chapter 2 - Bentham Science

Synthesis and Biological Applications of 

Glycoconjugates 

Editors 

Olivier Renaudet and Nicolas Spinelli 

University of Grenoble 

France

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CONTENTS 

Foreword i 

Preface ii 

List of Contributors iii 

CHAPTERS 

1. Bacterial Lectins and Adhesins: Structures, Ligands and Functions 3 

Anne Imberty 

2. Ligands for FimH 12 

Thisbe K. Lindhorst 

3. Multivalent Glycocalixarenes 36 

Francesco Sansone, Gabriele Rispoli, Alessandro Casnati and Rocco Ungaro 

4. Solving Promiscuous Protein Carbohydrate Recognition Domains with Multivalent 

Glycofullerenes 64 

Yoann M. Chabre and René Roy 

5. Monovalent and Multivalent Inhibitors of Bacterial Toxins 78 

Edward D. Hayes and W. Bruce Turnbull 

6. Monovalent and Multivalent Glycoconjugates as High Affinity Ligands for Galectins 92 

Sébastien Vidal 

7. Combinatorial Libraires of Dendritic Glycoclusters 116 

Jean-Louis Reymond and Tamis Darbre 

8. Cyclopeptide-Based Glycoclusters 129 

Olivier Renaudet, Didier Boturyn and Pascal Dumy 

9. Oligonucleotide-Carbohydrate Conjugates 145 

Nicolas Spinelli and Eric Defrancq 

10. Glycoliposomes and Metallic Glyconanoparticles in Glycoscience 164 

Marco Marradi, Fabrizio Chiodo, Isabel García and Soledad Penadés 

11. Chemoselective Glycosylation Techniques for the Synthesis of Bioactive Neoglycoconjugates, 

Glyconanoparticles and Glycoarrays 203 

Francesco Peri 

12. Glycosidases in Synthesis of Glycomimetics and Unnatural Carbohydrates 226 

Vladimír Křen 

13. Diels-Alder Based Synthesis of Glycomimetics 240 

Cristina Nativi, Elisa Dragoni, Barbara Richichi and Stefano Roelens

14. Analytical Tools for Protein-Carbohydrate Interaction Studies 255 

Cédric Goyer and Pierre Labbé 

15. Index 267

FOREWORD 

Attachment of carbohydrates in all its forms is a strategy exploited by both nature and those that study nature. Since the 

first reviews over 70 years ago the importance of this critically modulating and even defining role has long been 

recognized by who those study them. The very presence of carbohydrate units in naturally occurring structures and their 

mimetics has a dramatic effect on their physical, chemical and biological properties. Glycoscience is by necessity broad 

in the range of techniques that it encompasses and I have argued for over ten years that in this context the sometimesapplied 

and somewhat artificial distinction between "chemical" and "biological" techniques is unhelpful. 

This has never been the case – we are reaching a stage in the field where remarkable techniques are able to make 

even some very large naturally relevant glycoconjugates. Moreover, through these methods and the design of some 

remarkable molecular experimental approaches, we are using these glycoconjugates to unpick the many dazzling 

and exciting roles that carbohydrates play in Biology and Medicine. 

This book highlights this superbly. Drs Renaudet and Spinelli have assembled an outstanding collection of excellent 

and relevant reviews written by leading protagonists in the field that complement each other superbly. Together they 

provide a wonderful account of the current state-of-the-art. 

Vital current targets illustrate what might and will be achieved through both analysis and understanding. Many 

pathogens exploit the display of carbohydrates on host cell surfaces to gain entry. Imberty and Lindhorst, in their 

chapters, have described some of the most current and relevant examples. Integral to the approaches they have 

analyzed is a fundamental molecular analysis of the binding of sugars to proteins. This has been excellently 

amplified by the chapter of Goyer and Labbé, which describes some of the most pertinent quantitative techniques in 

current use, and by the several chapters that show how this understanding can be directly related to application. 

These include the design of inhibitors of bacterial toxins, superbly analyzed by Turnbull and Hayes, and ligands for 

the important mammalian galectins, the study of which Vidal summarizes superbly. 

Use of well-defined scaffolds is an essential element if this study and exploitation is to be precise. Various platforms 

have been analyzed here: fullerenes (Roy and Chabre), dendritic clusters (Reymond and Darbre), calixarenes (Casnati et 

al.), cyclic peptides (Renaudet et al.) and even nucleic acids (Spinelli and Defrancq). All highlight longer-term 

applications, especially in applications that may allow probing of interactions in whole organisms, and these are 

especially elegantly drawn out in the chapter by Penadés et al. that consider particles of many relevant types. 

Typically syntheses of glycoconjugates adopt one of two strategies. The first strategy is the formation of the glycanaglycone 

link early, to form, for example, glycosylated building blocks such as glycosides, glycopeptides or 

glycosylated dendritic wedges, that may then be assembled. While, of course, chemical methods can create 

glycosidic bonds in a number of ways, the use of biocatalysts can bring with it advantages of selectivity and 

efficiency; Křen’s chapter highlights this utility well. The second strategy is formation of the link later on in the 

synthesis once the scaffold for its presentation is in place. Given the instability that may be associated with the link 

and the requirements for protection that need to be considered in the use of glycosylated building blocks, it is clear 

why the latter has often seemed the most attractive option. Peri has deftly summarized a broad range of methods for 

this late stage approach as applied to many useful platform types. Nativi et al. have delineated an emerging area that 

could allow access to glycomimetics based on the use of glycals in cycloadditions. 

Together, these chapters give a powerful insight into the elucidation of the mechanism by which sugar-binding takes 

place, the tools used to probe into it and its consequences. This science has driven and continues to drive the 

synthesis and technology of glycoconjugates. These are the new tools of the glycobiology trade and I believe only 

the very start of an era of emerging diagnostics and therapeutics. 

i 

Prof. Ben Davis 

University of Oxford 

UK

ii 

PREFACE 

The interactions between carbohydrates and proteins are involved in major physiological and pathological events. 

With the recent emergence of glycomics, an increasing number of sophisticated glycosylated structures capable of 

mimicking the multivalent display of the cell surface glycocalix has been reported. Besides giving precious 

guidelines on the binding parameters that govern these complex biological processes, synthetic glycoconjugates 

often revealed considerable interests for diagnostic and therapeutic applications. 

Our main motivation to edit Synthesis and biological applications of glycoconjugates was to update the major 

advancements in this field through several chapters written by renowned experts who have largely contributed to the 

recent progresses. Illustrated with more than 200 colour figures and including literature citations mostly from the 

last decade, this book covers the recent chemical methods of glycoconjugates and clearly highlights their diverse 

biological properties. Chapter 1 analyzes the structure of lectins from pathogenic bacteria and gives 

structure/function relationships that are crucial for the development of high affinity ligands. In Chapter 2 

carbohydrate binding by FimH is discussed and modern means for its investigation including photoaffinity labelling 

are described. Very recent crystallographic work is also documented that provides an explanation for shearenhanced 

binding of type 1 fimbriated E. coli. Chapter 3 summarizes the most convenient methodologies for the 

synthesis of glycocalixarenes and describes their impressive aggregation properties and many other biological 

applications. Chapter 4 illustrates examples of bacterial and human lectins together with bacterial toxins having 

varied number of carbohydrate recognition domains that necessitated multivalent glycoconjugates. It focuses on 

glycofullerenes that have been used to describe novel synthetic strategies and possible fit with concomitant lectins. 

In Chapter 5 the structures of the cholera and E. coli heat-labile toxins are described. The authors also summarize 

the main strategies that have led to the development of monovalent and multivalent inhibitors of these toxins and 

they discuss the importance of chelation and protein aggregation as mechanisms of multivalent inhibition. Chapter 6 

focuses on the design of high affinity ligands for galectins and provides a better knowledge of the implications of 

these proteins in biology. Chapter 7 demonstrates that combinatorial chemistry led to the discovery of glycopeptide 

dendrimers as strong ligands for lectins or drug-delivery systems and shows how the amino acid composition of the 

dendrimer branches can influence their biological activity. Chapter 8 focuses on the synthesis of a recent class of 

glycoclusters displayed on a cyclopeptide platform and highlights their promising biological properties, in particular 

as inhibitors or synthetic vaccines. Chapter 9 shows that the cellular delivery and bioavailability of oligonucleotides 

can be improved by their conjugation with carbohydrates and also describes the construction of carbohydrate 

biochips or glycoclusters using these conjugates. Chapter 10 focuses on glycoliposomes and covalentlyfunctionalized 

glyconanoparticles which make use of the “glyco-code” to address specifically pathogens or 

pathological-related problems. Chapter 11 focuses on the synthesis of glycoproteins, glycopeptides, glycosylated 

natural compounds, carbohydrate-functionalized surfaces and nanoparticles using chemoselective glycosylation. In 

Chapter 12 the recent developments in glycosidase-catalyzed synthesis of unnatural semi-synthetic carbohydrate 

structures are presented. Chapter 13 reports the synthesis of glycomimetics and glycopeptidomimetics using hetero- 

Diels Alder reactions between ,’-dioxothiones and glycals. Chapter 14 aims at describing the usual methods to 

characterize protein-carbohydrate interactions, namely inhibition of hemagglutination assay, enzyme-linked lectin 

assays, isothermal titration calorimetry, surface plasmon resonance and the more recent atomic force microscopy. 

We believe that this Ebook will be of particular interest to a large community of graduate students, researchers and 

professionals in academia or industry involved in Glycoscience. We would like to express our sincerest gratitude to 

all of the authors who accepted to contribute to this exciting project, by sharing their strong experiences and 

knowledge in this research area. 

Olivier Renaudet 

Nicolas Spinelli 

University of Grenoble 

France

List of Contributors 

Didier Boturyn 

Département de Chimie Moléculaire, UMR-CNRS 5250 & ICMG FR 2607, Université Joseph Fourier, 38041 

Grenoble Cedex 9, France. 

