printer (Piezorray, PerkinElmer) to print the microarrays. 2. For each sample, 5 mL of the 200 mM solution described above is mixed with 5 mL 2× printing buffer and loaded into a 384well source plate with V-shape wells. The plate is fitted onto a metal adaptor for controlling the temperature during printing and set up securely in the printer. 3. According to the printer software, input the sample names, IDs, and positions. The printing pattern is programmed using the software according to manufacturer’s instructions, including

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Fig. 1. Purification of phosphodiesters of AEAB-derivatized glycans (GAEABs) by HPLC on PGC-HPLC. The GAEABs prepared from Man5-9 released from RNase B are converted to phosphodiesters by GlcNAc phosphotransferase as described in the text. The starting material (upper profiles) and the corresponding products of the phosphotransferase reactions (lower profiles) were applied to PGC-HPLC, and eluted with a gradient of acetonitrile and dH2O.

subarray number and sample replicate number. In this case, a microarray includes 14 identical subarrays. Each subarray incorporates eight GAEABs, eight phosphodiesters and eight phosphomonoesters along with two control glycans in six replicates. The structures and chart IDs are shown in Fig. 2. The printer software generates a GAL file based on the input, which is used by the Scanner software in the image processing step. 4. Bring NHS-activated slides from −20°C to room temperature in vacuum desiccators before opening the package. Then open the package, and set the desired number of NHS-slides on the printing platform. 5. Tune the four tips of the printer so that ideal morphology and size dispersion are reached according to manufacturer’s instructions. Start the printing process. 6. Perform a pin washing step to avoid carry over contamination from different samples. Pins are washed by pushing out sample with two cycles of water washes. The outside of the pins are washed with flowing water. Pins are then submerged in 1% Tween 20 in printing buffer and then sonicated. Two more full round of wash cycles with water are performed again.

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Fig. 2. The chart IDs of the individual glycans printed on the microarray.

7. After the printing is finished, the slides are set in a box in a 55°C water bath without direct water contact for 1  h. The water bath serves as a high humidity chamber. 8. The slides are washed with 0.05% Tween-20 in PBS, then dH2O, and then blocked with blocking buffer for 1 h, washed again with 0.05% Tween-20 in PBS and subsequently dried by centrifugation and stored desiccated at −20°C until use. 3.4. Interrogation of Phosphorylated Glycan Microarray with CD-MPR and CI-MPRs

1. The binding assay on the microarray includes binding, washing, and scanning steps. The proteins interact and specifically bind to their ligands printed on the microarray during the binding step. Nonspecific bound proteins and excess reagents are washed away in the washing step. The fluorescence-based detection is carried out using the microarray scanner in the last step to give a fluorescence image. In this experiment, biotinylated ConA and RCA I are used as controls to validate printing of the microarray, since they bind to specific types of glycans on the array. RCA-I binds to terminal Galb1, 4-GlcNAc, therefore it should only bind to LNnT-AEAB and NA2-AEAB on the array; ConA binds to the trimannosyl core of N-glycans, therefore it should bind to all glycans printed on the array except LNnT-AEAB, which is not an N-glycan. 2. The bound lectins are detected by a secondary incubation with Cy5-streptavidin. The CD-MPR and CI-MPRs, on the other hand, are detected with specific rabbit polyclonal antibodies followed by Alexa Fluor488-labeled goat anti-rabbit IgG.

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3. Remove slide(s) from desiccators and label slide as necessary on the top of the slide. 4. Do not write/touch on the slide where the glycans are printed. 5. Hydrate the slide by placing in a glass Coplin staining jar containing 100 mL of assay buffer for 5 min (see Note 6). 6. For multipanel experiments on a single slide, affix the multichamber adaptor on the slide to separate a single slide into 14 chambers sealed from each other during the assay. This allows for simultaneous multiple assays on up to 14 identical subarrays on the same slide. Apply sample (for example, biotinylated lectin, 50–100 mL) into each chamber and incubate the slide on a shaker at 60 rpm for 1 h. If some subarrays on one slide are not used, save the slide at −20°C for use at a later time. 7. For evaluation of printing efficiency, dissolve biotinylated lectins (ConA and RCA-I) in binding buffer (assay buffer containing 1% BSA and 0.05% Tween-20) at appropriate concentrations. Add the lectin solutions (50–100 mL) to separate chambers and incubate for 1 h at room temperature. Wash the chambers three times with the following buffers: (a) assay buffer containing 0.05% Tween-20 and (b) assay buffer. Detect bound lectins by a secondary incubation with 1 mg/mL streptavidin labeled with Cy5 and subsequently washed as described above. To remove salts prior to drying, wash the chambers three times with dH2O. 8. For assay with MPRs, apply proteins at the indicated concentrations in 70 mL of binding buffer to glycan arrays separated by the 16-chamber adapter, incubated for 1  h at room temperature, and processed as described above. After washing, apply 70 mL of 1:250 dilutions of rabbit polyclonal antibodies specific for CD-MPR and CI-MPR in binding buffer to appropriate subarrays and incubate for 1 h at room temperature. To detect MPRs, wash the subarrays as described, and add 70 mL of goat anti-rabbit IgG labeled with Alexa Fluor488 to each subarray and incubate for 1 h at room temperature. After washing, air-dry the slides. 9. Scan slide with a fluorescent scanner at the appropriate wavelengths. For Alexa Fluor488-labeled secondary antibodies, scan with 488 nm lamp, and to detect Cy5-streptavidin, scan with a 633 nm lamp. Save each image separately. 10. Process the image with Scanarray quantitation software to generate an excel worksheet as the raw data. Histograms with X-axis representing glycan chart IDs and Y-axis representing relative fluorescent units are generated for each protein sample. The binding of plant lectins and MPRs are shown in Figs. 3 and 4, respectively.

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Fig. 3. Binding of plant lectins to the phosphorylated high-mannose glycan microarray. The glycan microarray was printed as described in the text and the individual glycans are identified by their glycan number as indicated in Fig. 2. (a) Biotinylated ConA (0.5 mg/mL) detected with 5 mg/mL of Cy5-labeled streptavidin; (b) Biotinylated RCA-I (0.1 mg/mL) detected with 5 mg/mL of Cy5-labeled streptavidin. Error bars indicate ±1 standard deviation.

4. Notes 1. In this protocol, PNGase F digestion is applied directly to denatured RNaseB. For other glycoproteins, especially when larger amounts of glycoprotein are needed, the glycoproteins can be reduced and alkylated, then trypsinized. The dialyzed tryptic peptides are digested with PNGase F, after which the C18-carbograph purification procedure can be applied. This procedure increases the efficiency of PNGase F. 2. The capacity of C18 Sep-pak and carbograph varies toward various glycans. Typically, for 100  mg glycoprotein starting material, a 2 g C18 cartridge and a 1 g carbograph cartridge are used to obtain the free reducing glycans. 3. PGC-HPLC (Hypercarb) is a well-suited reverse phase system for the purification of phosphomonoesters and phosphodiesters. It has a wide pH range (0–14) and high capacity. The fractions collected can be dried and directly used for printing.

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Fig. 4. Binding of CD-MPR and CI-MPR to the phosphorylated high-mannose glycan microarray. (a) CD-MPR (50 mg/mL) detected with rabbit antibody B3.5 (1:250) and Alexa Fluor488-labeled goat anti-rabbit IgG (5  mg/mL); (b) CI-MPR (50  mg/mL) detected with rabbit antibody B14.5 (1:250) and Alexa Fluor488-labeled goat anti-rabbit IgG (5 mg/mL).

4. Phosphodiesters are relatively unstable toward acidic conditions, therefore need to be handled more carefully. However, the phosphomonoesters are fairly stable. 5. Extreme caution should be used to avoid contamination of buffers or glassware with alkaline phosphatase that might come into contact with phosphorylated glycans. 6. We have also used Tris buffers and MES buffers at pH 6.5 which is the pH optimum for ligand binding by the MPRs. Binding of the CD-MPR is influenced by divalent cations, with highest binding affinities observed in the presence of MnCl2, whereas the binding affinity of the CI-MPR is not significantly affected by divalent cations. References 1. de Paz JL, and Seeberger PH. (2006) Recent advances in carbohydrate microarrays. QSAR & Combinatorial Science 25, 1027–1032. 2. Feizi T, and Chai W. (2004) Oligosaccharide microarrays to decipher the glyco code. Nat Rev Mol Cell Biol 5, 582–588.

3. Hirabayashi J. (2003) Oligosaccharide microarrays for glycomics. Trends Biotechnol 21, 141–143. 4. Park S, Lee MR, and Shin I. (2008) Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes. Chem Commun (Camb), 4389–4399.

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5. Blixt O, Head S, Mondala T, Scanlan C, Huflejt ME, Alvarez R, Bryan MC, Fazio F, Calarese D, Stevens J, Razi N, Stevens DJ, Skehel JJ, van Die I, Burton DR, Wilson IA, Cummings R, Bovin N, Wong CH, and Paulson JC. (2004) Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci U S A 101, 17033–17038. 6. Ratner DM, Adams EW, Su J, O’Keefe BR, Mrksich M, and Seeberger PH. (2004) Probing protein-carbohydrate interactions with microarrays of synthetic oligosaccharides. Chembiochem 5, 379–382. 7. de Boer AR, Hokke CH, Deelder AM, and Wuhrer M. (2007) General microarray technique for immobilization and screening of natural glycans. Anal Chem 79, 8107–13. 8. Song X, Lasanajak Y, Xia B, Smith DF, and Cummings RD. (2009) Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays. ACS Chem Biol 4, 741–750. 9. Song X, Xia B, Stowell SR, Lasanajak Y, Smith DF, and Cummings RD. (2009) Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem Biol 16, 36–47. 10. Bohorov O, Andersson-Sand H, Hoffmann J, and Blixt O. (2006) Arraying glycomics: a novel bi-functional spacer for one-step microscale derivatization of free reducing glycans. Glycobiology 16, 21C-27C. 11. Park S, Lee MR, and Shin I. (2009) Construction of carbohydrate microarrays by using one-step, direct immobilizations of diverse unmodified glycans on solid surfaces. Bioconjug Chem 20, 155–162. 12. Feizi T, Fazio F, Chai W, and Wong CH. (2003) Carbohydrate microarrays – a new set of technologies at the frontiers of glycomics. Curr Opin Struct Biol 13, 637–645. 13. Liu Y, Feizi T, Campanero-Rhodes MA, Childs RA, Zhang Y, Mulloy B, Evans PG, Osborn HM, Otto D, Crocker PR, and Chai W. (2007) Neoglycolipid probes prepared via oxime ligation

for microarray analysis of oligosaccharide-protein interactions. Chem Biol 14, 847–859. 14. Song X, Lasanajak Y, Olson LJ, Boonen M, Dahms NM, Kornfeld S, Cummings RD, and Smith DF. (2009) Glycan microarray analysis of P-type lectins reveals distinct phospho-mannose glycan recognition. J Biol Chem. 284, 35201–35214. 15. Kudo M, and Canfield WM. (2006) Structural requirements for efficient processing and activation of recombinant human UDP-Nacetylglucosamine:lysosomal-enzyme-Nacetylglucosamine-1-phosphotran sferase. J Biol Chem 281, 11761–11768. 16. Dahms NM, and Hancock MK. (2002) P-type lectins. Biochim Biophys Acta 1572, 317–340. 17. Dahms NM, Olson LJ, and Kim JJ. (2008) Strategies for carbohydrate recognition by the mannose 6-phosphate receptors. Glycobiology 18, 664–678. 18. Ghosh P, Dahms NM, and Kornfeld S. (2003) Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol 4, 202–212. 19. Kornfeld S, and Mellman I. (1989) The biogenesis of lysosomes. Annu Rev Cell Biol 5, 483–525. 20. Chavez CA, Bohnsack RN, Kudo M, Gotschall RR, Canfield WM, and Dahms NM. (2007) Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester). Biochemistry 46, 12604–12617. 21. Bohnsack RN, Song X, Olson LJ, Kudo M, Gotschall RR, Canfield WM, Cummings RD, Smith DF, and Dahms NM. (2009) Cationindependent mannose 6-phosphate receptor: A composite of distinct phosphomannosyl binding sites. J Biol Chem. 284, 35215–35226. 22. Gordon JA, Blumberg S, Lis H, and Sharon N. (1972) Purification of soybean agglutinin by affinity chromatography on sepharose-N-epsilonaminocaproyl-beta-D-galactopyranosylamine. FEBS Lett 24, 193–196.

Chapter 10 Production of Fluorous-Based Microarrays with Uncharged Carbohydrates Sahana K. Nagappayya and Nicola L.B. Pohl Abstract Fluorous-based carbohydrate microarrays provide an alternative to traditional covalent microarray ­platforms for probing protein–carbohydrate-binding interactions. The most studied plant lectin, concanavalin A  (ConA), is known to bind to terminally a-linked mannose. In the studies presented, the binding of ConA with a-mannose is analyzed using a microarray formed on a fluorous-coated glass slide with the sugar containing a fluorous tag at the anomeric position. Key words: Fluorous, Microarray, Carbohydrate, Lectin, FITC-ConA

1. Introduction Carbohydrate microarrays are a facile method to qualitatively and quantitatively study carbohydrate binding with various binding partners like proteins and antibodies (1). Given the difficulty in obtaining large amounts of pure oligosaccharides from either synthesis or isolation from natural sources, microarray platforms are a particularly good method to study the interaction of these saccharides with other molecules as only a milligram of sugar can be used to form dozens of microarray spots. Whereas most microarray platforms require covalent attachment of the analyte to the slide, an alternate noncovalent approach relies on the hydrophobic, or “fluorophobic” interactions of a fluorocarbontagged compound with a perfluoroalkylsilane-coated glass slide surface. Such fluorous-based microarrays have seen use for probing carbohydrates as well as noncarbohydrate molecules (2, 3). In the case of carbohydrates, a fluorous-tagged carbohydrate – obtained through direct reaction of the reducing end of an unmodified sugar

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Fig. 1. Schematic of work process for fluorous-based carbohydrate microarray production and screening.

with a tag (4) or through synthesis in which the fluorous tag aids the purification steps throughout (5) is spotted onto the fluorous glass slide using a standard robot used for DNA arraying (Fig.  1). The fluorous tail of the carbohydrate aligns itself onto the glass in a humidified chamber and then the slide is incubated with a protein of interest, for example the jack bean lectin concanavalin A (ConA) labeled with fluorescein isothiocyanate (FITC) (5, 6). After the slide is washed to remove nonbinding proteins, the binding of FITCConA with a-mannose is then observed under a standard fluorescence slide scanner (5, 6).

2. Materials 2.1. Printing on Fluorous-Coated Glass Slide

1. Fluorous-tagged carbohydrates are synthesized (7). The fluorous tail for each carbohydrate contains a four-carbon alkyl spacer at the anomeric position linking 3-(perfluorooctyl) propanol tag to the sugar. The solutions of the carbohydrate for printing are prepared in 40% methanol, 40% DMSO, 20% water (5) (see Note 1). These solutions are placed in a 384well plate in a pattern defined in the arrayer.

