Carbohydrate chemistry volume 38

Specialist Periodical Reports

Edited by A Pilar Rauter

Carbohydrate Chemistry
Chemical and Biological Approaches
Volume 38


Carbohydrate Chemistry
Chemical and Biological Approaches

Volume 38



A Specialist Periodical Report

Carbohydrate Chemistry
Chemical and Biological
Approaches
Volume 38
A Review of the Literature Published between
January 2011 and February 2012
Editors
Amelia Pilar Rauter, Universidade de Lisboa, Portugal
Thisbe K. Lindhorst, Christiana Albertina University of Kiel, Germany
Authors
Tiina Alama¨e, University of Tartu, Estonia
Marta M. Andrade, Faculdade de Cieˆncias da Universidade de Lisboa, Portugal
Ana Arda´, Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain

Juan M. Benito, Instituto de Investigaciones Quı´micas, CSIC - Universidad de
Sevilla, Spain
M. A´lvaro Berbı´s, Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain
Nele Berghmans, Rega Institute for Medical Research, Belgium
Davide Bini, University of Milano-Bicocca, Milan, Italy
Pilar Blasco, Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain
Karin Bodewits, Ludwig-Maximilians-Universita¨t, Munich, Germany
Paz Briones, CSIC, IBC, Seccio´n de Errores Conge´nitos del Metabolismo,
Barcelona, Spain
Vasco Cachatra, University of Lisbon, Portugal
Vale´rie Calabro, Aix-Marseille University, France
Fernando Calais, Hospital Sa˜o Jose´, Lisboa, Portugal
Angeles Canales, Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain
F. Javier Can˜ada, Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain
Alice Capitoli, University of Milano-Bicocca, Milan, Italy
Myle`ne A. Carrascal, Universidade Nova de Lisboa, Portugal
Laura Cipolla, University of Milano-Bicocca, Milan, Italy
Fabio Dall’Olio, Universita` di Bologna, Italy
Anthony De Soyza, Newcastle University, UK
Flaviana Di Lorenzo, Universita` di Napoli Federico II, Italy
Sandrine Donadio-Andre´i, Aix-Marseille University, France
Nassima El Maı¨, Aix-Marseille University, France
Amalia M. Este´vez, CIQUS, University of Santiago de Compostela, Spain
Juan C. Este´vez, CIQUS, University of Santiago de Compostela, Spain
Ramo´n J. Este´vez, CIQUS, University of Santiago de Compostela, Spain
Ma Carmen Ferna´ndez-Alonso, Centro de Investigaciones Biolo´gicas, CSIC,
Madrid, Spain


Jose´ G. Ferna´ndez-Bolan˜os, Universidad de Sevilla, Spain

Vanessa Ferreira, Portuguese Association for CDG and other Rare Metabolic
Diseases, Portugal
Luca Gabrielli, University of Milano-Bicocca, Milan, Italy
Jose´ M. Garcı´a Ferna´ndez, Instituto de Investigaciones Quı´micas,
CSIC - Universidad de Sevilla, Spain
Ana M. Go´mez, IQOG-CSIC, Madrid, Spain
Alejandro Gonza´lez-Benjumea, Universidad de Sevilla, Spain
Chloe´ Iss, Aix-Marseille University, France
Jesu´s Jime´nez-Barbero, Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain
Rosa Lanzetta, Universita` di Napoli Federico II, Italy
Sandra Li, Rega Institute for Medical Research, Belgium
J. Cristo´bal Lo´pez, IQOG-CSIC, Madrid, Spain
´ scar Lo´pez, Universidad de Sevilla, Spain
O
Cristina Lupo, University of Milano-Bicocca, Milan, Italy
Andres Ma¨e, University of Tartu, Estonia
Filipa Marcelo, Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain
Karin Mardo, University of Tartu, Estonia
Sergio Martos, Universidad de Sevilla, Spain
Ine´s Maya, Universidad de Sevilla, Spain
Pene´lope Merino-Montiel, Universidad de Sevilla, Spain
Antonio Molinaro, Universita` di Napoli Federico II, Italy
Francesco Nicotra, University of Milano-Bicocca, Milan, Italy
Ana Oliete, Universidad de Sevilla, Spain
Ghislain Opdenakker, Rega Institute for Medical Research, Belgium
Carmen Ortiz Mellet, Universidad de Sevilla, Spain
Stefan Oscarson, University College Dublin, Ireland
Jose´ M. Otero, CIQUS, University of Santiago de Compostela, Spain
Ame´lia P. Rauter, University of Lisbon, Portugal
Catherine Ronin, Aix-Marseille University, France

Laura Russo, University of Milano-Bicocca, Milan, Italy
Paulo F. Severino, Universidade Nova de Lisboa, Portugal and Universita` di
Bologna, Italy
Alba Silipo, Universita` di Napoli Federico II, Italy
Mariana Silva, Universidade Nova de Lisboa, Portugal
Raquel G. Soengas, University of Aveiro, Portugal
Markus Sperandio, Ludwig-Maximilians-Universita¨t, Munich, Germany
Francesca Taraballi, University of Milano-Bicocca, Milan, Italy
Clara Uriel, IQOG-CSIC, Madrid, Spain
Jo Van Damme, Rega Institute for Medical Research, Belgium
Paula A. Videira, Universidade Nova de Lisboa, Portugal
Maria-Antonia Vilaseca, Guia Metabo´lica, Esplugues de Llobregat, Spain
Triinu Visnapuu, University of Tartu, Estonia
Ulrika Westerlind, Leibniz Institute for Analytical Sciences, Dortmund, Germany
Alina D. Zamfir, National Institute for Research and Development in Electrochemistry and Condensed Matter, and Aurel Vlaicu University of Arad, Romania


If you buy this title on standing order, you will be given FREE access
to the chapters online. Please contact E-mail: with
proof of purchase to arrange access to be set up.
Thank you.

