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COMPREHENSIVE
GLYCOSCIENCE
SECOND EDITION
VOLUME 1
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COMPREHENSIVE
GLYCOSCIENCE
SECOND EDITION
EDITOR IN CHIEF AND VOLUME EDITOR
JOSEPH J. BARCHI JR.

Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute,
Frederick, Maryland, United States
VOLUME 1
INTRODUCTION TO GLYCOSCIENCE, GLYCAN STRUCTURE AND FUNCTION

Elsevier
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Notices
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A catalogue record for this book is available from the British Library
ISBN 978-0-12-819475-1
For information on all publications visit our

website at http://store.elsevier.com
Publisher: Oliver Walter

Acquisitions Editor: Blerina Osmanaj
Content Project Manager: Michael Nicholls
Associate Content Project Manager: Fahmida Sultana
Designer: Greg Harris
FRONT COVER IMAGES: (Upper Left) Cartoon of a positively charged glycopolymer-coated nanoparticle (thanks to Diana Diaz-Dussan
for help with this image). (Lower Left) 3D structure (PDB 4N3C) of a truncated form of O-GlcNAc transferase (OCT4.5 derived from
Chapter 3.06 by Jiang). (Upper Right) Conformational free energy landscape of pyranosyl oxocarbenium ions, depicting global and local
minima; from Chapter 2.03 by Codée. (Lower Right) Logos from various glycan informatic websites that have been curated or expanded
since the first edition. The figure shows them radiating from Earth, indicating the multi-national aspect of the sites that now connect
scientists of all disciplines from around the globe. (Center) Structure of the SARS-CoV-2 spike protein ectodomain derived from extended
molecular dynamics simulations, including purported glycans from all included glycosylation sites. The snapshot is an overhead view of
the protein with the viral membrane as the background; the lipids in the bilayer are rendered in gray van der Waals spheres; the editors
thanks Elisa Fadda, National University of Ireland, Maynooth, for this figure.
EDITOR BIOGRAPHIES
Editor-in-Chief
Joe Barchi received his PhD in synthetic/marine natural products chemistry from
the University of Hawaii and was a postdoctoral fellow at Duke University.
He then joined the National Cancer Institute as a staff fellow in the Laboratory
of Medicinal Chemistry in 1988 where he rose to his current position of senior
scientist/principal investigator and NMR facility head at the newly formed Chem-
ical Biology Laboratory. His main research interests are in synthetic medicinal
chemistry as it relates to carbohydrate-based drug design, the development of
novel sugar-conjugated nanoparticles, and the high-resolution structural analysis
of sugars, glycopeptides, and small molecule drug candidates by NMR spectros-
copy. Dr Barchi’s career at the NCI has spanned a wide breadth of drug discovery
efforts, including the synthesis of PKC and reverse transcriptase inhibitors, the
development of glycopeptide antigens as vaccine candidates against HIV and
cancer, as well as the discovery of several glycopeptide analogues of antiprolifera-
tive factor (APF), a negative growth factor isolated from patients with interstitial
cystitis. Current focus in the lab is the discovery of novel nanoplatforms for the delivery and therapeutic
applications of immunogenic glycopeptides, the search for selective antitumor agents based on APF, and the
conformational analysis of these synthetic analogues by NMR spectroscopy to guide further discovery efforts. He is
on the editorial board of PlosOne, Current Cancer Drug Targets (Bentham Science Publishers) and Carbohydra-
teResearch (Elsevier). He has edited books and journal issues on topics such as Glyconanotechnology and Drug
Discovery.
Volume Editors
Ravin Narain, PhD, P.Eng., FRSC is a professor in the Department of Chemical

and Materials Engineering, University of Alberta, Canada. Prof. Narain has
made significant contributions to research on the design, fabrication, and
characterization of novel carbohydrate-based materials (glycopolymers, hydro-
gels, and nanomaterials) for a wide range of applications. His research has
also covered biomaterials, nanomedicine, and regenerative medicine, with
an emphasis on developing advanced materials as cancer therapeutics, anti-
fouling and antimicrobial uses, and cell/tissue engineering advances. He has
published over 180 articles and has edited several books namely Engineered
Carbohydrate-Based Materials for Biomedical Applications (Wiley), Chemistry of
Bioconjugates (Wiley), Glycopolymers: Synthesis and Applications (Smithers &
Rapra), and Polymers and Nanomaterials for Gene Therapy (Woodhead Publish-
ing & Elsevier). He is also on the Editorial Board for Polymer Chemistry (RSC),
Biomacromolecules (ACS) and Polymers (MDPI). He was the recipient for a
Distinguished Visiting Scientist Award from CSIRO (Manufacturing),
Melbourne, Australia (2017–18).
v
vi Editor Biographies
Naoyuki Taniguchi graduated from the Faculty of Medicine, Hokkaido Univer-

sity, and obtained his MD in 1967 and then PhD in 1972 from the same
university. He became assistant professor of the Department of Preventive
Medicine, Hokkaido University, and visiting associate professor at the Depart-
ment of Biochemistry, Connell University Medical School, New York. In 1977 he
became associate professor at Cancer Institute, Hokkaido University Faculty of
Medicine. In 1986, he became professor and chair of the Department of Bio-
chemistry at the Osaka University Medical School. In 2006, after retirement he
became Professor Emeritus, Osaka University, and an endowed chair professor
of Osaka University. Meanwhile, he launched the Systems Glycobiology
Research Group at RIKEN in 2007 and served as the group director until 2018.
Then he was recruited to the head of Department of Glyco-Oncology, Research
Center of Osaka International Cancer Institute. Since 2019 he served as the
director of the Research Center. His research interest is mainly focused on the
functional and structural changes of glycans in relation to various diseases including cancer. He has received
several distinguished awards, such as IGO (International Glycoconjugate Organization) Award in 2001, Medal
with Purple Ribbon from the Emperor of Japan in 2005, HUPO (Human Proteome Organization) Distin-
guished Service Award in 2009, and Japan Academy Prize in 2011. He also served as president in the 75th
Annual Meeting of the Japanese Biochemical Society in 2005 and as secretary general in the 20th IUBMB
11FAOBMB Congress in 2006 and the President of the Society for Glycobiology in 2014.
Sébastien Vidal received his PhD in organic chemistry in 2000 (University of

Montpellier, France – Prof. Jean-Louis Montero and Prof. Alain Morère) on the
synthesis of mannose 6-phosphate analogues. He then moved to UCLA with Sir
J. Fraser Stoddart to study glycodendrimers. In 2003, he joined NREL (Golden,
Colorado) with Prof. Joseph Bozell and studied the combination of organome-
tallic and carbohydrate chemistries. He obtained a CNRS position at University
of Lyon in 2004 and started his independent career on carbohydrate chemistry
and applications in biology. In 2019, he has joined the Department of Chemical
Biology at the Institut de Chimie des Substances Naturelles (Université Paris-
Saclay). Sébastien Vidal’s research interests are primarily on carbohydrate-lectin
interactions through the design of multivalent glycoclusters and their applica-
tion in anti-infectious therapeutic strategies. Another aspect is dealing with the
design of synthetic methodologies to construct bio-active glycoconjugates from
(C-)glycosylation to protecting group strategies and conjugation techniques
(e.g. azide-alkyne cycloaddition).
Spencer Williams was born and raised in Albany, Western Australia.

He graduated from the University of Western Australia with a B.Sc. (Hons.)
(1994) and studied for his PhD (1998) in carbohydrate chemistry at the same
institution with Prof. Robert (Bob) Stick. He received postdoctoral training in
the laboratories of Prof. Stephen Withers at the University of British Columbia
and Prof. Carolyn Bertozzi at the University of California at Berkeley, where he
gained a solid grounding in enzyme mechanism and glycobiology. He was
appointed in 2002 at the University of Melbourne, where he is now professor
of chemistry. His interests include carbohydrate chemistry and biochemistry,
pathways of carbohydrate metabolism, medicinal chemistry, enzyme mecha-
nism, and glycoimmunology.
LIST OF CONTRIBUTORS FOR VOLUME 1
Marya Ahmed Robert S Haltiwanger

Department of Chemistry; Faculty of Sustainable Design Department of Biochemistry and Molecular Biology,
Engineering, University of Prince Edward Island, Complex Carbohydrate Research Center, University of
Charlottetown, PE, Canada Georgia, Athens, GA, United States
Kiyoko F Aoki-Kinoshita Mark R Hardy
Glycan & Life Systems Integration Center (GaLSIC) and Mark R. Hardy Consulting LLC, Meriden, NH, United
Faculty of Science and Engineering, Soka University, States
Tokyo, Japan
Lisa A Holland
Joseph J Barchi Jr Department of Chemistry, West Virginia University,
Head, Glycoconjugate and NMR Section, Chemical Biology Morgantown, WV, United States
Laboratory, Center for Cancer Research, Frederick, MD,
Hsu Chen Hsu
United States
Institute of Atomic and Molecular Sciences, Academia
C Eugene Bennett Sinica, Taipei, Taiwan
Department of Chemistry, West Virginia University,
Shih-Pei Huang
Morgantown, WV, United States
Institute of Atomic and Molecular Sciences, Academia
Shreya Choudhary Sinica, Taipei, Taiwan
Department of Biotechnology, RV College of Engineering,
Yoshito Ihara
Bangalore, Karnataka, India
Department of Biochemistry, Wakayama Medical
Steve W Cui University, Wakayama, Japan
Guelph Research and Development Centre, Agriculture and
Yoko Inai
Agri-Food Canada, Guelph, ON, Canada
Department of Biochemistry, Wakayama Medical
Richard D Cummings University, Wakayama, Japan
Beth Israel Deaconess Medical Center, Department of
Yukishige Ito
Surgery, Harvard Medical School, Boston, MA,
RIKEN, Saitama; Graduate School of Science, Osaka
United States
University, Osaka, Japan
Tamara L Doering
Shweta Sudam Kallapur
Department of Molecular Microbiology, Washington
University in St. Louis, St. Louis, MO, United States
Elisa Fadda
Anusha Mysore Keerthi
Department of Chemistry and Hamilton Institute,
Maynooth University, Maynooth, Ireland
Qingbin Guo
Se-Kwon Kim
State Key Laboratory of Food Nutrition and Safety, College
Department of Marine Science & Convergence
of Food Science and Technology, Tianjin University of
Engineering, College of Science & Technology, Hanyang
Science and Technology, Tianjin, China
University Erica, Gyeonggi-do, South Korea
Praveen Kumar Gupta
Yuriy A Knirel
N. D. Zelinsky Institute of Organic Chemistry, Moscow,
Russia
vii
viii List of Contributors for Volume 1
Petra Larsen Raja Mazumder

Department of Chemistry, University of Prince Edward Department of Biochemistry and Molecular Medicine,
Island, Charlottetown, PE, Canada School of Medicine & Health Sciences, The George
Washington University, Washington, DC, United States
Gordan Lauc
Genos Glycoscience Research Laboratory; Faculty of Chi-Kung Ni
Pharmacy and Biochemistry, University of Zagreb, Zagreb, Institute of Atomic and Molecular Sciences, Academia
Croatia Sinica, Taipei, Taiwan
Chia Yen Liew Naohito Ohno
Institute of Atomic and Molecular Sciences, Academia Tokyo University of Pharmacy and Life Sciences, Tokyo,
Sinica, Taipei, Taiwan Japan
Frederique Lisacek Serge Perez
SIB Swiss Institute of Bioinformatics and Faculty of Centre de Recherche sur les Macromolecules Vegetales,
Sciences, University of Geneva, Geneva, Switzerland University of Grenoble Alpes, Centre National de la
Recherche Scientifique, Grenoble, France
Yan Liu
State Key Laboratory of Food Nutrition and Safety, College Marija Pezer
of Food Science and Technology, Tianjin University of Genos Glycoscience Research Laboratory, Zagreb, Croatia
Science and Technology, Tianjin, China
Ashwini Prabhu
Liza C Loza Yenepoya Research Centre, Yenepoya (Deemed to be
Department of Molecular Microbiology, University), Mangalore, Karnataka, India
Washington University in St. Louis, St. Louis, MO,
Vinitha Rani
United States
Yenepoya Research Centre, Yenepoya (Deemed to be
Kelvin B Luther University), Mangalore, Karnataka, India
Department of Biochemistry and Molecular Biology,
Jeffrey S Rohrer
Complex Carbohydrate Research Center, University of
Thermo Fisher Scientific, Sunnyvale, CA, United States
Georgia, Athens, GA, United States
Roberta Salinas-Marín
Thomas Lütteke
Laboratorio de Glicobiologí a y Diagnóstico Molecular,
GIP GmbH, Offenbach, Germany
Centro de Investigación en Dinámica Celular, Universidad
Olga Makshakova Autónoma del Estado de Morelos, Cuernavaca, Morelos,
Kazan Institute of Biochemistry and Biophysics, FRC Kazan Mexico
Scientific Center of Russian Academy of Sciences, Kazan,
David F Smith
Russia
NatGlycan, LLC, Atlanta, GA, United States
Shino Manabe
Xuezheng Song
RIKEN, Saitama; Laboratory of Functional Molecule
Department of Biochemistry, Emory University School of
Chemistry, Pharmaceutical Department and Institute of
Medicine, Atlanta, GA, United States
Medicinal Chemistry, Hoshi University, Tokyo; Research
Center for Pharmaceutical Development, Graduate School Mike Tiemeyer
of Pharmaceutical Sciences & Faculty of Pharmaceutical Complex Carbohydrate Research Center and Department of
Sciences, Tohoku University, Miyagi, Japan Biochemistry and Molecular Biology, University of Georgia,
Athens, GA, United States
Iván Martínez-Duncker
Laboratorio de Glicobiologí a y Diagnóstico Molecular, Shang-Ting Tsai
Centro de Investigación en Dinámica Celular, Universidad Institute of Atomic and Molecular Sciences, Academia
Autónoma del Estado de Morelos, Cuernavaca, Morelos, Sinica, Taipei, Taiwan
Mexico
Marie-Rose Van Calsteren
Marina Martinic Kavur Saint-Hyacinthe Research and Development Centre,
Genos Glycoscience Research Laboratory, Agriculture and Agri-Food Canada, Saint-Hyacinthe, QC,
Zagreb, Croatia Canada
List of Contributors for Volume 1 ix
Jayachandran Venkatesan Jeet Kiran Vora

Yenepoya Research Centre, Yenepoya (Deemed to be Department of Biochemistry and Molecular Medicine,
University), Mangalore, Karnataka, India School of Medicine & Health Sciences, The George
Washington University, Washington, DC, United States
Tania M Villanueva-Cabello
Laboratorio de Glicobiologí a y Diagnóstico Molecular, Göran Widmalm
Centro de Investigación en Dinámica Celular, Universidad Department of Organic Chemistry, Stockholm University,
Autónoma del Estado de Morelos, Cuernavaca, Morelos, Stockholm, Sweden
Mexico
PREFACE
While it is always difficult to categorize a period in history as defined by a unique area of study, the first two
decades of the 21st century can truly be considered a groundbreaking era in Glycoscience. Many researchers,
societies, and governments around the globe have contributed to this, but I will bias this paragraph by listing
three major initiatives in my home country, the United States, that have greatly contributed to this progress:
(1) a “glue grant” from the National Institute of General Medical Sciences of the National Institutes of Health
that led to the formation of the Consortium for Functional Glycomics; (2) a report by the National Research
Council of the National Academy of Sciences entitled “Transforming Glycoscience: A Roadmap for the Future”
that outlined a plan as to where Glycoscience should be in 10–15 years; and (3) the funding of seminal research
to achieve some of these goals by the NIH Common Fund, where grants were awarded to teams of scientists to
advance three seminal areas: (a) Facile Glycan Synthesis; (b) Tool Development for analysis, tracking, and
manipulation of glycans; and (c) data integration and bioinformatics tools to annotate and search all known
glycomes across gene, protein, and lipid data. The timelines for many of these initiatives are expiring, and thus
the timing of this second edition of Comprehensive Glycoscience could not be more appropriate. Although these
programs have reached their natural end, work in this field is continuing at a fevered pitch. These initiatives—as
well as the many unmentioned ones from other countries around the world—have both ramped up excitement
in traditional glycoscientists and ignited interest in those uninitiated to redirect their focus on more
glycan-related chemistry and biology projects.
This second edition of Comprehensive Glycoscience follows a similar pattern as one that shaped the first edition
in 2007, with a few logistical changes. The first edition was comprised of 4 volumes which dealt with
introductory material (nomenclature, glycan composition of various organisms, carbohydrate analysis tech-
niques), glycan synthesis, glycan biochemistry and carbohydrate interactions and glycans in disease. Since the
field has made major strides in the past 14 years, many of the original authors were asked to update their
chapters, while many new researchers were asked to contribute. This second edition has expanded to 5 volumes,
all with very focused content. Volume 1 sees some of the introductory material deleted, while focusing on new
advances in glycomics and carbohydrate analysis in 21 chapters. The 24 chapters of Volume 2 are dedicated
entirely to synthesis: Basic and modern glycosylation methods, protecting groups, synthesis of various sugar
types such as sialic acids and furanoses and several articles on the newly minted use of automation in
carbohydrate synthesis. Volume 3 is outlined similar to the first edition, with 22 chapters where subjects of
high research interest in the intervening years have been included, such as O-GlcNAc chemical biology,
metabolic engineering of glycans, and zwitterionic polysaccharides. Volume 4 is a new volume, with its
22 chapters dedicated to glycan materials, nanoparticles, and arrays. Very little of this subject material was
covered in the first edition, and it was decided to dedicate an entire volume to the subject, where much of this
research was in its infancy in 2006–07. Chapters on polymeric and metallic nanomaterials, glycopolymers
themselves and their applications, various array formats that have been developed, glyconanomaterials in
biosensing, and applications of these constructions to treating diseases are presented. Volume 5 is comprised
of 31 chapters on the functions of glycans in disease, with highlights on bacterial infections, neurological
disorders, IgG glycosylation, and immune effector functions along with two articles that describe the use of
human milk oligosaccharides in therapeutic applications.
The goal of this edition was not to simply update the first edition but to focus on the phenomenal
achievements that have advanced Glycoscience since the initial printing. While there are a similar number of
chapters, the content is more targeted to these advancements and to hopefully inspire the next generation of
glycoscientists. Thus, although there is a teaching element to this second edition, it is more geared toward the
xi
xii Preface
advanced undergraduate or graduate student who may be involved in a “glyco”-related science or has the
background to understand the concepts and who may be drawn into this highly intriguing and still somewhat
fledging field. The established professor or investigator should find this series a welcome addition to their
collection and will beautifully complement other treatises in glycoscience. The content, along with the
comprehensive list of references, should ground virtually any researcher with a foundation to explore and
expand this exciting area.
Lastly, it should be noted that the bulk of this edition has been assembled in the middle of once-in-100-years
pandemic. COVID-19 has affected all our lives in mostly detrimental ways, and it is a true testament to the
publisher, the editors, and the authors of the 120 chapters who all have fought through unprecedented adversity
to see this project to completion. I owe all involved a debt of gratitude that a project of this scope could be
completed under these unusual circumstances.
Joseph J. Barchi, Jr.