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2. Fluorous-coated glass slides coated with perfluoroocty­lethylsilyl chains are commercially available from Fluorous Technologies Inc (Pittsburgh, PA). 3. 0.1–0.2 mM solution of FITC-ConA in 1× phosphate-­buffered saline (PBS) (5) (see Note 2). 4. The spotting is carried out using a robotic spotter with pin ­lifting technology (Cartesian PixSys 5500 Arrayer, Cartesian Technologies, Inc., Irvine, CA) greater than 60% relative humidity. 2.2. Microarray Incubation, Washing, and Screening

1. 1 mM CaCl2 and 1 mM MnCl2 solution in deionized water. 2. For incubation, 10 mL 1 mM CaCl2, 10 mL 1 mM MnCl2 and 200  mL of FITC-ConA solution are added to 780  mL of 1× PBS buffer set at pH 7.2 (see Note 2). 3. PC500 CoverWell incubation chamber (Grace Biolabs, Bend, OR) to incubate the array. 4. For washing, 1× PBS buffer containing 1% BSA solution.

2.3. Scanning of the Glass Slide

1. Fluorous-coated glass slides are scanned using general scanning on a ProScan Array HT (Perkin Elmer Life and Analytical Sciences, Shelter, CT) set at 488 nm, the common wavelength to detect the FITC label at 10 mm resolution.

3. Methods Fluorous-tagged carbohydrates are dissolved in 40% methanol, 40% DMSO, and 20% water. This solvent combination was chosen for the unique role of each solvent. Methanol is used to dissolve fully deprotected fluorous-tagged carbohydrates; DMSO is used to decrease the speed of solvent evaporation allowing enough time for the fluorous tail to orient itself on the glass slide. The role of water is to facilitate the hydrophobic fluorous–fluorous interaction. After printing, the glass slide is allowed to dry in a humidified chamber to further enforce the orientation of the fluorous tags toward the slides and the hydrophilic sugars on the surface. (Note that this process does not likely form a monolayer, though.) The slide is then placed in a PC500 CoverWell incubation chamber. For the detection of protein–carbohydrate binding, FITC-ConA in PBS buffer is applied to the printed glass slide and the slide is incubated for an hour. The carbohydrate that recognizes the protein ConA will noncovalently bind to it. Washing is carried out using PBS buffer and deionized water to get rid of unbound protein; the glass slide is dried and stored in a dark chamber and scanned for the protein–carbohydrate interaction.

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3.1. Carbohydrate Spotting

1. Fluorous-tagged carbohydrates are synthesized. The fluorous tail for each carbohydrate contains a perfluorooctyl propane (or ethane, which is less reliably made) tag attached to the anomeric position of the sugar separated by a four-carbon alkyl linker (6, 7). 2. Completely deprotected fluorous-tagged carbohydrate is dissolved in 40% methanol, 40% DMSO, and 20% water. The solution of the carbohydrate is prepared in methanol and diluted with DMSO and water to make the concentration of the carbohydrate from 125 to 450 mM (5). 3. 30  mL of the fluorous-tagged carbohydrate is deposited in a 384-well plate using a micropipette. 4. Using the robotic spotter with pin lifting technology the carbohydrate solution is spotted onto the 12 × 2 cm2 fluorinated glass slide coated with perfluorooctylethylsilyl chains at a relative humidity of 60%. The spotting pin is dipped into the carbohydrate solution for one second, and then the robotic pin takes 25  ms to spot each spot on the slide at a distance of 400  mm apart (5, 6). The robotic spotter pin-head is programmed to be washed with pure distilled water after each spotting. 5. The glass slide is allowed to dry in a humidified chamber for 18–24 h after spotting.

3.2. Incubation and Washing

1. The printed glass slide is carefully placed in a PC500 CoverWell incubation chamber. 2. 200 mL solution of FITC-ConA (1 mM) incubation solution is applied onto the printed glass slide. This glass slide is incubated in the incubation chamber for 1 h (see Note 3) (4). 3. The incubated glass slide is washed once with 1× PBS buffer containing 0.01% Tween-20 and then with deionized water two to three times. Air is blown gently over this slide to let it dry and then it is placed in a dark chamber for ~2 h to let it completely dry (see Note 4).

3.3. Scanning

1. The washed glass slide is stored in a dark chamber and then scanned using a General Scanning ProScan Array HT set at 488 nm (see Note 5).

4. Notes 1. After preparation all carbohydrate solutions are stored at 4°C. 2. The PBS buffer is prepared by diluting the commercially available 10× PBS with deionized water. The pH of the PBS buffer is set at 7.26 and checked each time using a pH meter.

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3. The FITC-ConA solution will decompose if exposed to light or allowed to stand at room temperature for more than 4 h. This solution is prepared in vials covered with aluminum foil immediately before carrying out the arrays. 4. The number of washings of the glass slides with PBS buffer and deionized water can greatly affect the outcome of the fluorescent scan. A few initial washings as a trial have to be carried out to find a suitable washing protocol for the carbohydrateprotein to be studied and both positive and negative control compounds should be spotted and tested in the same well. Generally, each compound is also spotted 8–12 times to ascertain the consistency of the particular microarray spotter. (Such multiple spotting is particularly important for quantitative binding experiments.) 5. This general protocol can be adapted to various noncharged mono- and oligosaccharides tagged with a variety of fluorous tails to find their binding with other carbohydrate-binding proteins, such as lectins and antibodies.

Acknowledgments We are grateful to Iowa State University, the US National Science Foundation (MCB-0349139 CAREER grant and CHE-0911123) and the US National Institutes of Health (R41 GM075436-01, R42 GM075436, and R42 GM075436-03  S1) with Fluorous Technologies, Inc. for their support of our microarray development work. References 1. Pohl NL, (2008) Fluorous tags catching on microarrays. Angew Chem Int Ed 47:3868–3870 2. Nicholson RL, Ladlow ML, Spring DL (2007) Fluorous tagged small molecule microarrays. Chem Commun 3906–3908 3. Vegas AJ, Bradner JE, Tang W, MvPherson OM, Greenberg EF, Koehler AN, Schreiber SL (2007) Fuorous-based small-molecule microarrays for the discovery of histone deacetyase inhibitors. Angew Chem Int Ed 46: 7960–7964 4. Chen G, Pohl NL (2007) Synthesis of fluorous tags for incorporation of reducing sugars in

quantitative microarray platform. Org. Lett 10: 785–788 5. Jaipuri FA, Collet B Y M, Pohl NL (2008) Synthesis and quantitative evaluation of glycero-D-mannoheptose binding to Concanavalin A by fluorous-tag assistance. Angew Chem Int Ed 47:1708–1710 6. Ko K-S, Jaipuri FA, Pohl NL (2005) Fluorousbased carbohydrate microarrays. J Am Chem Soc 127: 12162–13163 7. Mamidyala SK, Ko K-S, Jaipuri FA, Park G, Pohl NL (2006) Noncovalent fluorous interactions for the synthesis of carbohydrate microarrays. J Flourine Chem. 127: 571–579

Chapter 11 General Procedure for the Synthesis of Neoglycoproteins and Immobilization on Epoxide-Modified Glass Slides Yalong Zhang and Jeffrey C. Gildersleeve Abstract Neoglycoproteins, such as BSA-glycosides, contain carbohydrates covalently attached to a protein carrier via nonnaturally occurring linkages. These conjugates have been used for decades to study carbohydrate–protein interactions and are frequently used as immunogens to raise antibodies to carbohydrate antigens. In fact, neoglycoproteins have been used extensively as vaccine antigens and several have obtained FDA approval. More recently, neoglycoproteins have been used in the construction of glycan arrays to produce “neoglycoprotein microarrays.” In this chapter, two methods for preparing neoglycoproteins are described along with methods to immobilize these conjugates on epoxide-coated glass microscope slides to produce arrays. Key words: BSA-glycosides, Neoglycoprotein, Glycan array, Lectin inhibitors, Cancer biomarker

1. Introduction Carbohydrate–protein interactions play fundamental roles in many biological processes, such as fertilization, inflammation, viral and bacterial infection, and cancer metastasis (1). Therefore, there have been significant efforts to identify and characterize carbohydrate– protein interactions and to develop agonists/antagonists of these interactions. For these objectives, neoglycoproteins have been extremely useful (2, 3). Neoglycoproteins contain one or more copies of a carbohydrate covalently attached to a carrier protein, such as albumin. They are distinct from natural glycoproteins in that the linkage between the carbohydrate and protein is nonnatural. More importantly, neoglycoproteins are normally much more homogeneous than natural glycoproteins, which are usually produced as complex mixtures of glycoforms having different glycan chains attached to the protein core and/or have varying degrees of

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Fig. 1. Coupling via reductive amination.

glycosylation site occupancy. Neoglycoproteins can be used in many common assays developed for proteins/glycoproteins, such as ELISAs and Western blots. They have also been used extensively as multivalent inhibitors of carbohydrate–protein interactions and as vaccine antigens. Many methods have been developed for conjugating carbohydrates to proteins to produce neoglycoproteins. Polysaccharides and oligosaccharides that contain a free reducing end can be coupled directly to lysine residues via reductive amination (see Fig. 1) (4, 5). The process results in ring opening of the reducing-end monosaccharide residue, but this is often acceptable, especially for longer glycan chains. Alternatively, carbohydrates can be synthesized or derivatized with a linker to facilitate conjugation. For example, linkers containing a free carboxylic acid can be coupled to free amines on proteins via formation of an activated ester, such as an N-hydroxysuccinimide ester (see Fig. 2) (6) or p-nitrophenyl esters (7). Linkers containing a free thiol can be conjugated to maleimide-derivatized proteins or dehydroalanine-containing proteins (8). Other conjugation methods include copper(I)-catalyzed azide–alkyne cycloaddition (9) and conjugation of two amines with a squaric decyl ester (10). Typically, the resulting product contains a distribution of conjugates, with varying attachment sites and conjugation ratios. For example, coupling to lysines might produce a product with an average conju­gation ratio of 20 carbohydrates per molecule of protein. However, there is a significant proportion of conjugates with 18, 19, 21, or 22 carbohydrates per molecule of protein, and the exact lysines that are modified vary from one molecule of protein to another. Nonetheless, they each have only one type of carbohydrate structure attached. Methods for sitespecific attachment of glycan chains to proteins have also been developed (11–14). Glycan arrays have recently been developed as high-throughput tools for studying carbohydrate–protein interactions (15–17).

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Fig. 2. Coupling via EDC activation. (a) Examples of sugar acids used for coupling to albumin and (b) reaction sequence for coupling.

Glycan arrays contain many different carbohydrates immobilized on a solid support in a spatially defined arrangement. They can be constructed in many ways, such as immobilization of linkermodified carbohydrates, lipid-modified carbohydrates, and neoglycoproteins (15–18). Our group has focused on printing neoglycoproteins on epoxide-coated glass microscope slides to produce neoglycoprotein microarrays (6, 19, 20). Since many neoglycoproteins are commercially available and others are readily prepared from oligosaccharides, this approach provides a convenient method to produce glycan arrays. In addition, the neoglycoproteins used on the array surface can be used in solution as agonists/ antagonists of carbohydrate–protein interactions and can be used in many other assays, enabling one to readily transition from array results to other applications. In this chapter, we describe two methods for preparing and characterizing neoglycoproteins. The first method involves reductive amination of oligosaccharides containing a free reducing end. The second involves activation of glycans that contain a linker with a free carboxylic acid. In addition, we describe methods to immobilize neoglycoproteins on epoxide-coated glass microscope slides to prepare glycan arrays, along with quality control checks to verify effective printing.

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2. Materials 2.1. Solutions for Reductive Amination Method

1. BSA solution (10×): 150 mg/mL bovine serum albumin (BSA, essentially protease free, ³98%, Sigma, St. Louis, MO). 2. Borate buffer (10×): 400  mM sodium tetraborate (Sigma), adjusted with 1 M NaOH to pH 8.5. Store at room temperature. If a precipitate forms, heat at 70°C until the solution is clear. 3. Sodium sulfate solution (6×): Approximately 3 M sodium sulfate (Sigma) was prepared at a temperature of 50°C to produce a saturated solution. Solutions are stored at room temperature but are reheated to 50°C prior to use. 4. Oligosaccharide solutions: 20  mM oligosaccharides in water. Solutions were stored frozen at −20°C until used. 5. Sodium cyanoborohydride solution (10×): 3 M sodium cyanoborohydride (Sigma). Prepare a fresh solution prior to reactions. 6. Dialysis buffer: 6 mM NaCl (Sigma) solution. 7. Dialysis units (MWCO 10 000, Slide-A-Lyzer MINI Dialysis Unit, Pierce, Rockford, IL). 8. Applied Biosystem Voyager-DE Pro MALDI-time of flight (TOF) mass spectrometry (International Equipment Trading Ltd., Vernon Hills, IL) for evaluating the content of glycans on BSA. 9. MALDI matrix solution: Sinapinic acid (3,5-dimethoxy-4hydroxycinnamic acid, Sigma) as matrix for all experiments. Matrix was prepared as a saturated solution in 50% acetonitrile/ H2O (0.1% TFA) (v/v).

2.2. Solutions for Active Ester Method

1. 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC; Anaspec, Fremont, CA) was dissolved in water and then diluted with an equal volume of DMF to give a final concentration of 300 mM. Prepare a fresh solution prior to reactions. 2. 300  mM N-Hydroxysuccinimide (NHS; Aldrich, St. Louis, MO). Prepare a fresh solution prior to reactions. 3. 4  mg/mL BSA in sodium borate buffer (10  mM sodium borate, 90 mM NaCl, pH 8.0). 4. Glycan solutions: Glycans containing a linker with a free carboxylic acid (sugar acids) were dissolved in water at a final concentration of 150 mM. Solutions were stored frozen at −20°C until used. Sugar acids are typically obtained by chemical synthesis. For example, glycopeptides that contain a free C-terminal carboxylic acid can be prepared via solid-phase peptide synthesis, deprotection, and purification.

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1. Printing buffer: 1× PBS, 2.5% glycerol, 0.006% Triton-X 100. 2. PBS buffer (20×): 2.74  M NaCl, 60  mM KCl, 200  mM Na2HPO4, 36  mM KH2PO4, pH 7.4. Store at room temperature. 3. Fluorophore-labeled BSA (Invitrogen Corporation, Carlsbad, CA) and BSA-glycosides were diluted with printing buffer to a concentration of 125 mg/mL. 4. ArrayIt SuperEpoxy 2 glass slides (TeleChem International, Inc., Sunnyvale, CA). 5. PBST0.05 washing buffer: 1× PBS, 0.05% tween 20. 6. Robotic microarrayer: MicroGrid II 600/610 (Genomic Solutions, BioRobotics, Ann Arbor, MI). 7. ArrayIt stealth pins: SMP2 pins (uptake volume 0.25  mL, delivery volume 0.5  nL) were purchased from TeleChem International, Inc. (Sunnyvale, CA). 8. Sample plates: 384-well V-bottom sample plates (Genetix, San Jose, CA). 9. 16-well slide modules (Grace Bio-Labs, Bend, OR). 10. Genepix 4000A microarray scanner (Molecular Devices Corporation, Union City, CA).