Alessandro Casnati 

Dip.to di Chimica Organica e Industriale, Università degli Studi, Parco Area delle Scienze 17/A, 43124 Parma, Italy. 

Yoann M. Chabre 

Pharmaqam – Groupe de Recherche en Chimie Thérapeutique, Université du Québec à Montréal, P.O. Box 8888, 

Succ. Centre-ville, Montréal, Québec, Canada H3C 3P8. 

Fabrizio Chiodo 

Laboratory of GlycoNanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE, Parque Tecnológico de 

San Sebastián, Pº de Miramón 182, 20009 San Sebastián, Spain. 

Tamis Darbre 

Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012, Berne, Switzerland. 

Eric Defrancq 



Elisa Dragoni 

ProtEra, Polo Scientifico Universita’ di Firenze, viale delle Idee, 22 I-50019 Sesto F.no (FI), Italy. 

Pascal Dumy 



Isabel García 

Laboratory of GlycoNanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE and Biomedical 

Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Parque 

Tecnológico de San Sebastián, Pº de Miramón 182, 20009 San Sebastián, Spain. 

Cédric Goyer 



Edward D. Hayes 

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. 

Anne Imberty 

CERMAV-CNRS (affiliated to Université Joseph Fourier and ICMG), BP 53, 38041 Grenoble cedex 9, France. 

Vladimír Křen 

Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of the Czech 

Republic, Vídeňská 1083, CZ 142 20 Prague 4, Czech Republic. 

Pierre Labbé 



iii

iv 

Thisbe K. Lindhorst 

Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, D-24098 Kiel, Germany. 

Marco Marradi 




Cristina Nativi 

Dipartimento di Chimica, Universita’ di Firenze, via della Lastruccia, 3,13 I-50019 Sesto F.no (FI), Italy. 

Soledad Penadés 




Francesco Peri 

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, 2; 20126 

Milano, Italy. 

Olivier Renaudet 



Jean-Louis Reymond 

Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012, Berne, Switzerland. 

Barbara Richichi 

Dipartimento di Chimica, Universita’ di Firenze, via della Lastruccia, 3,13 I-50019 Sesto F.no (FI), Italy. 

Stefano Roelens 

Istituto Metodologie Chimiche (IMC), CNR, Dipartimento di Chimica, Universita’ di Firenze, via della Lastruccia, 

3,13 I-50019 Sesto F.no (FI), Italy. 

René Roy 


Succ. Centre-ville, Montréal, Québec, Canada H3C 3P8. 

Francesco Sansone 


W. Bruce Turnbull 

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. 

Nicolas Spinelli 



Rocco Ungaro 


Sébastien Vidal 

Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie Organique 2, UMR 5246, 

Université Lyon 1 and CNRS, F 69622 Villeurbanne, France.

Synthesis and Biological Applications of Glycoconjugates, 2011, 3-11 3 

Bacterial Lectins and Adhesins: Structures, Ligands and Functions 

Anne Imberty * 

Olivier Renaudet and Nicolas Spinelli (Eds) 

All rights reserved - © 2011 Bentham Science Publishers 

CHAPTER 1 

CERMAV-CNRS (affiliated to Université Joseph Fourier and ICMG), BP 53, 38041 Grenoble cedex 9, France 

Abstract: Infection by bacteria is often initiated by the specific recognition of host epithelial surfaces by adhesins 

and lectins. These glycan binding proteins (GBP) are therefore virulence factors that play a role in the first step of 

adhesion and invasion. The adhesins are part of organelles, they are generally located at the tip of pili or fimbriae. 

On the opposite, soluble lectins occur either as individual proteins, or in association with toxins or enzymes. The 

human targets for bacterial adhesins and lectins are mostly fucosylated human histo-blood groups and/or 

sialylated epitopes. The binding of lectins and adhesins to host epithelial glycoconjugates is amplified by the 

multivalent presentation of the binding sites. The analysis of the available crystal structures of bacterial lectins 

and adhesins helps deciphering the structure/function relationship for this important class of proteins. It is a 

prerequisite for the development of multivalent high affinity ligands in antibacterial strategies. 

Keywords: Lectin, adhesin, crystal structure, bacteria, infection. 

LECTINS: UBIQUITOUS PROTEIN WITH A TASTE FOR SUGARS 

Lectins are proteins of non-immune origin that bind to specific carbohydrates without modifying them [1]. These 

proteins are ubiquitous as they have been identified in all living organisms, from viruses to bacteria, fungi, plants 

and animals. As a result of their interactions with glycoproteins, glycolipids and oligosaccharides, lectins are 

involved in cell-cell communication; they have the crucial role of deciphering the “glycocode”. Their biological 

functions are very diverse. Lectins are important players in the innate immunity system of many invertebrates as 

they are able to differentiate between self and non-self carbohydrates. Lectins are particularly abundant in plants, 

where they are proposed to be involved in protection against pathogens or feeders. The biological roles of animal 

lectins have been more thoroughly investigated; they involve glycoprotein trafficking and clearance, development, 

immune defence, fertility and many others. 

In pathogenic micro-organims, lectins are mostly involved in host recognition and tissue adhesion [2]. They can be 

present as subunit parts of toxins where they target the toxic catalytic unit towards host tissues carrying some 

specific glycoconjugates. They can also be part of multiprotein organelles, such as bacterial fimbria (pili), flagella 

and viral capsids. Therefore they participate to the adhesion process at the early stage of infection. Soluble lectins 

are also expressed as virulence factors by opportunistic bacteria. 

Structural studies of lectins and of their binding sites started with the elucidation of the crystal structure of 

Concanavalin A, a well characterized legume lectin specific for mannose and glucose [3]. Such structural 

investigations yield both the detailed knowledge of the protein structure and the identification of the most important 

contacts that are established in the binding pocket between the polypeptide chain and the carbohydrate. This is a 

prerequisite for a better understanding of the biological and physical processes of recognition [4]. Three dimensional 

structures are also the starting points for many developments in the biotechnological and therapeutical applications 

of lectin/carbohydrate interactions. 

Lectins are relatively easy to purify due to their carbohydrate binding properties that can be exploited for affinity 

purification. Lectins from plants are generally abundant and can be extracted from natural sources, whereas animal 

lectins are nowadays generally produced by recombinant approaches. A large number of lectins has therefore been 

used for crystallization assays, preferably in the presence of their carbohydrate ligand(s).More than 870 threedimensional 

structures (corresponding to 187 different lectins) are now available and gathered in the 3D-lectin 

database (http://www.cermav.cnrs.fr/lectines/). 

*Address corresponding to Anne Imberty: CERMAV-CNRS (affiliated to Université Joseph Fourier and ICMG), BP 53, 38041 Grenoble 

cedex 9, France; E-mail: Anne.Imberty@cermav.cnrs.fr

4 Synthesis and Biological Applications of Glycoconjugates Anne Imberty 

Inspection of the database content indicates that most crystal structures have been for the time being, obtained for 

plant and animal lectins (Fig. 1). Nevertheless, the number of structural works dealing with viral and bacterial 

materials increases rapidly. Interestingly, a strong bias is observed in the nature of the architecture of the lectins. As 

indicated in Fig. 1, most lectins adopt a -sandwich fold. The several -sandwiches topologies adopted by lectins are 

very different one to each others, but some interesting structural convergences are observed. For example legume 

lectins and those intracellular animal lectins which are involved in quality control of glycoprotein synthesis share the 

same protein fold [5]. 

Figure 1: Repartition of the 187 different lectins with structures available in the Protein data bank and accessible in the lectin 

database (http://www.cermav.cnrs.fr/lectines/). Lectins are classified as a function of: A) origin; B) fold. 

STRUCTURES OF LECTINS FROM PATHOGENIC BACTERIA 

Adhesins 

Adhesins refer to the lectin domains which are present at the tip of bacterial fimbriae (or pili). Although different 

types of pili are observed on bacteria, structural data have been obtained only for those lectins belonging to the twodomain 

adhesins (TDA)s from different pathogenic strains of Escherichia coli. These adhesins consist of a pilin 

domain and a lectin domain, both presenting an immunoglobulin-fold joined via a short interdomain linker [6]. The 

adhesins reported in Table 1 have been characterized in enteropathogenic E. coli (GafD on F17 fimbriae) and 

uropathogenic ones (FimH on type 1 fimbriae and PapG on P-pili). 

Table 1: Crystal structures representative of the different classes of bacterial adhesins and their specificity. 

Bacteria Adhesin Carbohydrate specificity 

E. coli PapG 

GalNAc13Gal14Gal14Glc 

(GbO4 oligosacch.) 

Structure 

PDB 

Ref. 

1J8R [7] 

E. coli GafD/F17-G GlcNAc 1O9W, 1OIO [8, 9] 

E. coli FimH Oligomannose 2VCO [10] 

Flexible F17 fimbriae of enterotoxigenic E. coli are capped with GafD adhesins specific for GlcNAc-terminated 

glycoconjugates. Uropathogenic E. coli adhesins bind to different parts of the human urinary tract. The long and flexible 

P fimbriae expose PapG adhesins specific for galabiose containing the oligosaccharide (Gal1-4Gal) motif whereas 

shorter type 1 pili are terminated by FimH that binds oligomannose. Despite the lack of any sequence identity, all these 

lectins share the same immunoglobulin-like fold of the two structural components; the corresponding pili are assembled 

by the chaperone-usher pathway [11]. The three fimbrial lectins, GafD, FimH and PapG share similar beta-barrel folds 

but display different ligand-binding regions and disulfide-bond patterns (Fig. 2).

Bacterial Lectins and Adhesins Synthesis and Biological Applications of Glycoconjugates 5 

Figure 2: Crystal structure of the three bacterial adhesins obtained in complex with carbohydrate ligands. A) PapG complexed 

with GbO4 (code 1J8R); B) GafD complexed with GlcNAc (1O9W); C) FimH complexed with Man3GlcNAc2 (2VCO). 