Editor in Chief
CONTENTS OF VOLUME 1
1.01 Introduction to Comprehensive Glycoscience: The Good, the Better and What's to Come 1
Joseph J Barchi Jr
1.02 Bacterial Exopolysaccharides 21

Yuriy A Knirel and Marie-Rose Van Calsteren
1.03 Fungal Polysaccharides 96

Naohito Ohno
1.04 Seaweed Polysaccharides: Promising Molecules for Biotechnological Applications 131

Vinitha Rani, Ashwini Prabhu, Jayachandran Venkatesan, and Se-Kwon Kim
1.05 Common Cellular Glycans: Biosynthesis, Modifications and Functions in Cancer and
Inflammation 142
Petra Larsen and Marya Ahmed
1.06 C-Mannosyl Tryptophan: From Chemistry to Cell Biology 163

Yoshito Ihara, Shino Manabe, Yoko Inai, and Yukishige Ito
1.07 O-Fucosylation of Proteins 182

Kelvin B Luther and Robert S Haltiwanger
1.08 Structure, Classification and Modification of Polysaccharides 204

Qingbin Guo, Yan Liu, and Steve W Cui
1.09 Overview of Cellulose Types and Applications 220

Praveen Kumar Gupta, Anusha Mysore Keerthi, Shweta Sudam Kallapur, and Shreya Choudhary
1.10 Mucins: Structure and Function 237

Roberta Salinas-Marí n, Tania M Villanueva-Cabello, and Iván Martí nez-Duncker
1.11 High-pH Anion-Exchange Chromatography (HPAEC) and Pulsed Amperometric

Detection (PAD) for Carbohydrate Analysis 266
Mark R Hardy and Jeffrey S Rohrer
1.12 Capillary Electrophoresis 290

Lisa A Holland and C Eugene Bennett
1.13 Modern Mass Spectrometry Techniques for Oligosaccharide Structure Determination:

Logically Derived Sequence Tandem Mass Spectrometry for Automatic Oligosaccharide
Structural Determination 309
Chi-Kung Ni, Hsu Chen Hsu, Chia Yen Liew, Shih-Pei Huang, and Shang-Ting Tsai
1.14 General NMR Spectroscopy of Carbohydrates and Conformational Analysis in Solution 340
Göran Widmalm
1.15 Computational Modeling in Glycoscience 374

Serge Perez, Elisa Fadda, and Olga Makshakova
1.16 Understanding the Structure and Function of Viral Glycosylation by Molecular Simulations:
State-of-the-Art and Recent Case Studies 405
Elisa Fadda
xiii
xiv Contents of Volume 1
1.17 Tools and Methods to Study the Human Glycome 416

Xuezheng Song, Richard D Cummings, and David F Smith
1.18 Glycosciences.de: Databases and Tools to Support Research in Glycomics and

Glycoproteomics 432
Thomas Lütteke
1.19 Systems Glycobiology: Immunoglobulin G Glycans as Biomarkers and Functional

Effectors in Aging and Diseases 439
Marina Martinic Kavur, Gordan Lauc, and Marija Pezer
1.20 Glycans of the Pathogenic Yeast Cryptococcus neoformans and Related Opportunities
for Therapeutic Advances 479
Liza C Loza and Tamara L Doering
1.21 Glycoinformatics Resources Integrated Through the GlySpace Alliance 507

Frederique Lisacek, Kiyoko F Aoki-Kinoshita, Jeet Kiran Vora, Raja Mazumder, and Mike Tiemeyer
PERMISSION ACKNOWLEDGEMENT
The following material is reproduced with kind permission of Oxford University press
Figure 2 of Glycan-based Materials in Cancer and Inflammation
Table 1 of Glycan-based Materials in Cancer and Inflammation
www.oup.com
The following material is reproduced with kind permission of Nature Publishing Group
Figure 29 of Surface Immobilized Glycopolymers
Figure 11 of Controls on Element Partitioning Behavior
Figure 5 of Overview of Cellulose Types
Figure 19 of Molecular and Mechanistic Basis of Lectin-glycan Interactions
Figure 6 of Overview of Cellulose Types and Applications
Figure 11 of Capillary Electrophoresis
Figure 8 of Glycan-based Materials in Cancer and Inflammation
http://www.nature.com
i
1.01 Introduction to Comprehensive Glycoscience: The Good, the Better and
What’s to Come
Joseph J Barchi Jr, Head, Glycoconjugate and NMR Section, Chemical Biology Laboratory, Center for Cancer Research, Frederick, MD,
United States
© 2021 Elsevier B.V. All rights reserved.
1.01.1 Introduction 1
1.01.1.1 Brief history of glycoscience 2
1.01.1.2 What are carbohydrates/glycans and what do they look like? 2
1.01.2 Analysis of glycans 9
1.01.2.1 High pH anion-exchange chromatography (HPAEC) and pulsed amperometric detection (PAD) 9
1.01.2.2 Capillary electrophoresis (CE) 9
1.01.2.3 Mass spectrometry 9
1.01.2.4 NMR spectroscopy and molecular simulations 10
1.01.3 Glycan synthesis 11
1.01.4 Cellular glycan functions 12
1.01.4.1 Glycan binding proteins 12
1.01.4.2 Glycans in disease 14
1.01.5 Summary and perspective 15
Appendix 16
References 16
1.01.1 Introduction
Although scientific studies of sugars date back hundreds of years, one could posit that the past three decades constitutes what could
be considered the “new era” of Glycoscience, where appreciation of the relevance of cellular carbohydrates is now indelibly stamped
in the mindset of both physical and life scientists. In fact, a search of the literature reveals that the word “Glycoscience” has only
been coined within those last 30 years with the first instance of the term coming (arguably) around 1994.1 The biology and
chemistry of carbohydrates had traditionally been relegated to a few select and highly specialized research groups that were bold
enough to tackle the complexities of this family of biomolecules. And complex they are: Oligo- and polysaccharides encompass
more “information density” than the other essential cellular information stores of nucleic acids, proteins and lipids.2–5 The
challenges with the analysis, structure, synthesis and biochemistry of cellular carbohydrates is aptly summarized in the forward
to an issue of Chemical Reviews, published in 2000. In it, James K. Bashkin writes6:
“I believe it was George Bernard Shaw who was asked if he knew that “sugar” was the only word in the English language where “su” was pronounced
“sh”. He replied, “Sure”. Sugars remain ubiquitous yet enigmatic, combining the simple with the complex. Carbohydrates are everywhere, from bulk
sucrose in the kitchen to cross-linked peptidoglycans that comprise cell walls, from microdiverse, posttranscriptionally modified cell-surface receptors to
the paper that this issue is printed on. Carbohydrates have often been relegated to the sidelines of chemistry, one example being the complete omission
of essential and challenging sugar portions from many papers on “natural product synthesis.” One can almost hear the collective sigh, “Difficult
chemistry, difficult biology, difficult analysis, I don’t want to see another protecting group, let’s leave the sugar off.”
It seemed that many in the research community thought that sugars just “gum up the works” and studying them made one’s life
difficult. In the early days of protein isolation and purification, sugars were enzymatically stripped off to insure better purity and
ease of handling. When recombinant expression of proteins came of age, the bacteria used for production, primarily E. coli, lacked
glycosylation machinery and hence proteins were produced without any covalent attachment of any post-translational oligosac-
charide chains. This certainly did not upset those working with these proteins, as it made purification and analysis much simpler.
Little did they understand the importance of many of these oligosaccharide chains that, had they been present, could not only
modulate, but actually dictate the function of that particular macromolecule. The research community has come to realize the
overarching importance of cellular glycosylation and today the field of Glycoscience has taken its rightful place among other
scientific disciplines that for years had overshadowed the relevance of cellular and naturally occurring mono-, di-, oligo- and
polysaccharides. There is a fevered interest in most all areas of glycosciences today, including, but not limited to: (1) Glycomics,7,8
the analysis of the full repertoire of the glycans of a particular cell type, which necessitated the development of the new field of
“Glycoinformatics”9,10; (2) glycan synthesis, both chemical and enzymatic, to allow access to all types of both eukaryotic and
prokaryotic saccharides that are known or will be through developments in area #1, (3) technology development, such as the design
and development of glycan/lectin arrays and spectroscopic techniques for glycan analysis and (4) advancement of glycan engineer-
ing techniques for both medical and biotechnology-related applications.
Comprehensive Glycoscience, 2nd edition https://doi.org/10.1016/B978-0-12-819475-1.00108-5 1

2 Introduction to Comprehensive Glycoscience: The Good, the Better and What’s to Come
1.01.1.1 Brief history of glycoscience

As can be inferred from the above discussion, Glycoscience research is a very vibrant field of study. Hence, the number papers and
reviews related to glycoscience has also expanded greatly in the intervening years since the first edition of the Comprehensive
Glycoscience series was published in 2007. There have been a number of books and monographs written on the subject which the
reader should refer to for detailed background information. A list of these are available at the end of this article. An excellent
reference guide to most all aspects of glycoscience is “Essentials of Glycobiology, 3rd edition” which can be found free of charge at
the National Center for Biotechnology Information.11
A quick SciFinder search (Fig. 1) shows the increase in publications with the term “Glycoscience” as the search criterion, with the
highest frequency in the past decade. Alternatively, the word “Glycan,” which seems to have originated in the literature in the late
1940s, has had a precipitous rise in usage and publications with the term in the title, abstract or keyword. In addition, Glycomics,
the study of the “Glycome” (the complete repertoire of glycans produced and presented by a specific organism) took flight just as the
human genome was revealed in 2000. A seminal editorial by Hirabayashi and Kasai, and possibly the first use of the word glycomics
in the literature, suggested that a field be started to define the full set of glycans in different organisms.12 They argued that the study of
glycans, being the third set of important biomolecules after nucleic acids and proteins, was essential to fully understand the function of
the genome and hence, the proteome. This was a truly prescient article that even outlined a bulleted plan as how the field of glycomics
could take shape. Since that time, no fewer than 2400 articles have the word “glycomics” included in the title or abstract. The reader is
referred to chapters by Smith (Chapter 1.17) and Lauc (Chapter 1.19) for a modern discussion of glycomics and the techniques used in
the field. Table 1 lists a few of the early discoveries in carbohydrate/glycoscience that set the stage for the vibrant field it has become.
The exponential growth in research related to an ever-widening breadth of Glycoscience-related topics has consequently led to
a concomitant rise in reviews, treatises and book series on these subjects. The seminal review published in 1993 by Varki suggested
that, regarding the biological roles of oligosaccharides, “all the theories are correct.”13 That is, the roles of saccharides ran the gamut
from trivial to essential in many biological systems, and while many roles are operational, exceptions to all of these are also found.
An update to that review published in 2017 recapitulated this argument, while bringing the idea up to a modern and expanded
version of its original self. As should now be evident, a “comprehensive” review of the field is beyond the scope of any series; and
while both reviews had over 1000 references, it was stated that in the 2017 version that “only a few examples will be cited”!14 Thus,
although one could ask “why write another one,” the previous sentence makes the answer obvious: Keeping pace with this field
warrants frequent and detailed refinements since it is now officially considered part of the mainstream of both the physical and
biological sciences.
The remainder of this section will very briefly introduce the structures of sugars for the uninitiated and continue to the describe
the progressive accomplishments in the fields of Glycoscience as they relate to the chapters here in the second edition of this series.
Brevity will be the operative word here; every effort will be made to refer concepts represented in this introductory chapter to
an appropriate chapter in one of the 5 volumes of this updated series.
1.01.1.2 What are carbohydrates/glycans and what do they look like?