3. Methods 3.1. Preparation of BSA-Glycosides by Reductive Amination Method

1. Mix BSA (2  mL, 150  mg/mL) with sodium borate (5.5 mL, 400 mM, pH 8.5), sodium sulfate (3.7 mL, saturated at 50°C), oligosaccharide (3.3 mL, 20 mM solution for 15 equiv), and water (5.3  mL) in a 200-mL PCR tube. Add sodium cyanoborohydride (2.2 mL, 3 M) and cap the tube (see Notes 1 and 2). 2. Incubate the solution in a PCR thermal cycler at 56°C for 96 h with a heated lid (see Note 3). 3. Dilute the reaction with H2O (100 mL), transfer to a 500-mL dialysis tube, and dialyze against H2O (2.5 L) containing 6 mM NaCl three times. 4. Dilute the dialyzed BSA conjugates to a final concentration of 1.0 mg/mL and store at −20°C.

3.2. Preparation of BSA-Glycosides by Active Ester Method

1. Mix sugar acid, NHS (300 mM), and then EDC (300 mM) in an Eppendorf tube at a volume ratio of 2:1:1 and incubate at room temperature with occasional gentle mixing to form the NHS ester in situ (see Note 4). 2. After 1 h, add the solution containing the NHS ester to a solution of BSA (4  mg/mL in sodium borate buffer, pH 8.0). Precool the solution to 4°C.

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3. After 15  min, warm the solution to room temperature and incubate for another 1 h. 4. Dialyze BSA-conjugates against H2O (2.5 L) containing 6 mM NaCl three times using dialysis units. 3.3. MALDI MS Analysis of BSA-Glycoside

1. BSA conjugates are analyzed by MALDI-TOF MS and the extent of conjugation is determined by subtracting the average mass of BSA (66431) from the average mass of the conjugate and then dividing by the molecular weight of the sugar acid minus 18 (see Notes 5 and 6). 2. The instrument is operated in linear mode under positive condition with the accelerating voltage of 25 kV, guide wire 0.15%, and grid voltage 91.5%. 3. A nitrogen laser should be used at 337 nm with 250 laser shots averaged per spectrum. 4. Sample preparation is a modified “dried droplet” procedure, whereby 0.3 mL of samples are spotted on a MALDI sample plate followed by 0.3 mL of matrix. The mixture is then allowed to air dry prior to analysis. 5. Data analysis is carried out using Data Explorer software resident on Voyager mass spectrometer and used BSA as a standard for external calibration.

3.4. Carbohydrate Microarray Fabrication

1. Transfer 15 mL of fluorophore-labeled BSA and neoglycoprotein solution (125 mg/mL) to wells of a 384-well V-bottom sample plates (see Notes 7 and 8). 2. Print carbohydrate microarrays on epoxide-modified glass slides using a robotic microarrayer fitted with SMP2 Microspotting Pins (see Note 9). 3. The humidity level should be maintained at ~50% with an ultrasonic humidifier. 4. Each BSA-glycoside component should be printed in duplicate (see Figs. 3 and 4). The diameter of each spot is around 90 mm and the pitch is 200 mm (see Note 10). 5. Printed slides are allowed to dry at room temperature for an additional few hours (keeping the total exposure time less than 36 h) and then stored at −20°C until use (see Notes 11 and 12).

3.5. Quality Assessment of Microarray Slides

Most slides have a complete array grid. Toward the end of a print run, pins may occasionally run out of liquid resulting in missing spots. In addition, pins may occasionally get clogged, resulting in missing spots. Tolerance for missing spots depends on the particular application. For highest quality data, only slides containing a complete grid should be used. For less-critical experiments, such as

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Fig. 3. Microarray printing. Hollow pins are dipped into wells of a 384-well plate containing print solutions. The filled pins are then moved to the slides and lightly tapped on the glass to print array spots.

Fig. 4. Immobilization of neoglycoproteins on epoxide-coated slides.

optimizing reaction parameters, missing spots are acceptable. Below, we describe two procedures for quality assessment of microarray slides. 3.5.1. Physical Inspection of Printed Arrays

1. Inspect each printed slide under a microscope at 10× magnification (see Fig. 5). 2. Note the slide, block, column, and row number for any missing spots. 3. Evaporation can complicate the analysis. Inspect slides immediately after printing.

3.5.2. Evaluate Binding with One or More Lectins

1. Fit slides with 16-well slide modules. 2. Block slides by addition of 3% BSA/PBS (200 mL per well) and incubate at room temperature for 2.0 h. 3. After removing block solution, incubate slides with one or more lectins (diluted in 1% BSA/PBS; 100  mL per well) at room temperature for 2  h. Lectins are typically assayed at a concentration of 1–10 mg/mL (see Note 13).

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Fig. 5. View of microarray spots after printing. A magnified view of liquid spots after printing showing uniform spots with consistent spacing and no missing spots.

4. After washing 3× with PBST0.05 (200 mL per well), incubate slides with Cy3–streptavidin (2  mg/mL in 1% BSA/PBS; 100 mL per well) at room temperature for 2 h. 5. After washing 3× with PBST0.05 (200 mL per well), remove slides from the well module and immerse in PBST0.05 for 15 min. 6. Place the slide in a 50-mL conical tube and centrifuge for 5 min at 200 × g to dry the slide. 7. Scan slides using a Genepix 4000A microarray scanner at PMT voltage settings, where no saturated pixels are obtained. Image analysis is carried out with Genepix Pro 6.0 analysis software from the same company. Spots are defined as circular features with maximum diameter of 100 mm. The background-corrected median feature intensity, F532median-B532, should be used for data processing. 8. Binding data is compared with results from previous batches of slides and with published binding data for each lectin.

4. Notes Reductive amination 1. The reaction conditions have been optimized for BSA. Other proteins may be more or less tolerant of the high salt conditions and high temperature. 2. Reactions occasionally result in precipitation, especially at higher temperatures.

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3. Evaporation and condensation of liquid on the lid of the reaction tube can be a significant problem, especially for small scale reactions. The use of a PCR tube and a PCR thermal cycler with a heated lid minimizes this problem. EDC/NHS coupling 4. EDC should be added last to the reaction mixture. MALDI MS analysis 5. The ratio of sugar acid to protein can be modulated to produce conjugates with varying numbers of carbohydrates. For the active ester method, at a ratio of 30:1, we typically get about 15 oligosaccharides per molecule of BSA. At 7:1, the final ratio is typically around 4/BSA. 6. For the reductive amination method, at a starting ratio of 9:1, the product typically has about 4-5 oligosaccharides per molecule of BSA. At a ratio of 35:1, the final conjugate should have around 15 oligosaccharides per molecule of BSA. Printing 7. We typically print Cy5–BSA as the first component and Cy3– BSA toward the end of the grid. These fluorophore–BSA conjugates simplify alignment of the array grid when analyzing experiments and serve as quality control checks for the print procedure. For example, carryover due to insufficient washing of pins is easily detected. 8. Print solutions in the source plate evaporate over time. Water should be added to compensate for evaporation. For example, when a source plate is in use for 4–5 h, we would add about 2 mL of water to each well. The exact amount depends on the total time, humidity, temperature, and arrayer. 9. Clean pins thoroughly to avoid carryover from one print component to the next. The optimal conditions should be determined experimentally. For our recommended print solutions and a MicroGrid II microarrayer, we recommend at least two complete wash cycles with a 5-mm submission height, a 3-s dwell time in each of two wash baths, and an 8-s drain time (where the water in the pins is removed by vacuum). 10. The optimal printing conditions are dependent on the type of microarrayer. For a MicroGridII fitted with SMP2 pins, our preferred conditions are listed in Tables 1 and 2. 11. Keep the total time for the printing process below 36  h to avoid protein degradation. 12. Cy5 is sensitive to degradation and photobleaching. Slides containing Cy5–BSA or Cy5-labeled proteins should be stored in the dark at −20°C.

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Table 1 Prespotting settings (to remove excess liquid on the pins) Parameter

Description

Recommended value

Number of prespots

Spotting times of a freshly loaded pin

6

Delay before spotting

Time of suspended pins above slide

0.5 s

Target height

Distance of pin into contact with slide

1.5 mm

Speed

Speed of pin into contact with slide

4 mm/s

Surface height

Thickness of slide

1 mm

Dwell time

Time tool is held in place at its target height

0.250 s

Multiple strikes

Number of tapping for each spot

1

Pitch

Distance between the centers of adjacent spots

0.45 mm

Table 2 Printing parameters Parameter

Description

Recommended value

Number of slides

High-print-quality slides

12–15

Delay before spotting

Time of suspended pins above slide

0 s

Target height

Distance of pin into contact with slide

1 mm

Speed

Speed of pin into contact with slide

4 mm/s

Surface height

Thickness of slide

1 mm

Dwell time

Time tool is held in place at its target height

0 s

Pitch

Distance between the center of spots

0.2 mm

Quality control checks 13. The choice of lectin(s) for quality control assessment depends on the composition of the array. We frequently use wheat germ agglutinin (WGA), ricinus communis agglutinin (RCA120), bauhinia purpurea agglutinin (BPA), and concanavalin A (ConA).

Acknowledgments This research was supported by the Intramural Research Program of the NIH, NCI.

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References 1. Dwek, R. A. (1996) Glycobiology: Toward Understanding the Function of Sugars, Chem. Rev.96(2), 683–720. 2. Roy, R. (1996) Syntheses and some applications of chemically defined multivalent glycoconjugates, Curr. Opin. Struc. Biol. 6, 692–702. 3. Stowell, C. P., and Lee, Y. C. (1980) Neoglyco­ proteins: the preparation and application of synthetic glycoproteins, Adv. Carbohyd. Chem. Bi. 37, 225–281. 4. Gildersleeve, J. C., Oyelaran, O., Simpson, J. T., and Allred, B. (2008) Improved Procedure for Direct Coupling of Carbohydrates to Proteins via Reductive Amination, Bioconjugate Chem.19(2), 1485–1490. 5. Clausen, M., and Madsen, R. (2004) Synthesis of oligogalacturonates conjugated to BSA, Carbohyd. Res. 339, 2159–2169. 6. Manimala, J. C., Roach, T. A., Li, Z., and Gildersleeve, J. C. (2006) High-throughput carbohydrate microarray analysis of 24 lectins, Angew. Chem. Int. Edit. 45, 3607–3610. 7. Wu, X., Ling, C.-C., and Bundle, D. R. (2004) A New Homobifunctional p-Nitro Phenyl Ester Coupling Reagent for the Preparation of Neoglycoproteins, Org. Lett. 6, 4407–4410. 8. Wang, J., Schiller, S. M., and Schultz, P. G. (2007) A biosynthetic route to dehydroalaninecontaining proteins, Angew. Chem. Int. Edit. 46, 6849–6851. 9. van Kasteren, S. I., Kramer, H. B., Jensen, H. H., Campbell, S. J., Kirkpatrick, J., Oldham, N. J., Anthony, D. C., and Davis, B. G. (2007) Expanding the diversity of chemical protein modification allows post-translational mimicry, Nature 446, 1105–1109. 10. Karelin, A. A., Tsvetkov, Y. E., Paulovicova, L., Bystricky, S., Paulovicova, E., and Nifantiev, N. E. (2009) Synthesis of a heptasaccharide fragment of the mannan from Candida guilliermondii cell wall and its conjugate with BSA, Carbohydr. Res. 344, 29–35.

11. Liu, H., Wang, L., Brock, A., Wong, C.-H., and Schultz, P. G. (2003) A method for the generation of glycoprotein mimetics, J. Am. Chem. Soc. 125, 1702–1703. 12. van Kasteren, S. I., Kramer, H. B., Gamblin, D. P., and Davis, B. G. (2007) Site-selective glycosylation of proteins: creating synthetic glycoproteins, Nat. Protoc. 2, 3185–3194. 13. Zhang, Y., Bhatt, V. S., Sun, G., Wang, P. G., and Palmer, A. F. (2008) Site-Selective Glycosylation of Hemoglobin on Cys b93, Bioconjugate Chem. 19, 2221–2230. 14. Chen, R., and Tolbert, T. J. (2010) Study of On-Resin Convergent Synthesis of N-Linked Glycopeptides Containing a Large High Mannose N-Linked Oligosaccharide, J. Am. Chem. Soc. 132, 3211–3216. 15. Bernardes, G. J. L., Castagner, B., and Seeberger, P. H. (2009) Combined Approaches to the Synthesis and Study of Glycoproteins, ACS Chem.Biol. 4, 703–713. 16. Liang, C.-H., and Wu, C.-Y. (2009) Glycan array: a powerful tool for glycomics studies, Expert Rev. Proteomics 6, 631–645. 17. Oyelaran, O., and Gildersleeve, J. C. (2009) Glycan arrays: recent advances and future challenges, Curr. Opin. Chem. Biol. 13, 406–413. 18. Zhao, J., Patwa, T. H., Lubman, D. M., and Simeone, D. M. (2008) Protein biomarkers in cancer: natural glycoprotein microarray approaches, Curr. Opin. Mol. Ther. 10, 602–610. 19. Zhang, Y., Li, Q., Rodriguez, L. G., and Gildersleeve, J. C. (2010) An Array-Based Method To Identify Multivalent Inhibitors, J. Am. Chem. Soc. 132, 9653–9662. 20. Oyelaran, O., Li, Q., Farnsworth, D., and Gildersleeve, J. C. (2009) Microarrays with Varying Carbohydrate Density Reveal Distinct Subpopulations of Serum Antibodies, J. Proteome Res. 8, 3529–3538.