References as in Table 1. All figures have been prepared using PyMol (DeLano Scientific). 

Toxin-Associated Lectins 

Bacteria use lectin domains to target another subunit which generally displays some enzymatic activity towards host 

cell types. The crystal structures that have been solved illustrate how neural or digestive tissues can be targeted by 

lectin domains (Table 2). 

Clostridia produce a large number of toxins such as neurotoxins and pore-forming toxins which are responsible for 

gangrenes and gastrointestinal diseases. Among these, two families of toxins contain lectin domains specific of 

human glycoconjugates. 

- Clostridia neurotoxins are well known because of the severe diseases that are caused by the 

corresponding bacteria: i.e. tetanus by Clostridium tetani and botulism by C. botulinum. Indeed clostridial 

neurotoxins are the most lethal protein toxins for humans but they are also used as therapeutic agents. 

These toxins have the particularity to inhibit neurotransmission. The lectin domain, at the C-term of the 

holotoxin, binds to complex polysialo-gangliosides present on neurons such as GT1b (Fig. 3A) [12]. After 

its translocation, the enzymatically active light chain specifically hydrolyses SNARE proteins thereby 

preventing SNARE complex assembly and consequently blocks the exocytosis of neurotransmitter [13]. 

- The large clostridial cytotoxins (LCT) represent a family of structurally and functionally related 

exotoxins from different Clostridium species. It includes C. difficile which is responsible for many cases 

of nosocomial antibiotic-associated diarrhea [15]. Toxins A and B (TcdA and TcdB) mono-O-glucosylate 

(or mono-O-GlcNAcylate) contain a specific threonine residue of Rho/Ras-proteins, which is essential for 

the function of the molecular switches. The LCTs are single-chain protein toxins, comprising three 

domains involved in host-binding, translocation and catalysis. The lectin domain is located at the Cterminus 

of the toxin and is characterized by polypeptide repeats folds that organize in a solenoid-like 

structure [16]. A portion of TcdA repetitive domain has been crystallized complexed to Galα1-3Galβ1- 

4GlcNAc – a non human glycan (Fig. 3B) [17]. It has been reported to recognize a variety of other 

carbohydrate structures having a lactosamine backbone such as blood group antigens Lewis X and Lewis 

Y, both of which being present in human intestinal epithelium. 

The human pathogen Staphylococcus aureus secretes many toxins that subvert the human immune system. The 

family of staphylococcal superantigen-like proteins (SSLs) contains 14 proteins. Their structures are very similar to 

that of the superantigens (SAgs) but they do not stimulate T cells [18]. Two toxins, SSL5 and SSL11, bind to sialic 

acid-containing glycoconjugates present on granulocytes and monocytes. The preferred ligand is the

12 Synthesis and Biological Applications of Glycoconjugates, 2011, 12-35 

Ligands for FimH 

Thisbe K. Lindhorst * 



CHAPTER 2 

Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, D-24098 Kiel, Germany 

Abstract: Adhesion of bacteria to glycosylated cells and surfaces is largely facilitated through adhesive organells 

projecting from the bacterial surface, which are called fimbriae. The most important fimbriae in Escherichia coli and 

in most of the other enterobacteria are the type 1 fimbriae which mediate adhesion in an -D-mannoside-specific 

manner. The lectin that is responsible for binding terminal -D-mannosyl residues is called FimH and is located at the 

tips of type 1 fimbriae. FimH is known to mediate shear-enhanced adhesion of bacteria to mannosylated cells or 

surfaces. Like many adhesins, FimH is a two-domain protein consisting of a pilin domain, that anchors FimH to the 

fimbrial shaft, and a distal, N-terminal lectin domain, harboring the carbohydrate binding pocket. A variety of natural 

as well as synthetic ligands for type 1 fimbriated bacteria and FimH, respectively, have been tested in order to improve 

our understanding of this protein-carbohydrate interaction in cellular adhesion and in an attempt to develop effective 

low-molecular weight compounds for an anti-adhesion therapy. Besides tailor-made ligands that have been designed 

based on the computer docking, various multivalent cluster mannosides have revealed interesting activities. In this 

review, carbohydrate binding by FimH will be discussed and modern means for its investigation including 

photoaffinity labelling will be described. In addition, very recent crystallographic work will be documented that 

provides an explanation for shear-enhanced binding of type 1 fimbriated E. coli. 

Keywords: Bacterial adhesion, type 1 fimbriae, FimH, mannosides, antiadhesives. 

INTRODUCTION 

In the first half of the 20th century, the term lectin was originally proposed for plant proteins that can agglutinate 

erythrocytes (“phytohemagglutinins”) and has later been broadened to include all sugar-specific proteins of nonimmune 

origin and lacking any enzymatic activity. Lectins are not only found in plants, but are ubiquitous in 

Nature,. Already during the first half of the 20th century it was discovered that many bacteria have the ability to 

agglutinate erythrocytes and these findings have prompted more systematic studies on bacterial lectins, that are also 

often referred to as adhesins or agglutinins [1]. It was shown that hemagglutination activity is a property that is 

expressed by many bacterial species, most commonly by those of the Enterobacteriaceae family. Hemagglutination 

activity of bacteria is almost always associated with the presence of multiple filamentous protein appendages 

projecting from the surface of bacteria that are called fimbriae (from the Latin word for ‘thread’) or pili (from the 

Latin word for ‘hair’). Gram-negative bacteria carry several hundreds of these adhesive organelles on their surface 

which might be of different Nature, and specificity, respectively. 

Studies on bacterial fimbriae have been ongoing since the 1950ies since most bacteria depend on expression of 

specialized adhesive organelles on the bacterial cell surface to mediate attachment to target tissues [2]. Several types of 

fimbriae have been identified and classified according to their carbohydrate specificity [3]. In Escherichia coli, besides 

type 1 fimbriae, P fimbriae (specific for Gal1,4Gal), S fimbriae (specific for Neu5Ac2,3Gal) and type G fimbriae 

(specific for GalNAc) are found. Oral Actinomyces express type 2 fimbriae that are specific for -galactosides. The best 

characterized pili, however, are type 1 fimbriae, that are one of the most abundant surface structures both in pathogenic 

and non-pathogenic Gram-negative bacteria and specific for terminal -mannosyl residues. Mannose specificity is 

mediated by a lectin called FimH forming a minor component of the fimbrial protein complex [4, 5]. 

TYPE 1 FIMBRIAE 

Type 1 fimbriae serve as extremely efficient adhesion tools for bacteria inhabiting diverse environments, including 

biotic and abiotic surfaces [6]. They are common throughout the Enterobacteriaceae and found in Escherichia coli, 

*Address correspondence to Thisbe K. Lindhorst: Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, D-24098 

Kiel, Germany; E-mail: tklind@oc.uni-kiel.de

Ligands for FimH Synthesis and Biological Applications of Glycoconjugates 13 

Klebsiella pneumoniae, Pseudomonas aruginosa, Salmonella spp., Serratia marcencens, and Shigella felneri. Type 

1 pili are present in at least 90% of all known uropathogenic strains of Escherichia coli (UPEC), which are the main 

cause of urinary tract infections in humans, and they have been shown to be important virulence and pathogenicity 

factors [7, 8]. They mediate agglutination of guinea pig red blood cells in an -mannose-inhibitable manner and are 

responsible for bacterial binding to a wide range of glycoproteins carrying one or more N-linked high-mannose type 

oligosaccharides [9]. 

Type 1 fimbriae are uniformly distributed on the bacteria, commonly between 100 and 400 per cell. Their length 

varies between 0.1 to 2 micrometer, their width is ~7 nm. They are composite structures in which a short tip-fibrillar 

structure containing the mannose-specific adhesin FimH is joined to a rod composed predominantly of FimA 

subunits. At least two genetically distinct type 1 fimbriae with distinct molecular compositions and receptor binding 

profiles exist, the E. coli type 1 fimbriae (type 1 E ) and Salmonella type 1 fimbriae (type 1 S ) [10]. In this review, the 

E. coli type 1 E fimbriae will be referred to simply as type 1 fimbriae. 

Type 1 fimbriae are relatively rigid, highly oligomeric, proteinaceous appendages, anchored to the outer membrane 

of Gram-negative bacteria. Individual fimbriae can be seen extending at various angles from the cell surface in 

electron micrographs of type 1 fimbriated bacteria. They consist of a cylindrical rod comprising repeating 

immunoglobulin-like (Ig) FimA pilin subunits, and a short and stubby tip, named fibrillum [11]. They are assembled 

by the chaperone/usher pathway (vide infra) and in their mature form the Ig fold of every subunit is completed by an 

amino-terminal extension from a neighbouring subunit in a process termed “donor strand exchange” (DSE). 

Type 1 fimbriae can easily be visualized in the electron microscope as thin structures extending from the bacterial 

cell surface (Fig. 1). The rod of type 1 fimbriae is a right-handed helical structure with 27 FimA subunits in the 19.3 

nm helical repeat (eight turns) and a 2.1-2.5 nm-wide central axial hole. Type 1 fimbriae consist of some hundreds 

to thousands of FimA subunits and of a few percent minor fimbrial components including the fimbrial adhesin FimH 

that is responsible for receptor binding. Like the other components of type 1 fimbriae the FimH adhesin is encoded 

in the fim gene cluster. 

Figure 1: Electron micrograph of type 1 fimbriated Escherichia coli bacteria; zoomed in on the right. 

THE fim GENE CLUSTER 

Type 1 fimbriae are encoded by the fim gene cluster including all genes that are necessary for the expression and 

assembly of type 1 fimbriae. The fim operon contains an invertible region and 9 genes, fimA to fimI, of which four 

gene products, FimA, FimF, FimG, and FimH, form the fimbrial rod (Fig. 2) [12, 13]. FimA is the major subunit of 

type 1 fimbriae and FimH is the adhesin. Proteins FimB, FimC, FimD, and FimE are needed in pilus assembly. The 

function of FimI is somewhat uncertain so far, however, when lesions in fimI were created by site-directed 

mutagenesis a piliation-negative phenotype was obtained. This finding suggests that the product of the fimI gene is 

necessary for E. coli type 1 pilus biosynthesis [14].