Carbohydrates, in various presentations, are present in all cells from all phyla and species. The word carbohydrate (or as are used
interchangeably: sugar, glycan and saccharide) derives from “carbon” and “hydrate” as all carbons of (most) sugars are “hydrated”,
i.e., contain the elements of water (H-OH). Hence, the formula can be written as such; Cn(H2O)n. Emil Fischer is often cited as the
“father” of carbohydrate chemistry, since it was Fischer who is credited with elucidation of the structure of glucose. The determi-
nation of glucose stereochemistry was a monumental achievement at the time, and a brilliant treatise on the steps involved in this
determination, as well as other sugars such as mannose, galactose and arabinose have been detailed by Claude S Hudson in a series
of papers from 1935–48.15–18 A wonderful account and centennial testament to his research was written by Lichtenthäler in
1992.19,20 The structure of glucose in its various forms is shown in Fig. 2.
Fischer depicted the hexose in a straight vertical chain with the aldehydic carbon on top. This presentation could be thought of
as the carbon chain “bowing into” of the plane of the paper (right structure in the blue box). These views are termed projections,
Fig. 1 Rise in the publications with the word “Glycoscience” (A) and “Glycan” (B). The graph in B extends back to 1894 since the search engine in Scopus
evidently equates the word “polysaccharide” with “Glycan”. The word glycan does not actually enter the scientific literature lexicon until about 1943.
Introduction to Comprehensive Glycoscience: The Good, the Better and What’s to Come 3
Table 1 A few of the early discoveries in carbohydrate/Glycoscience that set the stage for the vibrant field it is today.
Scientist (country of origin) Notable Discovery Year
Andreas Sigismund Marggraf (Germany) Discovered sucrose from sugar beets; Glucose from raisins 1747
Jean Baptiste André Dumas (France) Coined the term “glucose” (from the Greek for sweet) 1834
Jacobus Henrikus van’t Hoff (Netherlands) Proposed the concept of the tetrahedral carbon 1874
Nobel Prize in Chemistry 1901 Chirality, stereochemistry
Joseph Achille Le Bel (France)
Bernhard Tollens (Germany) Recognized the cyclic hemiacetal form of carbohydrates 1883
Emil Fischer (Germany) Determined the molecular structure and stereochemistry of glucose, and other sugars 1891
Nobel Prize in Chemistry 1902
Sir Walter Norman Haworth (United Kingdom) Structure and synthesis of ascorbic acid; novel way of drawing carbohydrate cyclic forms 1933
Nobel Prize in Chemistry 1937
Carl Cori and Gerty Cori (USA) Metabolism of glucose; Determined how the body produces and stores energy 1920’s, 1930’s
Nobel Prize in Physiology and Medicine 1947
Luis Leloir (France) Discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates 1949
Nobel in Chemistry 1970 (Prize in 1970)
Morgan and Watkins Discovery that ABO blood group antigens are sugars 1969
Fig. 2 Glucose as represented by a Fischer projection, Haworth projection and in three-dimensional forms that are primarily shown in the literature. The pyranose
form is a 6-membered ring whereas the furanose ring is 5-membered.
as they involve squashing a dimensional molecule onto a two-dimensional page. Most textbooks will initially use a Fischer
projection to differentiate between “D” and “L” sugars. Looking at the Fischer projection, the stereogenic carbon atom furthest
from the carbonyl carbon traditionally defined “D” glucose as the molecule with this hydroxy group to the right (Fig. 2, blue square)
and “L” with the hydroxyl pointing to the left. It should be noted that this now somewhat archaic description was based on the
arbitrary glyceraldehyde stereochemistry assigned by Rosanoff and this nomenclature was later referred to as the Fischer-Rosanoff
convention.21 Since “D” and “L” glucose (and any other D and L pair for that matter) are enantiomers, all the stereocenters need to be
reversed to produce “L” glucose, not simply C-5.
If one rotates the molecule 90 degrees to the right and curls the carbon chain to allow proximity of the 5th hydroxyl (counting
from the carbonyl carbon) to the aldehyde, it becomes evident how the hemiacetal carbon can be formed. This yields the Haworth
projection in the yellow box. Sugars are often depicted in the literature in this form, although the three-dimensional representation
shown in the lower half of Fig. 2 is more akin to a true Organic Chemistry structure where the stereochemistry of the carbon atoms
can easily be seen. The stereochemistry of the hemiacetal hydroxyl group can be either up (beta) or down (alpha) in what is referred
to as a “reducing sugar.” This designation comes from the fact that the aldehyde can act as a “reducing agent” toward entities such
as metals; this property is the basis for the Tollens test, where silver salts are reduced to elemental silver with simultaneous oxidation
of the reducing sugar aldehyde to a carboxylic acid.22–24
As mentioned above, carbohydrates are found on all cells from both eukaryotic and prokaryotic organisms. The sets of
carbohydrate structures that are biosynthesized and presented on cell surfaces between different species can differ dramatically.
Thus, the aforementioned complexities of sugars structures compared with nucleic acids and peptides comes from the following
facts: (1) there are actually 100s and predicted to be 1000s of structurally unique monomeric sugars that span all organisms from
bacteria to humans as opposed to 5 bases that comprise DNA and RNA and 21 natural amino acids that form most proteins; (2) the
anomeric center can exist in two epimeric forms; (3) all sugars have more than one functional group that can be modified/
conjugated. This makes the diversity of carbohydrate structures infinitely more complex than the simple connections that are made
between two amino acids to form peptides or two DNA/RNA base pairs to form a dinucleotide. Glucose is an example of
a monosaccharide, i.e., “one sugar unit.” This can be extended to di-, tri-, tetra- and what are considered “oligo”-saccharides
which constitute up to 10–20 monosaccharide units. Beyond that, these coupled chains are can now be considered
“poly”-saccharides (for example, cellulose and chrysolaminarin, are polymers of glucose, and are arguably the most abundant
materials on earth).25 Consider that a simple glucose monosaccharide contains five hydroxyl groups in five chiral centers, and
a choice of either an alpha or beta anomeric center. If you had 20 pyranose glycan units similar to glucose, and compared the
combinatorial number of hexamers that could be constructed compared to those from 4 nucleotides (46 ¼ 4096) or 20 natural
amino acids (206 ¼ 64,000,000), a total of 192,780,943, 360 hexasaccharides are possible.5 The information content in carbohy-
drate monomers and oligomers is truly vast and as yet, not fully appreciated (vide infra).
Descriptions of glycans from all species is beyond the scope of this article, thus, groups of various saccharides that are more
common in mammalian, plant and bacterial systems will be briefly discussed. It is generally recognized that there are 10 (the
number may be 11 or 12) basic mammalian sugar building blocks that are used to assemble the various cellular glycans that have
thus far been determined.4 These are shown in Fig. 3 with their standard abbreviations. The six-membered pyranoses are thought to
exist in a typical chair conformation that was learned from cyclohexane structures. In sugars, there is a notation that describes the
spatial relationship of atoms in a particular form. As shown in the figure, a “4C1” chair has Carbon-4 puckered “up” out of the
central plane that would bisect each bond, and the C-1 atom flexed “down.” Alternatively, a “1C4” pucker (fucose) has those atoms
Fig. 3 The 10 most common mammalian monosaccharides used to construct various cellular glycans and their abbreviations in parentheses. Numbering and
puckered forms are in colored numbers and circles.
flipped between up and down. As opposed to all other in the diagram, fucose is the only L-sugar. Sometimes one may see the
notation written “4C1” since that more accurately shows where the atoms are in space. N-Acetylneuraminic acid, more commonly
known as sialic acid, is a 9-carbon sugar acid were the carboxylate group is pointed “up” while the pyranose is in a “2C5” pucker,
since the numbering of this 9-carbon sugar starts at the carboxyl carbon (see bottom of Fig. 3).
These monosaccharides make up most of the glycans that are displayed on the surface (or now, within) a mammalian cell.
Cell-surface glycans are presented in a variety of forms, each with their own structural idiosyncrasies and dispositions. One of the
best depictions of this is from the introductory sections of “Essentials of Glycobiology” thus there is no need to reinvent this figure.
This schematic of the cell surface glycocalyx, as it is often called, is adapted and modified in Fig. 4 for this article. It shows in generic
form, the various structures that would be found, for example, on one of the cells of human tissue. Either the initial (reducing end)
sugar or the entire structure of each entity is illustrated for each type of glycan and arrows point to either the macromolecule, lipid or
polymer whose context that structure is associated. A listing of how these structures are arranged are as follows, and the chapters that
discuss the various roles these modifications play in biological function or disease in this series are shown in parentheses:
1. N-Linked glycans: Attached to asparagine in specific amino acid triplet motifs of Asn-Ser/Thr-X, where X ¼ any amino acid
except proline. b-Amide-linked anomeric center to a chitobiose unit (i.e., -GlcNAcb1,4-GlcNAcb-N-CO-CH2-amino acid)26 (Gu,
Chapter 5.25)
2. Glycolipids: Glycans from mono- to oligosaccharides attached to a lipid, can be a glycerol, sphingosine or ceramide unit.
Most contain a reducing end b-glucose, some with b-galactose. Can have varied composition of sugars based on cell and species
of origin.27 (Schnaar, Chapter 3.05, Savage, Chapter 3.22)
3. O-Linked mucin-type glycosylation: Glycans attached to either serine or threonine on cell-surface mucins through an initial
a-Galactosamine monomer; glycans can be anywhere from 2–12 monosaccharides, When “O-linked” saccharides are discussed,
it usually refers to this type of protein glycosylation. (Brockhausen, Chapter 3.10, and Ramirez, Chapter 1.10)
4. O-GlcNAc modification: Discovered about 30 years ago. widespread modification of cytoplasmic and nuclear proteins.
Molecular “switch” similar to phosphorylation but only one transferase and hydrolase known for attaching and releasing
the modification. (Hart, Chapter 5.08; Zachara, Chapter 3.13 and Jiang, Chapter 3.06)
5. C-Mannosyl tryptophan: Thought to be a unique protein modification and the only natural carbon-linked glycan known where
an a-mannose attaches to the indole C2 carbon atom of a tryptophan amino acid. Usually attached at a consensus sequence W-x-
x-W.28 (Ihara, Chapter 1.06)
6. Glycophosphatidylinositol (GPI anchor): Complex lipid/inositol glycan that anchors various proteins to cell membranes.
A phosphatidylinositol lipid embeds in the membrane with core glycans that comprise one glucosamine and three mannoses;
the terminal non-reducing end mannose contains an O6-linked phosphoethanolamine, which is amide-bonded to the carboxyl
terminus of a protein. Common form of cellular protein attachment in Protozoa. (Fujita, Chapter 3.04 and Murakami,
Chapter 5.21)
7. O-Fucosylation (O-glucose, O-mannose): Discovered around 1993, O-fucose and fucosylated glycans are a-linked to serine and
threonine residues and found on Epidermal Growth Factor (EGF) repeats on several proteins and occurs in a putative consensus
sequence (CXXGG(S/T)C, where S/T is the modified residue. These along with O-glucose and O-mannose have been found on
thrombospondin-type repeat protein motifs. O-Mannose is found highly concentrated in brain glycoproteins. (Haltiwanger,
Chapter 1.07)
8. Glycosaminoglycans (GAGs): Charged, sulfated, long linear polysaccharide chains primarily attached to proteins. Chondroitin
sulfate, dermatan sulfate, keratan sulfate and heparan sulfate are typical GAGs. Attached to proteins through a serine hydroxyl
with a consensus Ser-Gly/Ala-X-Gly motif in the core protein. A tetrasaccharide linker, -GlcAb1,3Galb1,3Galb1,4Xylb1-O-(Ser)-
begins the construction of the linear polysaccharide. The polysaccharides are built from disaccharide repeats that usually consist
of a sugar acid and an amino sugar. Proteins heavily glycosylated with GAGs are referred to as Proteoglycans. (Ramirez,
Chapter 3.03; Kitagawa, Chapter 3.02 and Liu, Chapter 2.24)
9. Hyaluronic acid (Hyaluronan): A GAG that is biosynthesized but immediately secreted into the intercellular space, thus not
formally a surface modification, and not covalently attached to proteoglycans. It is a component of the extracellular matrix.
(Kitagawa, Chapter 3.02)
Many of these glycans, especially N- and O-linked glycans, are structurally complex, where linear chains are built of many different
monosaccharides, and branching and “capping” (vide infra) are common where additional multi-antennae arrangements are
possible. Often, a single glycosylation site will be dynamically occupied by different oligosaccharides that may differ by one or
more monosaccharides. These different glycans at one site are called “glycoforms,”29,30 and it is obvious how this adds to the almost
overwhelming complexity of cellular glycosylation. The presence of various glycoforms results from the differential expression of
glycosyltransferases (enzymes that couple one sugar to another) and glycoside hydrolases (enzymes that cleave a sugar from
a oligosaccharide) that can arise in a tissue-, organ-, temporal- or spatial-specific manner. The presence of glycoforms is one of the
many ways to rationalize the “underwhelming” result of mapping the human genome. It surprised everyone when it was discovered
that we only harbor 21,000 protein-encoding genes, where the thought was closer to 100,000 in early estimates.31 However, the
one-gene/one-protein model is a gross oversimplification; alternative splicing can generate many proteins from a single gene and
post-translational modifications (phosphorylation, farnesylation, acylation, alkylation, sulfation and glycosylation, just to name
a few) can impart “fine-tuning” of functions to a single protein. These and many other changes that take place during development
can lead to a complex organism from a relatively small number of coding sequences.
6
Introduction to Comprehensive Glycoscience: The Good, the Better and What’s to Come
Fig. 4 A schematic of the various glycan forms that are primarily displayed on a mammalian cell surface along with nuclear/cytoplasmic O-GlcNAc, as adapted from “Essentials of Glycobiology”, third edition, Chapter 1,
Fig. 1.6. The carbohydrate structures that begin a particular glycan are shown for mucin type O-linked glycosylation (upper left, a-D-Galactosamine), N-linked glycosylation (chitobiose structure) and glycolipids (b-glucose
attached to a ceramide lipid). Also depicted is the disaccharide repeating unit of hyaluronic acid (hyaluronan, -GlcAb1,4GlcNAc-), glycosaminoglycan; b-O-GlcNAc, a prominent modification to nuclear and cytoplasmic proteins;
an entire structure of a glycophosphatidylinositol (GPI anchor), a complex, charged glycolipid that is an important connector between the cell membrane and various cell-surface proteins and the sole C-linked glycan modification,
C-mannosyl tryptophan, that is found in motifs containing the WxxW tetrapeptide stretch in proteins. Originally modified and updated from Varki, A. FASEB J. 1997, 11: 248–255; Fuster, M; Esko, J.D. Nat. Rev. Cancer 2005, 7,
526–542, originally with permission from Macmillan; and Stanley P. Cold Spring Harb. Perspect. Biol. 2011, 3, a005199.
Without explicitly depicting the many glycan variants, on perusing Fig. 4, the reader can imagine how complicated the cell
surface can be with the presence of 1000s of proteins each with their own glycosylation network. In addition, since the cell is
utilizing a vast amount of energy to assemble these complex structures, it follows that there must be some highly relevant functions
that these modifications impart to proteins and lipids (vide infra).
Whereas these structures make up the majority of the glycans in many eukaryotic glycomes, various database and bioinformatic
analyzes of bacterial glycomes suggest that there are over 700 different monosaccharides that are used to biosynthesize the
incredibly diverse array of glycans in bacteria species.32 In fact, a variety of unusual sugar structures come from organisms other
than humans and other mammals. Many of these structures are not six-carbon hexoses, but glycosides made up of different
numbers of carbon atoms from pentoses (5-carbon) to nonoses (9-carbon) and even longer. As the complexity of the glycan
repertoire grew, the early full chemical drawing depictions of sugars was thought to have become somewhat unwieldy. As early
as 1978, Kornfeld proposed a simplified presentation of the various sugar structures based on geometrical shapes, thought to be
a purposeful substitute for full stereochemical drawings. Future incarnations of this new mode of glycans representation matured
to what is now known as the Symbol Nomenclature for Glycans (SNFG).33 This code is evident in Fig. 4 with a small legend at the
bottom left. Many papers in the Glycoscience field will use this notation, as it has been used throughout this 2nd edition of
Comprehensive Glycoscience. Fig. A.1 that shows the latest listing of symbols to represent a large array of glycans is shown in the
Appendix section.
Bacteria, fungi and plants contain a rich array of glycans with monomer structures that vary from those in higher organisms.
Some of the more relevant monosaccharides are higher carbon (8- and 9-carbon) sugars. One of these structures, 3-deoxy-D-
manno-octulosonic acid (Kdo), is a component of the lipopolysaccharide portion of the outer surface of gram-negative bacteria.34
This extended covalent unit of lipids and sugars is referred to as endotoxin, and Kdo is attached to a portion called Lipid A. Lipid A is
released from the bacterium after infection or lysis in serum and is a powerful biological response modifier, and this glycosylated
assembly has been one of the more intensely studied portions of the bacterial cell membrane. This complicated glycolipid is a signal
to turn on aspects of the immune system that cause inflammation and other symptoms of a bacterial infection. Further study
revealed that Kdo is thought to be important for bacterial cell viability and survival. A schematic of the composition of the inner and
outer membrane structures from gram-negative bacteria are shown in Fig. 5.35 Examination of panels A, B and C shows increasing
detail as to the structure of LPS and its toxic component, Lipid A. Glycoscientists have learned an enormous amount about
saccharide biosynthesis, assembly and function through work on bacteria. As stated, there are a plethora of unusual sugars presented
in bacteria, and thoughts about their evolution and function have spurred additional work where for example, new synthetic and
analysis techniques have been developed. The reader is referred to excellent chapters by Brockhausen (Chapter 3.07), Molinaro
(Chapter 5.13), and Knirel (Chapter 1.02) for details of the structure and virulence of bacterial saccharides.
Bacteria have evolved to produce a variety of different glycans with modifications such as deoxygenation of different hydroxyl
groups, hydroxyl-to-amine substitutions, many L-sugars and stereochemically unique arrangements of functional groups.
Fig. 5 (A) Schematic depiction of the outer and inner membrane of gram negative bacteria; (B) an expansion of the features of the lipopolysaccharide (LPS) portion
of the outer membrane in a molecular schematic form, and (C) a further expansion of the lipid A portion of LPS in pictorial form (top), molecular schematic (middle)
and molecular formula form (bottom). Figure was derived from Zamyatina, A. Aminosugar-Based Immunomodulator Lipid A: Synthetic Approaches. Beilstein J. Org.
Chem. 2018, 14, 25–53.
Fig. 6 Sampling of some unusual saccharides found in various species of bacteria.
An extremely small sampling of the interesting diversity of bacterial saccharides is shown in Fig. 6. Both enantiomers of fucosamine
are found in gram negative bacteria.36,37 The deoxy-gulose molecules are found in bacteria but also are glycan constituents of cardiac
glycosides found in plants, most often the Foxglove species.38 Altrose is found in certain anaerobic bacteria strains such
as Butyrivibrio fibrisolvens strain CF3.39 Glycero-mannoheptoses are common constituents of the LPS core region saccharides from
a variety of bacterial species.40–46
A few words should be said about sialic acids. This 9-carbon sugar is a common glycan found in all vertebrates and has myriad
functions in cells. Referring to Fig. 4, most N- and O-linked glycans in normal cells will be “capped” by sialic acids; this final
addition to a glycan instructs the cell to stop conjugating additional sugars to the formed glycan. In addition, this adds a large
negatively charged coat to the cell surface that has defined effects on cellular properties.47 There are more than 50 sialic acid analogs
known to date across many species.48 Some are also found in bacteria such as those depicted in Fig. 7. These structures illustrate
the diverse biosynthetic pathways that are taken in bacteria as opposed to higher organisms. While Kdn, the “5-deaminated” version
of Neu5Ac, has been found both in bacteria and higher animals, pseudaminic, legionaminic and acinetaminic acids are all found in
bacteria.49
It should be noted that of all the major groups of biological macro/molecules that make up the composition of every cell—
nucleic acids, proteins, carbohydrates and lipids—each one either embodies or is associated with glycan structures: Base pairs of
nucleic acids are composed of a pyrimidine or purine base and a ribose (RNA) or deoxyribose (DNA) ring; the most abundant
post-translational modification of a protein is glycosylation and lipids can be of many forms, including highly important
glycolipids.
To date, the study of cellular glycans has shown that they are present on all cells, from every domain from Archaea—Bacteria—
Eukarya. Thus, we most likely have only scratched the surface of the number and types of glycans that are biosynthesized by various
Fig. 7 Analogs of sialic acid found in various species of bacteria.

organisms. This introduction serves to ground the reader for further exploration into glycans from their organism of choice. Further
reading in this series will find chapters dedicated to seaweed polysaccharides (Kim, Chapter 1.04), Mucin type (Brockhausen,
Chapter 3.10), yeast and fungal polysaccharides (Ohno, Chapter 1.03), bacterial exopolysaccharides (Knirel, Chapter 1.02),
nucleocytoplasmic protein glycosylation (Jiang, Chapter 3.06), bacterial zwitterionic polysaccharides (Andreana, Chapter 3.21),
vertebrate N-linked glycans (Suzuki, Chapter 3.01), Proteoglycans and GAGs (Ramirez, Chapter 3.03 and Kitagawa, Chapter 3.02),
Protozoan glycans (McConville, Chapter 3.08), Drosophila melanogaster (Nishihara, Chapter 5.01), Caenorhabditis elegans (Wilson,
Chapter 5.02), plant glycans (Misaki, Chapter 5.05) and human milk oligosaccharides (Garrido, Chapter 5.23 and Urashima).
1.01.2 Analysis of glycans
It may seem obvious from the above discussion that isolating pure glycans, analyzing their composition, determining monomeric
structures, correct linkages and stereochemistry is a daunting task. Many traditional methods are still used today and updates to
instrumentation and experimental techniques have allowed higher resolution and extension to different glycans. Newer analytical
tools and modernization and adaptation of existing tools has allowed carbohydrate analysis and glycomics research to take some
very large leaps in the years since the first edition of this series. This section will very briefly touch on primary analysis methods and
the discovery of some new methods to facilitate cellular glycan structure determination. Some of the unique challenges that the field
is attempting to address are:
1. Monomer separations
2. Oligosaccharide composition
3. Glycoforms
4. Linkage analysis
5. Low abundance of glycosylated proteins (sensitivity)
6. Specificity of reagent probes
7. Reagents for efficient release of various glycan types (O-linked, N-linked)
8. No simple sequencing procedures, unlike nucleic acids and proteins
A few examples from this series are briefly mentioned below.
1.01.2.1 High pH anion-exchange chromatography (HPAEC) and pulsed amperometric detection (PAD)
This technique has been available for more than 4 decades and is still a method of choice for the separation and detection of
carbohydrates.50,51 There are several challenges to carbohydrate analysis but one major for chromatography is there is no distinct
chromophore for simple UV detection at standard wavelengths. HPAEC-PAD is based on the property that carbohydrates are weak
acids and ionize at high pH. Carbohydrates are run through an anion exchange column where separation can be achieved at high
pH. Carbohydrates are also electrochemically active, that is they contain functional groups that can be oxidized (anomeric position,
hydroxyls, see Tollens test, vide supra). The effluent of the column can be sent to detector where a voltage is applied across electrodes;
a change in current occurs when an analyte is oxidized or reduced, facilitating detection of the separated sugars. The chapter 1.11 by
Rohrer outlines the process in an update to his chapter from 2007.
1.01.2.2 Capillary electrophoresis (CE)

This technique is based on the motion of analyte particles relative to a flow fluid under the influence of an electric field. This has
been used for carbohydrate analysis and quality control applications in the pharmaceutical industry for many years.52–56 This
technique could be thought of gel electrophoresis transferred to a narrow bore capillary. This in turns increases resistance, allowing
the use of lower electric current for separation and a higher voltage to be applied resulting in increased separation efficiency and
lower run times. Often, unlabeled carbohydrates will be tagged with a fluorescent moiety, usually attached at the reducing end, to
facilitate detection.57 In the last several years, CE has been coupled to mass spectrometry for simplified and high resolution
detection and identification of carbohydrates.58–64 Chapter 1.12 by Holland in this series is a thorough review of this technique
with applications to modern carbohydrate analysis.
1.01.2.3 Mass spectrometry