ISBN: 978-1-84973-439-4
ISSN: 0306-0713
DOI: 10.1039/9781849734769
A catalogue record for this book is available from the British Library
& The Royal Society of Chemistry 2012
All rights reserved
Apart from fair dealing for the purposes of research or private study for
non-commercial purposes, or for private study, criticism or review, as

permitted under the Copyright, Designs and Patents Act, 1988 and the
Copyright and Related Rights Regulations 2003, this publication may not be
reproduced, stored or transmitted, in any form or by any means, without the
prior permission in writing of The Royal Society of Chemistry, or in the case of
reproduction in accordance with the terms of the licences issued by the
Copyright Licensing Agency in the UK, or in accordance with the terms of the
licences issued by the appropriate Reproduction Rights Organization outside
the UK. Enquiries concerning reproduction outside the terms stated here should
be sent to The Royal Society of Chemistry at the address printed on this page.
Published by The Royal Society of Chemistry,
Thomas Graham House, Science Park, Milton Road,
Cambridge CB4 0WF, UK
Registered Charity Number 207890
For further information see our web site at www.rsc.org



Preface
DOI: 10.1039/9781849734769-FP005

Carbohydrate research plays a remarkable role in chemistry and biology
owing to the unique molecular features of the saccharides. They are multifunctional and stereochemically enriched molecules offering a superb
structural diversity to serve as molecular scaffolds or key intermediates in
the development of new drugs and novel carbohydrate-based or -functionalized materials for a variety of applications. In addition, carbohydrates
are the molecular basis for a multitude of biological processes that occur in
states of health as well as disease. Understanding the chemistry and biology
of this class of molecules will facilitate new options for the treatment of
medical conditions for which no cure exists to date. Thus in this volume,
glycochemistry and glycobiology topics have been combined to document
the latest findings in carbohydrate research and demonstrate the contributions of organic chemistry, modern analytics, biological and biochemical

expertise to the increasingly important field of glycomics.
Firstly a modified polysaccharide, chlorite-oxidized oxyamylose, is
described as an immunomodulator with therapeutic implications for acute
and chronic inflammation, and also cancer. A chapter that focuses
on lipopolysaccharide in cystic fibrosis (CF)-related pathogens, namely
Pseudomonas aeruginosa and Burkholderia spp. reports on the structural
investigation of these biomolecules, their structural adaptation to the host
tissues as well as the importance of their structural features in the clinical
management of CF.
Development of carbohydrate vaccines against infections and cancer
continues to be a challenge for glycoexperts all over the world. Synthesis of
inner core lipopolysaccharides for vaccines against Gram-negative bacterial
infections and that of MUC1 glycopeptides conjugated to different immunostimulants with promising results regarding the antibodies induced with
these synthetic vaccines against cancer will be highlighted here.
The immune response to invading pathogens during inflammation is
crucial in cell biology. Thus, the role of carbohydrate decorations in
leukocyte recruitment will be reviewed, putting a strong focus on posttranslational modification by sialic acids. New findings emphasizing the
influence of carbohydrate decoration on the regulation of inflammatory
response will be discussed. New interesting therapeutic approaches in the
treatment of acute and chronic inflammatory diseases are being offered.
Recent progress on glycoengineering, an advanced technology based on a
glycosylation strategy to optimize protein drugs, is reported. Impact of
N-glycosylation on therapeutic proteins, namely that of glycans on drug
bioavailability, glycoprotein biopotency, drug immunogenicity, protein
folding and epitope expression as well as production, safety and regulatory
aspects are discussed. Major advances in glycoengineering are reported with
an emphasis on N-glycosylation control to identify the conditions that
promote an optimal glycoform profile and reproducibility of glycomodification. In addition, identification and cloning of glycosyltransferase genes
Carbohydr. Chem., 2012, 38, vii–viii | vii


c

The Royal Society of Chemistry 2012


are described as new tools for manipulation of expression systems in order
to further improve the glycoform profile, in particular engineering of core
fucosylation and sialylation.
Glycosylation has significant implications in health and disease. Hence,
one chapter is dedicated to congenital disorders of glycosylation, which are
a group of disorders of abnormal glycosylation of N-linked oligosaccharides caused by deficiency in 29 different enzymes in the N-linked oligosaccharide synthetic pathway. The relevance and key aspects of the
glycosylation changes associated with bladder cancer are highlighted in
another chapter of this volume.
Interdisciplinarity of the glycosciences is also demonstrated by a contribution on novel approaches for the production of levansucrases, bacterial
extracellular enzymes that act on sucrose producing b-2,6-linked fructans.
Biochemical characterization of this protein encoded in the genome from
Pseudomonas bacteria, and an innovative mass spectrometric study of the
reaction products permits to identify the produced potentially prebiotic
fructooligosaccharides from sucrose or raffinose.
NMR is currently a potent methodology to analyse sugar conformation
and to study binding interactions. New advances from the NMR methodological viewpoint for analysis of saccharide conformation are described in
a chapter, in which examples are given for oligo- and polysaccharides,
glycopeptides, glycomimetics, and also carbohydrate-protein, carbohydrate-carbohydrate, and carbohydrate-nucleic acid interactions.
The contribution of glycochemistry to innovation in glycosciences is
shown in chapters 10–17. Here imino sugar glycosidase inhibitors, carbasugars, multivalent glycoconjugates, including glycodendrimers, glyconanotubes, and glyconanoparticles, and their uses in medicinal chemistry, as
well as artificial saccharide-based and saccharide-functionalized gene
delivery systems are presented.
Highly functionalized exo-glycals used for the generation of molecular
diversity in a chemoselective manner, namely for the preparation of furanose-based libraries with three or more sites for molecular diversity are
discussed. A chapter on siderophores that are based on monosaccharides

that have proven effective for Gram-negative bacteria and mycobacteria,
and the chapter on biomaterials, in particular on the so-called smart
materials, that can modulate and control cell behaviour, complete the
volume.
Volume 38 of Carbohydrate Chemistry – Chemical and Biological
Approaches contains contributions ranging from glycochemistry to glycobiology. They have been authored mostly by scientists that are members of
the European Science Foundation Network Euroglycoforum. This collection demonstrates in a meaningful way how the interdisciplinary approach
of an international glyconetwork can advance the field of carbohydrate
research in Europe and worldwide.
We hope you will enjoy the beauty of the ‘‘sweet’’ chemistry and biology
presented herein!
Ame´lia Pilar Rauter and
Thisbe K. Lindhorst

viii | Carbohydr. Chem., 2012, 38, vii–viii


CONTENTS
Cover
Tetrahydropyran-enclosed
ball-and-stick depiction of a glucose
molecule, and (in the background)
part of an a-glycosyl-(1-4)-D-glucose
oligosaccharide and a glycosidase,
all representative of the topics
covered in Carbohydrate Chemistry –
Chemical and Biological Approaches.
Cover prepared by R. G. dos Santos.