14 Synthesis and Biological Applications of Glycoconjugates Thisbe K. Lindhorst 

Figure 2: The gene cluster that is required for type 1 fimbriae assembly comprises 9 genes, fimA to fimH. The genes depicted in 

bold encode the proteins FimA, FimF, FimG, and the adhesin FimH, which together constitute the fimbrial fibre. 

Each type 1 pilus is composed of 500 to 3000 copies of FimA and a short linear tip fibrillum formed by the adhesin 

FimH and the minor subunits FimF and FimG. Unlike all other subunits of type 1 fimbriae, FimH features an aminoterminal 

lectin domain, which mediates -D-mannose-specific binding to glycoprotein receptors. 

PILUS ASSEMBLY: THE CHAPERONE/USHER PATHWAY 

Attachment to host cells via adhesive surface structures is a prerequisite for the pathogenesis of many bacteria. 

Uropathogenic Escherichia coli assemble P and type 1 pili for attachment to the host urothelium. Assembly of these 

pili, like assembly of over 25 other adhesive organelles in Gram-negative bacteria, requires the conserved 

chaperone/usher pathway, in which a periplasmic chaperone controls the folding of pilus subunits and an outer 

membrane usher provides a platform for pilus assembly and secretion. 

Figure 3: Schematic representation of the chaperone-usher pathway for assembly of E. coli type 1 pili. Subunits are represented 

as oval shapes colored by subunit type. The two-domain FimH adhesin at the fimbrial tip is colored in green, FimA in light blue 

and the periplasmic chaperone and the OM usher are colored in yellow and blue, respectively. Blowout topology diagrams of 

donor-strand complemented (lower right) and donor-strand exchanged (upper right) subunits are shown. The subunit’s Nterminal 

extension is labeled (Nte). Reprinted from Cell, 133 / May 16, H. Remaut, C. Tang, N. S. Henderson, J. S. Pinkner, T. 

Wang, S. J. Hultgren, D. G. Thanassi, G. Waksman, H. Li, Fiber Formation across the Bacterial Outer Membrane by the 

Chaperone/Usher Pathway / 641, Copyright (2008), with permision from Elsevier.


Multivalent Glycocalixarenes 

Francesco Sansone, Gabriele Rispoli, Alessandro Casnati * and Rocco Ungaro 



CHAPTER 3 

Dip.to di Chimica Organica e Industriale, Università degli Studi, Parco Area delle Scienze 17/A, 43124 Parma, Italy 

Abstract: Calixarenes, the cyclic oligomers obtained by condensation of phenols or resorcinols with aldehydes, are 

ideal scaffolds for the construction of multivalent glycosylated ligands with unique properties. Thanks to the 

possibility that we can vary their size, valency and conformation and finely tune the topology of the saccharide units 

in the space, a wide variety of glycocalixarenes could have been prepared in the last 15 years. In this account, after a 

brief review on the basic chemistry of calixarenes, we will focus the attention on the most convenient methodologies 

used so far for the synthesis of glycocalixarenes and on their aggregation and biological properties. Glycocalixarenes 

often show, in fact, an impressive ability in the recognition and inhibition of carbohydrate binding proteins (lectins) 

disclosing remarkable multivalent effects. The amphiphilic character, due to the simultaneous presence of highly 

hydrophilic saccharide units and of a lipophilic aromatic backbone, confers to glycocalixarenes a marked tendency to 

self-aggregate in water solution. A quite unique peculiarity of these glycoclusters is related to their combined ability 

to firmly bind small organic molecules and to strongly and selectively interact with lectins, which makes them ideal 

candidates for tomorrow’s site-specific drug-delivery systems. Other important biological functions have been 

envisaged to be influenced by the multivalency of glycocalixarenes which span gene-delivery, inhibition of tumor 

cell migration and proliferation to stimulation of the immunogenic system. 

Keywords: calixarenes, multivalent effect, lectin inhibition, tumor targeting, cell transfection. 

INTRODUCTION 

Saccharides are not only fundamental chemical energy sources and structurally important elements, but also play a 

fundamental role in a wide range of biological processes [1]. Many important physiological and pathological events, 

such as intercellular communication, cell trafficking, immune response, infections by bacteria and viruses, growth and 

metastasis of tumour cells, occur due to the recognition of carbohydrate moieties by the corresponding cell protein 

receptors. These proteins, which lack immune and enzymatic activity, are known as lectins [2]. The communication 

between carbohydrates and lectins is so specific that it is frequently referred to as Sugar Code [3]. Interestingly, lectins 

are frequently characterized by the presence of equivalent binding sites for carbohydrates so that, although the affinity of 

a single saccharide unit for the receptor is usually low, the simultaneous presentation of several proper and identical 

glycoside units to lectins, ensures a strong and specific interaction. This phenomenon has been named glycoside cluster 

effect [4], a particular and widely spread case of multivalency [5], and the multivalent neoglycoconjugates which are 

synthesised to interfere with all these important phenomena are also called glycoclusters. 

MULTIVALENCY 

Multivalency is the ability of a particle (or molecule) to bind another particle (or molecule) via multiple, and 

simultaneous noncovalent interactions (Fig. 1) [5]. The valency is the number of ligating functionalities of the same 

or similar type connected to each of these entities. 

Even when the single interactions are rather weak (with dissociation constants Kd in the mM range), thanks to the 

simultaneous operation of multiple attractive interactions, multivalency is usually characterised by high 

thermodynamic (Kd in the subnanomolar range) and kinetic stability. Nowadays it is clearly ascertained that 

multivalency controls many important biological processes [1, 5]. Nature exploits multivalency to convert relatively 

weak interactions (e.g. carbohydrate-protein interactions) into strong and specific recognition events. It is therefore 

clear why, in the latest years, Supramolecular Chemistry, the chemistry of noncovalent interactions, became more 

*Address correspondence to Alessandro Casnati: Dip.to di Chimica Organica e Industriale, Università degli Studi, Parco Area delle Scienze 

17/A, 43124 Parma, Italy; E-mail: casnati@unipr.it

Multivalent Glycocalixarenes Synthesis and Biological Applications of Glycoconjugates 37 

and more interested in applying the multivalency concepts to the recognition of biological important molecules [6-8] 

and to nanotechnology [9]. Interestingly, supramolecular systems, often being simpler than the natural ones, can help 

the understanding and quantitative description of the multivalent effect [10]. Although multivalent ligands may 

remarkably differ for their topology (Fig. 2), they generally consist of a main core, called scaffold, which bears several 

covalent connections, linkers or spacers, to the peripheral ligating (binding) units. In principle, any multivalent scaffold 

can be used, from those having low valency such as benzene derivatives, monosaccharides, transition metal complexes, 

azamacrocycles, cyclodextrins or calixarenes to high valency ones such as dendrimers, polymers, peptoids, proteins, 

micelles, liposomes, and self-assembled monolayers (SAMs) on nanoparticles or surfaces (Fig. 2). 

Figure 1: a) Monovalent and b) multivalent complex formation. 

Figure 2: Different types of multivalent ligands. a) 1-D finite and linear arrangement; b) polymers/peptoids; c) 2-D 

cyclic/macrocyclic structures; d) 2-D self-assembled monolayers (SAM) on Au/quartz; e) 3-D cavity containing scaffolds 

(cyclodextrins/calixarenes); f) nanoparticles/dendrimers/liposomes. 

A quite useful approach to describe multivalent binding and based on the additivity of the free energies [11, 12] was 

proposed, where the standard binding free energy for the multivalent interaction G ° multi is 

G ° multi = nG ° mono + G ° interaction 

(eq. 1) 

where G ° mono is the standard binding free energy of the corresponding monovalent interaction, n is the valency of 

the complex and G ° interaction is the balance between favourable and unfavourable effects of tethering. 

A more qualitative but often rather helpful parameter, named or enhancement factor, was introduced by 

Whitesides [5]. 

It is defined as = Kmulti/Kmono 

(eq. 2) 

where Kmulti and Kmono are the association constants for the multivalent and monovalent complexes, respectively.

38 Synthesis and Biological Applications of Glycoconjugates Sansone et al. 

A similar parameter is the relative potency (rp) often used to compare the IC50 (concentration of a 

substance/inhibitor needed to inhibit a given biological process by half) of a multivalent (IC50 mult ) and a monovalent 

(IC50 mono ) ligand. It is defined as 

rp = IC50 multi /IC50 mono 

(eq. 3) 

If the valency n of the cluster is known, the enhancement factor and the relative potency rp can be normalised to n, 

giving rise to the parameters /n and rp/n which are also quite useful to compare the efficiency of ligands having 

different topology, valency and linker. Ligands exhibiting high /n or rp/n values (> 1) are efficient 

ligands/inhibitors and show a positive multivalent effect, while low /n or rp/n values (< 1) clearly indicate a lower 

affinity/inhibition than the monovalent ligand and therefore are characterised by a negative multivalent effect. 

Kitov and Bundle [12] described a more rigorous approach to evaluate a multivalent binding process (Fig. 3). In this 

model, the standard free energy of the multivalent interaction, G ° avidity, is a function of three terms G ° inter (for the 

first intermolecular step), G ° intra (for the second intramolecular step) and a statistical term S ° avidity, namely avidity 

entropy, calculated on the basis of the topology and valency of the complex. The avidity entropy can increase 

rapidly with the valency of the complex, always favouring binding and widely counterbalancing the loss of 

conformational entropy which takes place during multivalent complex formation. Very important is the choice of the 

linker between the scaffold and the ligating units. It should be of the proper length to allow the simultaneous binding 

of all the ligating groups, without generating enthalpic unfavourable strains (enthalpically diminished binding). 