Probably the most important and relevant technique for glycomics/glycoproteomic studies, and one that has advanced more than
many others in the past 15–20 years is mass spectrometry.58,59,61,65–88 “Mass spec” (MS) is highly sensitive and can determine
a variety of properties associated with glycans, dependent on the ionization mode, number of stages, specific labeling and
enrichment methods. Single stage MS can only reveal oligosaccharide composition but combination of MS with various fragmen-
tation methods have been used for more refined structural determination. Mass spec has traditionally been used with derivatization
methods such as reduction and permethylation of oligosaccharides to facilitate the use of ionization methods such as
collision-induced dissociation in line with tandem mass spectrometry (MS-MS) to determine the primary structure of
oligosaccharides. The past 20 years has seen enormous progress in ionization techniques and methods for glycoproteomic analysis
along with an increase in the database information available and bioinformatics platforms for analysis. In this series, the chapter by
Ni et al., (Chapter 1.13) describes a MS technique called logically derived sequence (LODES) multistage tandem mass spectrometry
(MSn). This technique can determine the primary structure of oligosaccharides, with stereochemical information. The chapters by
Smith (Chapter 1.17), Lauc (Chapter 1.19), and others in Volume 3 also refer to mass spec techniques for glycomics research.
1.01.2.4 NMR spectroscopy and molecular simulations

Nuclear Magnetic Resonance (NMR) spectroscopy is arguably one of the most important and information-rich technique in
chemistry and biochemistry research. When molecules such as glycans, proteins or nucleic acids are placed in a magnetic field,
certain atomic isotopes especially those with a spin quantum number of 1/2, such as 1H, 13C, 15N, 31P and 19F, act like little bar
magnets and align with (or against) the external magnetic field. Application of a radio frequency at the “resonance” energy causes
spins to flip between aligned/unaligned states, and the energy used to cause this is different for atoms in different chemical and
electronic environments (the “chemical shift”). Detection of those states leads to a fingerprint of those atoms in the molecule,
leading to determination of their molecular arrangement. When atoms such as protons and carbons are within 1–5 bonds of one
another, they interact through the electron that make up these bonds and “couple” to each other, causing splits in the energy levels
of their neighbors, leading to more complicated spectra. These coupling constants are different depending on the angle between the
coupling partners, and thus can report on defined arrangements in space. One can see that the information gained from NMR such
as chemical shift and coupling constants leads to assignments of both molecular environment and stereochemistry. In addition,
atoms can “couple” to one another through dipole-dipole interactions through space, and this property, called the nuclear
Overhauser enhancement (NOE), can detect protons that are not coupled through bonds but are close in space. This method
along with through-bond interactions between protons and carbons allows the determination of linkage information between
conjugated monosaccharide units. Reviews of NMR in carbohydrate structure determination are plentiful, and the reader is referred
to these monographs.89–100 A simple NMR spectrum of the disaccharide methyl b-maltoside, taken from Chapter 1.14 by
Widmalm, is shown in Fig. 8.
It is beyond the scope of this chapter to describe NMR theory and practice in detail; suffice it to say, NMR has advanced over the
years to expand to an armamentarium of experiments that include multidimensional NMR that can simultaneously correlate several
NMR-active nuclei that are coupled vicinal, coupled long range or coupled through space. The use of what is known as residual
dipolar couplings has made a tremendous impact on the protein structural biology and that has extended to carbohydrate
structures.93 In addition, many techniques such as Saturation Transfer Difference (STD), WaterLogsy and T2 filtering can detect
these interactions in complexes of carbohydrates and macromolecules (proteins/DNA/RNA).99,101–104 Chapter 3.15 by Jimenez-
Barbero, details the more modern aspects of these techniques.
The composite of all the many sets of information one can glean from NMR can be used to help construct three-dimensional
conformations of glycans. However, from years of structural work on various oligosaccharides, it has been shown that glycosidic
linkages are relatively flexible, and glycans conformations can be highly dynamic. Thus, information obtained by NMR experiments
can be very useful, but insufficient to fully define the conformational space mapped by large oligosaccharides since it reports on
a time-averaged population of conformations. Molecular modeling simulations based on defined force fields that have been curated
by structural information over the course of many decades, can fill this gap.
Molecular mechanics and dynamics are techniques that use what is called a force field to define various atomic and molecular
parameters to closely mimic an actual real-world molecule have evolved dramatically over the years. The availability of structural
data by X-ray crystallography and NMR spectroscopy have fueled the progress to adapt more refined force fields and electronic
Fig. 8 NMR spectrum (left) of methyl b-maltoside (right) with color-coded assignments of the two anomeric protons. HDO is protonated residual solvent from
D2O. The methyl singlet is labeled.
parameters that may yield improved outcomes for defining molecular conformations. Some of the most recognized force field used
in carbohydrate simulations are GAFF1,105,106 CHARMM107–116 and GLYCAM106,117,118 which have been parameterized for the
very unique linkages (anomeric, amino acid-carbohydrate) that are present in oligosaccharides and carbohydrate-protein conju-
gates. Often, NMR data is combined with molecular modeling to “match” each other’s calculations. For example, through-space
interactions defined by NOE’s can be included as “restraints” in simulated structures to maintain specific conformational
adjustments and to insure the fluctuating, calculated conformations do not violate these experimentally-derived limits. An in-depth
discussion of these concepts is available in the chapters by Perez (Chapter 1.15) and Fadda (Chapter 1.16).
1.01.3 Glycan synthesis
The synthesis of glycans of all kinds is of course an entire field to itself. Since the days of Fischer and Hudson, researchers have tried
to synthesize carbohydrate analogs. It was not until perhaps 1953 that the first disaccharide, sucrose, was synthesized by Ray
Lemieux. This was considered a landmark synthesis and his 5% yield was “tops” at the time! Since then, our ability to synthesize
complex glycans has also progressed as other areas of the field. Volume 2 has a full array of chapters to get anyone up to speed on
modern carbohydrate synthesis.
The synthetic routes used to produce oligosaccharides have a few key features that have remained unchanged for generations.
The coupling of two sugar units has commonly been comprised of one saccharide as the glycosyl “donor” and the other the
“acceptor.” The donor will “accept” the second sugar at its anomeric center. Hence the donor needs a leaving group at C1. The
acceptor uses a free hydroxyl as a nucleophile to react with the donor under the auspices of an activating agent that primes the donor
for nucleophilic attack. Since sugars have several free hydroxyl groups, the ones that are not acting as the acceptor nucleophile need
to be masked, or “protected”. After the coupling, a selective deprotection of a separate hydroxyl group frees a second nucleophile to
react with another donor molecule. A schematic of this type of transformation is shown in Fig. 9, taken from Chapter 2.01 by
Oscarson. In the brackets are what are thought to be the intermediate species in the reaction: The top structure is called
an oxocarbenium ion, and through the years, glycosylations were thought to go through this intermediate, making the reaction
an Sn1 like mechanism. There has been some controversy as to the existence of this intermediate and a beautiful discussion of the
theoretical and experimental data can be found in Chapter 2.02 by Blériot.
Glycan synthesis has evolved where there is now an extensive list of functional groups that can be used as donors (thioglycosides,
trichloroacetimidates, xanthates, phosphates, halogens and pentenyl glycosides, to name just a few), an equally large list of
promotors/catalysts and a variety of methods to string together many diverse monosaccharides where reaction economy is
exploited. In addition to chemical synthesis, enzymatic synthesis, through the use of glycosyltransferases and hydrolases, have
made a distinct mark on sugar chemistry in recent years.118 There are several “one-pot” procedures that have been developed that
utilize differential reactivity of donors and acceptors to certain specialized reaction conditions. These were pioneered by
Wong119,120 and have been increasing in popularity. One ingenious method has been developed into an extremely useful technique
in glycan synthesis is the use of a one-pot multi enzyme (OPME) system of Chen.121–124 This system has been expanded to
glycolipids and glycopeptides (Fig. 10).
The one-pot methods were a precursor to the development of automated oligosaccharide synthesis. Prior discussions in this article
confirmed the difficulties in automating glycan assembly. However, great strides have been made in this area since the first edition, and
the chapters by Demchenko (Chapter 2.20), Hurevich (Chapter 2.17) and Downey (Chapter 2.18) outline the latest in this research,
with the development of the first commercial automated oligosaccharide synthesizer, the Glyconeer® (Hurevich Chapter 2.17).
Fig. 9 The glycosylation reaction in schematic form. A donor and an acceptor are coupled through the use of a promotor or catalyst, and a disaccharide is formed,
typically through the intermediacy of the oxocarbenium ion (brackets, top structure).
Fig. 10 OPME strategy: Carbohydrate substrates and starting materials can be chemically modified followed by enzymatic synthesis. The multi-enzyme system
uses enzymes that can recycle the natural nucleotide diphosphate donors. Subsequent modifications may take place and the process repeated.
One mention should be given to the assembly of a sugar from non-sugar precursors, or the de novo synthesis of glycans
(O’Doherty, Chapter 2.14).
1.01.4 Cellular glycan functions
Most reviews on glycans will begin with a paragraph something like this:
“Carbohydrates are the most abundant and structurally diverse naturally occurring organic compounds. They are presented on the surfaces of all types of
cells from different organisms [1]. The cell surface carbohydrate coating (or glycocalyx) can be readily observed under microscopes [2]. Carbohydrates
on mammalian cells are involved in numerous biological and pathological processes including homeostasis, cell-cell interaction, cell migration,
development, bacterial and viral infection, inflammation, immunology, cancer metastasis, etc. [3,4]. The variety of these properties are the results of the
structural diversity of carbohydrates. Unlike proteins and nucleic acids, carbohydrates are not the products of template-driven biosynthesis but are
directly dependent on the expression and substrate specificity of glycosyltransferases as well as the availability of corresponding sugar nucleotides [5].
Diverse monosaccharide building blocks and various stereo- and regiochemistry in glycosidic linkages contribute to the complexity of the linear and
branched structures of carbohydrates. . ...”
The above paragraph recapitulates most of the details of this article: Complexity, multifunctional, non-template biosynthesis,
difficult to isolate or make, etc. The biological functions of glycans are nearly endless, as new roles are discovered often. This series
and other reviews such as the seminal one by Varki13 and his update in 2017 are testaments to how important every variety of
cellular glycan, from bacteria to plants to man are critical for survival. Conversely, they can cause infection and exacerbate illness,
contribute to non-productive cell adhesion and inflammation and drive tumors to be more aggressive and metastasize. As alluded
to above, the first thing any other cell, tissue or molecule “sees” when encountering another cell is a forest of glycans often referred to
as the glycocalyx, and this dense composition of sugars is often involved in orchestration of binding event, permeability of ions and
can be involved in the progression of disease.125–129 This is a collective function of what may be referred to as the “macro” cell
surface glycosylation environment. Each individual glycan may also contribute to structure and function in myriad ways. Recall the
list of glycans that were described in Section 1.01.1.2. Fig. 11 depicts these structures and the wheel lists at least two functions of
each family of glycans. This is simply to set the stage for the fact that all cellular glycans contribute some role in the tertiary structure,
adhesion or biological function of that particular cell type. The importance of this initial meeting between cells is highlighted by
what could be considered the “ultimate” first: We are all aware that life begins by the recognition of eggs by sperm, but it may not be
widely known that this recognition is through a lectin-carbohydrate interaction!130–132
In lieu of attempting any semblance of complete listing of functions for glycans of various forms and from different species, the
reader is referred to the aforementioned reviews and to Volumes 3 and 5 in this series which are dedicated to the biochemistry of
glycans and the role that glycans play in many disease states, respectively. However, a sampling of concepts will be introduced in
Section 1.01.4.2.
1.01.4.1 Glycan binding proteins

While most of this article has been dedicated to the structure of the sugars that are biosynthesized, another group of molecules is
equally important in the function of cellular glycans and these are their binding partners or Glycan Binding Proteins (GBPs).133,134
Fig. 11 Function “wheel” of the most prominent vertebrate glycan types. Inner wheel lists the glycan type and outer wheel lists two of the many functions that
each of the depicted glycans (outside of the wheel) possess.
These make up a huge array of macromolecules that help choreograph the interactions that glycans have with their intrinsic and
extrinsic environs. Most often referred to as lectins, these proteins contain a non-catalytic domain that can reversibly bind to
different carbohydrates, and their specificities can be for glycans from their own or other species. They mediate interactions of
glycans either within their own cellular space, between cells (adhesion) and they may act as a bridge to higher order structures
mediated by the Glycan-Protein interaction. Lectins usually bind their carbohydrate determinants in a shallow groove on the
protein surface, hence the binding affinities for monosaccharides is consequently quite weak; compensation for this weak
interaction is often accomplished by multivalency, the binding of several glycans and proteins simultaneously (cluster glycoside
[“Velcro”] effect).135–137 Chapter 3.16 by Brewer and Dam provides detailed information on the molecular basis of these inter-
actions. Many of these binding events mediate cellular signaling pathways that are essential for cellular survival and/or cell
death.134,138
Much of our knowledge of lectins stems from the proteins that were originally found in plants.139 As far back as the late 19th
century, it was known that some substances from plants could facilitate agglutination of erythrocytes and hence they were termed
hemagglutinins, or phytohemagglutinins since they were derived from plants.140 Work in the 1940s and 1950s by Boyd, Renkonen,
Morgan and Watkins showed that the agglutination properties of these plant molecules were found to be inhibited by certain sugars,
and it was thought that the function of the plant agents was through the binding of carbohydrate moieties on the red blood cells.
This was the beginning and the basis of what would be the work that differentiated various blood types in to their agglutination
properties from plant lectins. Thus the blood group antigens that we know today as A, B, O and AB are defined by carbohydrate
determinants. There are a host of plant lectins that are used as tools in glycobiology in a variety of different ways. A listing of a small
sample of these and their carbohydrate specificities are shown in Table 2.141 Plant lectins have evolved to mediate many essential
roles in the organism, such as their involvement in defense mechanisms, immunity and growth stimulation.142 Since the
recognition of many carbohydrate determinants of plant lectins is transmissible to many types of glycan presentations, these
proteins have been used for decades as tools in early glycomics and glycobiology research, and Chapter 3.18 by Enam some of the
uses of lectins as analytical tools.
Table 2 List of various plant lectins and their carbohydrate specificities.
Abbreviations Origin Binding specificity
ConA Concanavalia ensiformis a-Linked Man, branched and terminal

LCA Lens culinaris Core fucosylated terminal LacNAc, Man, GlcNAc
PSA Pisum sativum Core fucosylated terminal LacNAc, Man, GlcNAc
GNL Galanthus nivalis Man
AAL Aleuria aurantia Fuc-a-(1,6)-GlcNAc; Fuc-a-(1,3)-Gal-b-(1,4)-GlcNAc
UEA-1 Ulex europaeus Fuc-a-(1–2)-LacNAc
PHA-E Phaseolus vulgaris Biantennary complex N-linked glycans
PHA-L Phaseolus vulgaris Tri- and tetra-antennary complex oligosaccharides
sWGA (succinylated WGA) Triticum vulgaris GlcNAc
Jacalin Artocarpus integrifolia GlcNAc-b-(1,3)-Gal; Gal-b-(1,3)-GalNAc-a-Thr/Ser
WGA Triticum vulgaris GlcNAc; Neu5Ac
GSL-II Griffonia simplicifolia GlcNAc
SBA Soybean Terminal GalNAc
DBA Dolichos biflorus a-GalNAc
VVL Vicia villosa Terminal GalNAc
GSL-I Griffonia simplicifolia a-GalNAc; GalNAc a-Thr/Ser; a-Gal
MAL-II Maackia amurensis Neu5Ac-a(2,3)-Gal(NAc); sulphated glycans
MAL-I Maackia amurensis LacNAc; Neu5Ac-a-(2,3)-Gal(NAc)
SNA-I Sambucus nigra Neu5Ac-a-(2,6)-Gal(NAc)
PNA Arachis hypogaea Gal-b-(1,3)-GalNAc-a-Thr/Ser
RCA120 Ricinus communis LacNAc
ECL Erythrina cristagalli LacNAc
Adapted from Ravida, A.; Cwiklinski, K.; Aldridge, A.M.; Clarke, P.; Thompson, R.; Gerlach, J.Q.; Kilcoyne, M.; Hokke, C.H.; Dalton, J.P.; O’Neill, S.M.,
Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host. Mol. Cell. Proteomics 2016, 15 (10), 3139–3153.
Mammalian lectins are as diverse in their functions and those in plants, and play integral roles in the immune system. There are
several diverse groups of lectins that are also involved in critical cellular signaling events. Although too numerous to discuss in
detail, four families should be mentioned here. These are the Selectins,143 Galectins, Siglecs and C-Type Lectins. Selectins are
proteins expressed on platelets (P-Selectin), leukocytes (L-Selectin) and endothelial cells (E-Selectin). These proteins mediate white
blood cell migration and adhesion to sites of inflammation where their carbohydrate determinants are fucosylated and sialylated
saccharides of the Lewis blood group type, such as sialyl Lewis X.144,145 They also are involved in tumor metastasis by binding
overexpressed blood group ligands on tumor cells. Galectins are a series of 15 or so proteins that bind b-galactoside-containing
glycans on a number of cell types.146 Expression and distribution of galectins is tissue and disease state-dependent. Their expression
and activity has been linked to many processes such as tumor progression147–150 and fibrotic diseases.151–153 Siglecs (sialic
acid-binding immunoglobulin-type lectins) are a family of sialic acid-binding proteins found on the cell surface of immune and
are critical for specific functions.154–156 Their role is to bind to sialic-acid-containing glycoconjugates to mediate roles such as cell
adhesion and signal transduction. They can act either in a cis (bind glycoconjugates on the same cell) or trans (bind glycans from
another cell) fashion. The C-type lectin (CTL) receptors on antigen presenting cells are a series of calcium-dependent (“C-Type,”
as are the selectins) lectins that are involved in endocytosis and facilitate uptake of foreign epitopes from various microbes such
as fungi, viruses and bacteria.157–159 They are known bridges between the innate and adaptive immune system as they recognize
pathogen-associated molecular patterns (PAMPs). Each CTL has a different carbohydrate ligand, and this recognition capability has
been employed in vaccine design to target immunogenic epitopes to APCs.157,160–163 A discussion of selected CTLs in vaccine design
and antigen presentation is described in the Chapter 3.19 by van Kooyk.
1.01.4.2 Glycans in disease