Chapter 12 Immobilization of Polyacrylamide-Based Glycoconjugates on Solid Phase in Immunosorbent Assays Oxana E. Galanina, Alexander A. Chinarev, Nadezhda V. Shilova, Marina A. Sablina, and Nicolai V. Bovin Abstract Our experience in coating of solid surfaces with glycans, mainly for obtaining routine glycoarrays based on immunological plates, is summarized. Three polystyrene coating techniques are described: direct physical adsorption, covalent binding, and immobilization using the biotin tag. Protocols for studies on anticarbohydrate antibodies are considered, with special emphasis on the application niches of different immobilization techniques as related to the specificity of each method of glycan-binding protein assay, as well as the problems of background binding and quantitative estimation of the results. Key words: Glycoarrays, ELISA, Glycopolymers, Immobilization techniques, Physical adsorption, Covalent immobilization, Biotin–streptavidin bridge, Polyacrylamide

Abbreviations Atri AP Btri Biot BSA ELISA Glyc GPC Ig HRPO m.w. PAA PBS pNPA pNSA Str TLC

A trisaccharide, GalNAca1-3(Fuca1-2)GalbAlkaline phosphatase B trisaccharide, Gala1-3(Fuca1-2)GalBiotin Bovine serum albumin Enzyme-linked immunosorbent assay Glycoside residue Gel-permeation Anti-mouse antibodies Horse radish peroxidase Molecular weight Poly(acrylamide) Phosphate saline buffer poly(4-nitrophenylacrylate) poly(N-oxisuccinimidylacrylate) Streptavidin Thin-layer chromatography

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1. Introduction Glycans constitute a complex and diverse family of biomolecules involved in a broad range of physiological (cell development and differentiation, cell recognition and cell-to-cell adhesion, cell motility, etc.) and pathological (malignant transformations, autoimmune diseases, host–pathogen interactions, etc.) processes, many important characteristics of which remain poorly studied (1–3). For profiling glycan-binding proteins and studying interactions involving them, enzyme-linked immunosorbent assay (ELISA) and related assays based on the classical multiwell plates are generally used. Although many researchers note that ELISA is not sensitive enough and time consuming, its major advantages are simplicity and flexibility of the procedure. This allows the method to be adjusted to a specific research task, e.g., selection of the direct binding or inhibition conditions; choice of the optimal detection method (colorimetric or fluorescent); instant variation of glycan combinations, coating concentrations, and surface density; and choice between the monovalent and multivalent types of their presentation. ELISA requires at least one order of magnitude lower concentrations of the analyzed proteins to be than, e.g., the printed glycan array (PGA) and suspension (Bioplex) array. It is noteworthy that the assay does not require sophisticated equipment. Glycan ELISA has its own application niche; it is optimal for miniformat glycan profiling when 5–15 glycans are necessary for characterization of a glycan-binding protein. In practice, ELISA has proved to be a good complementary “partner” for PGA, when PGA is used as a screening tool, followed by ELISA for accurate verification of the results. Apart from research purposes, ELISA can be easily adapted for medical applications. The assay can be performed manually or, if necessary, robotized (4–7). Typically, interactions involving glycans are weak; this impels researchers to use carbohydrates in the multivalent form. In particular, glycoconjugates based on linear polyacrylamides (PAAs) with attached-side carbohydrate groups (Glyc) solve this problem. The polymer backbone plays two roles: first, a flexible linker between Glyc residues facilitating their optimal arrangement for multivalent binding to an antibody or lectin irrespective of the distance between their carbohydrate-binding domains (8, 9); second, a long spacer between glycans and the support. On the contrary, monomeric glycans arrayed on a solid surface are often hardly capable of multivalent binding with glycan-recognizing proteins because of suboptimal density of Glyc residues and length of their bonds with the surface; i.e., this type of glycan arrays can be only technically regarded as multivalent (10–12).

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Fig. 1. The use of glycopolymers in ELISA. A glycan from the library of w-aminoalkyl ­glycosides is quantitatively coupled to fully activated polyacrylic acid prepared by radical polymerization of nitrophenylacrylate or N-acryloyl succinimide. There are three ways of further use of the resultant glycopolymers containing active ester groups. First, they can be covalently immobilized on aminated surfaces (including polystyrene) through the ­formation of amide bonds. Second, they can be physically adsorbed in the form of ­Glyc–PAA. Third, they can be coated onto a streptavidin-modified surface in the form of a biotinylated glycopolymer.

In this paper, we describe a set of techniques using various types of PAA-based glycoconjugates for coating polystyrene ELISA plates; the general strategy of the glycopolymer use in ELISA is shown in Fig. 1.

2. Materials 2.1. Carbohydrates

Atri-O(CH2)3NH2 and Btri-O(CH2)3NH2 were obtained from Lectinity, Inc. (Russia).

2.2. Biotin

Biot-NH(CH2)6NH2 was obtained from Lectinity, Inc. (Russia).

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2.3. Activated Polyacrylic Acids

Poly(4-nitrophenylacrylates), pNPA, biot1-pNPA, and poly (N-oxisuccinimidyl acrylate), pNSA, were prepared as described before (11, 13).

2.4. Other Commercial Reagents and Solvents

Regular chemicals were obtained from Fluka (USA), Aldrich (USA), and Merck (Germany).

2.5. Buffers

1. PBS: 137  mM NaCl, 2.7  mM KCl, 10.0  mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4. 2. ELISA-coating buffer: 15  mM Na2CO3, 35  mM NaHCO3, pH 9.6. 3. ELISA washing buffer: 0.1% Tween-20 in PBS. 4. ELISA blocking buffer: 3% BSA in PBS. 5. Colorimetric solution: 0.1 M sodium phosphate, 0.1 M citric acid buffer, 0.04% O-phenylenediamine, pH 4.7. 6. Fluorescent-revealing solution: 10−4  M 4-methylumbellyferyl phosphate disodium salt in the coating buffer.

2.6. Antibodies

1. Mouse monoclonal antibodies B8 (against Btri) and A16 (against Atri) were obtained from All-Russian Hematology Research Center (Moscow, Russia). 2. Streptavidin–horseradish peroxidase (Str–HRPO) conjugate was the product of GE Healthcare (UK). 3. Anti-mouse IgG + IgM (H + L)–alkaline phosphatase (Ig–AP) conjugate was the product of AP Biotech. Inc. (UK).

2.7. 96-Well Plates

1. Reacti-Bind TM Streptavidin coated High Binding Capacity black plates (#15503) were purchased from ThermoScientific (USA). 2. MaxiSorp transparent immunoplates (#439454) were purchased from NUNC (Denmark). 3. NH2-modules were obtained from Costar (USA).

2.8. Chromatography

1. Thin layer chromatography (TLC) aluminum sheets covered with silicagel 60 (Merck, Germany). 2. Sephadex LH-20 gel (Pharmacia BioTech, Austria). 3. Eluent A for 10:1:1:1:0.3.

TLC:

EtOH/BuOH/Py/H2O/AcOH,

4. Eluent B for TLC: MeOH/1 M Py·AcOH, 3:1. 5. Eluent C for gel-permeating chromatography (GPC): MeCN/ H2O, 1:1. 6. Charring reagent for TLC plates: 7% H3PO4. 2.9. Equipment

Victor2 multilabel counter (PerkinElmer, USA).

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3. Methods Three approaches to immobilization of Glyc–PAA are considered here: physical (noncovalent) adsorption, chemical (covalent) immobilization, and immobilization via biotin–Str bridge. Physical adsorption due to Van der Waals interactions between glycopolymer molecules and the plate surface is the simplest and the least sophisticated method. The adsorption efficiency depends on the m.w. of the glycopolymer; the number of contacts and interaction strength between glycopolymer molecules and the surface increase with an increase in the m.w. (13). Two glycoconjugates differing in m.w., Glyc–PAA30 and Glyc– PAA2000 (see Notes 1 and 2), were synthesized (the protocols were developed for blood group A trisaccharide derivatives, Atri–PAA) as shown in Fig. 2a and coated onto the plates. The coating procedure included incubation of the glycoconjugate solutions in wells and subsequent washing off of nonadsorbed material. It was found that only as little as about 2% of the added low-m.w. glycopolymer is adsorbed, whereas this value for high-m.w. ­glycopolymer is ~20% (11). The difference in the efficiency of immobilization for low- and high-m.w. Glyc–PAAs is illustrated in Fig.  3. Addition of equal amounts of Glyc into wells yields a stronger optical signal in the case of Glyc–PAA2000; the deference between glycoconjugate signals is greater at a low coating concentration (<50 pmol Glyc per well). The maximum adsorption for the high- and low-m.w. glycoconjugates is observed, respectively, at 50 and 200 pmol of Glyc residue per well. Thus, the use of the high-m.w. glycoconjugate allows reducing the amount of carbohydrates required for coating by a factor of 10–20. The difference observed in mAb binding with glycopolymers is explained as follows. The average size of coiled glycopolymer molecules adsorbed on polystyrene is known to be ~100 Å for 30-kDa conjugates and ~500 Å for 2,000-kDa ones (11), whereas the distance between Fab fragments of antibodies varies from 100 to 150 Å for IgG and may be even more for IgM molecules (14). Thus, in the case of Atri–PAA30, binding of an antibody with several neighboring glycopolymer molecules on the surface is much more probable compared to an antibody with only one glycopolymer (Fig. 2b). An increase in the density of Atri–PAA30 molecules on the surface leads to an increase in the detected signal. On the contrary, the size of Atri–PAA2000 molecules allows them to bind one or several antibodies simultaneously: a signal is detectable even at a low density of Atri–PAA2000 (Fig. 2c). Chemical immobilization of glycopolymers on the surface of aminated polystyrene is accomplished via formation of covalent

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Fig. 2. Polyacrylamide glycoconjugates for physical adsorption on polystyrene plates. (a) Synthesis: One of activated polyacrylic acids differing in m.w. (pNPA or pNSA) is bound with Glyc-O(CH2)3NH2 and then treated with ethanolamine to quench the remaining ­activated ester groups. The average sizes of molecules and distances for (b) low- and (c) high-m.w. glycoconjugates on the surface are shown.

amide bonds (Fig. 4). Activated polyacrylic acids with pendant Glyc residues, Glyc–pNPA30 and Glyc–pNSA2000 (see Notes 1 and  2), are used without isolation. The coating procedure consists of ­consecutive incubations of plates in a glycopolymer solution (the coupling stage) and then in an aqueous ammonia solution (the quenching stage), which is followed by washing off of the unbound

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Fig. 3. Physical adsorption of (a) high- and (b) low-m.w. glycoconjugates (Atri–PAA2000 and Atri–PAA30, respectively) on 96-well, transparent polystyrene plates. The efficacy of adsorption was demonstrated by colorimetric detection using anti-Atri mAbs.

Fig. 4. Coating of an aminated surface with glycopolymers.

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material. As a result, the plates are coated with PAA glycoconjugates differing in m.w. (30 and 2,000 kDa). A stronger binding is observed for the high-m.w. glycoconjugate; the sorption is maximum at a concentration of 75 pmol of Glyc per well, whereas in the case of the low-m.w. glycoconjugate, a concentration of 200 pmol per well is required (Fig. 5). A similar immobilization protocol allows glycopolymers to be attached to other aminated surfaces, e.g., coronary angioplasty stents made of stainless steel coated with nanodiamonds and then aminated plasmachemically over the diamond layer (Plasmachem GmbH, Germany). To solve the problem of smooth muscle cell overgrowth and to make the stent surface attractive for selectinbearing endothelial cells, the stent surface is modified with carbohydrates capable of mediating endothelial cell adhesion (15). The efficacy of chemical coating of the stents with SiaLea–pNPA30 was proved by ELISA with anti-SiaLea mAbs (Fig. 6). A convenient method of glycopolymer immobilization on plates is based on the use of biotin–Str bridge, when a glycopolymer bearing a biotin tag is anchored to a Str-coated plate. We used two approaches to the synthesis of biotinylated glycopolymers. According to the first one, a biotin derivative containing an amino group in the linker (biot-NH(CH2)6NH2) and an w-aminoalkyl glycoside (Glyc-O(CH2)3NH2) are consecutively coupled to pNPA. Finally, the remaining active ester groups in the polymer are quenched using ethanolamine (Fig. 7a). The resultant glycopolymer contains several side-pendant Glyc and biotin residues (see Notes 2 and 3). In the second method, a biotin residue is introduced into the ­polymer scaffold as a single end group (Fig. 7b) with a fragment of

Fig. 5. Chemical immobilization of (a) high- and (b) low-m.w. glycoconjugates (Btri–PAA2000 and Btri–PAA30, respectively) on aminated polystyrene plates. The efficacy of coating was demonstrated using anti-Btri mAbs.

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Fig. 6. Photograph of a SiaLea-modified stent placed into a well of a nonadsorbing plate (as a vessel) and stained with ­anti-SiaLea mAbs; the color is developed due to HRPOlabeled secondary antibodies.

Fig. 7. Biotinylated glycopolyacrylamides for coating onto Str plates: Synthesis of the ­glycopolymers with (a) side- and (b) end-pendant biotin residues. (c) Anchoring of glycopolymers with one and several pendant biotins on the surface and their presentation for interaction with antibodies.

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biotinylated initiator of polymerization. The resultant activated polyacrylic acid, pNPA–biot1, interacts with Glyc-O(CH2)3NH2, which is followed by treatment with ethanolamine. The resultant glycopolymer, Glyc–PAA30–biot1, contains several side-pendant Glyc residues and the only one end biotin (see Note 2). The immobilization procedure includes incubation of the biotinylated glycopolymers, Glyc–PAA30–biot5 and Glyc–PAA30– biot1, with Str plates and washing off of nonadsorbed material; the procedure has been shown to give a quantitative yield (16). The monobiotinylated glycopolymer Glyc–PAA30–biot1 slightly better bound mAbs compared to an analogue bearing several-side biotins, Glyc–PAA30–biot5 (Fig.  8). The adsorption was maximal for the monobiotinylated conjugate at a concentration of 70  pmol/well, whereas for the pauci–biot analog it was maximal at 100 pmol/well. As the glycopolymers have the same m.w., the observed difference is likely to have resulted from differences in the anchoring on the surface and, hence, in the presentation of the glycan for mAbs (16). In immobilized molecules of Glyc–PAA30–biot1, the single end biotin is hidden inside the streptavidin matrix. The monobiotinylated glycopolymer is recommended for plate coating when additional biotin residues are expected to affect the results of the assay, i.e., when hydrophobicity of extra biotin residues could critically elevate the background signal of the assay or when (strept)avidin-based reagents are used not only for immobilization, but also at one of the next stages, as illustrated in Fig. 9. Finally, it should be noted that immobilization through Str–biotin bridge has appeared to be a less reagentconsuming method.

Fig. 8. Binding of anti-Btri mAbs by glycopolymers with (a) end- and (b) side-pendant biotin residues (Btri–PAA30–biot1 and Btri–PAA30–biot5) immobilized on an Str plate; fluorimetric detection.

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Fig. 9. Demonstration of the lack of available biot residues in the composition of Str ­complexed with end-biotinylated glycopolymer. Str plates are coated with a glycopolymer with side- or end-pendant biotin residues, Glyc–PAA30–biot5 (open bars) and Glyc–PAA30– biot1 (hatched bars), and developed with Str–AP.

In summary, three approaches can be used for coating immunological plates with glycans in the form of Glyc–PAA; in all cases, the immobilized molecules are practically the same. What is the reason for selecting one of the methods for design of a specific assay? Physical adsorption has proved to be the simplest protocol; however, it has a serious drawback, namely, a high consumption of carbohydrates to be coated. Therefore, it is impracticable when only a minute quantity of glycan is available or the cost of the assay is the critical point. Biotin-mediated coating eliminates this disadvantage and, in addition, is methodologically simple. One important characteristic of biot–Str immobilization should be noted. In the case of physical adsorption or the aforementioned covalent binding, the amount of glycoconjugate immobilized on the plate surface is not strictly proportional to their amount added. In contrast, biotinylated glycoconjugate can be immobilized quantitatively when the biot-to-Str ratio is <1 (in assays using the same plates as in this study, at concentrations of biotin of 0–50  pmol Glyc/well). Therefore, immobilization via biot–Str bridge allows a researcher to know the absolute amount of the coated material, which is necessary, e.g., when a correct affinity constant is calculated. Limitations of the biot–Str technique are the high cost of commercially available plates and, sometimes, a low stability of the streptavidin monolayer. Chemical immobilization is undoubtedly the most sophisticated and time-consuming technique. It could be recommended for nonroutine assays, when the use of other methods may lead to desorption because of unusual conditions, or the plate should be regenerated for repeated assays without loss of the coating glycoconjugate.