Preface

vii

Ame´lia Pilar Rauter and Thisbe K. Lindhorst

Applications of glycobiology: biological and immunological effects of a
chemically modified amylose-derivative
Ghislain Opdenakker, Sandra Li, Nele Berghmans and Jo Van Damme
1 Introduction
2 Historical breakthroughs and examples
3 Polysaccharides and derivatives
4 An historical finding in virology?
5 COAM does not induce interferon
6 COAM is an immunomodulator
7 Therapeutic implications for acute and chronic
inflammation and cancer
8 Conclusions and future perspectives
Abbreviations
Acknowledgements
References

Lipopolysaccharide structure and biological activity from the cystic
fibrosis pathogens Burkholderia cepacia complex
Anthony De Soyza, Flaviana Di Lorenzo, Alba Silipo, Rosa Lanzetta
and Antonio Molinaro
1 Introduction

1

1

2
5
5
6
7
8
9
10
10
10

13

13

Carbohydr. Chem., 2012, 38, ix–xiv | ix

c

The Royal Society of Chemistry 2012


2
3

Basic Lipopolysaccharide structure
The detailed chemical structure of the P. aeruginosa
LPS
4 Burkholderia cepacia complex and Cystic Fibrosis
5 General structural features of the core region of BCC

LPS
6 O-chain of BCC LPS
7 Conclusion
Acknowledegments
References

Synthesis of bacterial carbohydrate surface structures containing Kdo
and glycero-D-manno-heptose linkages
Stefan Oscarson
1 Introduction
2 Kdo-containing structures and Kdo donors
3 Glycero-D-manno-heptose-containing structures
References

Synthetic glycopeptides in vaccine development and antibody epitope
mapping
Ulrika Westerlind
1 Introduction
2 Protein conjugate vaccines
3 Build-in adjuvant vaccines
4 Dendrimer vaccines
5 Antibody epitope mapping
6 Conclusions
References

Posttranslational sialylation and its impact on leukocyte recruitment
during inflammation
Karin Bodewits and Markus Sperandio
1 Leukocyte recruitment cascade
2 Posttranslational modifications

3 Sialic acids and sialyltransferases (ST)
4 Sialylation-dependent functions of signalling and adhesion
relevant molecules
5 Conclusion and outlook
References
x | Carbohydr. Chem., 2012, 38, ix–xiv

16
20
23
28
30
33
34
34

40

40
40
48
59

61

61
62
65
68
69

72
73

75

75
78
82
88
91
91


Glycoengineering of protein-based therapeutics
Sandrine Donadio-Andre´i, Chloe´ Iss, Nassima El Maı¨,
Vale´rie Calabro and Catherine Ronin
Introduction
1 Impact of N-glycosylation on therapeutic proteins
2 Glycosylation optimization
3 Conclusions
References

Congenital Disorders of Glycosylation (CDG): from glycoproteins
to patient care
Vanessa Ferreira, Paz Briones and Maria-Antonia Vilaseca
1 Introduction
2 Basic principles of protein glycosylation
3 Congenital disorders of glycosylation (CDG)
4 Concluding remarks and future perspectives: CDG as a
challenge for glycobiomedicine in the next decade!

5 Highlights
6 Further information
Abbreviations
Acknowledgements
References

Bladder cancer–glycosylation insights
Paulo F. Severino, Mariana Silva, Myle`ne A. Carrascal,
Fernando Calais, Fabio Dall’Olio and Paula A. Videira
1 Introduction
2 Bladder cancer: epidemiology and current treatment
3 General aspects of glycosylation
4 Final considerations/conclusions
Abbreviations
References

94

94
95
105
113
115

124

124
124
128
144

145
146
146
147
147

156

156
157
158
168
169
169

Levansucrases of Pseudomonas bacteria: novel approaches for protein 176
expression, assay of enzymes, fructooligosaccharides and
heterooligofructans
Tiina Alama¨e, Triinu Visnapuu, Karin Mardo, Andres Ma¨e and
Alina D. Zamfir
1 Levansucrase genes and proteins
176
2 Heterologous expression of levansucrases from Pseudomonas 177
syringae pv. tomato with two different expression systems
Carbohydr. Chem., 2012, 38, ix–xiv | xi


3

Biochemical properties of the levansucrases: substrate

specificity and kinetic parameters
4 Predicted catalytic triad residues and 3D structures of Lsc1,
Lsc2, Lsc3 and LscA proteins
5 Lsc2, Lsc3 and LscA produce not only levan, but also
FOS that can be detected by a novel mass spectrometric
method
6 Isolation and novel screening methods for levansucrase
mutants
7 Concluding remarks and future perspectives
Acknowledegements
References

178

Recent advances on the application of NMR methods to study the
conformation and recognition properties of carbohydrates
Ana Arda´, M. A´lvaro Berbı´s, Pilar Blasco, Angeles Canales,
F. Javier Can˜ada, Ma Carmen Ferna´ndez-Alonso, Filipa Marcelo and
Jesu´s Jime´nez-Barbero
1 Introduction
2 The access to new NMR parameters and methodological
developments
3 Applications. Saccharides in solution
4 Applications. The interaction of saccharides with other
natural and synthetic molecules
Acknowledgments
References

192

Glycosidase inhibitors: versatile tools in glycobiology
O´scar Lo´pez, Pene´lope Merino-Montiel, Sergio Martos and
Alejandro Gonza´lez-Benjumea
1 Introduction
2 Polyhydroxylated pyrrolidines
3 Six-membered ring-based imino sugars
4 Azepanes and azetidines
5 Bicyclic imino sugars
6 Thio, seleno and carbasugars as glycosidase
inhibitors
7 Miscellaneous
8 Pharmacological activities of imino sugars
9 Concluding remarks
Acknowledgements
References
xii | Carbohydr. Chem., 2012, 38, ix–xiv