Much of the above discussion has outlined some of the roles that glycans play in disease. Volume 5 of this series is dedicated to this
topic and some of the disease-related functions presented are simply listed here: Congenital disorders of glycosylation (Freeze,
Chapter 5.19), a series of disorders where there is a genetic defect in the expression of an enzyme that is critical for the assembly of
a relevant glycan, such as those in GPI anchor construction (Murakami, Chapter 5.21); glycan’s role in demyelination (Kitazume,
Chapter 5.16); Glycation-related disorders (Takahashi, Chapter 5.07); biomarkers in pancreatic cancer (Miyoshi, Chapter 5.27);
role of glycans in chronic obstructive pulmonary disease (Taniguchi, Chapter 5.20); lung cancer (Honke, Chapter 5.22) and
diabetes (Hart, Chapter 5.08). The number of reports that find novel functions for glycans and glycan-binding-related in many
pathological states is ever rising and has become a major area of clinical-related research.
1.01.5 Summary and perspective
This introduction is meant to initiate the reader into this amazing world and encourage others to join this group of researchers in the
pursuit of future breakthroughs and discoveries. It is also meant to orient the reader to the outstanding array of monographs in this
series that describe the latest developments in this ever-expanding area. Although not discussed in detail above, the area of glycan
materials and polymers, glyconanotechnology and array-based tools development is one of the most recently developed but could
be one of the most provocative areas of glycoscience. This is the subject of many of the fantastic collection of chapters in Volume 4.
Glycoscience is a unique field that has matured over the past few decades as a result of the ubiquitous nature of glycans and ever
expanding roles they play in nearly all facets of biology.
Since the first edition, there have been several worldwide testaments to the field’s relevance. There are several countries that have
initiated glycoscience-related societies and consortia.
Japan has always been a leader in glycoscience and some of their initiatives are: The Japan Consortium for Glycobiology and
Glycotechnology gives awards, plans symposia, publishes their proceedings and charts future progress. The Mizutani Foundation for
Glycoscience has given out grants to up and coming glycoscientists for more than 30 years. Naoyuki Taniguchi (Editor, Volume 5) is
one of Japan’s leading glycoscientists and has spearheaded many of the initiatives and publications there.
Canada has also led the way in glycoscience beginning at the University of Alberta with the group of Raymond Lemieux. His
research helped evolved the department into the Alberta Ingenuity Centre for Carbohydrate Science. The Canadian Glycomics
Network, or commonly called Glyconet, is a consortium of over 170 researchers across many universities and commercial entities.
Todd Lowary (Chapters 2.08 and 4.09) is actually the director of Glyconet.
In Denmark, the University of Copenhagen Center for Glycomics and the Carlsberg Research Center have been at the forefront of
glycochemistry and glycobiochemistry research.
The USA has a rich history of glycoscience initiatives and the past 20 years has seen this increase exponentially. In 2001, The
National Institute of General Medical Sciences at the National Institutes of Health awarded a “glue grant,” or a collaborative award
to fund what was to become the Consortium for Functional Glycomics (CFG). This was a group of scientist who assembled a series
of core facilities to solve some of the questions in glycoscience and curate data from all of these cores as a public resource. In 2012,
the US National Research Council of the National Academy of Sciences convened an ad hoc committee to: “Conduct an in-depth
analysis of the current state of research in glycoscience and glycomics in the U.S.” This report that was funded by the NIH mapped
out the future of glycoscience and suggested the best course of action and use of resources to advance the glycosciences. Finally, The
NIH Common Fund, a resource of programs that “address emerging scientific opportunities and pressing challenges in biomedical
research that no single NIH Institute or Center (IC) can address on its own, but are of high priority for the NIH as a whole” funded
three initiatives in glycoscience and awarded several cooperative grants to fund research to:
• develop methods and technologies for synthesis of biomedically relevant carbohydrates
• develop accessible tools for probing and analyzing carbohydrates and their interaction partners
• develop data integration and analysis tools
These programs have seen great success and will continue in the future.
Several centers and departments around the country are dedicated to advancing glycoscience such as The Comprehensive
Carbohydrate Research Center, a center of excellence in glycoscience at the University of Georgia, Glycoworld at the University of
Missouri (Demchenko, Chapter 2.20) and the Center for Functional Glycomics at Harvard University (Cummings, Chapter 1.17).
All these endeavors and resources have both advanced the field and have brought non-specialists in glycoscience “into the fold”.
This is critical to the future success of glycoscience since many scientists may work in an area where a glyco-centric problem may
arise and dispensed with due to lack of expertise. Thus, we need to train the next generation of glycoscientists to continue a tradition
started by many of the authors in these volumes. We need to continue to develop tools that will make interogatation of the glycome
easier; synthetic methods to make all the known glycans available to all that need them and compile the data in repositories and
databases that are easily searchable, well curated and updated continuously. This field can be considered the “DNA/RNA/Protein of
the Future”. It is time to indoctrinate both scientists and laymen to see the wonders of glycoscience and the benefits it can have for
mankind in the future.
Appendix
Fig. A.1 Latest version of the SNFG listing. Specific shapes are indicative of the type of sugar depicted (Hex, HexNAc, HexA, deoxyHex, etc.). From the
Carbohydrate Structure Database (CSDB)164 website http://csdb.glycoscience.ru/plant_fungal/index.html?help¼eog.
Websites for glycoscience related data and information are listed in the Relevant Websites section (see also chapters in
Volume 1).
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Relevant Websites
http://www.glycopedia.eu/resources/online-databases-tools/article/databases—Listing of many databases.
Databases and Bioinformatic Tools for Glycobiology and Glycoproteomics:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556027/—Databases and Bioinformatic Tools for Glycobiology and Glycoproteomics.
https://glycosmos.org/ —Japanese Society for Carbohydrate Research.
http://www.glycome-db.org/ —Japanese Ministry of Education, Culture, Sports, Science & Technology developed by researchers at the Soka University and the Complex Carbohydrate
Research Center. University of Georgia.
http://www.glycosciences.de/database/ —Originally the German Cancer Research Center (DKFZ), now the University of Gießen maintained in the group of Thomas Lütteke (author of
a chapter in Volume 1).
1.02 Bacterial Exopolysaccharides☆
Yuriy A Knirela and Marie-Rose Van Calsterenb, aN. D. Zelinsky Institute of Organic Chemistry, Moscow, Russia; bSaint-Hyacinthe
Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, QC, Canada
© 2021 Elsevier B.V. All rights reserved.
1.02.1 Introduction 22
1.02.2 Composition, structure, and biosynthesis of exopolysaccharides: General aspects 23
1.02.3 Exopolysaccharides in biofilms 26
1.02.4 Exopolysaccharides of gram-negative bacteria 27
1.02.4.1 Gammaproteobacteria—Enterobacteriales 27
1.02.4.1.1 Enterobacteriaceae: Escherichia coli 27
1.02.4.1.2 Enterobacteriaceae: Klebsiella and Raoultella 28
1.02.4.1.3 Enterobacteriaceae: Other genera 36
1.02.4.1.4 Other Enterobacteriales 36
1.02.4.2 Other Gammaproteobacteria 39
1.02.4.2.1 Vibrionaceae 39
1.02.4.2.2 Pasteurellaceae 42
1.02.4.2.3 Pseudomonadaceae 43
1.02.4.2.4 Moraxellaceae: Acinetobacter baumannii 43
1.02.4.2.5 Other Moraxellaceae 45
1.02.4.2.6 Other families of Gammaproteobacteria 51
1.02.4.3 Alphaproteobacteria 51
1.02.4.3.1 Rhizobacteria and agrobacteria 51
1.02.4.3.2 Sphingomonadaceae 52
1.02.4.3.3 Acetobacteraceae 56
1.02.4.3.4 Other Alphaproteobacteria 56
1.02.4.4 Betaproteobacteria 56
1.02.4.4.1 Burkholderiaceae 56
1.02.4.4.2 Neisseriaceae 59
1.02.4.4.3 Other Betaproteobacteria 59
1.02.4.5 Epsilonproteobacteria 59
1.02.4.5.1 Campylobacteraceae 59
1.02.4.6 FCB group 61
1.02.4.6.1 Bacteroidaceae 61
1.02.4.6.2 Other families of FCB group 62
1.02.5 Exopolysaccharides of gram-positive bacteria 62
1.02.5.1 Bacilli—Lactobacillales: Pathogenic Streptococcus 62
1.02.5.1.1 Streptococcus pneumoniae 62
1.02.5.1.2 Streptococcus agalactiae 69
1.02.5.1.3 Streptococcus suis 69
1.02.5.1.4 Other pathogenic Streptococcus 71
1.02.5.2 Bacilli—Lactobacillales: Lactic acid bacteria 71
1.02.5.2.1 Food Streptococcaceae 72
1.02.5.2.2 Lactobacillaceae and Leuconostocaceae 72
1.02.5.3 Other bacilli 79
1.02.5.3.1 Other Lactobacillales 79
1.02.5.3.2 Bacillales 79
1.02.5.4 Clostridia—Clostridiales 81
1.02.5.4.1 Lachnospiraceae 81
1.02.5.4.2 Clostridiaceae 81
1.02.5.5 Actinobacteria: Food bacteria 83
1.02.5.5.1 Bifidobacteriales 83
1.02.5.5.2 Propionibacteriales 83
1.02.5.6 Other Actinobacteria 84
1.02.5.6.1 Corynebacteriales 84
1.02.5.6.2 Micrococcales 84
☆
Change History: January 2020. Y. Knirel and M.-R. Van Calsteren completely updated this chapter from the version in the previous edition.
This is an update of I.W. Sutherland, Bacterial Exopolysaccharides, Comprehensive Glycoscience, edited by Hans Kamerling, Elsevier, 2007, pp. 521–558.
Comprehensive Glycoscience, 2nd edition https://doi.org/10.1016/B978-0-12-819475-1.00005-5 21

22 Bacterial Exopolysaccharides
1.02.5.6.3 Streptomycetales 87
1.02.6 Conclusions 87
References 87
Glossary
ABC transporter- and synthase-dependent pathways Pathways of biosynthesis of homopolysaccharides and
heteropolysaccharides with a small repeating unit. According to the former pathway, polymerization is accomplished by
sequential glycosyl transfer on an acceptor and followed by export of the polymer by the ATP-binding cassette (ABC)
transporter. Synthase-dependent pathway employs a single glycosyltransferase or a multi-protein complex consisting of
a glycosyltransferase and a co-polymerase.
Biofilm A slimy polymeric matrix composed of extracellular polysaccharides, proteins, lipids, and DNA, in which bacterial
cells stick to each other and often also to biotic or abiotic surfaces.
Biological repeating unit An oligosaccharide that is assembled on a lipid carrier and then polymerized according to the Wzy-
dependent pathway.
Capsule A protective polysaccharide layer outside the bacterial cell associated with the cell envelope.
Chemical repeating unit Any oligosaccharide repeat of a polysaccharide that may coincide with the biological repeating unit
or differ from it by cyclic permutation of the constituent monosaccharides.
Sheath A protective extracellular microtube, in which a chain of bacterial cells is enclosed.
Slime A viscous material excreted from the bacterial cell into the surrounding medium. Slime consists mostly of
exopolysaccharides and may also contain glycoproteins and glycolipids.
Wzy-dependent pathway A pathway of biosynthesis of heteropolysaccharides, which is distinguished by the assembly of
an oligosaccharide on a lipid carrier followed by polymerization.
1.02.1 Introduction
The present chapter is devoted to bacterial exopolysaccharides (EPSs). These biopolymers can be divided into two types: cell-bound
capsular polysaccharides (CPSs) and non-bound polysaccharides or slime, which are excreted into the environment. When there is
not enough data to distinguish between these two types, a more general term “exopolysaccharide” can be used. Polysaccharides
reported as cell wall or teichoic acids were not included, contrary to some presumably extracellular or capsular polysaccharides that
employed similar extraction procedures or were extracted from whole cells.
Bacterial EPSs are important components of biofilms implicated in cell adhesion to biotic and abiotic surfaces. They provide
resistance to desiccation and protect bacteria from the external milieu and host immune defenses, such as complement mediated
killing and phagocytosis, by minimizing complement deposition on the bacterial surface. Some bacteria form a protective sheath,
which represents an extracellular microtube, in which a chain of cells is enclosed.
The EPSs are also involved in interactions of bacteria with plants and bacteriophages and play an important role in the host-
symbiont specificity. Fine CPS structure defines the serological specificity of bacterial strains, and CPSs are widely used for
serotyping of bacteria. Recently, application of CPS-based vaccines, including the 23-valent pneumococcal vaccine, the tetravalent
meningococcal vaccine, and the Hib glycoconjugate vaccine, has dramatically reduced the rate of infectious diseases caused by the
bacterial pathogens.
Many bacterial EPSs possess rheological properties and produce highly viscous solutions or gels. Some of them, such as bacterial
cellulose, hyaluronic acid, dextran, xanthan, emulsan, gellan, and some other EPSs, represent valuable renewable resources having
commercial applications in medicine, agriculture, food industry, soil remediation, concrete, and oil fields. This issue is covered in
a review by I. Sutherland published in the 1st edition of Comprehensive Glycoscience.1
Chemical, physicochemical, and biological studies of EPSs help better understanding the pathogenesis of infectious diseases and
provide information that is necessary for development of vaccines and diagnostic reagents. Understanding their biosynthesis is
required for developing the knack of its regulation to increase production yields of commercially important EPSs.
The present article focuses mainly on EPSs of medical bacteria, which have been recognized as the major surface-associated
virulence factor, as well as plant-associated and food bacteria. EPSs of marine bacteria of the orders Alteromonadales, Cellvibrio-
nales, Oceanospirillales (all Gammaproteobacteria), Rhodobacterales (Alphaproteobacteria), and Rhodocyclales
(Betaproteobacteria) are not included as they were the subject of several recent reviews.2–4 It is not excluded that some other
contributions in the field of bacterial EPSs have been overlooked.
Composition and structure of bacterial polysaccharides, including EPSs, have been surveyed repeatedly,5–9 and an annually
updated Bacterial Carbohydrate Structure Database (BCSDB) is available online at http://csdb.glycoscience.ru/bacterial. Whenever
EPS structures of particular bacteria have been presented in a previous review or summarized in a research article, only these papers
are referenced here to avoid extensive citation of papers published earlier. For revised structures, only the publication reporting the
final structure is cited. Some more published structures may be incorrect and require revision. Structures of some
Bacterial Exopolysaccharides 23
heteropolysaccharides are not displayed here as in the original article but permuted to present a more likely biological repeating
unit, which is assembled on a lipid carrier and then polymerized according to the Wzy-dependent pathway of bacterial polysac-
charide synthesis. When known, various biosynthesis pathways of the EPSs are discussed in the corresponding sections.
Classification of bacteria is subject to change. In this review, the current names for bacterial classes, families, genera, and species
are used according to the NCBI Taxonomy Browser (http://www.ncbi.nlm.nih.gov/Taxonomy/). When an EPS structure was
reported under a different bacterial name, the old name is indicated in parentheses.
1.02.2 Composition, structure, and biosynthesis of exopolysaccharides: General aspects
Monosaccharide composition of bacterial EPSs is highly diverse (Table 1). Most EPS components are monosaccharides that are
widely distributed in nature. These are pentoses, hexoses, hexosamines, their 6-deoxy derivatives, and hexuronic acids. Sugars that
have not been found outside the Bacteria can be exemplified by various stereoisomers of heptoses and 6-deoxyheptoses in
Campylobacter spp. and aldulosonic acids, such as isomers of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in Acinetobacter
baumannii, D-lyxo-hex-2-ulosonic acid and rhodaminic acid (an isomer of neuraminic acid) in Rhodococcus spp., and branched
monosaccharides named yersiniose and erwiniose in Bacteroides fragilis and Pectobacterium atrosepticum, respectively (Fig. 1). In most
Table 1 Monosaccharide components of bacterial exopolysaccharides.
Aldoses and ketoses

D-, L-Arabinose (D-Ara, L-Ara) D-, L-Rhamnose (D-Rha, L-Rha)
D-Xylose (D-Xyl) D-, L-Fucose (D-Fuc, L-Fuc)
D-Ribose (D-Rib) 6-Deoxy-L-altrose (L-6dAlt)
D-Glucose (D-Glc) 6-Deoxy-D-, L-talose (D-6dTal, L-6dTal)
D-, L-Mannose (D-Man, L-Man) 3,6-Dideoxy-L-xylo-hexose (colitose, Col)
D-Galactose (D-Gal) 6-Deoxy-D-manno-heptose (D-6dmanHep)
L-Altrose (L-Alt) 6-Deoxy-D-altro-heptose (D-6daltHep)
D-glycero-D-manno-Heptose (DD-gromanHep) 6-Deoxy-D-talo-heptose (D-6dtalHep)
D-glycero-D-altro-Heptose (DD-groaltHep) 6-Deoxy-D-ido-heptose (D-6didoHep)
D-glycero-L-gluco-Heptose (DL-groglcHep) 6-Deoxy-L-gulo-heptose (L-6dgulHep)
D-Fructose 4-Deoxy-L-threo-hex-4-enose
L-Sorbose D-threo-Pent-2-ulose (D-xylulose, D-Xul)
Amino sugars
D-Glucosamine (D-GlcN) 3-Amino-3-deoxy-D-quinovose (D-Qui3N)
D-Galactosamine (D-GalN) 3-Amino-3-deoxy-D-fucose (D-Fuc3N)
D-Mannosamine (D-ManN) 4-Amino-4-deoxy-D-quinovose (D-Qui4N)
D-, L-Quinovosamine (D-QuiN, L-QuiN) 4-Amino-4-deoxy-D-rhamnose (D-Rha4N)
L-Rhamnosamine (L-RhaN) 2,4-Diamino-2,4-dideoxy-D-quinovose (D-QuiN4N)
D-, L-Fucosamine (D-FucN, L-FucN) 2,4-Diamino-2,4-dideoxy-D-fucose (D-FucN4N)
2-Amino-2,6-dideoxy-L-talose (L-6dTalN) 2-Amino-2,6-dideoxy-D-xylo-hexos-4-ulosea
Alduronic acids
D-Riburonic acid (D-RibA) D-Glucosaminuronic acid (D-GlcNA)
D-, L-Glucuronic acid (D-GlcA, L-GlcA) D-Mannosaminuronic acid (D-ManNA)
D-Mannuronic acid (D-ManA) D-, L-Galactosaminuronic acid (D-GalNA, L-GalNA)
D-Galacturonic acid (D-GalA) L-Gulosaminuronic acid (L-GulNA)
L-Guluronic acid (L-GulA) 2,3-Diamino-2,3-dideoxy-D-, L-glucuronic acid (D-GlcN3NA, L-GlcN3NA)
L-Altruronic acid (L-AltA) 2,3-Diamino-2,3-dideoxy-D-mannuronic acid (D-ManN3NA)
2-Deoxy-D-arabino-hexuronic acid
Aldulosonic acids
D-lyxo-Hex-2-ulosonic acid
3-Deoxy-D-threo-hex-2-ulosonic acid
3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo)
3-Deoxy-D-glycero-D-manno-non-2-ulosonic acid (Kdn)
5-Amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid (neuraminic acid, Neu)b
5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (pseudaminic acid, Pse)
5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid, Leg)
5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid (8-epilegionaminic acid, 8eLeg)
5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid (acinetaminic acid, Aci)
5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-L-altro-non-2-ulosonic acid (8-epiacinetaminic acid, 8eAci)
Branched sugars
3,6-Dideoxy-4-C-[(S)-1-hydroxyethyl]-D-xylo-hexose (yersiniose)
3,6,8-Trideoxy-4-C-[(R)-1-hydroxyethyl]-D-gulo-octose (erwiniose)
Two isomers of this class, one having the arabino configuration, the other with unknown configuration, have also been reported.
a
Another isomer of this class (rhodaminic acid), having the lyxo configuration of the C-4−C-6 fragment, has been found.
b
CH2OH
HO
OH OH OH
O HOH2C CO2H
HO O
HO CO2H HO
HO
OH
1 2
OH OH OH OH
HOH2C CO2H H3C CO2H