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3.1. Preparation of Polyacrylamide-Based Glycoconjugates 3.1.1. Synthesis of Atri–PAA30 for Physical Adsorption on Polystyrene Plates (See Note 4)

1. Add a mixture of 3.2 mg (5.5 mmol) of Atri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 in 100 mL of DMSO to a solution of 4.7 mg (27.5 mmol) of pNPA in 400 mL of DMSO. 2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.35 for Atri-O(CH2)3NH2, Rf 0 for Atri–PAA30 (see Subheading 3.2). 3. Add an excess of ethanolamine (90 mL) to the reaction mixture and keep it for 24 h at 40°C. 4. Purify the product by GPC (see Subheading 3.3). 5. Yield 85–95%; white solid.

3.1.2. Synthesis of Atri–PAA2000 for Physical Adsorption on Polystyrene Plates

1. Add a mixture of 3.2 mg (5.5 mmol) of Atri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 4.1  mg (27.5  mmol) of pNSA in 400  mL of DMSO. 2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.35 for Atri-O(CH2)3NH2, Rf 0 for Atri–PAA2000 (see Subheading 3.2). 3. Add an excess of ethanolamine (90 mL) to the reaction mixture and keep it for 24 h at 40°C. 4. Purify the product by GPC (see Subheading 3.3). 5. Yield 85–95%; white solid.

3.1.3. Synthesis of Btri–pNPA30 for Chemical Immobilization on Aminated Polystyrene Plates

1. Add a mixture of 1.4 mg (2.6 mmol) of Btri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 1.9  mg (12.8  mmol) of pNPA in 400  mL of DMSO. 2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.3 for Btri-O(CH2)3NH2, Rf 0 for Btri–pNPA30 (see Subheading 3.2). 3. Dilute 100 mL of Btri–pNPA30 reaction mixture with 900 mL of DMSO containing 0.2 mL of NEt3 (to concentration 0.94 nmol Btri/mL) and use the obtained solution for coating of aminated polystyrene plates (see Subheading 3.4.2).

3.1.4. Synthesis of Btri–pNSA2000 for Chemical Immobilization on Aminated Polystyrene Plates

1. Add a mixture of 1.4 mg (2.6 mmol) of Btri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 1.7  mg (12.8  mmol) of pNSA in 400  mL of DMSO. 2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.3 for Btri-O(CH2)3NH2, Rf 0 for Btri–pNSA2000 (see Subheading 3.2). 3. Dilute 100 mL of Btri–pNSA2000 reaction mixture with 900 mL DMSO containing 0.2 mL of NEt3 (to concentration 0.94 nmol Btri/mL) and use the obtained solution for coating of aminated polystyrene plates (see Subheading 3.4.2).

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1. Add a mixture of 500 mg (1.3 mmol) of biot-NH(CH2)6NH2 and 0.4 mL (2.6 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 4.7  mg (27.5  mmol) of pNPA in 300  mL of DMSO. 2. Add a mixture of 3 mg (5.5 mmol) of Btri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 in 100 mL of DMSO to the obtained solution and keep it for 24 h at 40°C; absence of the free aminoligands in the reaction mixture was confirmed by TLC; eluent A: Rf 0.3 for Btri-O(CH2)3NH2, Rf 0 for Btri–PAA30–biot5; eluent B: Rf 0.54 for biot-NH(CH2)6NH2; Rf 0 for Btri–PAA30– biot5 (see Subheading 3.2). 3. Add an excess of ethanolamine (90 mL) to the reaction mixture and keep it for 24 h at 40°C. 4. Purify the product by GPC (see Subheading 3.3). 5. Yield 85–95%; white solid.

3.1.6. Synthesis of Btri–PAA30–biot1 for Immobilization on Str Plates

1. Add a mixture of 3 mg (5.5 mmol) of Btri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 4.7 mg (27.5 mmol) of pNPA–biot1 in 300 mL of DMSO. 2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.3 for Btri-O(CH2)3NH2, Rf 0 for Btri–PAA30–biot1; eluent B: Rf 0.54 for biot-NH(CH2)6NH2; Rf 0 for Btri–PAA30–biot1 (see Subheading 3.2). 3. Add an excess of ethanolamine (90 mL) to the reaction mixture and keep it for 24 h at 40°C. 4. Purify the product by GPC (see Subheading 3.3). 5. Yield 85–95%; white solid.

3.2. Thin-Layer Chromatography

1. Apply 5 mL of reaction mixture on a TLC plate and develop the plate using appropriate eluent (see Subheading 3.1.1–3.1.6). 2. Spray the charring reagent and heat the plate for 10  min at 150°C to visualize spots.

3.3. Preparative GPC

1. Apply reaction mixture on a glass column (75 × 1.5 cm) packed with Sephadex LH-20. 2. Elute the column with the eluent C. 3. Collect the first 50 mL of eluate and evaporate it to dryness. 4. Dissolve the residue in water and freeze dry the obtained solution.

3.4. Plate Coating with Glycopolymers 3.4.1. Physical Adsorption

1. Add serially twofold diluted Atri–PAA solution in coating buffer (concentration 0.02–200 mg/mL or 2–2 × 105 pmol Atri per well) to the plate (100 mL per well); keep the plate for 1 h at 37°C (see Notes 5, 6, and 7).

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2. Wash the plate (hereinafter, the washing buffer, 200  mL per well). 3. Block the plate (the blocking buffer, 200  mL per well) and wash. 3.4.2. Chemical Immobilization

1. Add serially twofold diluted Btri–pNPA30 or Btri–pNSA2000 solution in DMSO (concentration 0.02–200 mg/mL or 2–188 pmol Btri per well) to the aminated plate (100 mL per well) and incubate the plate for 24 h at room temperature. 2. Add 3 M aqueous ammonia to the plate (10 mL per well) and incubate for 24 h at room temperature. 3. Wash the plate (hereinafter, the washing buffer, 200  mL per well). 4. Block the plate (the blocking buffer, 200  mL per well) and wash (See Note 7).

3.4.3. Coating via Biotin–Str Bridge

1. Rinse streptavidin-coated, black, 96-well plate (see Note 7) twice with PBS. 2. Add serial twofold dilution of coating solution in PBS (containing 0.002–80  mg/mL biotinylated glycoconjugate or, respectively, 2–9 × 104 pmol Btri per well) to the plate (100 mL per well); keep the plate for 1 h at 37°C. 3. Wash the plate (hereinafter, the washing buffer, 200  mL per well). 4. Block the plate (the blocking buffer, 200  mL per well) and wash (See Note 7).

3.5. ELISA 3.5.1. ELISA with Colorimetric Detection

1. Add A16 mAbs (1:4,000 in PBS containing 0.3% BSA) to the 96-well transparent plate (100 mL per well). 2. Incubate the plate for 1 h at 37°C and wash with the washing buffer. 3. Add Ig–Biot (1:2,000 in PBS containing 0.3% BSA) to the plate (100 mL per well), incubate the plate for 1 h at 37°C, and wash with the washing buffer. 4. Add Str-HRPO (1:2,000 in PBS containing 0.3% BSA) to the plate (100 mL per well), incubate the plate for 1 h at 37°C, and wash with the washing buffer. 5. Just before use, add 0.03% H2O2 to a colorimetric solution and apply the obtained solution to the plate. Incubate the plate for 30 min at room temperature; stop the reaction by addition of 50 ml 1 M aqueous H2SO4. Read an absorbance at 492 nm in Victor2 counter. 6. Measure optical density (492  nm) by Victor2 counter. Carry out each assay in duplicate, and omit mAbs to obtain blank reaction.

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1. Add B8 mAbs (1:100 in PBS containing 0.3% BSA) to the 96-well black plate (100 mL per well). 2. Incubate the plate for 1 h at 37°C and wash. 3. Add Ig–AP (1:5,000 in PBS containing 0.3% BSA) to the plate (100  mL per well); incubate the plate for 1  h at 37°C and wash. 4. Add fluorescent-revealing solution to the plate (100  mL per well); incubate the plate for 30 min at room temperature. 5. Measure fluorescence intensity (355/460 nm) by Victor2 multilabel counter (PerkinElmer, USA). Carry out each assay in duplicate, and perform blank reaction by omitting the mAbs. Subtract the blank reading from the final fluorescence to provide the corrected fluorescence intensity values.

4. Notes 1. The meanings of apparent m.w. (in kDa) are shown in the glycopolymer notations as the upper index. Degree of polymerization in the case of high-m.w. activated polyacrylic acid is reproduced with high accuracy; it could be monitored by analytical GPC. For details, see ref. 16. 2. Molar fraction of the monomer units substituted with Glyc is 20 mol% for all the glycopolymers considered here; according to our previous experience, this is the optimal loading of polymer scaffolds affording to better binding with carbohydraterecognizing antibodies (8, 9). 3. Molar fraction of the monomer units substituted with biotin in Glyc–PAA30–biot5 is 5 mol%, which corresponds to five biotin residues per molecule of the glycopolymer. 4. 30-kDa Glyc–PAAs can be available via home pages of CFG (http://www.functionalglycomics.org/), Lectinity Corp. (http://www.lectinity.com/), or GlycoTech Corp. (http:// www.glycotech.com/). 5. Physical immobilization of Glyc–PAA is carried out at 37°C for 1 h or alternatively at 4°C for 12 h. These two regimens give rise to the same efficiency. 6. Coating buffer pH basically does not affect the efficiency of Glyc–PAA immobilization in the pH interval from 5.0 to 9.6. Salts-free water as a solvent gives twofold less rate of coating. 7. Serially diluted coating antigen and constant concentration of antibodies is recommended as a protocol optimization stage. Then, when the optimal antigen coating has been selected, the antibodies in test are serially diluted.

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8. Storage and use of Str-coated plates should be in strict accordance with manufacturer’s manual in order to avoid loss of capacity. Otherwise, plates with similar quality and capacity could be prepared in-house by coating Str onto NUNC Maxisorb polystyrene. 384-well avidin-coated plates could be used as well (6).

Acknowledgments This study was supported by the Program of the Presidium of the RAS “Molecular and Cell Biology” (FP6 Project SP5B-CT2007-044512) and the RFBR grant #10-04-01693-a. References 1. Varki, A., Cummings, R.D., Esko, J.D., et  al. (2009) Essentials of Glycobiology, 2nd ed. CSH Press, New York. 2. Fukuda, M., Hindsgaul, O. (2003) Introduction to Glycobiology. Oxford University Press, New York. 3. Brockhausen, I., Kuhns, W. (1997) Glycoproteins and Human Disease. Springer, Heidelberg, Germany. 4. Katrlik, J., Švitel, J., Gemeiner, P., Kožár, J., Tkac, J. (2010) Glycan and lectin microarrays for glycomics and medicinal applications. Med Res Rev 30: 394–418. 5. Liang, P.H., Wu, C.Y., Greenberg, W.A., Wong, C.H. (2008) Glycan arrays: Biological and medical applications. Curr Opin Chem Biol 12: 86–92. 6. Alvarez, R.A., Blixt, O. (2006) Identification of ligand specificities for glycan-binding proteins using glycan arrays. Meth Enzym 415: 292–410. 7. Larsen, K., Thygesen, M.B., Guillaumie, F., Willats.,W.G.T., Jensen, K.J. (2006) Solidphase chemical tools for glycobiology. Carb Res 341: 1209–1234. 8. Bovin, N.V. (2003) Neoglycoconjugates as probes in glycobiology. In Chemical Probes in Biology (M. P. Schneider Ed.), Kluwer Academic Publishers, Dordrecht, Germany, pp. 207–225.

9. Bovin, N.V. (1998) Polyacrylamide-based ­glycoconjugates as tools in glycobiology. Glycoconj J 15: 431–446. 10. Galanina, O.E., Mecklenburg, M., Nifant’ev, N.E., et  al. (2003) GlycoChip: multiarray for the study of carbohydrate-binding proteins. Lab Chip 3: 260–265. 11. Shilova, N.V., Galanina, O.E., Pochechueva, T.V., et al. (2005) High molecular weight neoglycoconjugates for solid phase assays. Glycoconj J 22: 43–51. 12. Dyukova, V.I., Shilova, N.V., Galanina, O.E., et al. (2006) Design of carbohydrate multiarrays. Biochem Biophys Acta 1760: 603–609. 13. Esser, P. (2010) Principles in Adsorption to Polystyrene. ThermoScientific Bulletin 6a. http://www.nuncbrand.com/en/frame. aspx?ID=579. Accessed August 2010. 14. Stryer, L. (1995) Biochemistry, 4th ed. W. H. Freeman and Company, New York, pp. 361–390. 15. Berg, E.L., Robinson, M.K., Mansson, O., Butcher, E.C., Magnani, J.L. (1991) A carbohydrate domain common to both sialyl Le(a) and sialyl Le(x) is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1. Biol Chem 266: 14869–72. 16. Chinarev, A.A., Galanina, O.E., Bovin, N.V. (2010) Biotinylated multivalent conjugates for surface coating. In: Functional Glycomics (J. Li Ed.), Meth Mol Biol 600, Humana Press, 67–78.