Figure 3: Inter- and intramolecular steps for the formation of a multivalent complex or of an intermolecular aggregate. 

CALIXARENES AS MULTIVALENT SCAFFOLDS 

Calixarenes recently turned out to be an important class of scaffolds for the synthesis of multivalent ligands to be 

used both in bioorganic chemistry and nanotechnology. The name calixarene [13], originally attributed by David C. 

Gutsche in 1978 to indicate the [1n]-metacyclophane (I) obtained by the condensation of para-substituted phenols 

and formaldehyde (Fig. 4), is due to the cup-like shape of these macrocycle which resembles that of a greek vase 

(Fig. 5b) named calix in latin. This name was later on associated also to other macrocyclic compounds having 

similar structure such as resorcinarenes (or calixresorcinarenes or resorcarenes), thiacalixarenes, oxacalixarenes, 

azacalixarenes and, more recently, calixpyrroles and pillarcalixarenes [6, 13-17].




CHAPTER 4 

Solving Promiscuous Protein Carbohydrate Recognition Domains with 

Multivalent Glycofullerenes 

Yoann M. Chabre and René Roy * 


Succ. Centre-ville, Montréal, Québec, Canada H3C 3P8 

Abstract: Studies on multivalent carbohydrate protein interactions critically depend on the nature of protein’s binding 

sites, their number and also their relative three dimensional orientations. Although a wide range of neoglycoconjugates 

including glycodendrimers has been synthesized to address this issue, no systematic rules exist yet that can predict the 

optimal shapes, size, and number of required exposed carbohydrate ligands. This chapter will illustrate a few examples 

of bacterial and human lectins together with bacterial toxins having varied number of carbohydrate recognition domains 

that necessitated multivalent glycoconjugates. In particular, fullerenes, because of their particular physical properties, 

have been used to describe novel synthetic strategies and possible fit with concomitant lectins. 

Keywords: glycofullerenes, multivalent inhibitors, glycosidase inhibitors, Bingel's cyclopropanation, Prato reaction, 

Click chemistry. 

INTRODUCTION 

The past few years have witnessed the discovery, development and, in some cases, large-scale production and 

manufacturing of novel materials that lie within the nanometer size scale. More particularly, the incorporation of 

nanotechnologies in early-stage development of new drugs, diagnostics, or therapeutics constitutes a powerful addition 

to the arsenal of classical macromolecular structures. Consequently, this strategy opens new avenues for the original 

and efficient design of adapted nanomedicines. In this context and owing to their three-dimensional architectures, 

fullerenes constitute promising candidates for novel nanometric constructs having biological properties and offering 

challenging scaffolds toward multivalent presentation of biologically relevant saccharide motifs. 

Fullerenes, the third allotropic form of carbon along with graphite and diamond, are an original class of spherically 

shaped molecules made exclusively of carbon atoms. They have generated much enthusiasm and numerous research 

efforts during the past few years [1]. Hence, the chemical and physical features of C60, also named 

Buckminsterfullerene, the most representative example among the fullerenes, have extensively been explored. Their 

intrinsic properties such as their size, hydrophobicity, three-dimensionality, and electronic properties have made 

them very promising nanostructures, offering interesting features at the interface of various scientific disciplines, 

ranging from material sciences [2] to biological and medicinal chemistry [3-5]. 

In order to demonstrate and illustrate their attractive potential in the foregoing applications, an array of studies has 

been recorded, including cytotoxicity investigations on both unmodified and functionalized fullerenes. Initial results 

have indicated that these novel and fascinating architectures were not carcinogenic when applied to the skin, nor did 

they affect the proliferation and the viability of cells when they are internalized. Hence, despite cases of dosedependent 

toxicity reported for certain related derivatives, early observations point to pledge for applications in 

several arenas such as: DNA cleavage, photodynamic therapy, enzymatic inhibition, antiviral, antibacterial, and 

antiapoptotic activity [4-6]. However, the total lack of solubility in aqueous or physiological media constitutes 

severe drawbacks for their quick and efficient development as suitable biomolecular carriers. In order to circumvent 

the natural repulsion of fullerenes towards water, numerous methodologies have been adopted, including their 

entrapment into tailored microcapsules, suspension with the help of co-solvents, and chemical derivatization, 

notably their introduction onto peripheral solubilising appendages. Furthermore, it has been shown that the 

*Address correspondence to Rene Roy: Pharmaqam – Groupe de Recherche en Chimie Thérapeutique, Université du Québec à Montréal, P.O. 

Box 8888, Succ. Centre-ville, Montréal, Québec, Canada H3C 3P8; E-mail: roy.rene@uqam.ca

Solving Promiscuous Protein Carbohydrate Recognition Domains Synthesis and Biological Applications of Glycoconjugates 65 

multivalent presentation of polar groups around the fullerene spheres can prevent clustering phenomena in 

reasonably dilute solutions and consequently increase the hydrosolubility of the resulting conjugates. In this context, 

a variety of chemical functionalities has been utilized to increase both the hydrophilicity (with groups such as OH, 

CO2H, NH2, quaternary ammonium, and cyclodextrin) and to prepare novel compounds possessing valuable 

biological and pharmacological activity. Diversified common reactions, including oxygenations, halogenations, 

radical and nucleophilic additions, and cycloadditions have been utilized toward this goal [7-9]. 

THE LOGIC OF MAKING MULTIVALENT CARBOHYDRATE LIGANDS 

Amongst the extensive panel of neoglycoconjugates [10], intricate fullerenoglyco-conjugates (also termed 

glycofullerenes) in particular display an exquisite combination of appealing properties regarding both watersolubility 

and biological significance. The spherical character of fullerenes has provided suitable scaffolds for 

multivalent carbohydrate presentation at the periphery of the molecules and valuable reactions have been adapted for 

their efficient and controlled covalent attachment. 

Since most carbohydrate binding proteins display rather low affinity and generally narrow carbohydrate recognition 

domains (CRDs) [11-15] involving fewer than a tetrasaccharide residue, their intrinsic specificities often reside in their 

valencies (number of CRDs per protein) together with their various topologies. The number of CRD in this ubiquitous 

family of proteins may vary as much as from one (Galectin-3) [16-18] up to fifteen (Shiga toxin) (Fig. 1) [19-21]. 

Figure 1: Monovalent Galectin-3 with bound LacNAc (PDB 1A3K). Linear dimeric Burkholderia cenocepacia lectin (Bcla, 

PDB 2WRA). Rectangular tetrameric Pseudomas aeruginosa PAIL lectin bound to Gal1-3Gal1-4Glc trisaccharide (PDB 

2VXJ). Tetrahedral tetrameric Pseudomonas aeruginosa PA-IIL bound to trisaccharide Lewis a (PDB 1W8H). Pentameric 

Cholera toxin B subunit (CTB5) bound to five GM1-oligosaccharide (PDB 3CHB). Shiga-like toxin-I complexed with 15 P k 

trisaccharide ligands (PDB 1BOS).

66 Synthesis and Biological Applications of Glycoconjugates Chabre and Roy 

Hence, the design of glycofullerenes possessing numerous copies and orientation of carbohydrate ligands may help 

decipher our understanding of challenging and promiscuous CRDs and to provide assistance in the observed mixed 

and matched QSAR-multivalency phenomenon. 

The first glycofullerene syntheses were directed toward monovalent architectures that were subsequently tailored 

and optimized to generate multivalent glyconanostructures based on C60 where biological properties were enhanced 

by taking advantage of the “glycoside cluster effect” [22]. 

MONOVALENT C60 

Vasella et al. first reported the elegant synthesis of monoglycosylated fullerenes via the glycosylidene carbene 

precursor 1 which generated enantiomerically pure spiro C-linked glycosylated-C60 derivative 2 (Scheme 1) [23]. A 

similarly direct conversion of C60 with various glycosyl azides 3 provided monovalent fullerene glycoconjugates 4-8 

via thermal cycloaddition in boiling chlorobenzene [24]. Although this method afforded mixtures of two inseparable 

stereoisomers in relatively poor yields (13 to 28%), the generality of the reaction has been demonstrated with a 

series of useful mono- (4, D-glucopyranose and 5, D-galactopyranose), di- (6, lactose and 7, maltose), and trisaccharide 

(8) glycofullerenes. The adaptation of this direct cycloaddition-based strategy has also furnished 

interesting results under ultrasonication conditions, leading to various glycosyl C70 derivatives from various 2azidoethyl 

per-O-acetyl glycopyranosides [25]. However, yields for major closed [5,6]-bridged isomers remained 

lower than 10% and mixtures of inseparable stereoisomers were unavoidably obtained. 

RO RO 

RO RO 

1 

OR 

O 

OR 

OR 

O 

RO 

N 

N 

2 

PhMe 

r.t. or 45°C 

55% 

R 2 

AcO 

R 1 

R 2 

AcO 

R 1 

AcO 

OAc 

O 

AcO 

OAc 

O 

N 

C 60 

N 3 

PhCl, , 7-10h 

MeNHCH 2CO 2H (10) 

PhMe, 

4 R 1 = H , R 2 = OAc (28%) 

5 R 1 = OAc , R 2 = H (18%) 

6 R 1 = H , R 2 = O--Gal(OAc) 4 (13%) 

7 R 1 = H , R 2 = O--Glc(OAc) 4 (18%) 

8 R 1 = H , R 2 = O--Mal(OAc) 7 (16%) 

Scheme 1: Direct syntheses of monovalent glycofullerenes introduced onto C 60. 