HO O H2N O
H2N
HO HO
NH2
3 4
OH OH OH OH
H3C CO2H H3C CO2H

H2N O H2N O
H2N H2N
HO HO
5 6
OH OH OH OH
H3C CO2H H3C CO2H

H2N O H2N O
H2N H2N
HO HO
7 8
OH OH
H3C H3C H3C H3C
O O
OH OH OH
OH OH OH OH
9 10
Fig. 1 Higher sugar components of bacterial exopolysaccharides. (1) Ketodeoxyoctonic acid (Kdo); (2) ketodeoxynononic acid (Kdn); (3) neuraminic acid (Neu); (4)
pseudaminic acid (Pse); (5) legionaminic acid (Leg); (6) 8-epilegionaminic acid (8eLeg); (7) acinetaminic acid (Aci); (8) 8-epiacinetaminic acid (8eAci); (9) yersiniose;
(10) erwiniose. For the systematic names of the monosaccharides see Table 1.
cases, the absolute configuration of monosaccharides has been unambiguously determined, but in a few cases it remains unknown
or (for L-glucuronic and 2,3-diamino-2,3-dideoxy-L-glucuronic acids), in our opinion, requires revision.
Most monosaccharides exist exclusively in the pyranose form, but ketoses, such as D-threo-pent-2-ulose (xylulose), L-sorbose, and
D-fructose, as well as 6-deoxy-D-altro-heptose are present only as furanosides, for xylulose this being the only possible form. Some
other monosaccharides, including L-arabinose, D-ribose, D-galactose, and D-fucose, occur in both forms.
N-Acetyl and O-acetyl groups are widespread in EPSs, whereas other N-acyl (e.g., formyl, acetimidoyl, 3-hydroxybutanoyl) and
O-acyl (L-glyceroyl, succinoyl) substituents are much less common (Table 2). In some EPSs, hexuronic acids exist as a primary amide
Table 2 Non-sugar components of bacterial exopolysaccharides.
Component Mode of linkage Component Mode of linkage
Methanol (Me) O-Alkyl D-Alanine (D-Ala) Hexuronamide

Lactic acid (Lac) [(R), (S)] O-Alkyl L-Threonine (L-Thr) Hexuronamide
3,9-Dideoxy-D-threo-D-altro-nononic acid, O-Alkyl L-Glutamic acid (L-Glu) Hexuronamide
2-linked
Acetic acid (Ac) O-Acyl, N-acyl Taurine (Tau) Hexuronamide
Succinic acid (Suc) O-Acyl Pyruvic acid (Pyr) Acetal [(R), (S)]
L-Glyceric acid (L-GroA) O-Acyl 3-Hydroxypropanoic acid Hexuronic ester
Formic acid (Fo) N-Acyl 2-Aminoethanol (EtN) Hexuronamide, phosphoric ester
Acetimidic acid (Am) N-Acyl Glycerol (Gro) Phosphoric ester
3-Hydroxybutanoic acid (3Hb) N-Acyl Ribitol (Rib-ol) Phosphoric ester
[(R), (S)]
3-Hydroxy-3-methylbutanoic acid N-Acyl D-Arabinitol (D-Ara-ol) Phosphoric ester
L-Cysteine (L-Cys) N-Acyl Glucitol (Glc-ol) Phosphoric ester
L-Aspartic acid, 4-linked (L-Asp) N-Acyl Mannitol (Man-ol) Phosphoric ester
Glycine (Gly) N-Acyl, hexuronamide Choline (Cho) Phosphoric ester
L-Alanine (L-Ala) N-Acyl, hexuronamide O-Methyl phosphoramidic acid (MeOPN) Ester
D-, L-Serine (D-Ser, L-Ser) N-Acyl, hexuronamide 2-(2-Hydroxyethylamino)ethylphosphonic Ester
acid
Ammonia (NH2) Hexuronamide Phosphoric acid (P) Ester, diester
2-Amino-1,3-propanediol (GroN) Hexuronamide Sulfuric acid Ester
Where known, the absolute configuration of lactic acid, 3-hydroxybutanoic acid, and pyruvic acid acetal is indicated in polysaccharide structures.
or an amide with an amino compound, like 2-aminoethanol, 2-amino-1,3-propanediol, or an amino acid. A number of EPSs are
acidic due to the presence of ether-linked lactic acid or acetalically linked pyruvic acid. Some other EPSs contain a phosphate group
that attaches 2-aminoethanol, choline, or various alditols or interlinks two monosaccharides. Rarely occurring are a cyclic sugar
phosphate and derivatives of a phosphonate and a phosphoramidate.
EPSs are typically made of oligosaccharide repeats (K units), which may be linear or branched with one or several mono- or
oligosaccharide side chains and consist of two to ten monosaccharide residues. Some bacteria produce extracellular homopoly-
saccharides, mainly glucans and fructans, other homoglycans being less common. More than one structurally related or sometimes
unrelated EPSs may occur in one strain. The length of the EPS chain varies considerably from several oligosaccharide repeats to
hundreds and even more of them.
Biosynthesis of EPSs of gram-negative bacteria is accomplished by one of three known pathways: the Wzy-dependent pathway,
the ATP-binding cassette (ABC) transporter-dependent pathway, and the synthase-dependent pathway.10,11 Gram-positive bacteria
use the Wzy- and synthase-dependent pathways, but not the ABC transporter-dependent pathway, for EPS biosynthesis.
Heteropolysaccharides usually are synthesized by the so-called Wzy-dependent pathway, which is initiated by the transfer of
a sugar 1-phosphate from a nucleotide (UDP) derivative to undecaprenyl phosphate on the cytoplasmic side of the inner
membrane. Then an oligosaccharide that corresponds to the biological repeating unit of the EPS is assembled by sequential transfer
of other constituent monosaccharides by glycosyltransferases, which are specific to sugars and linkages and thus dictate the
composition and structure of the repeating unit. Further pathways may diverge: after translocation across the inner membrane
with the help of flippase Wzx, the repeating unit can either be polymerized to a polysaccharide or ligated to lipid A or a protein to
give a short-chain lipopolysaccharide (LPS) or a glycoprotein, respectively. Polymerization is accomplished by another transmem-
brane protein, polymerase Wzy, with participation of modal chain length regulator (copolymerase) Wzz.
In heteroglycans that are synthesized by the Wzy-dependent pathway, the first monosaccharide of the biological repeating unit
possesses the D-gluco or D-galacto configuration and may be D-Glc, D-Gal, or an N-acyl derivative of a 2-amino-2-deoxy-D-hexose
(D-GlcN, D-GalN) or a 2-amino-2,6-dideoxy-D-hexose (D-QuiN, D-FucN, D-QuiN4N, D-FucN4N). Data on bacterial EPS often are
limited to the structure of the so-called chemical repeating unit, which may either agree with the structure of the biological repeating
unit or be any cyclic permutation thereof. One can assume that in heteropolysaccharides whose biosynthesis pathway has not been
elucidated, a D-gluco- or a D-galacto-configured monosaccharide also is the first in the repeating unit.
In the Wzy-dependent pathway, the CPS biosynthesis is regulated by a phosphoregulatory system, whose main components
consist of bacterial tyrosine kinases and their cognate phosphatases.12 The ability to regulate capsule biosynthesis has been shown
to be vital for pathogenicity, making the phosphoregulatory proteins suitable as drug targets.
Homopolysaccharides and some heteropolysaccharides that have relatively small repeating units (three monosaccharide
residues in the main chain at most) often are synthesized by the ABC transporter-dependent pathway. In this case, polymerization
is accomplished by sequential glycosyl transfer on an acceptor composed of an oligosaccharide primer linked to a lipid carrier that is
embedded into the inner membrane. The completed polymer molecule is then exported by the ABC transporter, but it is not clear
whether chain extension must be completed before the translocation across the inner membrane begins.
Synthase-dependent pathway employs an inner membrane-embedded multi-protein complex typically consisting of

a glycosyltransferase and a co-polymerase, which polymerizes the EPS and facilitates its translocation across the membrane.
Some glucans and fructans are synthesized extracellularly by transglucosylation with the help of a single sucrase protein, which
elongates the polymer by direct addition of monosaccharides (glucose or fructose) obtained by cleavage of sucrose.
The repetitive EPS structure may be masked by a non-stoichiometric modification, most commonly glycosylation and/or
O-acetylation. It is usually introduced after polymerization or accompanies it being added to the growing polysaccharide chain.
A typical postpolymerization modification in alginates is epimerization at C-5 of D-ManA to give L-GulA. The modifications
contribute to structural heterogeneity of EPSs, which may also be due to another feature, such as alternation of N-acetyl and
N-(3-hydroxybutanoyl) groups on an amino sugar.13
In some bacteria (e.g., Escherichia coli14), CPSs possess a terminal reducing-end domain with a structure different from that of the
repeating unit. Such domain that evidently serves to link the EPS to a membrane-embedded lipid anchor may be much more
common than anticipated.
1.02.3 Exopolysaccharides in biofilms
Biofilm is a slimy extracellular matrix composed of EPSs, proteins, lipids, and DNA, in which bacterial cells stick to each other and
often also to a biotic or abiotic surface. It plays an important role in the persistence of chronic infections by increasing tolerance to
antibiotics and endowing resistance to phagocytosis and other components of the immune system. Biofilms often contain EPSs
common for groups of bacteria and having relatively simple structures, such as dextran, levan, alginate, poly-b-(1!6)-N-acetyl-D-
glucosamine (PNAG), cellulose, and the Pel polysaccharide.
Dextran, that mainly consists of a-(1!6)-linked D-glucose, and levan, a predominantly b-(2!6)-linked D-fructan, contribute to
biofilm production by a wide range of bacteria, including human and plant pathogens. Both polymers may be branched to different
degrees.
Alginate is the major component of the biofilm matrix produced by mucous strains of the opportunistic pathogen Pseudomonas
aeruginosa, which colonize the lungs and cause chronic infections in cystic fibrosis patients. In a soil bacterium Azotobacter vinelandii,
this polymer is an integral part of the layers surrounding the desiccation-resistant cysts formed by the bacteria under unfavorable
environmental conditions. Alginic acid is a linear polymer composed of b-D-ManpA and a-L-GulpA residues randomly distributed
along the polymer chain. It is first synthesized as a homopolymer of the former, and then a part of the monosaccharide residues are
epimerized at C-5 to give the latter. The degree of epimerization varies between bacterial species and strains of the same species.
Some D-ManA residues, but not L-GulA residues, are O-acetylated at position 2 and/or 3.
Cellulose, a linear b-(1!4)-linked homopolymer of D-Glcp, is produced by Acetobacter xylinum and a variety of other Proteo-
bacteria, including members of the families Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae. In biofilm of Pseudomonas
fluorescens SBW25, acetylated cellulose was found with 14% of D-Glc residues carrying a single O-acetyl group at any position.15
Cellulose modified with phosphoethanolamine is present in E. coli.16 Due to the absence of lignin and hemicellulose, bacterial
cellulose is considered as a biocompatible material having potential application in regenerative medicine.
In PNAG, a b-(1!6)-linked polymer of D-GlcpNAc, some amino sugar residues lack the N-acetyl substituent owing to partial
de-N-acetylation, which takes place after synthesis of a fully N-acetylated glycan. PNAG has been found in a number of gram-negative
bacteria, including Escherichia coli, Yersinia pestis, Bordetella spp., Aggregatibacter actinomycetemcomitans, Actinobacillus pleuropneumoniae,
Acinetobacter baumannii, as well as some gram-positive pathogens (Staphylococcus aureus, S. epidermidis). Due to a cationic character,
this polymer favors interactions between polysaccharide strands and the cell wall and/or lectin-like proteins, leading to intercellular
adhesion.17 Conjugates based on a partially de-N-acetylated form of PNAG or synthetic b-(1!6)-linked D-GlcpN oligomers18 have
potential of vaccines capable to elicit protective immunity to the broad range of PNAG-producing pathogens.
The Pel polysaccharide occurs in biofilms formed by P. aeruginosa and Ralstonia solanacearum. It is composed predominantly -
of (1!4)-linked D-GalpNAc as well as D-GlcpNAc residues randomly distributed along the linear polymer chain19 (configurations of
the glycosidic linkages of the monosaccharides remain to be determined). The polymer is positively charged due to de-N-acetylation
of some amino sugar residues, which enables interaction of the Pel polysaccharide with other key biofilm matrix components.
All EPSs mentioned above are produced by the synthase-dependent pathway.11 The synthesis of alginate, cellulose, PNAG, and
Pel is post-translationally regulated by binding the secondary messenger bis-(3-50 )-cyclic dimeric guanosine monophosphate to the
synthase complex consisting of a glycosyltransferase and a co-polymerase. After polymerization and transport across the membrane,
the EPSs are frequently subjected to modifications by additional enzymes present in the periplasm, which are critical for the
formation of biofilms, bacterial virulence, and persistence. Enzymes for regulation of the synthesis, polymerization, translocation,
modifications, and export of these EPSs are encoded in a single operon alg, bcs, pga, and pel, respectively.
Dextran and levan are synthesized outside the cell by dextransucrase and levansucrase classified as glycoside hydrolases, which
catalyze the transfer of glucose or fructose, respectively, from sucrose onto a growing oligo- or polysaccharide chain.10 The enzymes
are secreted and anchored to the cell wall, but the exact enzymatic mechanisms of chain initiation and elongation of the sucrase-
dependent polymers remain to be determined.
In addition to the C-5-epimerization by AlgG and O-acetylation with the help of four proteins AlgIJFX, alginate is degraded by
alginate lyase AlgL, which is an exo-enzyme, cleaving sugars at the polysaccharide end by the mechanism of b-elimination. Cellulose
is degraded by a cellulose hydrolase BcsZ, which is an endo-acting enzyme. Multi-domain proteins PgaB and PelA involved in the
biosynthesis of PNAG and Pel, respectively, exhibit both hydrolase and de-N-acetylase activities. The hydrolases and alginate lyase
can be involved in regulation of polymer length and export, but their exact biological role remains unknown. Due to the ability to
disrupt the preformed biofilms and to prevent biofilm formation, these enzymes are considered as potential therapeutics for the
treatment of chronic biofilm-related infections.
The current understanding of the molecular mechanisms involved in the synthesis of the EPSs described in this section, including
the similarities and differences of the corresponding synthase systems, is described in recent reviews.10,11
1.02.4 Exopolysaccharides of gram-negative bacteria

1.02.4.1 Gammaproteobacteria—Enterobacteriales
The order Enterobacteriales includes genera of the family Enterobacteriaceae and those classified formerly into this family but
recently moved to other families (Morganellaceae, Yersiniaceae, Erwiniaceae, Pectobacteriaceae).
1.02.4.1.1 Enterobacteriaceae: Escherichia coli