Chapter 13 Surface Plasmon Resonance Imaging Analysis of Protein Binding to a Sialoside-Based Carbohydrate Microarray Matthew J. Linman, Hai Yu, Xi Chen, and Quan Cheng Abstract Monitoring multiple biological interactions in a multiplexed array format has numerous advantages. However, converting well-developed surface chemistry for spectroscopic measurements to array-based, high-throughput screening is not a trivial process and often proves to be the bottleneck in method development. This chapter reports the fabrication and characterization of a new carbohydrate microarray with synthetic sialosides for surface plasmon resonance imaging analysis of lectin–carbohydrate interactions. Contact printing of functional sialosides on neutravidin-coated surfaces was carried out and the properties of the resulting elements were characterized by fluorescence microscopy. Sambucus nigra agglutinin (SNA) was used for testing on four different carbohydrate-functionalized surfaces and differential binding was analyzed. Multiplexed detection of SNA/biotinylated sialoside interactions on arrays up to 400 elements has been performed with good data correlation, demonstrating the effectiveness of the biotin–neutravidin-based biointerface to control probe orientation for reproducible and efficient protein binding to carbohydrates. Key words: Carbohydrate microarray, Surface plasmon resonance imaging, Biotinylated sialosides, Biointerface, Biosensor

1. Introduction Carbohydrates are key components in cell surface interactions with proteins, responsible for recognition, cell adhesion, and cell-to-cell signaling (1). Since they are structurally complex, there has been considerable effort in synthesizing carbohydrate analogs and investigating their structure–function properties. Monitoring multiple carbohydrate interactions is challenging but highly desirable for gaining affinity information in a timely and cost-effective manner, particularly for pharmaceutical research. This has led to the construction of carbohydrate microarrays for multiplexed detection of protein–carbohydrate interactions as early as in 2002 (2–5). Yann Chevolot (ed.), Carbohydrate Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 808, DOI 10.1007/978-1-61779-373-8_13, © Springer Science+Business Media, LLC 2012

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In the work presented in this chapter, the carbohydrate microarrays are created by contact printing. Generally, methods for array fabrication can be classified as either contact or noncontact printing. The former utilizes pin-type arrayers that transfer a defined volume of sample by directly touching the surface of the substrate, whereas the latter dispenses the sample droplets onto the substrate without a direct contact to the surface. We use contact printing with solid pins as there are a number of advantages to this technology, including easy deposition of viscous solutions (6), simple design that enables reproducible and efficient printing (7), and simple cleaning procedure for the pins (7). In this chapter, we employ the use of a pin and ring spotter known as the GMS 417 arrayer, which uses a large ring to hold the sample and the pass of a pin through the ring deposits a droplet (8). In the last portion of this chapter, biomolecular interaction using this carbohydrate microarray is described in detail. Biointeraction studies desire proper orientation of the ligands and probe density for functional binding. It is well-known that surface chemistry employed to immobilize the biomolecules of choice is of great importance. This is especially true when immobilizing carbohydrates in an array format. Among various covalent coupling approaches that are typically used, one main problem is the lack of control with regards to the orientation of the probe glycan relative to the surface (9). In addition, how this lack of control affects detection performance with new detection schemes, such as label-free methods, has not been investigated as the majority of detection is fluorescence based. Label-free detection with microarray has gained considerable acceptance in the past few years (10, 11) and there was a recent report that uses SPR and XPS to examine a self-assembled carbohydrate monolayer for array analysis (11). But in general, the effects of carbohydrate immobilization on microarray performance and reliability, especially that involving protein-based surface chemistries, have not been extensively studied. In this chapter, we describe in detail the fabrication of a carbohydrate microarray using contact printing with biotinylated sialosides. This array has been further characterized for its effectiveness in the study of interactions with lectins by surface plasmon resonance imaging (SPRi). SPRi allows multiplex detection of biological interactions with real-time kinetic analysis possible (10). We recently demonstrated an SPR spectroscopic analysis with surface chemistry based on synthetic sialosides for the detection of lectins (12). In this chapter, we use a homebuilt SPRi system (13) along with a commercial contact printing arrayer (the pin-and-ring arrayer) to reveal the morphological connection to biotin–neutravidin chemistry in the immobilization of carbohydrates and its impact on performance (14). The arrayer is employed to create upward of 400 individual array elements for demonstrating proof of principle

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for high-density fabrication with quality. The biointerface design described herein should be applicable to many biological systems for high-throughput bioanalysis.

2. Materials 2.1. Synthesis of Biotinylated Sialosides

1. Hexaethylene glycol (Sigma–Aldrich).

2.1.1. Reagents

4. KI (Fisher Scientific).

2. Ag2O (Sigma–Aldrich). 3. p-Toluenesulfonyl chloride (Sigma–Aldrich). 5. Celite (Fisher Scientific). 6. Sodium azide (Sigma–Aldrich). 7. TMSOTf (Sigma–Aldrich). 8. Biotin-NHS (Sigma–Aldrich). 9. Sodium methoxide (Fisher Scientific). 10. DOWEX HCR-W2 (H+) resin (Fisher Scientific). 11. 10% palladium on charcoal (Fisher Scientific). 12. 4 Å powdered molecular (Fisher Scientific). 13. Et3N (Fisher Scientific). 14. Hexane (Fisher Scientific). 15. 95% Ethanol (Fisher Scientific). 16. DMF (Fisher Scientific, cat. no. AC34843-0010). 17. CH2Cl2 (Fisher Scientific). 18. Ethyl acetate (Fisher Scientific). 19. Methanol (Fisher Scientific). 20. ManNAc (Sigma–Aldrich). 21. Mannose (Sigma–Aldrich). 22. Sodium pyruvate (Fisher Scientific). 23. Cytidine 5¢-triphosphate disodium salt (CTP; Sigma–Aldrich). 24. MgCl2⋅6H2O (EMD). 25. p-Anisaldehyde (Sigma–Aldrich). 26. H2SO4 (EM science). 27. Acetic acid (Fisher Scientific). 28. Tris base (Fisher Scientific).

2.1.2. Equipment

1. Buchi Rotary evaporator (Fisher Scientific). 2. C25KC incubator shaker (Fisher Scientific). 3. Model 2110 fraction collector (Bio-Rad).

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4. Sorvall legend T/RT benchtop centrifuge (Fisher Scientific). 5. Sterile 50-mL centrifuge tubes (Biologix Research Company). 6. Bio-Gel P-2 Gel, fine (Bio-Rad). 7. Thin-layer silica gel plates (Sorbent technologies). 8. Silica gel (Sorbent technologies). 2.1.3. Enzymes

1. Enzymes used in this protocol, including sialic acid aldolase from Escherichia coli K-12, CMP-sialic acid synthetase from Neisseria meningitidis (NmCSS), sialyltransferase from Pasteu­ rella multocida (PmST1), a2–6-sialyltransferase from Photo­ bacterium damsela (Pd2,6ST), are recombinant proteins cloned, expressed, and purified in Prof. Xi Chen’s laboratory (15–18). 2. p-Anisaldehyde sugar stain is prepared by adding p-anisaldehyde (25  mL) to ice-chilled methanol (425  mL). Concentrated H2SO4 (50 mL) was then cautiously added dropwise to the methanol solution incubated in an ice bath during a 60-min period with vigorous stirring without splashing. The staining solution prepared was stored at −20°C before use.

2.2. Preparation for Biotinylated Sialoside Surface

1. Running buffer: 20 mM phosphate-buffered saline (PBS), pH 7.4, 150 mM NaCl. 2. Prepare Sambucus nigra agglutinin (SNA) and Fluorescein tagged-SNA (FL-SNA) (Vector Laboratories, Burlingame, CA) in 20 mM PBS, pH 7.4, 150 mM NaCl at stock concentrations of 1 mg/mL and dilute thereafter for various assays. 3. Prepare Biotinylated bovine serum albumin (b-BSA), Neutravidin, Avidin, and Streptavidin (Pierce Biotechnology, Rockford, IL) in PBS in stock solutions of 1 mg/mL. 4. Prepare BSA (Sigma–Aldrich, St. Louis, MO) in a stock solution of 10  mg/mL to use as a control/blocking reagent to prevent nonspecific binding. 5. Prepare a piranha solution, H2SO4:H2O2 (70/30% w/v) by warming the solution in a large glass dish at 70°C for 30 min. This solution is used to clean the glass microscope slides.

2.3. Array Fabrication and SPR Imaging Characterization

1. Clean BK-7 Glass Slides (Corning, NY) coated with about 50 nm of gold via e-beam evaporation are the sensing substrates. 2. The sensing substrates are spotted with various carbohydrate probes via a 96-well EIA/RIA Plate (Corning, NY) in the GMS 417 Microarrayer (Genetic Microsystems) contact printer. 3. All substrates are analyzed for biomolecular binding with a homebuilt SPRi system (13). Attached to this imaging system is a homebuilt flow cell with area ~2  cm2 as defined by the polycarbonate O-ring.

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4. 60-mL Syringe with Luer-Lock tip (BD Biosciences, Sparks, MD) is used to store buffer solution which is delivered to the surface at a specific flow rate via a syringe pump (KD ScientificModel 100, Holston, MA). 5. Various analyte injections are made with a 6-Port Medium Pressure Injection Valve, Model V-451, PFA Tubing. The whole microfluidic system also combines 0.040″ diameter, fingertight PEEK fittings (F-120), flangeless fittings, and ferrules (Upchurch Scientific, Oak Harbor, WA).

3. Methods The preparation of biotinylated sialosides and fabrication of the array itself are both equally important steps. Presented herein, we cover the synthesis of the carbohydrates themselves and also the fabrication of the carbohydrate microarray. Finally, the SPRi experimental process is detailed. 3.1. Synthesis of Biotinylated Sialosides

1. Dissolve hexaethylene glycol 1 (25 g, 90 mmol) in anhydrous CH2Cl2 (300 mL) in a 1,000-mL round-bottom flask and add Ag2O (33.0 g, 135 mmol). Cool the flask to 0°C, stir the mixture for 15 min, and then follow with the addition of p-toluenesulfonyl chloride (20.0 g, 100 mmol) and KI (3.2 g, 20 mmol). Stir the reaction mixture at room temperature overnight. Remove the precipitated silver salts by filtration through Celite and wash the Celite with EtOAc. Evaporate the combined solution in a rotary evaporator and purify the compound by a silica gel column using a mixed solution, CH2Cl2:MeOH = 20:1, as an eluent providing the product hexaethylene glycol monotosylate 2 as a colorless oil (Fig. 1). 2. Add sodium azide (11 g, 169 mmol) to a solution of hexaethylene glycol monotosylate 2 (32  g, 73.3  mmol) in DMF (150 mL) in a 500-mL round-bottom flask. Stir the reaction mixture at 60°C for 12  h. Cool to room temperature, and remove the DMF by rotary evaporation at 40°C. Purify the residue by flash chromatography using a silica gel column and CH2Cl2:MeOH = 10:1 as an eluent to produce pure hexaethylene glycol azide 3 as a colorless oil. 3. Dissolve lactosyl trichloroacetimidate 4 (4.7  g, 6.02  mmol) (prepare by chemical synthesis as described (18) and store at −20°C before use) in dry CH2Cl2 (60 mL) containing 4 Å powdered molecular sieves (1 g) in a 250-mL round-bottom flask under argon atmosphere. Add hexaethylene glycol azide 3 (1.85 mL, 6.02 mmol) using a syringe and cool the reaction mixture to −30°C and stir for 30  min. Added TMSOTf

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Fig. 1. Chemoenzymatic synthesis of biotinylated sialosides 10–13. Aldolase recombinant Escherichia coli K-12 sialic acid aldolase, NmCSS recombinant Neisseria meningitidis CMP-sialic acid synthetase, PmST1 recombinant multifunctional Pasteurella multocida a2–3-sialyltransferase, Pd2,6ST recombinant multifunctional Photobacterium damsela a2–6sialyltransferase.

(3.0 mmol, 0.5 mL) dropwise for a duration of 10 min. Allow the reaction mixture to warm up to room temperature and stir it overnight. Then, add Et3N (1.0 mL) to quench the reaction. Filter and concentrate the reaction mixture over Celite. Finally, purify the crude material by a silica gel column using hexane:acetone = 1.5:1 as an eluent to produce peracetylated lactoside 5 as a white solid. 4. Dissolve lactoside 5 (2.0  g, 2.16  mmol) in dry methanol (30 mL) containing a catalytic amount of sodium methoxide. Stir the resulting mixture at room temperature for 12  h and neutralize it with DOWEX HCR-W2 (H+) resin. Filtration and concentration afforded deacetylated lactoside 6 as a white solid. 5. Dissolve compound 6 (1.1 g, 1.74 mmol) in water (15 mL) and stir the mixture under hydrogen atmosphere in the presence of 10% palladium on charcoal for 2  h. Next, filter and concentrate the solution to afford the reduced intermediate. Dissolve the residue (0.48 g, 1.08 mmol) in anhydrous DMF (15  mL) and biotin-NHS (0.41  g, 1.19  mmol) was added. After stirring overnight, concentrate and purify the reaction

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mixture by flash chromatography to produce biotintylated lactoside 7 as a white solid. 6. Chemoenzymatic sialylation of biotintylated lactoside 7 is achieved using a highly efficient and convenient one-pot, threeenzyme system established in the Chen laboratory (16–18). Dissolve N-Acetylmannosamine (ManNAc) 8 or mannose 9 (50–100 mg, 1.5 equiv), biotintylated lactoside 7 (1.0 equiv), sodium pyruvate (7.5 equiv), and CTP (1.5 equiv) in Tris–HCl buffer (5 mL, 100 mM, pH = 8.5) containing 20 mM of MgCl2 in a 50-mL centrifuge tube. After adding E. coli K-12 sialic acid aldolase (2.0 mg), N. meningitidis CMP-sialic acid synthetase (NmCSS) (0.8 mg), and PmST1 (1.2 mg for preparing a2–3 linked sialosides) or Pd2,6ST (0.5 mg for preparing a2–6 linked sialosides), the total volume of the reaction mixture should be brought to 10  mL by adding water. Incubate the reaction solution at 37°C with agitation at 140 rpm for 2–4 h. Monitor the reaction by TLC analysis (EtOAc:MeOH:H2O:HOAc = 4: 2:1:0.1, v/v) and stained with a p-anisaldehyde sugar stain. When the product formation reaches its optimum, add 10 mL of 95% EtOH to precipitate the enzymes in the reaction mixture. Then, remove the precipitate by centrifugation. Concentrate the supernatant by rotary evaporation and purify it by a Bio-Gel P-2 gel filtration column to produce pure sialoside products 10–13 (Fig. 1). The structures of sialoside products are characterized by 1H and 13C NMR as well as mass spectrometry. 3.2. Preparation of Gold Slides

1. Before array fabrication, immerse the sensor substrates for 10 min in a piranha solution (caution – see Note 1) and then rinse with both ethanol and DI water. After drying with N2 gas, the sensor substrates should be ready for use. 2. SPR gold chips with a 2-nm chromium adhesion layer and 51  nm of gold are deposited by e-beam evaporation onto cleaned BK-7 glass slides and can be subsequently used in all assays (see Note 2). E-beam evaporation of gold is based on a standardized procedure that should be available on any campus with a Class 100 clean room.

3.3. Surface Preparation with Biotinylated Sialosides

1. All SPR and SPRi experiments use a 20  mM PBS (pH 7.4, 150 mM NaCl) as both a running buffer and dilution buffer (see Note 3). 2. Place the buffer in a 60-mL syringe and pump at a rate of ~8  mL/h across the sensor surface via a homebuilt flow cell (area ca. 2  cm2) connected through to the syringe pump via PFA tubing and fittings/ferrules noted above. 3. The substrates for the arrays are generated by injecting 100 ml of 0.5 mg/mL biotin-BSA through a glass syringe via a six-port, medium-pressure valve. Anchor this valve to a ring stand and connect it to the flow cell.