179
180

186
189
190
190

192
192
194
202
210

210

215

215
216
219
226
231
245
250
251
256
256
256


An overview of key routes for the transformation of sugars into
carbasugars and related compounds

263

Raquel G. Soengas, Jose´ M. Otero, Amalia M. Este´vez,
Ame´lia P. Rauter, Vasco Cachatra, Juan C. Este´vez and
Ramo´n J. Este´vez
1 Introduction
2 Strategies for the synthesis of carbasugars and
pseudo-carbasugars
Acknowledgements
References

263
265

Multivalent glycoconjugates in medicinal chemistry

303

Jose´ G. Ferna´ndez-Bolan˜os, Ine´s Maya and Ana Oliete
1 Introduction
2 Glycoclusters with a flexible core
3 Glycoclusters with a rigid core
4 Multivalent glycopeptides in immunotherapy
5 Glycoconjugates on tubular scaffolds
6 Glycodendrimer chips
7 Glyconanoparticles
8 Conclusions
Acknowledgements
References

303
303
308
313
318
318
320
331
331
331

Glycotransporters for gene delivery

338

Carmen Ortiz Mellet, Jose´ M. Garcı´a Ferna´ndez and Juan M. Benito
1 Introduction
2 Carbohydrate-grafted cationic polymers and lipids
3 De novo designed carbohydrate-based polymers
4 Preorganized glycomaterials for gene delivery
5 Outlook and perspectives
Acknowledgments
References

338
341
346
354
368
368
369

Furanose-based templates in the chemoselective generation of
molecular diversity
Ana M. Go´mez, Clara Uriel and J. Cristo´bal Lo´pez
1 Introduction
2 Design and synthesis of functionalized furanose-based
precursors

296

297

376

376
377

Carbohydr. Chem., 2012, 38, ix–xiv | xiii


3
4

Reactivity of functionalized furanose-based precursor 4a
Reactivity of the alkenyl halide moiety in functionalized
furanose-based precursors 4b,c
5 Three components assembly of functionalized furanose
derivatives 4b,c
6 Generation of furanosidic libraries possessing three – or
more – sites for molecular diversity
7 Conclusions
Abbreviations
Acknowledgements
References

380
387
388
391
392

393
393
393

Synthesis of carbohydrate-based artificial siderophores and their
biological applications
Marta M. Andrade and Ame´lia P. Rauter
1 Introduction
2 Artificial siderophores - background
3 Synthesis of Carbohydrate-based artificial siderophores
4 Study of siderophore activity
5 Conclusions and perspectives
References

398
400
402
407
412
414

Smart biomaterials: the contribution of glycoscience

416

Laura Cipolla, Laura Russo, Francesca Taraballi, Cristina Lupo,
Davide Bini, Luca Gabrielli, Alice Capitoli and Francesco Nicotra
1 Introduction: biomaterials and tissue engineering
2 Glycoscience for biomaterials design
Conclusions

References

416
424
440
440

xiv | Carbohydr. Chem., 2012, 38, ix–xiv

398


Applications of glycobiology: biological and
immunological effects of a chemically
modified amylose-derivative
Ghislain Opdenakker,* Sandra Li, Nele Berghmans
and Jo Van Damme
DOI: 10.1039/9781849734769-00001

Carbohydrate chemistry, oligosaccharide sequencing, synthesis technologies and glyco-engineering have helped to establish glycobiology alongside
molecular biology. However, examples of therapeutic implications of glycobiology are limited to oligosaccharides. These include engineered antibodies containing sialic acids, inhibition of leukocyte rolling effect in the
interactions between mucins and selectins and antiviral imino sugars. The
best example of a successful glycodrug is oseltamivir, Tamiflus, as an
inhibitor of the influenza virus neuraminidase. Polysaccharides, although at
first sight structurally less complex and biologically less challenging, are
interesting molecules with unexplored possibilities in biology and medicine.
Polysaccharides may form protease-resistant scaffolds for tissue engineering
and chemically modified polysaccharides (CMPs) have potent immunomodulating activities. This is illustrated here with chlorite-oxidized oxyamylose (COAM), a broad-spectrum antiviral agent. COAM interacts with
the chemokine system and thus can be used to modulate leukocyte compartmentalization in vivo. This knowledge has been used to alter the clinical
course of acute viral infections, cancer and experimental autoimmune

encephalomyelitis (EAE), an animal model of multiple sclerosis. The latter
example is proof-of-concept that CMPs constitute important probes to
study immune functions and are novel drugs applicable for specific disease
entities.
1

Introduction

From semantic viewpoint the term molecular biology is a pleonastic
expression and does not correspond with the present day interpretations.
Indeed, all biological processes are driven by molecules. What is meant by
molecular biology are processes driven by nucleic acids. Thus, molecular
biologists are stricto sensu researchers using DNA and RNA tools for
sequencing and synthesis, for practical uses in genetic engineering and for
therapy with recombinant drugs. Glycobiology (the biology of glycans) is a
more strict term in that it defines, by its name, the molecules under study,
namely carbohydrates. Why does the field of glycobiology then still need
promotion? Why do the DNA/RNA biologists generate more appreciation
and the glycobiologists less recognition? This is certainly not because DNA
and RNA are sugar-phosphate polymers. Maybe it is because of discovery
of the beauty of the linear connection between codons and amino acids.
Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
E-mail:

Carbohydr. Chem., 2012, 38, 1–12 | 1

c

The Royal Society of Chemistry 2012

Maybe it is also because functional aspects of a branched glycan tree are not
yet understood. As long as the interconnections between RNA/DNA
biology with glycobiology are not yet hold by a solid stem, scientists need to
invest time and resources in finding important examples in glycobiology.
This is simply to state that the real basis of glycobiology, for instance
relating structures to functions, is a barrier for many scientists and needs
further promotion. The original concepts that glycoforms and glycotypes
confer different functions to proteins must be seen as eye-openers [1–2].
These concepts, well elaborated for immunoglobulin-G (IgG) and tissuetype plasminogen activator (t-PA), are becoming apparent for many other
glycoproteins. Aside effects of glycosylation on the specific activity of t-PA
and functional aspects of the glycans on the Fc part of IgG (vide infra),
glycoprotein folding, trafficking, recognition and involvement in the
immune system demonstrate that oligosaccharides are endowed with multiple functions [3–6].
The successes in technology progresses in nucleic acid and protein
sequencing and synthesis are evident and their impacts on medicine,
industry and society are obvious. The field of RNA/DNA biology has the
intrinsic advantage of a limited number of building blocks, with limited
natural chemical modifications of the five bases. Protein biology, based on
about 20 building blocks, is already more complex and subject to many
more natural chemical modifications: proteolytic processing, phosphorylation and dephosphorylation, citrullination, isoprenylation, acetylation/
deacetylation, to name a few. Glycosylation is nowadays recognized as the
most diverse and complex posttranslational modification of proteins.
Glycobiology forms the next, and perhaps the most challenging level.
Intrinsically, carbohydrate biochemistry is also based on a limited number
of natural monosaccharides, but their chemical modifications are multifold.
In addition, the glycosidic bonding of monosaccharides is subject to various
linkages and the involved carbon atoms form stereochemical centers. The
branching of oligo- and polysaccharides into arborized structures often
supersedes our imagination capacity, which is too much trained for linear

DNA, RNA and protein sequences. However, in practice in mammalian
systems a limited set of monosaccharides are used. Even so the linkage
variation creates an extraordinary diversity of structures.
2

Historical breakthroughs and examples

If glycobiology is to succeed as the science of the 21st century, then one
needs to define new breakthroughs. Happily one can walk in the footsteps of
those who prepared and enhanced the field with exoglycosidase oligosaccharide sequencing [1, 2], with mass spectrometry [7] and NMR analysis
of sugars [8], with lectin studies [9] and with glyco-engineering. The discovery of congenital disorders of glycosylation [10] not only points to future
therapeutic applications, but also has yielded a bridging function between
glycobiology and nucleic acid biology.
Oligosaccharide sequencing evolved over the last 30 years from an
extremely labor-intensive discipline towards an efficient analytical method
in the hands of experts. Pioneering work by the groups of Akira Kobata
2 | Carbohydr. Chem., 2012, 38, 1–12


(University of Tokyo) [11] and of Raymond Dwek (Oxford Glycobiology
Institute) [12] led to the understanding of the proinflammatory effects of
agalactosyl IgGs in rheumatoid arthritis [2]. This knowledge was complemented with the findings of anti-inflammatory sialic acids in IgGs [13].
Together, these breakthroughs can be summarized in one sentence. Oligosaccharides attached to proteins fine-tune the biological activities of these
molecules. This should be not compared with an on/off switch (as is often
the case with protein phosphorylation), but rather with the selection of a
specific program or tuning. Presently, oligosaccharide exoglycosidase
sequencing has been perfected to such a level [14, 15] that broad-spectrum
and high-throughput analysis and glycomics are within reach. One next
breakthrough will come from coupling of this type of sophisticated analysis
with proteomics, genomics and transcriptomics.

A second aspect of structural analysis is mass spectrometry and NMR
analysis. Whereas mass spectrometry per se is nowadays routinely used in
proteomic analysis [16], for oligosaccharide analysis the identification of
structural details by mass spectrometry remained more complicated for the
simple reason that, for example, the various hexoses maintain the same
molecular mass. Nevertheless, sophisticated mass spectrometry technology
for sugars has been developed, is presently the expertise of few specialists
[7, 17], but is becoming common practice, because the combination of
high-performance liquid chromatography and mass spectrometry of oligosaccharides is very powerful and rapid. Considerable growth is expected in
this area and basic mass spectrometry analysis is already becoming classical
technology for structural analysis. Unfortunately and as is the case for other
applications of mass spectrometry, high-end equipment is rather expensive
and a limited number of specialized centers, homing dedicated specialists
and equipped with such instruments, can answer easily any exotic demands
for the analysis of complex glycoconjugates. Similar economic considerations may apply for NMR analysis, with the additional practical point
that the amounts of purified glycoconjugates needed for NMR analysis
supersede considerably those for mass spectrometry identification.
A third aspect is related to practical aspects, which we best place under
the term glyco-engineering. Agalactosyl [2] and sialyl IgGs [13] have already
been mentioned as disease promoting and disease-limiting factors, respectively, in inflammation. Such knowledge is successfully addressed by
glyco-engineering companies making optimized recombinant glycoprotein
preparations [18]. Another example is related to the classical ABO blood
group system. Nobel Prize laureate Karl Landsteiner was a glycobiologist
avant-la-lettre. Present-day glyco-engineers have managed to modify the
ABO blood group antigens [19], heralding novel possibilities in transfusion
medicine. Because the immunological insights with blood transfusions
formed the basis of organ transplantation medicine, it is not excluded that
the glyco-engineering of the ABO blood group antigens forms the basis of
new applications in xenotransplantation, where galactosylation is a major
hinder in the application of pig donor organs [20].