3 

OR 1 

R 

RO 

2O Me 

N 

 

9 

RO 

OR 

O 

RO 

O 

RO 

11 R = R 1 = Bn , R 2 = H (14%) 

or 

R = R 2 = Bz , R 1 = H (10%) 

Shortly afterwards, a three-component approach involving C60, a carbohydrate aldehyde (9) and N-methylglycine 10 

(sarcosine) was developed leading to the formation of glycofullerenopyrrolidine monocycloadducts (11, Dgalactopyranoside 

or D-glucopyranoside) in low yields (10-14%). The reaction proceeded via 1,3-dipolar cycloaddition 

from the intermediate azomethine ylide to C60 (Prato reaction) [26]. The feasibility of this straightforward transformation 

was nevertheless demonstrated with the use of a family of 1-deoxy-1-C-formyl derivatives of galactopyranose, 

glucopyranose and mannofuranose. Additionally, this methodology has been advantageously adapted toward the 

syntheses of C60 derivatives containing a 6-(β-D-glycopyranosylamino)pyrimidin-4-one unit [27]. 

Alternative approaches using the initial introduction of suitable chemical functionalities onto the C60 fullerene have 

been adopted by other research groups, allowing conjugation of the saccharide moieties with complementary 

CHO 

OR 

OR2 OR 1


Monovalent and Multivalent Inhibitors of Bacterial Toxins 

Edward D. Hayes and W. Bruce Turnbull* 



CHAPTER 5 

School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK 

Abstract: Cholera and travellers’ diarrhoea are caused by AB 5 protein toxins that bind to ganglioside GM1 at the 

surface of the cells lining the intestine. Inhibition of this protein-carbohydrate interaction would prevent the toxin 

from entering the cells, and thus prevents toxin-induced diarrhoea. In this review we will describe the structures 

of the cholera and E. coli heat-labile toxins, and summarize the main strategies that have led to the development 

of monovalent and multivalent inhibitors of these toxins. A number of key design concepts emerge from these 

studies including the importance of pre-organization of the sugar residues within the monovalent ligands, and also 

the pre-organization of monovalent ligand groups within larger multivalent ligands. The importance of chelation 

and protein aggregation as mechanisms of multivalent inhibition is also discussed. 

Keywords: bacterial toxin, multivalency, dendrimer, cholera, inhibitor. 

INTRODUCTION 

Cholera is a potentially fatal bacterial infection, with symptoms that include ‘rice water’ diarrhoea and vomiting [1]. The 

associated fluid loss can reach up to 30 litres per day [2], resulting in severe dehydration and, without treatment, death 

can occur within hours [3]. It is still a major world health issue; for example, the recent epidemic in Zimbabwe lasted 

almost a year with over 98,000 cases and over 4,000 deaths recorded by July 2009 [4]. In 2007, an epidemic in Angola 

resulted in 82,204 cases and 3,092 deaths [5]. Cholera is not only an epidemic threat to the developing world, it is also 

endemic in South Asia and sub-Saharan Africa [6]. World Health Organisation (WHO) Figures show that there were 

190,103 reported cases of cholera in 2008; an increase of 7.6% compared to 2007, and there was also a 27% increase in 

fatal cases [7]. It is believed that reported cases may only correspond to 5-10% of the true number [8]. 

Cholera is caused by cholera toxin (CT) which is secreted by the Gram-negative bacterium Vibrio cholerae [9]. It is 

a water-borne bacterium, and infections generally occur from contaminated food and water sources. As water 

sources are often shared by large numbers of people, the disease can spread rapidly through whole communities [9], 

and across large geographical areas, thus increasing its impact. The 2008-2009 Zimbabwe outbreak affected all ten 

provinces in that country [7], and also spread to both Zambia and South Africa [4]. 

A closely related disease is travellers’ diarrhoea, which is largely caused by enterotoxigenic Escherichia coli [10]. 

These E. coli strains produce a protein toxin called heat-labile enterotoxin (LT) [11], which is very similar to cholera 

toxin in structure [12], mode of action [13], and in the resulting symptoms [10]. As with cholera toxin, LT causes 

diarrhoea, however the effects are less severe [14]. Nevertheless, heat-labile enterotoxin is believed to cause up to 1 

billion cases of diarrhoea a year, with 300-400 million of these cases in children under the age of five [2]. The 

resulting diarrhoeal dehydration is estimated to be the cause of 300,000-500,000 deaths annually [10]. 

TOXIN STRUCTURE AND FUNCTION 

Cholera toxin and heat labile enterotoxin both have an AB5 structure (Fig. 1) [15, 16], and share 80% sequence 

identity in both the A and B-subunits [17]. 

The A-subunit of CT (CTA) and LT (LTA) is responsible for the toxic activity of the proteins; it comprises two distinct 

parts A1 and A2 (Fig. 1b). The A1-component has a toxic enzyme activity, while the A2-component non-covalently 

links the A-subunit to the B-subunit (Fig. 1c) via an extended helix [18]. When cholera toxin binds to the cell, the whole 

toxin is transferred into the cell via receptor mediated endocytosis (Fig. 2). The toxin is then transported to the 

endoplasmic reticulum (ER), at which point the A1-subunit dissociates from the rest of the toxin assembly [19]. 

*Address correspondence to W. Bruce Turnbull: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of 

Leeds, Leeds LS2 9JT, UK; E-mail: w.b.turnbull@leeds.ac.uk

Monovalent and Multivalent Inhibitors of Bacterial Toxins Synthesis and Biological Applications of Glycoconjugates 79 

Figure 1: The crystal structure of the cholera toxin protein solved at a resolution of 1.90 Å (Protein Data Bank (PDB) accession 

code 1S5E). (a) Elevation view of the AB5 holotoxin (the enzymatically active A1 component is shown in blue, the A2 

component is green and the B-subunits are red; (b) The A-subunit showing the peptide that extends through the centre of the B 5 

pentamer; (c) plan view of the B5 pentamer. 

The process of toxin delivery to the cytosol requires that the A-subunit is first nicked by a protease, and then the 

disulfide group that still connects the A1- and A2-peptides must be reduced by peptide-disulfide isomerase in the ER 

[20, 21]. The origin of the protease differs for CT and LT. In the case of CT, proteolytic cleavage involves a 

protease secreted by V. cholerae [20, 22], whereas the A1-subunit of LT is cleaved by intestinal trypsin [23]. 

Following cleavage from the toxin assembly, the A1-subunit is translocated to the cytosol, where it catalyses the 

NAD-dependent ADP ribosylation of arginine 201 of the stimulatory G protein (Gsα) [21]. This modification locks 

the Gsα in its GTP-bound conformation resulting in the continual stimulation of adenylate cyclase. The increased 

production of cyclic AMP causes an electrolyte imbalance and water loss into the intestine, leading to potentially 

fatal diarrhoea [19, 24-30]. 

Figure 2: A cartoon depiction of the binding of cholera toxin to the cell and the subsequent transfer of the A-subunit into the cell. 

The B-Subunit Lectin and its Carbohydrate Ligand Ganglioside GM1 

The B-subunits of CT (CTB) and LT (LTB) are homo-pentamers. Each of the five monomer units comprise two 

alpha helices and two three-stranded beta sheets (Fig. 1c) [31, 32], which come together to form a doughnut-shaped 

structure, with a central pore into which the C-terminal helix of the A2-subunit extends (Fig. 1a) [33]. The natural 

cell surface ligand for both CT and LT is the GM1 ganglioside 1 (Fig. 3c), which comprises a branched 

pentasaccharide that is anchored to the surface of intestinal epithelial cells via a ceramide lipid [34]. Each B-subunit 

protomer has a single GM1 binding site, allowing CT and LT to bind up to five GM1 gangliosides simultaneously 

(Fig. 3a-b). The binding mode of GM1 1 to cholera toxin has been described as a “2-fingered grip” by Merrit et al. 

[35, 36]. The “thumb” is the sialic acid sugar residue (coloured red in Fig. 3c) and the “forefinger” is the terminal 

galactose (blue) and the N-acetylgalactosamine sugar residue (green) [21]. 

The binding interaction between GM1 and CTB/LTB has been studied extensively by several research groups [37- 

43]. A detailed isothermal titration calorimetry (ITC) study by Turnbull et al. [44] determined that the GM1os ligand 

binds CTB with a dissociation constant (Kd) of 40 nM, making it one of the strongest protein-carbohydrate 

interactions known. In contrast, all of the mono- or disaccahride fragments of GM1os bind much more weakly; for

80 Synthesis and Biological Applications of Glycoconjugates Hayes and Turnbull 

example, the terminal galactose residue displays a 15 mM Kd, which improves only two-fold in the case of the full 

Gal-GalNAc “forefinger”. The sialic acid residue binds even more weakly to the protein (Kd ca. 200 mM). Of all the 

individual sugar residues, it is the terminal galactose sugar residue that contributes the largest proportion of free 

energy of binding when CTB binds to GM1 [44]. 

Figure 3: The 1.25 Å crystal structure (PDB 3CHB) of the five B-subunits of cholera toxin with GM1 oligosaccharide (GM1os) 

bound (shown in blue): (a) plan view; (b) elevation view; (c) Ganglioside GM1 1: the pentasaccharide comprises two galactose 

residues (blue), an N-acetylgalactosamine sugar residue (green), a glucose residue (pink), and a sialic acid residue (red). The 

pentasaccharide is linked to the cell surface via a ceramide lipid. 

Structural Differences Between LT and CT 

The widely used abbreviation of heat labile enterotoxin (LT) is slightly ambiguous, as it is used to indentify two 

very similar, but distinct proteins: LTh and LTp [13, 45, 46]. LTh is the toxin produced by an E. coli strain isolated 

from a human sample [47, 48], while LTp originates from an E. coli strain isolated from porcine samples [49]. They 

both share 80% sequence identity with CT in both A- and B-subunits, and sequence analysis of the B-subunits of 

LTp (LTBp) and LTh (LTBh) show they have a sequence identity of 96%. From the perspective of designing CT/LT 

inhibitors, it is in the GM1 binding site where the most significant difference between the three proteins occurs; 

amino acid residue 13 is a histidine in both CT and LTh, whereas in LTp, residue 13 is an arginine (Fig. 4) [50, 51]. 