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4. Allow the sensor chips to incubate for 30 min (see Note 4). 5. Rinse the surface copiously with DI water to avoid salt buildup from the buffer. 6. Add neutravidin (0.5 mg/mL) to the substrate via injection, followed by incubation and rinsing as previously mentioned. 3.4. Array Fabrication Using the GMS 417 Microarrayer

1. Take the functionalized sensor chip from the flow cell on the SPR imager and dry it under a gentle stream of nitrogen gas. 2. Place the cleaned sensor chip in the GMS 417 Arrayer by clipping it into the arrayer platform. The GMS 417 Arrayer uses the pin-and-ring type of contact printing technology, which delivers highly reproducible dots (7). Set the parameters on the pattern such that the arrayed spots are approximately 150 mm in diameter and spot spacing (center to center) adjusted anywhere from 300 to 375 mm. 3. Next, fill the wells of a plastic microtiter plate (A1 to D4–16 spots total) with 50  mL of 2.0  mg/mL of each biotinylated sialoside and clamp into the well plate holder. 4. Fill up the DI water reservoir on the left side of the arrayer. This water is used to clean the pins before and after deposition (see Note 5). 5. Open the “GMS 417” software program and for the first set of arrays, create four small 2 × 2 arrays containing the four biotinylated sialosides. To do this, set the dot spacing at 300 mm and with all four arrays set them to be separated by 1 mm. 6. Start the arrayer run and the arrayer should spot the substrate with the four different biotinylated sialosides. Afterward, the pins should be automatically rinsed in the DI water reservoir which continuously is replaced from a large bottle of fresh DI water with contaminated water taken to a waste reservoir via a vacuum pump. 7. To create 400-spot, high-density arrays, perform the same steps as 1 through 5, but in this case fill all the spots in the microtiter plate and then run the program “test.” This creates a high-density, 400-spot array (see Note 6).

3.5. Setup of SPR Imaging Experiment for Carbohydrate Microarray

1. After creating the carbohydrate microarrays, take the arrayed chips from the GMS 417 microarrayer and clamp it carefully onto the flow cell. 2. Run PBS buffer across the surface of the arrayed substrate to establish a pre-lectin deposition baseline for about 15 min. 3. Once the signal is stable, rotate the polarizer to 90° to take an s-polarized image for background subtraction and then subsequently rotate back to its original position for p-polarized image acquisition (see Note 7).

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Fig. 2. Processing SPR images. (a) Image after spotting 1.0  mg/mL Neu5Aca2–6-LHEB carbohydrate on the surface; (b) 500 mg/mL SNA lectin; (c) difference image.

4. An image of the array under p-polarization with carbohydrate only is shown in Fig. 2b with buffer flowing. 5. While continuing to run PBS buffer across the sample surface to achieve a stable baseline, add 500 mg/mL SNA (or fluoresceintagged SNA) onto the substrate via flow injection. The images of the substrate should look similar to Fig. 2a. 6. The image before SNA binding (Fig. 2b) was subtracted from the image after binding (Fig. 2c) to obtain difference images (Fig. 2c). 7. For confirmation of binding events, FL-SNA was deposited on the carbohydrate-functionalized surface in the same manner and images of the arrayed substrate were obtained with a confocal fluorescence microscope (Fig. 3). 8. Fluorescence microscopy experiments can be carried out on a Zeiss LSM 510 confocal laser scanning microscope (CLSM) with 488-nm argon laser excitation, CCD camera, and 527-nmlong pass emission filter with a 10× dipping objective. The carbohydrate arrays are fabricated in the same manner as normal SNA arrays as noted above. 9. After FL-SNA is deposited, place the substrate in a Petri dish and cover with aluminum foil until analysis to avoid photodegradation. 10. Using the 10× dipping objective on the confocal fluorescence microscope, snapshot fluorescent images (like those shown in Fig. 3) can be taken of the arrayed substrates and presented without further optimization in an effort to confirm the specificity of lectin–carbohydrate binding.

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Fig. 3. Fluorescence image of Neu5Aca2–6-LHEB array with SNA interaction on the sensor surface.

Fig. 4.  SPR difference image of a high-density, carbohydrate microarray of (a) Neu5Aca2–6-LHEB interacting with 500 mg/ mL SNA; (b) a zoomed up SPR image of a 6 × 5 arrayed area; and (c) the corresponding reflection intensity profile for the array elements.

3.6. SPR Imaging Analysis

1. Analyze all SPR images using the freeware software ImageJ (available at http://rsbweb.nih.gov/ij/). Firstly, process all the images as 8-bit images with a max signal of 256. 2. Using the difference images, line profiles can be created by drawing a rectangular box around the region of interest. A sample result from this process is shown in Figs. 4.

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4. Notes 1. Piranha solution is an extremely strong oxidant and should be handled very carefully. When cleaning slides, always perform this step in a chemical fume hood. 2. After e-beam evaporation of gold, there is a simple method to identify the gold-laden side of the slide. Look down the short axis of the slide and if it lies flat, that is the gold side. If there is a “ledge,” then that is the non-gold side. 3. When making the buffer, we generally make 1 L; this makes the calculations easier and allows for enough buffer for a whole set of experiments. After making the buffer, it is recommended to filter the buffer via vacuum filtration and store it in a plastic container. Before use, we also recommend degassing the buffer with a vacuum hose in a sonicating bath to remove all air bubbles. This extra step helps prevent any air bubble in the microfluidics that could ruin experiments. 4. Biotin-BSA directly binds to gold. But to get high surface coverage to prevent further nonspecific adsorption, at least a 30-min incubation at this step is recommended. 5. When cleaning the pins on the GMS 417 microarrayer, fill up the waste reservoir to DI water. This reservoir should be connected to a pump that takes this dirty DI water to a waste container while constantly replenishing the waste reservoir water from a fresh DI water-holding container. This method always keeps the pins clean and prevents cross-contamination during the spotting process. 6. This “test” program on the GMS 417 microarrayer is an automated program, where the loop of the ring is completely filled and then the solution is spotted 400 times. This assay is generally used to figure out how much solution one would need to put in a given well to allow for 400 spots to be placed in adequate coverage. We found that with 100 ml or so in a given well on the microplate making a 400-spot array with uniform spot density was very reliable. 7. On conventional gold substrates, s-polarized light does not excite surface plasmons; so this s-polarize image serves as a great tool for background subtraction.

Acknowledgments The authors would like to acknowledge the financial supports from NSF grant CHE-0719224 (to QC) and NIH grant R01GM076360

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(to XC). MJL would like to acknowledge the support from the American Chemical Society, Division of Analytical Chemistry Fellowship, sponsored by Procter & Gamble. References 1. Hakomori, S. (2004) Carbohydrate-tocarbohydrate interaction, through glycosynapse, as a basis of cell recognition and membrane organization. Glycoconj. J. 21, 125–37. 2. Fukui, S., Feizi, T., Galustian, C., Lawson, A. M., and Chai, W. (2002) Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat. Biotechnol. 20, 1011–17. 3. Wang, D., Liu, S., Trummer, B. J., Deng, C., and Wang, A. (2002) Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nat. Biotechnol. 20, 275–81. 4. Park, S. J., and Shin, I. J. (2002) Fabrication of carbohydrate chips for studying protein-carbohydrate interactions. Angew. Chem.-Int. Edit. 41, 3180–82. 5. Houseman, B. T., and Mrksich, M. (2002) Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. Chem. Biol. 9, 443–54. 6. Weibel, C. (2002) “The spotting accelerator™”, customizable head assembly for advanced microarraying. JALA 7, 89–94. 7. Barbulovic-Nad, I., Lucente, M., Sun, Y., Zhang, M. J., Wheeler, A. R., and Bussmann, M. (2006) Bio-microarray fabrication techniques – A review. Crit. Rev. Biotechnol. 26, 237–59. 8. Mace, M. L., Montagu, J., Rose, S. D., and McGuinness, G. (2000) in “Microarray Biochip Technology”, pp. 39–64, Eaton Publishing, Natick, MA. 9. Liang, P. H., Wu, C. Y., Greenberg, W. A., and Wong, C. H. (2008) Glycan arrays: biological and medical applications. Curr. Opin. Chem. Biol. 12, 86–92. 10. Chen, Y. L., Nguyen, A., Niu, L. F., and Corn, R. M. (2009) Fabrication of DNA Microarrays with Poly(L-glutamic acid) Monolayers on Gold Substrates for SPR Imaging Measurements. Langmuir 25, 5054–60.

11. Dhayal, M., and Ratner, D. A. (2009) XPS and SPR analysis of glycoarray surface density. Langmuir 25, 2181–87. 12. Linman, M. J., Taylor, J. D., Yu, H., Chen, X., and Cheng, Q. (2008) Surface plasmon resonance study of protein–carbohydrate interactions using biotinylated sialosides. Anal. Chem. 80, 4007–13. 13. Wilkop, T., Wang, Z. Z., and Cheng, Q. (2004) Analysis of u-contact printed protein patterns by SPR imaging with a LED light source. Langmuir 20, 11141–48. 14. Linman, M. J., Yu, H., Chen, X., and Cheng, Q. (2009) Fabrication and characterization of a sialoside-based carbohydrate microarray biointerface for protein binding analysis with surface plasmon resonance imaging. ACS Appl. Mater. Interfaces 1, 1755–62. 15. Yu, H., Yu, H., Karpel, R., and Chen, X. (2004) Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases. Bioorg Med Chem 12, 6427–35. 16. Yu, H., Huang, S., Chokhawala, H., Sun, M., Zheng, H., and Chen, X. (2006) Highly efficient chemoenzymatic synthesis of naturally occurring and non-natural alpha-2,6-linked sialosides: a P. damsela alpha-2,6-sialyltransferase with extremely flexible donor-substrate specificity. Angew Chem Int Ed Engl 45, 3938–44. 17. Yu, H., Chokhawala, H. A., Huang, S., and Chen, X. (2006) One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities. Nat Protoc 1, 2485–92. 18. Yu, H., Chokhawala, H., Karpel, R., Wu, B., Zhang, J., Zhang, Y., Jia, Q., and Chen, X. (2005) A multifunctional Pasteurella multocida sialyltransferase: a powerful tool for the synthesis of sialoside libraries. J Am Chem Soc 127, 17618–9.

Chapter 14 Glycoarray by DNA-Directed Immobilization François Morvan, Yann Chevolot, Jing Zhang, Albert Meyer, Sébastien Vidal, Jean-Pierre Praly, Jean-Jacques Vasseur, and Eliane Souteyrand Abstract Glycoarrays have become a powerful platform to investigate the interactions of many biological events involving carbohydrates. The carbohydrates immobilization on the surface of the substrates is a key step of glycoarray fabrication. Plenty of strategies have been applied to the immobilization process. Herein a protocol for the synthesis of oligonucleotide glycomimetic conjugates is proposed. The resulting molecules are immobilized by hybridization on a DNA microarray (DNA-directed immobilization; DDI). DDI has been proved to be a very efficient and site-selective. This protocol provides detailed procedures for the preparation of fluorescent oligonucleotide trigalactosylmimetic conjugates and for the preparation of carbohydrate microarrays by DDI on glass slides. Key words: Glycoarrays, Click chemistry, Glycomimetics, DNA-directed immobilization

1. Introduction In recent years, considerable attention has been paid to the study of the interactions involving carbohydrates (1–5). Glycoarrays are very powerful investigation tools, which can realize high-throughput profiling and quantitative analysis by using minute amount of materials (6–18). The surface immobilization of oligosaccharides is one of the crucial steps during the processing of glycoarray fabrication. To set up an efficient glycoarray, many important factors should be considered during the immobilization step, such as the distance between each saccharide (surface density), the distance from the saccharide to the surface of the substrate, the orientation and spatial structure of the saccharide, and so on. Diverse methods of immobilization have been reported (19), which can be divided

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a

Cy5

b

Cy5 Lectins

Oligosaccharides Anchoring sequences

Lectins

Oligosaccharides Cy3 Cy3 Oligonucleotides

Fig. 1. Schematic map of recognition methods. (a) “On-chip” recognition. (b) “In-solution” recognition. Reproduced with permission from Wiley-VCH Verlag GmbH & Co. KGaA (36).

into three groups: (1) physisorption (14, 17, 20, 21), (2) chemical covalent grafting (22–33) and (3) specific biological-based interactions (34–38). The DNA-directed immobilization (DDI) belongs to the third group. The principle of DDI is to immobilize biomolecules coupled with a single-stranded DNA moiety, via its specific hybridization with its complementary sequence immobilized on a solid support (Fig.  1a). DDI was first introduced into protein microarray to study biological events involving proteins and peptides (39, 40), and more recently it has been applied to the fabrication of glycoarray (35–38). The DDI of glycoconjugates has been proved to be a very efficient, site-selective. The DNA platform is reusable (36). Furthermore, a fluorescent tag can be introduced at the free end of the carbohydrate–DNA conjugate for surface immobilization quality control (36). Finally this approach offers the possibility to perform carbohydrate–protein interaction in solution and to address the resulting complex on the surface according to the DNA sequence reference (36, 37) (Fig. 1b). In the following a protocol for the synthesis of fluorescent oligonucleotide galactosylmimetic conjugates is given. Next, the fabrication of the DNA anchoring platform is described: It includes microfabrication of microwells by wet etching on glass slides, surfaces functionalization and covalent immobilization of oligonucleotide. Next the immobilization of fluorescent oligonucleotide carbohydrate conjugates by DDI (Fig.  1a) is described. Finally, interaction of immobilized glycoconjugates with Ricinus communis Agglutinin 120 (RCA 120) is described. The interaction of the fluorescent oligonucleotide carbohydrate conjugate with RCA120 in the solution followed by the immobilization of the resulting complex on the DNA anchoring platform is also described (Fig. 1b).

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2. Materials 2.1. Equipment

Rotary evaporator. Thin layer chromatography (TLC) from Macherey-Nagel (Precoated TLC-sheets ALUGRAM SIL G UV254. Silica gel (0.04–0.06 nm). Vacuum desiccator with phosphorous pentoxide (P2O5). Magnetic stirrer. UV–Vis spectrophotometer. Microwave synthesizer with 0.2- to 0.5-mL vials, caps, and crimper. ABI 394 DNA synthesizer with synthesis columns. SpeedVac vacuum system. MALDI-TOF mass spectrometer. Reverse-phase HPLC. Reverse-phase C18 (5 mM) column (150 × 4.6 mm; for analyses). Reverse-phase C18 (15  mM) column (300 × 7.8  mm; for purifications). 822 sputtering system from Material Research Corporation. MJB3 Mask aligner from Zeiss. Nanodrop™ 2000 rim Thermo Scientific. Genepix personal 4100 A fluorescent scanner.

2.2. Solvents and Buffers for Synthesis of Glycoconjugates 2.2.1. Dry Solvents

All the solvents are stored at room temperature if not specified. Dry solvents were obtained as described later. Otherwise synthesis grade solvents were used as received from the manufacturers. 1. Dry Dimethylformamide (DMF) was used. 2. Dry pyridine was dried by distillation over CaH2. 3. Dry Methylene chloride (CH2Cl2) was obtained by distillation over P2O5. 4. Dry and extra dry acetonitrile (CH3CN) was obtained from Biosolve. 5. Dry Triethylamine (Et3N) was obtained by distillation over CaH2. 6. Pyridine Synthesis Grade. 7. Methylene chloride (CH2Cl2). 8. Acetonitrile (CH3CN). 9. Triethylamine (Et3N). 10. Cyclohexane.