Escherichia coli is considered a commensal microorganism and is widely used in microbiology and biochemistry research. However,
some strains may cause diseases in humans and animals, including gastrointestinal and urinary tract infections, septicemia, and
meningitis. Enterohemorrhagic strains, first of all E. coli O157, are responsible for foodborne illness, which sometimes causes
a severe complication called hemolytic-uremic syndrome.
Extraintestinal isolates of E. coli express acidic CPSs or K antigens (from Kapselantigene) with type-specific structure, which serve
as an anti-phagocytic barrier endowing serum resistance to the bacteria. There are about 80 serologically distinguishable E. coli
K types, which are divided into four groups based on genetic and biochemical data.20,21 The CPS structure and biosynthesis in E. coli
are being studied comprehensively (for reviews see refs. 20–22), and data on the structure of 72 K types from all four groups are
deposited in the EK3D database available through www.iith.ac.in/EK3D/.23
Briefly, group 2 and 3 CPSs are synthesized by the ABC transporter-dependent pathway on an acceptor composed of a b-Kdo
oligosaccharide primer linked to a phosphatidylglycerol group, which is believed to anchor the capsule to the cell surface.13,20
Group 2 capsules are distinguished by the temperature-regulated expression. CPSs of both groups have relatively small repeats
(K units) of one to three monosaccharide residues in the main chain. Their typical acidic components are Kdo, D-GlcA, and
a phosphate group that links a sugar to another sugar, ribitol, or glycerol. Less common are other acidic monosaccharides (Neu5Ac
and D-ManNAcA in group 2 or a 4-deoxyhex-2-ulosonic acid of unknown configuration in group 3) as well as non-carbohydrate
components (L-threonine carboxyl-linked to D-GlcA and malonic acid N-linked to D-Qui4N in group 3). In nine K types of group 2,
a !Ribf!Kdo! backbone is present, and Ribf and Kdop or Kdof occur separately in eight more K types. A !Ribf!Rib-
ol!P! backbone is found in three K types. L-Rha is common for most CPSs of group 3, and a side-chain D-Fruf residue is
found in several polysaccharides of both groups.
Group 1 and 4 CPSs are synthesized by the Wzy-dependent pathway. Group 1 CPSs synthesis is initiated by the transfer of
D-Galp-1-P or D-Glcp-1-P from the corresponding UDP derivative to undecaprenyl phosphate catalyzed by WbaP or WcaJ,
respectively. Group 4 CPSs are also called O-antigen capsules due to their similarity to O-antigens of LPSs. Synthesis of these
CPSs begins with the transfer of D-GlcpNAc-1-P to the carrier with the help of WecA and may be followed by enzymatic
4-epimerization of phospholipid-linked D-GlcpNAc to D-GalpNAc.
CPSs of E. coli are expressed in a proper capsule form or as a so-called KLPS. Group 1 strains lack functional Wzy polymerase, and
the polymer elongation is terminated by the transfer of the nascent short oligosaccharide chain of one or a few K units to the LPS
core–lipid A moiety, which anchors the KLPS molecule into the cell membrane. In group 4, KLPS resembles the full LPS with a long
polysaccharide chain. As opposite to group 1 strains, group 4 polysaccharide elongation is driven by Wzy under control of modal
chain length regulator Wzz.
Group 1 and 4 CPSs are linear or branched with K units varying from tri- to hexasaccharide. Each K unit contains one to three
acidic components, most typical of which are D-GlcA, D-GalA, and pyruvic acid linked as an acetal at various positions to
a constituent monosaccharide. In CPSs of group 4, ManNAc3NAcA (tentatively L-configured), Neu5Ac, glycerol phosphate, and
carboxyl-linked amino acids (L-Ser and L-Thr) occur as well. Each group 1 CPS includes D-Glc and/or D-Gal, and group 4 CPS
contains D-GlcNAc and/or D-GalNAc, and in accordance with the biosynthesis pathway (see above), one of these monosaccharides is
first in the K unit. Typical other neutral sugars are D-Man, L-Rha (in both groups), and L-Fuc (in group 1). Less common amino
sugars, including D-ManNAc, L-FucNAc, and D-Fuc3NAc, occur in group 4 CPSs.
O-Acetyl groups are rather common in E. coli CPSs of all four groups, and the O-acetylation pattern may be solely responsible for
classification of strains to different K types. Several CPSs are structurally related having the same composition, and some from them
(e.g., K27, K28, and K33 in group 1) share monosaccharide sequence but differ in the presence or absence of O-acetyl or pyruvate
groups and/or the position of substitution of monosaccharides.
Strains of all groups of E. coli, except for group 1, co-express colanic acid or M antigen, another protective extracellular
polysaccharide, which is also synthesized by other members of Enterobacteriaceae at low temperatures.20 This polymer is suggested
to be important for survival of bacteria outside the host and is involved in biofilm development and withstanding desiccation.20 As
opposite to the type-specific CPSs, colanic acid is loosely bound to the bacterial surface, and its substantial amount is secreted
as a slime into the growth medium.
Colanic acid is a polyanionic polysaccharide having a conserved structure. It contains D-Glc, D-Gal, L-Fuc, and two acidic
components, D-GlcpA and pyruvic acid acetal, as well as O-acetyl groups. In a highly mucoid strain E. coli K-12,
an oligosaccharide that corresponds to the colanic acid repeat is linked to the LPS core to give so-called MLPS.24 The data of this
oligosaccharide indicate that the reported colanic acid structure requires revision as follows:
→4)-α--Fucp-(1→4)-α--Fucp2|3Ac-(1→3)-β--Glcp-(1→
(R)-Pyr-(2=4,6)-α--Galp-(1→4)-β--GlcpA-(1→3)-α--Galp2Ac-(1→3)┘
In E. coli, the chromosomal gene cluster responsible for production of colanic acid is located downstream of the O-antigen gene
cluster. Being structurally similar to E. coli group 1 capsules, colanic acid is synthesized by an essentially identical process, including
translocation of a preassembled lipid-linked oligosaccharide unit across the inner membrane with the help of flippase Wzx followed
by its polymerization catalyzed by Wzy.25 Protein components Wza, Wzb, and Wzc involved with the export of the polysaccharides
are similar as well, but the expression of colanic acid and group 1 CPSs is regulated differently.20
1.02.4.1.2 Enterobacteriaceae: Klebsiella and Raoultella

Bacteria of the genus Klebsiella belong to ESKAPE, a group of six bacterial pathogens that are able to escape the effects of commonly
used antibiotics. They can cause a wide range of diseases, including such nosocomial infections as septicemia, pneumonia, and
urinary tract infections, as well as community-acquired infections complicated with meningitis and endophthalmitis. The majority
of human infections are associated with Klebsiella pneumoniae, followed by Klebsiella oxytoca.
A polysaccharide capsule plays an important role in the virulence of Klebsiella. At present, a total of 79 capsular (K) types have
been identified by serotyping and genotyping in different Klebsiella species, some of which having been reclassified into Raoultella
species.26,27 From them, K1 and K2 are the most virulent types compared to other K types, being associated with a higher
abundance of virulence genes. Thus, K1 is a predominant cause of pyogenic liver abscess, and K2 most commonly found among
Klebsiella isolates from infected individuals. Some carbohydrate polymer components of protective biofilm matrices produced
by Klebsiella are structurally identical to CPSs.28 Bacteriophages with specific capsule depolymerase activity are a valuable tool
in Klebsiella CPS structure analysis and also have potential for development of agents on their basis for diagnosis and treatment
of infections.29,30
CPS structures have been established for all Klebsiella K types, except for K29, K42, K65, KN1, and KN2. The structures known by
that time were summarized by L. Kenne and B. Lindberg in 1983.5 Later, some from them have been confirmed whereas some others
specified in respect to position of O-acetylation or revised. CPS structures of type strains and other Klebsiella strains studied in this
respect31–88 are presented in Table 3. Based on genetic data,27 structure of the biological repeating unit could be predicted for most
K types as shown in Table 3.
Oligosaccharide K units of the Klebsiella CPSs are linear or branched with one to three side chains. The size of the K unit varies
from tri- to heptasaccharide, the main chain from di- to hexasaccharide, and the side chains from mono- to trisaccharide. All CPSs
are acidic, and some contain two acidic components in each K unit. Most common are D-GlcA and pyruvic acid acetal. Less common
is D-GalA, and a few CPSs contain rarely occurring components, such as 3-deoxy-D-glycero-D-galacto-non-2-ulosonic (deaminoneur-
aminic) acid (K4), an unsaturated hexuronic acid (K38), an amide of D-glucuronic acid with L-glutamic acid (K82), and ethers of
(R)-lactic acid with D-glucose (K66) or (S)-lactic acid with D-glucuronic acid (K22 and K37). As for many other bacterial poly-
saccharides, neutral sugars, including D-Glc, D-Gal, D-Man, L-Rha, and L-Fuc, are common for Klebsiella CPSs. In contrast, amino
sugars are not present in any CPS. O-Acetylation of various monosaccharides occurs in CPSs of many K types and often is non-
stoichiometric. In some instances, the exact location of an O-acetyl group and the absolute configuration of a pyruvic acid acetal
remain unknown.
Klebsiella CPSs of different K types and non-reference strains may have the same composition and related structures, including
the same or similar main chains and/or side chains. Most close structurally are CPSs that have an identical carbohydrate backbone
and differ in the O-acetylation pattern only. Noteworthy is another mode of structure variation, namely different positions of
pyruvic acid acetal on the same CPS backbone (K30 and K33 versus K69). Structure similarity between other types (e.g., K1 and K58,
K2 and K13, K12 and K41, K57 and K68, K74 and K80) is also observed (Table 3) and has been discussed recently in view of their
serology and genetics.27
CPSs of Klebsiella spp. are similar in structure to those expressed by E. coli group 1 strains, but they do not occur in the form of
KLPS having a core–lipid A anchor. Moreover, some CPSs of these bacteria, namely Klebsiella K20 and E. coli K30, Klebsiella K63 and
E. coli K42, are pairwise identical.
Klebsliella spp. and E. coli group 1 strains possess highly similar capsule biosynthesis locus (cps) sequences, presumably as a result
of horizontal gene transfer between the bacteria mediated by IS elements, which occur close to the cps clusters.20 General features of
the Klebsliella spp. cps loci have been discussed (ref. 27 and refs. cited therein). Briefly, the type-specific region of the locus between
the conserved wzc and gnd genes contains genes encoding glycosyltransferases, processing proteins (flippase Wzx and polymerase
Wzy), and modifying enzymes, such as acetyltransferase and pyruvate transferase. The gnd-ugd region located downstream contains
genes for synthesis of nucleotide precursors of D-Man and/or L-Rha. For most K types, good correlation is observed between the CPS
structure and cps gene content. Atypical features have been revealed in strains of several K types, for instance, the absence of wzx-like
gene in K11 and K34 or wzy-like gene in K29 and K50. No structure has been established for K29, but the other three strains produce
typical CPSs, and it could be hypothesized that the missing genes are located outside the cps locus.
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iron filings instead of sand. This bath was heated by means of a
single burner as long as red fumes were observed, and then for
about five hours with a triple burner. Finally, the crucibles were
transferred to a nickel crucible in the bottom of which a plate of
unglazed porcelain was placed. The nickel crucible which had
previously been set tightly into a hole cut in an asbestos board was
then heated over the blast lamp for two hours. After this the
porcelain crucible and contents were weighed and then reheated for
half hour periods as before until three successive weighings
remained constant. This usually required from three to four hours of
blasting. In all determinations, the resulting product was tested for
oxides of nitrogen with potassium iodide, starch and hydrochloric
acid, but none was found. All weighings were reduced to the vacuum
standard on the assumption of 8.4 for the Sp. Gr. of brass, 21. for
platinum, 3.31 for the oxalate and 8.15 for cadmium oxide. The
results are:
Cadmium Oxalate. Cadmium Oxide. At. Wt. Cd.

I 1.97674 1.26414 111.73
II 1.94912 1.24682 111.82
III 1.96786 1.25886 111.77
IV 1.87099 1.19675 111.77
V 1.98941 1.27242 111.79
VI 1.37550 .87994 111.85
VII 1.33313 .85308 111.95
VIII 1.94450 1.24452 112.04
IX 2.01846 1.29210 112.09
A glance at these results shows that there is a variation of .36 of

a unit and that the atomic weight in general increases with the
number of determinations. In the first four determinations, there may
have been loss of cadmium by reduction and subsequent
volatilization, but in the later determinations this is not probable. It is
believed that the greater part of the variation was due to imperfect
dehydration of the oxalate. This and other sources of error in this
method will be referred to later. The nickel crucible used gave a
slight sublimate on heating, even after fifteen hours’ blasting. This
condensed on the porcelain crucible as a brownish coating but, as
both the crucible and its tare were blasted for the same length of
time, it did not seem to change the difference of their weights. More
than a dozen nickel crucibles were tried but none was found not to
give a sublimate. The amount was so slight that no attempt was
made to determine its nature.
The Sulphide Method.
This method is based on the conversion of cadmium oxalate into

cadmium sulphide by heating in a current of hydrogen sulphide. The
method has been used by Partridge. This result was 111.61 for the
atomic weight of cadmium.
Preparation of Hydrogen Sulphide
In the present work this gas was always prepared from
potassium hydrosulphide which was made from barium sulphide
(commercial). Barium sulphide was treated with dilute hydrochloric
acid and the resulting hydrogen sulphide washed thoroughly with a
solution of potassium hydrosulphide and then with pure water. It was
then passed into a strong solution of potassium hydroxide until the
later was saturated. When it was required, it was set free from this
solution by adding dilute sulphuric acid and again washing the
resulting gas with a strong solution of potassium hydrosulphide.
Preparation of Nitrogen.
Whenever a current of nitrogen was required, it was prepared by
passing air over a layer of hot copper gauze in a combustion tube. A
short layer of copper oxide was first introduced, then the copper
gauze and finally another layer of copper oxide. The air was dried
with caustic potash before entering the tube and the nitrogen
obtained was also passed through a long tube filled with lumps of
this substance before being used.
Mode of Procedure.
A number of weighing tubes 140 millimetres long and 13
millimetres internal diameter were made especially for this work.
They were always used in pairs, one being kept as a counterpoise. A
porcelain boat of such dimensions as just to slide into the tube was
placed in each one. For a determination, a tube and its boat were
tared with another tube and boat, glass against glass and porcelain
against porcelain until the difference in weight was less than two
tenths of a milligramme. Both boats were heated in a current of
hydrogen sulphide to incipient redness for about one hour. The
current of hydrogen sulphide was then replaced by one of nitrogen,
in which the boats were cooled, but while still warm they were
transferred to their weighing tubes and allowed to cool in a
dessiccator containing caustic potash and weighed. Before weighing,
the stoppers of the weighing tubes were loosened for a moment in
order to equalize the internal and external pressure. This treatment
was usually repeated two or three times and the difference in weight
remained perfectly constant. A portion of cadmium oxalate sufficient
for a determination was placed in the weighed boat and dried at
150°C. The oxalate had been prepared exactly like that used in the
oxalate method which has already been described. The gas
pressure in the laboratory varied very much while this method was
under investigation and great difficulty was experienced in
maintaining a constant temperature although a thermoregulator was
used. Sometimes a specimen of oxalate which was supposed to be
dry would lose several tenths of a milligramme when the
thermometer would only have gone up to 160°C or 165°C for an
hour by accident. Under these conditions the drying was so
uncertain that only four determinations were completed although
many were started. The boat containing the oxalate which had been
dried and weighed was placed on supports of unglazed porcelain in
a combustion tube and a current of dry hydrogen sulphide passed
over it. As soon as the air was expelled, the tube, which was in a
combustion furnace, was slowly heated until all the oxalate seemed
to be decomposed and then raised to dull redness. After this
temperature had been maintained for about an hour, the sulphide
was allowed to cool to a temperature of about 200°C. and the current
of hydrogen sulphide replaced by dry nitrogen, using a three-way
stopcock. When nearly cold the boat was slipped into its weighing-
tube and weighed, the same precautions being used as when
weighing the empty boat.
At this stage the sulphide was always from one to two
milligrammes lighter than at the end of the determination. It was
reheated for periods of one hour until the weight remained constant.
This generally required from three to five hours. All weighings were
reduced to the vacuum standard on the basis of 4.5 for the Sp. Gr. of
cadmium sulphide, 3.31 for the Sp. Gr. of cadmium oxalate, 8.4 for
the Sp. Gr. of brass weights and 21 for the Sp. Gr. of platinum
weights.
The results are as follows:
Cd C2O4 CdS At. Wt. Cd.

I. 2.56319 1.84716 112.25
II. 2.18364 1.57341 112.17
III. 2.11643 1.52462 112.05
IV. 3.13105 2.25582 112.12
The first three determinations were made exactly as above

described, the heating in hydrogen sulphide being done in a
Bohemian glass combustion tube. The hydrogen sulphide was dried
with calcium chloride.
The fourth determination was made under somewhat different
conditions. The boat containing the weighed oxalate was placed in a
combustion tube which passed through an asbestus covered air-
bath. The air was displaced by a current of dry hydrogen sulphide
and the bath slowly heated. When the temperature had risen to
210°C. it was maintained there for three hours, and then raised to
250°C. for three hours. The sulphide then weighed 2.27 grammes,
being 14 milligrammes heavier than when the determination was
finished. It was replaced in the tube and reheated in a current of
hydrogen sulphide at a temperature of 300°C. for four hours. It was
then transferred to a porcelain tube and heated to redness for one
hour. It then weighed 2.25437 grammes, being 1.45 milligrammes
lighter than at the end of the determination. The weight did not
become constant until it had been heated six hours more to redness
in a current of hydrogen sulphide. When this oxalate was slowly
heated in H2S, a small amount of oxalic acid sublimed to the colder
part of the tube, but, in the other cases where the heating was more
rapid, only carbon monoxide, carbon dioxide, and water were
observed.
Discussion of the Method.
When hydrogen sulphide is passed through a red-hot tube,
sulphur is deposited on the colder parts because at this temperature
hydrogen sulphide dissociates and the elements do not recombine
on cooling. In this work, a faint sublimate was noticed before coming
to the zone of sulphur deposit. On exposure to air, it deliquesced in a
few minutes forming small yellow drops which had a saline taste,
and gave tests for potassium and sulphur. The sublimate had a
yellow color and was evidently formed by the action of sulphur on
glass. It seemed to do no harm, but in the fourth determination an
effort was made to avoid it by using a porcelain tube instead of a
glass combustion tube for heating to redness in a current of
hydrogen sulphide.
The fact that sulphide of cadmium was always too light after the
first hour’s heating in hydrogen sulphide proves that it must have
contained some oxide of cadmium even after this heating. Oxide of
cadmium is readily absorbed by the glaze on porcelain, and some
error must have been introduced in this way because it would not be
converted into sulphide after forming a silicate.
The effect of this would be to give a low result for the atomic
weight of cadmium. To get some idea of the magnitude of this error,
the sulphide was poured out of the boats used in the first and second
determinations. They were then warmed with nitric acid for a few
moments, washed in water, and heated over the blast lamp for a few
minutes. The boats used as tares were treated in exactly the same
manner. On weighing, the boats in which the oxalate in
determinations I and II had been decomposed, were found to be
1.12 milligrammes and .82 milligrammes heavier respectively than at
the beginning of the determinations. This would only introduce an
error of .03 of a unit in the atomic weight on account of the small
difference in weight between these amounts of oxide and equivalent
amounts of sulphide. The boats were warmed, as above mentioned,
with nitric acid to remove any adhering sulphide. This might have
decomposed some cadmium silicate at the same time, and the error
due to cadmium oxide thus be found smaller than it really is.
The following experiment was made in the hope of avoiding the
formation of cadmium silicate. The glaze was removed from the
inside of a porcelain boat by hydrofluoric acid followed by a thorough
scouring with sand and water. The boat was then heated in the flame
of a blast lamp for several minutes, tared against another boat which
was not treated with hydrofluoric acid. Both were heated to redness
in a current of hydrogen sulphide for an hour, cooled, weighed, and
then heated in hydrogen sulphide for another hour, and weighed
again. The boat gained 1.7 milligrammes during this second heating,
showing that a boat whose glaze has been removed by hydrofluoric
acid could not be used in this method. Throughout this work, great
care was taken to exclude the oxygen of the air from the cadmium
sulphide, while hot. The current of hydrogen sulphide in which the
cadmium sulphide is heated must not be too slow, otherwise the
sulphur in the dissociated gas will diffuse to the colder parts of the
tube and condense, the residual gas becoming very rich in
hydrogen. The hydrogen will then reduce some of the sulphide to
metal, causing loss by volatilization. One determination was lost in
this way, over two milligrammes of the sulphide being sublimed out,
and it could easily be detected on the side of the tube. It is believed
that the cause of the variations in the four determinations made by
this method, is due to imperfect dehydration of the oxalate. It did not
seem advisable to continue this part of the work any farther;
therefore the chloride method was taken up.
The Chloride Method.
Huntington had determined the ratios of CdBr2 to AgBr and also