A fourth aspect to be recognized as historical in glycobiology is the study
of lectins as probes for sugars. Plant lectins, recognizing in defined ways
oligosaccharides, have been widely used in medical diagnostics, often before
Carbohydr. Chem., 2012, 38, 1–12 | 3


their mechanism of action was known [21, 22]. Lymphoblast transformation
with phytohaemagglutinin for karyotyping purposes and definition of
chromosomal aberrations was used long before the cytokines that are
induced in this process were identified as interleukin-2, interleukin-6 and
others. Lectins are also used for disease diagnosis in histochemical analysis
of cancer and, more recently, with the use of lectin microarrays [23]. The
literature on this topic is vast, exemplary reviews are available for detailed
and general information [24, 25]. In contrast, the information about lectins
as therapeutics is still rather sketchy. After the discovery of selectins, the
mammalian lectins that regulate rolling of leukocytes at an early step in the
inflammatory response, selectin knockout mice were developed to study
immunophenotypes (how these mice differ from wildtype animals with
reference to immunological parameters) and attempts were made to develop
novel anti-inflammatory agents on the basis of these lectin-glycan interactions [26–28]. A second example about glycotherapy relates to viral infections. Human immunodeficiency virus is enveloped in a glycocoat, limiting
access to protein (peptide) epitopes. One way to flag this virus is with
mannose-recognizing lectins, a strategy that is investigated with increasing
intensity [29].
An important way to enhance the field of glycobiology is to define
new medical applications. These may involve deficiencies, resulting from
aberrant glycosylation, in need for substitution therapy or storage diseases
with a pathogenic role of accumulated saccharides, in which reduction
forms a treatment strategy. An important class of rare diseases is the family
of congenital diseases of glycosylation (CDG) [10]. However, a critical
aspect to recognize is the fact that CDGs create the link between nucleic

acid and protein biology and glycobiology. Identified CDGs, resulting
from the mathematics of evolution (combinations of random mutations
that result in life offspring with clinical phenotypes), demonstrate that
glycosylation is a biological process of vital importance. In addition, the
creation of knockout mouse models of CDGs is an important step to
understand better the biological importance of sugars, but may also form a
solid basis to define treatments of these rare diseases, both in cases of
deficiencies [30] and of substrate reduction therapy in storage diseases, like
Gaucher disease [31].
Finally, we come to the level of commonly used drugs. One viral target
enzyme stands out as a glycobiological example: the influenza virus neuraminidase with its inhibitors oseltamivir (Tamiflus) and zanamivir
(Relenzas). The concept is simple, but the efforts done are considerable:
definition of a microbial target enzyme (sufficiently different from sister
enzymes in the host) and screening for inhibitors [32]. It does not take much
imagination that future antivirals and other antimicrobial agents may be
developed on the basis of other microbial sugar modifying enzymes. In
virology, such examples of interesting glycotarget enzymes may be restricted
because of the limitation of the coding capacity of viral genomes and,
therefore, the influenza neuraminidase will keep the lead for a while.
However, in the world of bacteria, fungi and parasites an enormous
potential exists to discover target enzymes, generate inhibitors and define
new medicines.
4 | Carbohydr. Chem., 2012, 38, 1–12


Most of the examples mentioned so far relate to oligosaccharides. What is
the status about polysaccharides?
3

Polysaccharides and derivatives

Although changes are emerging with the recognition of the importance of
polysaccharide vaccines, in biomedical education the time spent to study
polysaccharides is inversely correlated with their abundance in nature.
Cellulose [polyb(1-4)D-glucose], amylose [polya(1-4)D-glucose] and branched amylopectin in starch form most biomass and energy supply for most
organisms. Intestinal resorption of starch glucose (from amylose and
amylopectin) as monosaccharides and disaccharides and conversion into
glycogen is basic medical knowledge that can be linked to rare glycogen
storage diseases and to metabolic disease number one, i.e. diabetes. Deficiencies of amylase and disaccharidases lead to alterations of the gut
microbiomes and to gastrointestinal disorders as is the case with specific
polysaccharide derivatives. Indeed, dextran sodium sulphate (DSS) is
commonly used to induce inflammatory bowel disease in mice (used as an
animal model for Crohn’s disease or ulcerative colitis). In other words,
polysaccharide derivatives may be harmful or not, depending on their
degradability and their location.
4

An historical finding in virology?

After the discovery of interferon (IFN) in 1957, great hope was generated
for its use as therapy for viral diseases and mass production lines were
explored ending in the recombinant expression of IFN and use in the clinic
[33]. IFNs were not developed into first choice antivirals. Eventually, IFN-a
from leukocytes and IFN-b from fibroblasts became first choice antiinflammatory drugs for the treatment of multiple sclerosis. Recombinant
IFN-g, also dubbed immune interferon, because it is produced by lymphocytes and natural killer cells, found its way to the treatment of chronic
granulomatous disease [34].
Piet De Somer (1917–1985), founder of the Rega Institute (Fig. 1) was an
insightful man because he thought about and tried to solve the IFN production problem (before its cloning and expression [35]) by finding ways to
induce endogenous IFN. He admired and followed his scientist example,
Karl Landsteiner, by trying to use simple but practical rules. Karl

Landsteiner, who discovered the ABO blood group system, used simple
serological tests for discovery.
The aftermath of the second world war was characterized by discoveries
of many new mold-derived antibiotics, when antiviral IFN was discovered
upon its inducibility by viruses. IFNs protect neighbouring cells by inducing
an antiviral state and the virologists in the early 1960s started to search for
inducers that could replace viruses. In those booming days of DNA and
RNA research, screening of mold or bacterial extracts and synthetic
molecules for antiviral activity was a commonly used technique. Penicillium
products and synthetic polyanionic polymers were found to possess broad
spectrum antiviral activity and this incited De Somer to search for and test
other polyanions for antiviral activity. De Somer explored synthetic
Carbohydr. Chem., 2012, 38, 1–12 | 5


Fig. 1 Rector Piet De Somer (1917–1985) and the Rega Institute for Medical Research,
founded in 1954. Drawings are by Gerard Thijs, graphic artist, Leuven.

polyanions, such as polymethacrylates and polyacrylates, as antiviral agents
[36] and as inducers of interferon with some success [37]. Later it was found
that nucleic acids, such as synthetic double stranded RNA, induce the
production of IFN (most probably by mimicking the replicative intermediate forms of RNA viruses). Double-stranded RNA is a polyanionic
molecule and like polyriboinosinic acid:polyribocytidylic acid (poly IC),
synthetic polyanions were found to be toxic, presumably by their nondegradability. De Somer then gave the chemists of the Rega Institute the
task to manufacture a biodegradable polyanionic molecule that could be
used as an antiviral agent and/or interferon inducer: chlorite-oxidized
oxyamylose or COAM was born [38]. The chemists used amylose as starting
material in a stepwise oxidation reaction with openings of the glucose rings
(Fig. 2) to yield the polycarboxylic acid structure, now known as COAM.
Quickly, the product was tested against viruses and found to be a potent

and broad-spectrum antiviral agent [38–48]. In those days, this was often
not done with cell-based assays and permissive viruses, but instead by in vivo
testing (Table 1). COAM was supposed to induce IFN, although from the
time of the first studies onwards it was not clarified that the observed serum
IFN levels in mice treated with COAM and infected with virus were indeed
derived from induction by either the virus or by COAM. Because of some
inconclusive discrepancies in the COAM literature [39, 49], the question
whether COAM itself induces IFN or enhances IFN production by other
inducers remained open. In fact, data obtained already in 1976 from in vivo
experiments, corroborating earlier studies with COAM [42], remained
hidden in our laboratory notebooks till publication in 2010 [50].
5