Figure 4: Comparison of the GM1 binding sites of CTB (green, PDB 3CHB), LTBh (blue, PDB 2O2L) and LTBp (red, PDB 

1EFI). The difference at residue 13 is highlighted. 

As the GM1 binding sites of CTB and LTBh are identical, it can be assumed that inhibitors designed to inhibit the 

CTB-GM1 interaction will also inhibit the LTBh-GM1 interaction. There is also a high possibility that these 

inhibitors would also perturb the LTBp-GM1 interaction. This hypothesis is reinforced by work carried out by Fan 

et al. [52] who studied m-nitrophenyl α-D-galactopyranoside as an inhibitor of CTB and LTBp; the crystal structure 

of this ligand bound each protein was solved, and it was found that the ligand resided in identical positions in both 

cases (Fig. 5).




CHAPTER 6 

Monovalent and Multivalent Glycoconjugates as High Affinity Ligands for 

Galectins 

Sébastien Vidal * 

Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie Organique 2, UMR 

5246, Université Lyon 1 and CNRS, F 69622 Villeurbanne, France 

Abstract: The biological implications of lectins have prompted a large number of research projects at the interface 

between biology and chemistry for a better understanding of their roles. Several synthetic high affinity ligands have 

been designed in order to inhibit their negative effects such as bacterial or viral infections and cancer. Among these 

receptor proteins, galectins are galactose-binding lectins implicated in inflammation or cancer and are important 

biological targets for the design of treatment against cancer. The careful design of high affinity ligands for galectins 

has been investigated through several studies using either (1) a “medicinal chemistry” approach in which the native 

ligand (i.e. galactose) is modified on one or several positions or (2) based on a multivalent approach in which 

galactose is repeated n times at the periphery of a core scaffold. Both strategies yielded essential information about the 

fundamental aspects of galectin-ligand interactions and provided a better knowledge of the implications of galectins in 

biology. The present review with 130 references will focus particularly on the past decade and present the most recent 

results obtained in this field for monovalent and multivalent ligands of galectins. 

Keywords: Galectins, multivalency, inhibition, ligand, structure-activity relationship. 

INTRODUCTION 

Biological processes are frequently governed by the biomolecular interaction of host-guest systems either in a 

reaction pathway (e.g. enzymes transforming its substrate into a product) or in a recognition system between a 

protein and its ligand. Among these particularly important set of binding events, lectin-carbohydrate interactions are 

playing a major role in several pathologies such as viral or bacterial infection, cancer metastasis, cell-cell 

communication of even fecundation. Lectins [1] are multimeric protein capable of binding selectively to a 

carbohydrate motif (i.e. mono- or oligosaccharides) and do not display any enzymatic activity (e.g. hydrolysis of a 

glycosidic bond). They are classified in different families according to their binding properties for a specific 

carbohydrate. Lectins can be considered as biological targets for the design of therapeutic agents in cancer [2, 3] or 

in targeted drug delivery systems [4]. The galactose binding lectins are called galectins and play a prominent role in 

several biological pathways. The present review will cover the recent progress for the design of high affinity ligands 

of galectins and their potential therapeutical applications. Chemists have developed two main approaches to obtain 

potent ligands of these lectins through modifications of the carbohydrate scaffold in order to maximize the 

interaction with the lectin’s binding pocket or through multivalent glycoconjugates taking advantage of the “cluster 

glycoside effect” [5]. The different reviews cited in this manuscript should therefore be considered in order to reach 

a more general overview of this particular topic of galectins and their numerous implications in biology. 

GENERAL ASPECTS OF GALECTINS 

Different Classes of Galectins 

Galectins [6-11] were identified in the late 1970s and further characterized in the early 1980s [12]. They share a 

common sequence of roughly 130 amino acids and crystallography has been used to determine their structure [12]. 

Galectins are proteins defined by at least one carbohydrate recognition domain (CRD) capable of binding selectively 

to -galactosides and presenting a minimum sequence homology [10]. They usually bind to N-acetyllactosamine 

(LacNAc) or lactose mainly through the recognition of the -galactose residue [13]. There are about 15 mammalian 

galectins identified and they can be divided into three families if they display either one or two CRD(s) with the 

*Address correspondence to Sebastien Vidal: Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie 

Organique 2, UMR 5246, Université Lyon 1 and CNRS, F 69622 Villeurbanne, France; E-mail: sebastien.vidal@univ-lyon1.fr

Monovalent and Multivalent Glycoconjugates as High Affinity Synthesis and Biological Applications of Glycoconjugates 93 

additional chimera type [9, 14, 15]. Galectins are soluble cytosolic proteins and their quaternary structure depends 

on their concentration and environment [15]. Below a concentration of 10 µM, galectins are usually present as noncovalent 

symmetric dimers while some members may occur as trimers and tetramers (Fig. 1) [15, 16]. 

Figure 1: Schematic representation of galectins aggregation (adapted from [16]). 

Galectins-1, -2, -5, -7, -10, -11, -13, -14 and -15 are characterized as monomers but their biologically active form is 

a monomer for galectins-5, -7 and -10 while galectins-1, -2, -11, -13, -14 and -15 require the formation of soluble 

homodimers (Fig. 1A). Galectin-3 is a chimera protein [16] oligomerizing in solution with a C-terminus CRD and a 

100-150 proline-glycine rich peptide containing phosphorylation sites important for its biological activity (Fig. 1B). 

Nevertheless, in the presence of a glycoconjugate displaying -galactosides, galectin-3 will create multivalent 

aggregates at micromolar concentrations [17] as well as for other galectins [18]. Galectins-4, -6, -8, -9 and -12 are 

composed of two CRDs separated by a short linker peptide of various lengths (Fig. 1C). 

Biological Aspects of Galectins 

Galectins have numerous implications in biology and can function either extracellularly with cell-surface glycolipids 

or glycoproteins but also intracellularly with cytoplasmic or nuclear proteins. They are therefore involved in 

inflammatory diseases [19, 20] or protein trafficking [21] but the main implications of galectins are in cancer as 

modulators of tumour progression [22] or in cell growth and apoptosis [23, 24] as for instance urological cancer 

[25]. Recent reviews have reported the implications of galectin-1 [26], galectin-3 [27-29], galectin-4 [30], 

galectin-7 [31], galectin-8 [32, 33] and galectin-9 [34] in cancer biology. 

Potential Applications 

Several galectins have been identified as targets for the development of therapeutic agents due to their role in 

various pathological disorders. Galectin-1 [35] is expressed in normal and pathological tissues and is implicated in 

cell survival and proliferation [36] as well as regulation of immune response, inflammation, allergies, metastasis or 

even host-pathogen interactions [37]. Galectin-2 was discovered during the cloning of galectin-1 and is thought to 

play a similar function to galectin-1 but in different tissues [38]. The role of galectin-5 is not well established but its 

large expression in erythrocytes strongly suggest an implication in these cells [38]. Galectin-6 was discovered 

during the study of galectin-4 and both lectins cannot be distinguished in terms of tissue expression [38]. They play 

a role in cell adhesion or human colon carcinoma but can also be localized at the plasma membrane and act as a 

stabilizing agent [38]. Galectin-7 was initially identified in keratinocytes [39] but also in the oesophagus and oral

94 Synthesis and Biological Applications of Glycoconjugates Sébastien Vidal 

epithelia, cornea and thymus [40]. A recent study has highlighted potential applications for the design of anticancer 

drugs targeting galectin-7 [41]. Galectin-9 was isolated from spleen tissue of a patient with Hodgkin’s lymphoma 

and 50% of patients have antibodies against this galectin [38]. Among these galactose-binding lectins, galectin-3 is 

probably the most intensively investigated lectin due to its unambiguously identified role in cancer apoptosis [28, 

42], breast cancer [43], metastasis [29] or cancer resistance [44]. 

Design of High Affinity Ligands of Lectins 

The synthesis and discovery of high affinity ligands for lectins in general and galectins more particularly represents 

therefore a powerful and reliable approach for the design of potential therapeutic agents against several diseases 

such as viral of bacterial infection but also cancer as galectins are highly involved in this pathology. 

A first approach has been designed where small molecules can be synthesized and evaluated as ligands of lectins 

and their affinity will be improved by changing functional groups of the native carbohydrate recognition unit of the 

lectin in a medicinal chemistry approach with structure-activity relationships information. In parallel, multivalent 

glycoconjugates have been synthesized [45-47] and identified as powerful ligand of lectins taking advantage of the 

concept of multivalency [48-53] and also the so-called “cluster glycoside effect” [5] where several lectins and/or 

carbohydrates will come together to enhance the affinity between partners in order to reach improved avidities and 

specificities in comparison to monovalent interactions. In both cases, the three-dimensional description of the target 

lectin, ideally by crystallography, is almost required for the rational design of such high affinity ligands. 

The careful analysis of the binding properties for the ligands of galectins designed requires a large set of 

bioanalytical techniques [54]. Frontal affinity chromatography [55, 56] (FAC), hemagglutination inhibition assay 

[13, 57-59] (HIA), microarrays [60], flow cytometry [61] or ELISA [62] assays have been used for the 

determination of the binding properties of several carbohydrate derivatives towards galectins. Surface plasmon 

resonance (SPR) is a powerful technique for studying biomolecular interactions and determining the affinity 

properties of ligands [63]. Isothermal titration calorimetry (ITC) [13, 48, 64, 65] will also provide a large set of 

data for the comprehensive study of lectin-carbohydrate interactions. A recent study has been reported for the 

identification of the carbohydrate-binding preferences of eight human galectins using a carbohydrate microarray 

technology [66]. Seventeen oligosaccharides have been synthesized with a thiol-terminated tether at their anomeric 

center and then incorporated into maleimide-functionalized microarrays. This technology provides a rapid analysis 

and a reliable comparison technique for the determination of the binding properties of lectins in general and more 

particularly for the growing number of galectins identified in the recent years [6-11]. 