CdBr2 to Ag very carefully, obtaining the result 112.24 for the atomic
weight of cadmium. Morse and Jones had obtained 112.07 for this
constant by the oxide method. The object of the work about to be
described was to find the cause of this discrepancy if possible. It was
thought advisable however to make some determinations of the ratio
of CdCl2 to AgCl before beginning the bromide method.
Dumas, in 1859, used cadmium chloride to determine the
atomic weight of the metal. He did not establish its ratio to silver
chloride but to silver by titration. He prepared cadmium chloride by
dissolving the metal in hydrochloric acid and melting the resulting
product in a platinum capsule for five or six hours. He made two
series of three determinations. The chloride used in the first series
was yellow in places and not completely soluble. The result was
112.476. The second series was made with chloride which was
perfectly white and soluble and gave 112.007 for the atomic weight
of cadmium. It is evidently more reliable than the first series and
Dumas himself concluded that the atomic weight is very near 112.01.
Preparation of Cadmium Chloride
Four different specimens of cadmium chloride were used in this
work and from these specimens portions were taken for analysis.
These portions were treated differently in different analyses,
therefore it will be necessary to give a brief descriptions of them and
mention the number of the determinations, in which each one was
used. Chloride of cadmium was prepared in the following manner. A
solution of pure hydrochloric acid was prepared by passing a current
of hydrochloric acid gas into pure water which was contained in a
porcelain crucible until no more was absolved. The water used had
been purified by distilling against a platinum dish and the
hydrochloric acid gas was obtained by heating ordinary concentrated
chemically pure hydrochloric acid in a distilling bulb whose neck had
been closed by fusion in order to avoid the use of a cork or rubber
stopper. Hydrochloric acid thus prepared will leave no residue on
evaporation when air is excluded (Stas, Aronstein’s German
translation, p. 111). A piece of platinum foil freed from iron by heating
in the vapors of ammonium chloride as recommended by Stas
(Aronstein’s translation, p. 112) was introduced and a piece of
cadmium laid on it. Solution begins at once, the hydrogen being
liberated on the platinum foil. During the later part of the process,
heat was applied. After all of the metal had dissolved, the solution
was evaporated, the platinum foil having previously been removed.
The crystals of cadmium chloride which separated were not dried but
allowed to remain slightly moist with hydrochloric acid. If no platinum
foil is used, the solution of the pure metal becomes exceedingly
difficult, unless a very large excess of acid is used. No objection can
be raised to the use of platinum foil for in making fifty grammes of
cadmium chloride it cost less than a tenth of a milligramme and even
this could probably have been avoided by using a somewhat larger
amount of hydrochloric acid. The foil was always kept submerged in
the acid liquid. The moist crystals of cadmium chloride were
transferred to a combustion tube passing through an asbestus
covered air-bath, and dried in a current of hydrochloric acid gas for
several hours at 300°C. The hydrochloric acid gas had been passed
through a long calcium chloride tube to dry it, although calcium
chloride probably does not do this very thoroughly. The hydrochloric
acid gas was then replaced by a current of nitrogen prepared as has
already been described under the sulphide method. After the current
of nitrogen had been passing for about half an hour, the tube was
allowed to cool, and the chloride transferred to another combustion
tube, one end of which had been sealed in the flame of a blast lamp.
The other end was drawn out and attached to a Sprengel mercury
pump. After exhausting, the chloride was sublimed in the vacuum.
This takes place at a moderate temperature and the sublimate has a
beautiful crystalline structure and is perfectly white. The crystalline
mass exposes so much surface that water is taken up very rapidly
when exposed to the air. This action is so rapid that the crystals
cannot be transferred to a weighing-glass without introducing an
appreciable error. The whole sample was accordingly transferred to
a stoppered glass bottle which was kept under a bell jar with sticks of
caustic potash. Three samples were prepared in this manner, the
first being used in determination one, the second in determinations
two to seven inclusive, and the third in determinations eight to
nineteen inclusive. The samples used in determinations twenty and
twenty-one were prepared in the following manner: About three
grammes of cadmium were placed in a combustion tube in which
three bridges (as in the distillation of pure cadmium) had been made.
A section may be represented thus
The metal was placed in cavity A and a stream of chlorine

passed through the tube. The chlorine was prepared from potassium
bichromate and hydrochloric acid and dried by passing it through a
long tube containing calcium chloride. When the air had been
displaced, the cadmium was heated. It fused and began to burn to
the chloride which partly flowed over the bridge into cavity B and
partly distilled over into this cavity. When the reaction had ended, the
current of chlorine was replaced by one of dry nitrogen, and the tube
was allowed to cool and the chloride taken for analysis XX. The
specimen used in analysis twenty-one was prepared in exactly the
same way, only the chlorine used was obtained from manganese
dioxide, sodium chloride and sulphuric acid, and was dried with
phosphorous pentoxide instead of calcium chloride.
The special treatment of the portions taken for analysis was as
follows: Those taken for determinations I, II and from XI to XIX
inclusive were placed in a platinum boat and put into combustion
tube. A current of hydrochloric acid gas obtained by heating the
aqueous acid was passed through the tube. The gas had been dried
by calcium chloride. When the air was displaced, the chloride was
heated somewhat higher than its fusing-point i.e. to incipient
redness, and maintained there for a length of time varying from a few
minutes to more than an hour. The hydrochloric acid was then
displaced by a current of nitrogen, and the chloride allowed to cool.
The boat with the chloride, while still slightly warm, was placed in a
weighing-tube, cooled in a dedicator containing caustic potash and
weighed. The chloride thus prepared is transparent and presents
only a small surface to the air. It takes water up so slowly that no
error is introduced from this source. This was tested in one case by
allowing a boat containing some chloride thus prepared to stand in
the air for a certain length of time and noting the increase in weight.
It was quite slow. In several cases specimens of chloride were tested
for hydrochloric acid using tropaeolin as an indicator. It was always
found neutral. The portions used for determinations III and VI to X
inclusive were prepared in exactly the same manner as the
preceding ones except that the hydrochloric acid gas in which they
were fused was not dried but used just as it came from the aqueous
acid. In some cases the platinum boat in which the chloride was
fused was weighed before and after the fusion. The weight remained
unchanged.
For determinations IV and V, about six grammes of cadmium
chloride were placed in a platinum boat, and more than two-thirds of
it distilled out in a current of hydrochloric acid gas which had not
been dried. Part of the distillate was collected after cooling in
nitrogen and used in determination IV while the residue remaining in
the boat was used for determination V. The method of preparing the
chloride used in determinations XX and XXI has already been
described.
The Filters.
Thinking that a Gooch crucible with a platinum sponge on the
bottom in place of asbestus would be desirable for this work one was
accordingly made and answered the purpose very satisfactorily. All
determinations were made by using such filters. C. E. Munroe
(Chem. News, Vol 58, p. 101) has described the preparation of these
filters. A platinum Gooch crucible was placed on a filter paper and
some ammonium platonic chloride which had been thoroughly
washed introduced by suspending it in alcohol and then pouring this
into it. The precipitate settles to the bottom forming a uniform layer
and the alcohol drains, off through the filter paper. The crucible was
then dried slowly in an air-bath. After this it was transferred to a
porcelain crucible and slowly heated until decomposition was
complete. In this manner a layer of platinum felt is obtained which
acts as a very efficient filter. Another layer of double chloride was
then decomposed as before so that if there were any imperfections
in the first layer they would be covered by the second layer. The
surface was smoothed down by means of a glass rod. To prepare a
good filter the drying and subsequent heating should be very slow.
The heating must not be at too high a temperature, otherwise the felt
becomes very compact and is useless for filtering purposes.
Pressure produces the same effect. The filters were always treated
with strong nitric acid, washed and reheated before being used, but
in no case was chlorine detected in the nitric acid after the washing,
nor any loss in weight of the crucible. An objection to the use of
these crucibles for the purpose named was found in the course of
this work, but it will be discussed later. The crucibles were always set
in a large weighing-glass, and another weighing-glass containing an
equal amount of platinum foil used as a tare, in weighing. This
precaution was perhaps unnecessary, but at least it did no harm.
Analytical Process.
The weighed cadmium chloride was dissolved by placing the
boat containing it in an Erlenmeyer flask containing water. The boat
was then washed, dried and replaced in its weighing-tube. On
weighing again, the loss in weight is equal to the weight of cadmium
chloride taken. All samples gave a perfectly clear solution except
those used for determinations XX and XXI. A drop of nitric acid (1:3)
was added to each solution except in determination XIV where the
cubic centimetres were added, and in XVI where ten cubic
centimeters were added. A solution of silver nitrate was then added
to precipitate the chlorine. This as well as the subsequent washing
was done in a dark-room illuminated by a single gas light whose rays
had to pass through a strong solution of neutral potassium chromate.
The precipitate was contracted by warming on the water-bath. It was
then collected in the prepared Gooch crucibles and washed. Before
filtering, the flask containing the precipitate and mother-liquor was
allowed to cool. Silver chloride is soluble in water to a considerable
extent but is reprecipitated by adding an excess of either silver
nitrate or hydrochloric acid. Stas (Ann. de Chem. et Phys. [4], 25, 22;
[5], 3, 145; [5], 3, 289.) investigated this very thoroughly. Cooke also
did some work on it and used a dilute solution of silver nitrate to
wash the chloride thus preventing solution (Proc. Amer. Acad. 17,
7.). In the above work, therefore, a solution containing 0.10 grammes
of silver nitrate per liter was first used, followed by one only one-
tenth as strong, and finally pure water was used. Only two or three
washings could be made with water as the chloride went into
solution after this owing to the removal of the silver nitrate. The last
silver nitrate solution used is so weak that any error introduced by
not washing it out completely is insignificant. After washing, the silver
chloride was dried at temperatures varying from 150°C. to 300°C. to
constant weight. A glass air-bath was used in order to prevent
products from the burning gas from coming in contact with the
chloride. It was then weighed. The quantity of silver nitrate used in
the determinations was varied very much. The excess over what was
required to precipitate the chloride is given in the table of results in
those cases in which it is known. The quantity of water used in each
determination is also given where it is known. It is given in the
number of cubic centimetres used per gramme of cadmium chloride
and does not include wash water. All weighings are reduced to the
vacuum standard on the basis that Sp. Grs. of CdCl2 = 3.94 and
AgCl = 5.5. The results are:
No. CdCl2 AgCl H2O Excess At. Wt. Melted
per AgNO3 in
Grm.
I 3.09183 4.83856 112.339 Dry
HCl
II 2.26100 3.53854 112.329 „„
III 1.35729 2.12431 112.320 Moist
HCl
IV 2.05582 3.21727 112.339 „„
V 1.89774 2.97041 112.306 „„
VI 3.50367 5.48473 8.90 112.283 „„
VII 2.70292 4.23087 200 1.79 112.301 „„
VIII 4.24276 6.6398 300 8.10 112.387 „„
IX 3.40200 5.32314 300 18.95 112.368 „„
X 4.60659 7.20386 300 25.62 112.472 „„
XI 2.40832 112.434 Dry
HCl
XII 2.19114 3.42724 112.433 „„
XIII 2.84628 4.45477 300 4.45 + 112.319 „„
3cc.
HNO3
XIV 2.56748 4.01651 300 .10 112.399 „„
XV 2.31003 3.61370 300 .10 + 112.406 „„
10cc.
HNO3
XVI 1.25008 1.95652 300 4.66 112.319 „„
XVII 1.96015 3.06541 300 3.22 112.466 „„
XVIII 2.29787 3.59391 300 4.27 112.448 „„
XIX 1.94227 3.03811 300 3.61 112.423 Dry
HCl
XX 1.10976 1.73547 112.471 „„
XXI 1.63080 2.55016 112.476 „„
Average 112.383
Discussion of the Results.
In the first five determinations, the analytical operations were
conducted as nearly as possible alike, but the preparation of the
portions of cadmium chloride taken for analysis was varied very
much as will be seen by referring back to this part of this paper. The
results do not vary more than ±0.015 from their average. This is very
strong evidence of the purity of the chloride used for, if it contained
any impurity, we should have expected to vary the amount in the
different portions. After this, attention was paid especially to the
analytical process, for it was thought that there probably was some
serious error in the method, the result being higher than any that had
previously been obtained, if we exclude Dumas’ first series which he
himself did not accept. The conditions were varied in many ways to
see how much the result could be influenced, but under no
conditions were results as low as Huntington’s average (112.24)
obtained. A number of errors were found in the method during the
work, but they seem to neutralize each other to a great extent. The
more important ones will now be given. Nearly every filtrate including
the corresponding wash water was examined for chlorine after the
silver and cadmium had been precipitated by hydrogen sulphide.
The excess of hydrogen sulphide was expelled by boiling, after the
addition of some nitric acid. In two cases an inverted condenser was
used. On adding silver nitrate a precipitate was always obtained
showing the presence of chlorine. Care was always taken to filter off
sulphur formed by the oxidation of hydrogen sulphide, before adding
the silver nitrate. The precipitate was never very heavy, and was not
estimated quantitatively. It is evident that cadmium nitrate exerts a
solvent action on silver chloride. In some cases a very large excess
of silver nitrate was added but it did not change the results markedly.
Silver nitrate itself dissolved silver chloride to some extent. The
increase in insolubility, if any, on adding an excess of silver nitrate is
probably counterbalanced by the increased error due to occlusion of
nitrates in the silver chloride. Stas (Aronstein’s Trans. p. 156) says it

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Comprehensive Glycoscience: From

Chemistry to Systems Biology 2nd

JOSEPH J. BARCHI JR.

INTRODUCTION TO GLYCOSCIENCE, GLYCAN STRUCTURE AND FUNCTION

Copyright © 2021 Elsevier B.V. All rights reserved

Library of Congress Cataloging-in-Publication Data

British Library Cataloguing-in-Publication Data

For information on all publications visit our

Publisher: Oliver Walter

Ravin Narain, PhD, P.Eng., FRSC is a professor in the Department of Chemical

Naoyuki Taniguchi graduated from the Faculty of Medicine, Hokkaido Univer-

Sébastien Vidal received his PhD in organic chemistry in 2000 (University of

Spencer Williams was born and raised in Albany, Western Australia.

Marya Ahmed Robert S Haltiwanger

Petra Larsen Raja Mazumder

Jayachandran Venkatesan Jeet Kiran Vora

Joseph J. Barchi, Jr.

1.02 Bacterial Exopolysaccharides 21

1.03 Fungal Polysaccharides 96

1.04 Seaweed Polysaccharides: Promising Molecules for Biotechnological Applications 131

1.06 C-Mannosyl Tryptophan: From Chemistry to Cell Biology 163

1.07 O-Fucosylation of Proteins 182

1.08 Structure, Classification and Modification of Polysaccharides 204

1.09 Overview of Cellulose Types and Applications 220

1.10 Mucins: Structure and Function 237

1.11 High-pH Anion-Exchange Chromatography (HPAEC) and Pulsed Amperometric

1.12 Capillary Electrophoresis 290

1.13 Modern Mass Spectrometry Techniques for Oligosaccharide Structure Determination:

1.15 Computational Modeling in Glycoscience 374

1.17 Tools and Methods to Study the Human Glycome 416

1.18 Glycosciences.de: Databases and Tools to Support Research in Glycomics and

1.19 Systems Glycobiology: Immunoglobulin G Glycans as Biomarkers and Functional

1.21 Glycoinformatics Resources Integrated Through the GlySpace Alliance 507

Comprehensive Glycoscience, 2nd edition https://doi.org/10.1016/B978-0-12-819475-1.00108-5 1

1.01.1.1 Brief history of glycoscience

1.01.1.2 What are carbohydrates/glycans and what do they look like?

Scientist (country of origin) Notable Discovery Year

Fig. 6 Sampling of some unusual saccharides found in various species of bacteria.

Fig. 7 Analogs of sialic acid found in various species of bacteria.

1.01.2 Analysis of glycans

1.01.2.2 Capillary electrophoresis (CE)

1.01.2.3 Mass spectrometry

1.01.2.4 NMR spectroscopy and molecular simulations

1.01.3 Glycan synthesis

1.01.4 Cellular glycan functions

1.01.4.1 Glycan binding proteins

Table 2 List of various plant lectins and their carbohydrate specificities.

Abbreviations Origin Binding specificity

ConA Concanavalia ensiformis a-Linked Man, branched and terminal

1.01.4.2 Glycans in disease

1.01.5 Summary and perspective

Comprehensive Glycoscience, 2nd edition https://doi.org/10.1016/B978-0-12-819475-1.00005-5 21

1.02.2 Composition, structure, and biosynthesis of exopolysaccharides: General aspects

Table 1 Monosaccharide components of bacterial exopolysaccharides.

Aldoses and ketoses

HOH2C CO2H H3C CO2H

H3C CO2H H3C CO2H

H3C CO2H H3C CO2H

Table 2 Non-sugar components of bacterial exopolysaccharides.

Component Mode of linkage Component Mode of linkage

Methanol (Me) O-Alkyl D-Alanine (D-Ala) Hexuronamide

Synthase-dependent pathway employs an inner membrane-embedded multi-protein complex typically consisting of

1.02.3 Exopolysaccharides in biofilms