COAM does not induce interferon

About 35 years after its synthesis [38], we reiterated the COAM project in
2006 using the now available powerful techniques of genetic engineering.
First, we applied a simple in vitro virus infection model with the picornavirus mengovirus on mouse fibroblastoid L929 cells. COAM protected
6 | Carbohydr. Chem., 2012, 38, 1–12


Fig. 2 Chemical synthesis of COAM from amylose. The steps of glucose ring opening by
periodate and further oxidation with chlorite into a polycarboxylate structure is illustrated
(Adapted from P. Claes et al., 1970, reference 38).

dose-dependently the cell cultures against mengovirus, but only poly IC
induced an antiviral state by induction of IFN-b. We did not observe
induction of IFN bioactivity or of IFN mRNAs by COAM. By performing
time-of-addition experiments with COAM, we deduced that COAM
acted at the stage of virus entry. It was also noticed that rather high

concentrations (1 mg/ml) of COAM were needed to exert an in vitro
antiviral effect [51].
6

COAM is an immunomodulator

The in vivo efficacy of COAM against many viruses (Table 1) was much
better than what was expected on the basis of the in vitro antiviral profile.
For those reasons immune cells and molecules were thought to be involved
and were studied. The conclusions that were deduced from these studies
included (i) COAM acts mainly on myeloid cells (neutrophils and macrophages) rather than on lymphocytes and (ii) intraperitoneal injection of
COAM leads to massive neutrophil influx, hinting to an interaction with
neutrophil chemokines. Since the most potent neutrophil chemokine in the
mouse is granulocyte chemotactic protein-2 (GCP-2) [52], the induction and
binding of GCP-2 by COAM was investigated and the link with chemokines
was established. It was concluded that COAM induces and binds GCP-2
and thus establishes a potent chemokine gradient for myeloid cells to the
peritoneal cavity where the local virus infection is contained [50]. The
information about a CMP drug that protects against lethal virus infections
after prophylactic parenteral administration is perhaps not so important
for common acute virus infections. However, because rescue (more than 90
Carbohydr. Chem., 2012, 38, 1–12 | 7


Table 1 Antiviral effects of COAM
Virusa

Species (treatment)b

Dosis/

concentrationc

Effectd

Reference

Coxsackie B4
Foot-and-mouth ip.
Foot-and-mouth ip.
Friend leukemia
Hog cholera
HSV-1 in eye
HSV-1 in.
Influenza in.
Influenza ip.
Mengo ip.
Mengo ip.
Mengo ip.
Mengo
Moloney mouse
sarcoma im.
Newcastle disease iv.
PVX leaf discs
Rabies im.
Semliki Forrest ip.
TMV leaf discs
Vaccinia iv.
Vaccinia iv.
Vesicular stomatitis
Vesicular stomatitis

in.

Mice (profyl. 24 h)
Mice (profyl. 24 h)
Swine (profyl. 24 h)
Mice (profyl.)
Swine (profyl. 24 h)
Rabbit (profyl. 4 h)
Mice (profyl. 24 h)
Mice (profyl. 24 h)
Mice (profyl. 24 h)
Mice (profyl. 24 h)
Mice (profyl. W16 h)
Mice (profyl. 24 h)
L929 cells (in vitro)
Mice (profyl. 24 h)

2 mg/mouse ip.
5–60 mg/kg ip.
120 mg/kg ip.
0.5 mg/mouse
120 mg/kg ip.
50 mg/ml topic.
0.8–4 mg/mouse ip.
2 mg/mouse ip.
2 mg/mouse ip.
2 mg/mouse ip.
2 mg/mouse ip.
2 mg/mouse ip.
1 mg/ml

0.5 mg/mouse ip.

Protection
Protection
No effect
Improvement
Protection
Protection
Protection
Protection
Protection
Protection
Protection
Protection
CPE inhibition
Protection

53
40
40
46
40
40
47
39
41
40
42
50
51

43

Mice (profyl.)
Plant (posti.)
Mice (3 h posti.)
Mice (profyl. 24 h)
Plant (posti.)
Mice (profyl. 24 h)
Mice (profyl. 24 h)
MEF (in vitro)
Mice (profyl.)

1–10 mg/mouse ip.
1% solution
0.3 mg/mouse
2 mg/mouse ip.
1% solution
2 mg/mouse ip.
W55 mg/mouse iv.
10 mg/ml
10 mg/mouse

Protection
No effect
Worsening
Protection
Protection
Protection
Protection
CPE inhibition

No effect

44
48
45
39
48
38
39
38
44

a

HSV-1: Herpes simplex virus type 1; TMV: Tobacco mosaic virus; PVX: Potato virus X.
profyl.: prophylactic, before virus; posti.: postinfection.
c
iv.: intravenous; ip.: intraperitoneal; in.: intranasal; im.: intramuscular.
d
CPE: cytopathic effect.
b

percent survival without disease sequels) was observed after lethal virus
challenge by pretreatment with a purified COAM preparation, the CMP
preventive action may be crucial to contain clusters of lethal virus infections, including those with Ebola and other deadly viruses [50]. In another
model of Coxsackievirus B4 pancreatic infection it was corroborated that
COAM has potent antiviral effects, most probably mediated by myeloid
inflammatory cells [53].
7