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11. Ethyl acetate (EtOAc). 12. Methanol (MeOH). 13. Toluene. 14. Acetone. 15. Concentrated aqueous ammonia solution (NH4OH) was stored at 4°C. 16. Sodium bicarbonate saturated aqueous solution. 17. Sodium chloride solution (Brine) saturated aqueous. 18. 0.05 M triethylammonium acetate buffer, (pH 7) (TEAAc). 19. Sulfuric acid (H2SO4) stain solution (EtOH/H2SO4 90:10 v/v). 20. Vanillin stain solution was fabricated as follow: 5 g of vanillin were added in 25 mL of H2SO4, 225 mL of MeOH/H2O, 1:1, v/v. 21. Water was 18.2 MW. 2.3. Reagents for the Synthesis of Carbohydrates DNA Conjugates

In this section, reagents for the synthesis of DNA conjugates are listed. It comprises reagent for the synthesis of azido glycoside, H-phosphonate monoester linkers, DNA (H-phosphonate or phosphoramidate) cycles, and reagents for amidative oxidation and clik chemistry. Reagents were stored according to the manufacturer’s recommendations. Reagents recommended for automated solid-phase oligonucleotide synthesis. All these reagents are commercially available. (1-Dimethoxytrityloxy-propanediol-3-succinoyl)-long chain alkylamino-CPG from Glen Research or could be prepared according to Seela and Kaiser (41). 1. 2-[2-(2-Chloroethoxy)ethoxy]ethanol. 2. 1,2,3,4,6-Penta-O-acetyl-b-d-galactopyranose. 3. Tin(IV) chloride (SnCl4) as a 1 M solution in CH2Cl2. 4. Silver trifluoroacetate. 5. Sodium azide (NaN3) (see Note 1). 6. Tetra n-butylammonium iodide (nBu4NI). 7. Sodium sulfate (Na2SO4). 8. 4,4’-O,O’-Dimethoxytrityl chloride. 9. 1,4-cyclohexanedimethanol (cis- and trans-mixture). 10. Tetraethyleneglycol. 11. Diphenylphosphite. 12. Propargyl amine. 13. Carbon Tetrachloride. 14. Dry pyridine acetonitrile solution 1:1 v/v.

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15. Pivaloyl chloride (0.2 M) in dry pyridine acetonitrile solution 1:1 v/v. 16. 3% dichloroacetic acid (DCA) in dry CH2Cl2 was used as deblocking solution in H-phosphonate cycle. 17. Standard 2-cyanoethyl deoxyribonucleoside phosphoramidites. 18. Cy3 phosphoramidite from General Electric Healthcare. 19. 5-Benzylthio-1H-tetrazole (BMT) in solution in dry acetonitrile (0.3 M) was used as activator in the phosphoramidate cycle synthesis of DNA. 20. Oxidation solution: 0.1 M iodine in 70/10/20 (v/v/v) THF/ pyridine/water was used as oxidant Reagents recommended for automated solid-phase oligonucleotide synthesis. All these reagents are commercially available. 21. (1-Dimethoxytrityloxy-propanediol-3-succinoyl)-long chain alkylamino-CPG from Glen Research or could be prepared according to Seela and Kaiser (41). 22. Acetic anhydride in dry pyridine/THF 10/10/80 (v/v/v) was used as Cap A solution in the phosphoramidate cycle synthesis of DNA. 23. N-methylimidazole (10%) in dry THF was used as Cap B solution in the phosphoramidate cycle synthesis of DNA. 24. Dichloroacetic acid 3% (DCA) in dry CH2Cl2 as deblocking solution in the phosphoramidate cycle synthesis of DNA. 25. Copper sulfate (CuSO4 or CuSO4⋅5H2O). 26. Sodium ascorbate. 27. 1-Azido-3,6-dioxaoct-8-yl 2,3,4,6-tetra-O-acetyl-b-d-gala­ cto­pyranoside. 2.4. Fabrication of DNA Anchoring Platform

The glass slides (Borosilicate, Nexterion D, Schott GMBH, Germany) were used as the immobilization substrates. Microreactors are etched on the flat glass slides by means of photolithography and wet etching (42, 43). Each glass slide featured 52 round microreactors (2 mm diameter, 65–100 mm deep with less than 1% variation in depth for one etching lot) with a volume near 1 mL. Next the glass slides were silanized (35–38, 42, 44, 45) leading to ester functional bearing glass slides. After acidolysis of the ester function and activation of the corresponding carboxylic acid, amino-modified oligonucleotides were covalently grafted on the surfaces. Eventually, the DNA-carbohydrate conjugates could be immobilized by hybridization on the surface. Solvents Tetrahydrofuran (purum grade, THF) and Dichloromethan (DCM) (ACS reagent grade) (Riedel de Haen, Seelze, Germany) were dried and stored over molecular sieves when required.

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Deionized water 18.2 MW was fabricated by a PURELAB ultra from ELGA (see Note 12). 1. TFD4 detergent (Franklab SA, Billancourt, France). 2. Piranha solution: 96% Sulphuric acid (Riedel de Haen, Puriss, Seelze, Germany), 7 volumes is mixed with 35% hydrogen peroxide (Fluka, Puriss, Steinheim, Germany), 3 volumes (see Note 13). 3. SPR 220-4.5 photoresist (Rohm Haas electronic materials, Lucerne, CH). 4. Photolithographic mask were laser-printed plastic foils. 5. MF 26A developer (Rohm Haas electronic Materials, Lucerne, CH) was used. 6. Chromium Etchant for the selective etching of chromium (Merck, Darmstadt, Germany). 7. Glass etching solution: Buffered oxide etchant (abbreviated BOE, 7/1, Hydro fluoridric acid: ammonium fluoride, Honeywell), 37% hydrochloric acid (HCl, Riedel de Haen, Seelze, Germany) and water (18.2  MW) were mixed in a 1/2/2, v/v ratio. The etching solution is freshly prepared before use. 8. Ethanol and Acetone from Riedel de Haen were used or the removing of SPR 220-4.5 Photoresist. 9. Dry nitrogen (quality 5.0, Linde, Toulouse, France) was used. 10. Pentane (Riedel de Haen, Seelze, Germany). 11. Tetra Hydro furan (THF) was purchased from (quality purum (>99%), Riedel de Haen, Seelze, Germany). 12. Tert-butyl-11-(dimethylamino) silylundecanoate TDMU was synthesized according to Dugas et al. (45). 13. Solution for the conversion of the ester function into the corresponding carboxylic acid: Glacial formic acid from Riedel de Haen (>99%, Seelze, Germany). 14. Carboxylic acid activating solution: 0.1-M N-hydroxysuccinimide (Fluka, Steiheim, Germany) and 0.1-M di (isopropyl) carbodiimide (Fluka, Steinheim, Germany) were dissolved in dry THF. Activated ester glass slides should be used as soon as possible after activation. 15. 10× Phosphate buffer saline (abbreviated PBS 10×): 0.1-M phosphate, 1.38-M NaCl, and 0.027-M KCl. The 10× PBS was prepared by dissolving PBS powder (Sigma, Steinheim, Germany) in 100  mL of Dionised water (18.2  MW). The pH was adjusted to 8.5 by addition of 15-M sodium hydroxide (Sigma, Steinheim, Germany). The solution was stored at 4°C.

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16. 1× Phosphate buffer saline (abbreviated PBS 1×): 0.01-M phosphate, 0.138-M NaCl and 0.0027-M KCl. The solution was fabricated by dissolution of Phosphate buffer saline powder (Sigma, Steinheim, Germany) in 1,000 mL of Deionized Water. PBS 1× was stored at 4°C. 17. Amino-modified oligonucleotides solution: 100  mM Aminomodified oligonucleotides (Eurogentec) in PBS 1×. The mother solution was stored at −20°C. Allicuots were further diluted to 25 mM in PBS 10× pH 8.5 prior immobilization on activated ester glass slides. 18. 0.1% (w/v) sodium dodecyl sulfate (SDS, Sigma Steinheim, Germany) washing solution: 0.1% of SDS in DI water; the solution was stored in at RT. 19. Surface blocking solution: 4% Bovine Serum Albumin (BSA, Sigma, Steinheim) in PBS 1×. It was stored at 4°C. 20. Washing solution: 0.05% Tween20 (Roth, Karlsruhe, Germany) in PBS 1×. The solution was stored at 4°C. 21. 1-mM Glycoconjugates hybridization solution: 100-mM glycoconjugates in PBS 1× concentration. The mother solution was allicuoted and stored at −20°C. Allicuots were further diluted at 1 mM in PBS 1× pH 7.4 prior hybridization on glass slides (on-chip approach). 22. Sodium Saline Citrate (SSC) 2× with 0.1% Sodium Dodecyl Sulfate (SDS, Sigma Steinheim, Germany) and SSC 2× were fabricated by dilution of Saline Sodium Citrate 20× (named here after SSC 20×) powdered blend (Sigma, Steinheim, Germany). The SSC 20× was diluted with DI water (18.2 MW) four times to give at SSC 2×. Sodium Dodecyl Sulfate (SDS; Sigma, Steinhiem) was eventually added to give SSC 2×, SDS 0.1%. Solutions were stored at 4°C. 2.5. Biological Recognition

1. 1× Phosphate buffer saline: see Subheading 2.4, item 16. 2. R. communis Agglutinin 120 (RCA120,Sigma, Steiheim, Germany) was stored at 4°C. 3. Cy5 Ab Labeling Kit (Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) was used for the labeling of RCA120. Store at 4°C. Avoid light exposure. 4. Cy5 labeled RCA 120 incubation solution: Cy5 labeled RCA120 (at the desired concentration), 10 mM CaCl2 (Sigma, Steinheim, Germany) and 2% BSA (Sigma, Steiheim) in PBS 1×. The solution was stored at 4°C. 5. Washing solution: 0.02% v/v Tween 20 in PBS 1×. The solution was stored at 4°C.

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3. Methods 3.1. Synthesis of 5 ¢-Cy3-DNA-3 ¢Trigalacto­sylmimetics

This protocol describes the method to synthesize 5¢-fluorescent oligonucleotides conjugated with a glycomimetic at the 3¢ end. The glycomimetic is constituted of a scaffold bearing three galactose residues with two different linkers between each galactose residue (Fig. 2). We describe (1) the preparation of galactose azide derivative; (2) the H-phosphonate monoester linkers; (3) the introduction of the H-phosphonate diester linkages by H-phosphonate chemistry; (4) their oxidation into phosphoramidate linkage by carbon tetrachloride in presence of propargylamine allowing the preparation of the alkyne scaffold; (5) the conjugation by click chemistry with carbohydrate azide derivative under copper (I) catalysis by 1,3-dipolar cycloaddition affording the solid-supported fully acetylated glycomimetic; (6) the elongation and the labeling of the oligonucleotide by phosphoramidite chemistry; and (7) the deprotection, purification and characterization.

3.1.1. Synthesis of 1-Azido-3,6-Dioxaoct-8-yl 2,3,4,6-Tetra-O-Acetyl-bd-Galactopyranoside

1. Introduce the peracetylated galactose 1 (5 g, 12.8 mmol) and silver trifluoroacetate (4.24 g, 19.2 mmol) in a 250-mL roundbottom flask. Close the flask with a rubber septum and inert with Argon (Scheme 1). 2. Add dropwise 2-[2-(2-chloroethoxy)ethoxy]ethanol (2.80 mL, 19.2 mmol) and freshly distilled dichloromethane (120 mL) to obtain a solution. Keep an argon atmosphere on the reaction. 3. Add SnCl4 (1 M in CH2Cl2, 38.4 mL, 38.4 mmol) dropwise within ~30 min at 0°C (ice bath) to the stirred solution. Stir the solution under argon at room temperature. DNA anchoring sequence

Cy3 DNA

Linker-O

Fluorescent label HO HO

OH O

O P O NH

N N N O

OH

OH 3 Conjugation on solid-support through amidative oxidation

3

Selective chemical ligation through "click chemistry"

Interaction with lectins

Fig. 2. Structure of the 5¢-fluorescent oligonucleotide 3¢-tri-galactosylmimetic. Reproduced with permission from Wiley-VCH Verlag GmbH & Co. KGaA (36).

14  Glycoarray by DNA-Directed Immobilization AcO

OAc O

AcO 1

OAc

a, b OAc

60 %

AcO AcO

OAc O OAc

O

O

O

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N3

2

Scheme 1. Synthesis of azido-functionalized galactose derivative. Reagents and conditions: (a) SnCl4, CF3CO2Ag, H(OCH2CH2)3Cl, CH2Cl2, rt, 2–3 h, (b) NaN3, nBu4NI, DMF, 70°C, 16 h.

4. Follow the reaction by TLC (eluent: PE-EtOAc 1:1, Rf = 0.60 for 1 and 0.34 for 2). Disappearance of the starting material was observed within 1–3 h, occasionally with the mixture turning to a pale pink color. 5. Add a solution of saturated aqueous NaHCO3 (100  mL) to adjust pH above 8 and stir the solution vigorously for 20 min. 6. Dilute the resulting biphasic system with 500 mL of water (see Note 2) and extract the aqueous layer with CH2Cl2 (3 × 150 mL). 7. Combine the organic layers and wash with saturated aqueous NaHCO3 (150 mL), water (3 × 150 mL), brine (2 × 150 mL) and dry over sodium sulfate (Na2SO4). 8. Filter the solid on cotton and wash with CH2Cl2 (2 × 50 mL). Evaporate the solvent on a rotary evaporator to obtain a crude pale yellow gum and transfer the gum in a 250-mL roundbottom flask (dissolve and re-evaporate the solvent). 9. Add sodium azide (see Note 1) (4.16 g, 64.0 mmol) and tetran-butyl ammonium iodide (4.73  g, 12.8  mmol). Close the flask with a rubber septum and inert with Argon. 10. Add anhydrous DMF (150 mL) and stir the mixture at 70°C under argon for 16 h. 11. Cool the reaction mixture to room temperature, filter the solids off on a frit and wash with EtOAc (3 × 100 mL). 12. Dilute the filtrate with EtOAc to obtain a total volume of 1 L. Wash the organic layer with saturated aqueous NaHCO3 (3 × 300  mL), water (3 × 500  mL), brine (400  mL), and dry over sodium sulfate (Na2SO4). 13. Filter on cotton the solid and wash with EtOAc (2 × 50 mL). Evaporate the solvent on a rotary evaporator to obtain a crude yellow to orange gum. 14. Purify the product by silica gel column chromatography (eluent: PE-EtOAc 1:1, internal diameter = 45  mm; length =  250–300 mm) to obtain 2 (3.88 g, 60% yield) as a pale yellow gum (see Note 3). 1-Azido-3,6-dioxaoct-8-yl 2,3,4,6-tetra-O-acetyl-b-d-galactopyranoside 2, (36, 46, 47): Rf = 0.34 (1:1 PE-EtOAc). 1H NMR

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(CDCl3) d 1.99, 2.04, 2.07, 2.15 (4s, 4 × 3H, 4 × CH3CO), 3.40 (t, 2H, J = 5.0 Hz, CH2N