Therapeutic implications for acute and chronic inflammation and cancer

On the basis of the mentioned experimental data, the question was asked
whether the polysaccharide derivative COAM may be applicable in other
diseases. De Somer’s wish of using IFN-b as a potent antiviral agent did not
really materialize. Instead, IFN landed as a real drug for the treatment of
multiple sclerosis [33].
At the time when it was still controversial whether COAM induces IFN
and not yet clear that COAM acted in a different way, we investigated
whether COAM might protect against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. It is worth
8 | Carbohydr. Chem., 2012, 38, 1–12


mentioning that these experiments were also intended to investigate the
clinical effects of COAM in virus-free conditions.
The major conclusions from studies with a hyperacute model of EAE
formed the basis of a doctoral dissertation (Dr. N. Berghmans, 2008,
University of Leuven, Belgium, promoter Prof. H. Heremans). COAM did
not induce IFN-a, IFN-b or IFN-g in vivo, but possessed a significant
protective effect against hyperacute EAE. Intraperitoneal injection of
COAM was locally chemotactic for leukocytes with such potency that it
diminished the leukocyte numbers significantly in the central nervous system (brain and spinal cord). Because of these observations, we proposed a
new immunotherapeutic strategy to treat acute and chronic CNS inflammation and called this ‘‘cell deviation therapy with an immunostimulating
glycomimetic’’. This research is presently continued and firm evidence has
been established that COAM also protects against autoimmune EAE,
induced by myelin oligodendrocyte glycoprotein-derived peptide in
EAE-susceptible IFN-g knockout mice [54] and [Berghmans et al., in
preparation].
Another type of application was investigated in tumor biology. As
already described in Table 1, COAM protected against Moloney mouse

sarcoma virus infections [43]. This was interpreted as an antiretroviral
effect, and also as an antitumoral effect, because both tumor incidence and
lethality caused by tumor growth were reduced by COAM. Moreover, by
now it is clear that inflammation is a critical component in tumor biology
[55] and that myeloid cells, alongside lymphocytes, are key players in tumor
biology [56]. For these reasons, we took the simple approach of testing
COAM against melanoma in a syngeneic mouse model. It was found that
COAM reduced significantly the tumor burden of dermal melanomas when
locally injected early in tumor development [57].

8

Conclusions and future perspectives

In comparison with many studies on the biology of complex oligosaccharide
structures, polysaccharides and derivatives have not yet attracted much
interest from industry. Nevertheless several interesting principles may be
demonstrated with the example of the amylose-derivative COAM. First, old
literature and hypotheses (about polysaccharides and derivatives) should be
tested with all novel technologies at our disposal. For instance, structural
analysis can now be executed with the tools of exoglycosidase sequencing
and mass spectrometry and biological analyses may rely on the use of RTPCR, transcriptional micro-arrays and in vivo animal models. Second, on
the basis of biological discoveries new therapies may be invented. For
instance, on the basis of the discovery that COAM (induces and) binds
chemokines and thus may create unseen potent endogenous chemokine
gradients, we discovered that it is possible to call leukocytes out of the CNS
back into the circulation and the (immunological) periphery to cure mouse
EAE. Such proof of principle may pave the way to new treatments of acute
and chronic neuroinflammations and should definitely stimulate basic and
applied research on polysaccharides.

Carbohydr. Chem., 2012, 38, 1–12 | 9


Abbreviations
CMP
COAM
CDG
CPE
EAE
HSV-1
IgG
IFNim.
in.
ip.
iv.
poly IC
t-PA

chemically modified polysaccharide
chlorite-oxidized oxyamylose
congenital disorder of glycosylation
cytopathic effect
experimental autoimmune encephalomyelitis
herpes simplex virus type 1
immunoglobulin-G
interferonintramuscular
intranasal
intraperitoneal
intravenous
polyriboinosinic acid:polyribocytidylic acid

tissue-type plasminogen activator

Acknowledgements
This overview is a tribute to the late Professor Piet De Somer, founder of the
Rega Institute for Medical Research and an eminent scientist-entrepreneur
and university rector in Belgium. To commemorate his passing away, 25
years ago, the University of Leuven honored De Somer with a symposium
and the City of Leuven renamed its central place into ‘‘Piet De Somer
Square’’. The authors thank Professor Alfons Billiau (Leuven) and
Professor Raymond A. Dwek (Oxford) for inspiration and critically reading
and commenting on the manuscript and Professor Hubertine Heremans
(Leuven) for expertise in EAE and promotion of the COAM-project. This
work was funded by the ‘‘Geconcerteerde OnderzoeksActies’’ (GOA2012/
17) and by the Fund for Scientific Research of Flanders (FWO-Vlaanderen).
GO is a Steering Committee Member of the European Science Foundation
(ESF, Strasbourg, France) Research Network Programme ‘‘European
GlycoScience Forum’’ (EGSF).
References
1 T. W. Rademacher, R. B. Parekh and R. A. Dwek, Annu. Rev. Biochem., 1988,
57, 785.
2 R. B. Parekh, R. A. Dwek, B. J. Sutton, D. L. Fernandes, A. Leung, D.
Stanworth, T. W. Rademacher, T. Mizuochi, T. Taniguchi, K. Matsuta, F.
Takeuchi, Y. Nagano, T. Miyamoto and A. Kobata, Nature, 1985, 316, 452.
3 P. M. Rudd, T. Elliott, P. Cresswell, I. A. Wilson and R. A. Dwek, Science,
2001, 291, 2370.
4 G. Opdenakker, P. M. Rudd, C. P. Ponting and R. A. Dwek, FASEB J, 1993, 7,
1330.
5 P. M. Rudd and R. A. Dwek, Crit. Rev. Biochem. Mol. Biol., 1997, 32, 1.
6 P. Van den Steen, P. M. Rudd, R. A. Dwek and G. Opdenakker, Crit. Rev.
Biochem. Mol. Biol., 1998, 33, 151.

7 D. J. Harvey, Mass. Spectrom. Rev., 2012, 31, 183.
8 J. C. Paulson, J. Weinstein, L. Dorland, H. van Halbeek and J. F. Vliegenthart,
J. Biol. Chem., 1982, 257, 12734.
10 | Carbohydr. Chem., 2012, 38, 1–12