Galactose-Containing Mono- and Multivalent Ligands Targeting other Lectins 

Even though galectins are attractive biological target for potential therapeutical applications, several galactose-based 

ligands have been designed and their binding has been studied towards other lectins. Modified galactosides have 

been designed targeting cholera [67] or shiga toxins [68]. Nevertheless, the most abundant literature can be 

collected for monovalent galactose derivatives as ligands for galectin-3 (see Section II). Multivalent systems have 

also been designed where the positioning of the carbohydrate epitopes is dynamic and can adjust itself to the 

targeted lectin using a poly-rotaxane system [69], vesicles [70, 71] or magnetic glycoparticles [72, 73]. Another 

series of multivalent macromolecules have been described incorporating galactose units on a various scaffolds such 

as a phthalocyanine [74], a fullerene [75], -peptides [76], -peptoids [77], oligonucleotides [78-82] or cubic 

shaped silsesquioxanes [83, 84]. Additional multivalent architectures have been reported including a galactose 

epitope (sometimes included in a lactose or LacNAc residue) and biological interactions have been studied with 

agglutinins (PNA [85, 86], WGA/ECA [87], RCA120 [79-82]), HIV-1 rgp120 [88], selectins [89], bacterial 

toxins (CTB and LTBh) [90], bacterial lectins (e.g. PA-IL [91, 92]), the asialoglycoprotein receptor (ASGP-R) 

[93, 94] or as gene delivery systems [95, 96]. This short list is not exhaustive but highlights some recent reports in 

this field and also the possible applications of high affinity ligands of lectins in bacterial or viral infections. 

SMALL MOLECULE INHIBITORS OF GALECTIN-3 

Among the several galectins identified as putative biological targets for the design of potential drugs, the most 

abundant literature can be found for the study of galectin-3 as a key lectin in cancer. Several studies have been


Combinatorial Libraries of Dendritic Glycoclusters 

Jean-Louis Reymond * and Tamis Darbre 



CHAPTER 7 

Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012, Berne, Switzerland 

Abstract: Dendrimers, displaying a regularly branched tree-like structure, offer an optimal synthetic platform to 

explore multivalency effects, in particular to construct bioactive glycoclusters as ligands for lectins. We have 

used combinatorial peptide chemistry to prepare glycopeptide dendrimer libraries as glycoclusters of the general 

structure (sugar-XXX)4(LysXXX) 2LysXXXNH 2 (X = alpha-amino acid, Lys = branching lysine residue). Binding 

assay was performed on such combinatorial libraries bearing an alpha-C-fucoside endgroup, and led to the 

discovery of strong ligands for the fucose specific lectin LecB from Pseudomonas aeruginosa. These dendrimers 

are potent inhibitors of biofilms, and require a tetravalent ligand for activity. In a related approach, involving 

binding assay to cancer cells using galactosylated glycopeptide dendrimers led to drug-delivery dendrimers. In 

both cases the amino acid composition of the dendrimer branches strongly influences the biological activity of the 

glycoclusters in addition to the simple multivalency effects, demonstrating the utility of peptide chemistry for 

systematically optimizing physicochemical and biological properties of the dendrimers. 

Keywords: Combinatorial chemistry, peptides, solid-phase synthesis, C-glycosides, lectins, drug delivery, 

galactose, fucose. 

INTRODUCTION 

Dendrimers, displaying a regularly branched tree-like structure [1-3], offer an optimal synthetic platform to explore 

multivalency effects [4, 5]. Their potential to construct bioactive glycoclusters has been broadly recognized as 

reviewed in several other chapters of this book. Glycoclusters are particularly suitable as multivalent ligands for 

lectins, which are proteins binding to the terminal carbohydrates of glycoproteins or glycolipids by means of 

generally weak but specific binding sites [6, 7]. Lectins usually possess multiple binding sites and form oligomers, 

which enhances binding to carbohydrate epitopes displayed in polyvalent format on cell surfaces. Glycoclusters 

including dendrimers generally bind strongly to lectins by virtue of such multivalency effects [8-13]. 

In principle glycoclusters can be assembled from a variety of structures. Multivalency effects for such glycoclusters 

are computed by comparing their affinity to the specific lectin target with that of a reference monovalent ligand. 

However the higher affinity of a glycocluster might be caused not only by multivalency itself, but also by secondary 

binding interactions of the glycocluster with the lectin outside the carbohydrate group. Furthermore, the glycocluster 

backbone is likely to play a critical role in binding not only by setting the distance between individual carbohydrate 

epitopes, but also by determining their relative orientation, and by influencing the global physicochemical 

properties, in particular its charge, hydrophobicity and aqueous solubility. 

With the aim of addressing the optimization of these parameters in dendritic glycoclusters, we have used 

combinatorial peptide chemistry to prepare glycopeptide dendrimers as glycoclusters, expanding on our 

investigations of peptide dendrimers as synthetic protein models [14-16]. Peptide dendrimers were pioneered early 

on by Denkewalter and coworkers with poly-lysine dendrimers [17] and in the form of multiple antigenic peptides 

by Tam and coworkers [18-21]. However in these previous studies a poly-lysine tree was used for the only purpose 

of providing multivalency without any attempt to vary the peptide backbone for optimization. Our work performs 

such backbone variations in systematic manner using combinatorial solid-phase synthesis of peptide sequences 

featuring diamino acids branching points at every 2 nd , 3 rd or 4 th position. In this manner, peptide dendrimers are 

produced that can be easily varied at the core, within the branches, and at the branches’ ends (Fig. 1). 

*Address correspondence to Jean-Louis Reymond: Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012, 

Berne, Switzerland; E-mail: reymond@ioc.unibe.ch

Combinatorial Libraries of Dendritic Glycoclusters Synthesis and Biological Applications of Glycoconjugates 117 

FmocHN 

160 mg of Rink 

Amide Resin 

0.25 mmol/g 

1) piperidine, DMF 

2) FmocAAOH, BOP, HOBT 

3) repeat 1) and 2) 10 times 

4) 1), then Ac 2O, py 

5) CF 3CO 2H, then HPLC 

H 2N 

O 

HO 

X 7 

X 8 

X 7 

X 8 

X 7 

X 8 

X 7 

X 8 

L2K7 (28 mg, 10 % isolated yield) 

X 5 

X 6 

X 5 

X 6 

X 7 

X 8 

X 5 

X 6 

X 3 

X 4 

X 3 

X 4 

X 5 

X 6 

X 7 

X 8 

X 7 X 8 

X 1 

X 2 

X 7 X 8 

O 

NH 

HN 

O 

NH 

O 

HN H 

HN 

2N 

O 

HN 

H 

O 

O 

NH 

N 

NH 

NH H2N O O 

O NH 

HN O 

HN 

HN 

HN 

H 

O 

3N 

O NH 

O 

NH NH2 H 

O N 

O 

HN 

O NH2 HO 

HO 

NH 

HN 

HN 

O HN 

HO 

O 

O NH 

O 

NH 

O HN 

NH 

NH3 O 

NH 

O 

NH 

O 

HN 

O 

HN 

O 

HN NH 

O 

NH2 NH 

HN 

H2N NH 

O 

O 

HN 

NH2 HN NH2 H2N HN HN 

H 

NH O 

N 

O 

O 

HN 

NH O 

HN 

O 

OH 

NH O 

HN 

N O 

H 

NH H2N H3N 2 

H3N NH3 Figure 1: Synthesis and structure of the 3 rd generation peptide dendrimer L2K7. 

Our use of combinatorial libraries of synthetic glycoclusters in the form of peptide dendrimers to discover cytotoxic 

and lectin binding ligands is discussed below. 

COMBINATORIAL SYNTHESIS, SCREENING, AND DECODING OF DENDRITIC GLYCOCLUSTERS 

SPPS of Peptide Dendrimers and their Combinatorial Libraries 

Dendrimers are usually prepared by solution phase synthesis and convergent assembly to minimize sterical 

hindrance and purification problems. Our approach contradicts these principles: we use a divergent synthesis 

protocol on solid support under the standard conditions of solid phase peptide synthesis with Fmoc-protected amino 

acid building blocks [22-25] to prepare peptide dendrimers up to the fourth generation and up to 16 end groups [26- 

27]. The C-terminal amino acids which are coupled first to the solid support form the core of the dendrimer. 

Branches are grown as diamino acids are added along the sequence. A typical 11-step coupling sequence produces a 

dendrimer of 3 rd generation with branches two amino acids in length, which contains a total of 37 amino acids and 

an average molecular weight of 4,400Daltons, as shown for the aldolase dendrimer L2K7 (Fig. 1) [28]. 

The isolated yields of peptide dendrimers obtained from Fmoc-SPPS after side-chain deprotection, cleavage from 

the solid support and purification by reverse-phase HPLC are comparable to the yields for linear peptides of similar 

size. As for linear peptides, the isolated yields are influenced by the amino acid sequence and by the physicochemical 

properties of the product. In particular strongly hydrophobic sequences may be difficult or impossible to 

purify due to their lack of solubility in any solvent. Overall, many “well-behaved” peptide dendrimers comprising a 

good balance of charged, polar, and hydrophobic amino acids, are readily accessible by SPPS. Peptide dendrimer 

synthesis is also facilitated by the commercial availability of amino acid building blocks for Fmoc-SPPS at

118 Synthesis and Biological Applications of Glycoconjugates Reymond and Darbre 

reasonable prices, which renders the synthesis quite affordable and potentially scalable using the existing industrial 

infrastructure for SPPS. 

The split-and-mix protocol for assembling peptide libraries by SPPS [29-33], which was a founding experiment in 

combinatorial chemistry, can be applied to prepare the corresponding peptide dendrimer libraries on solid support 

[34-36]. In these so-called “one-bead-one-compound” (OBOC) libraries, each bead of the solid support carries only 

one possible sequence, yet a large number of different sequences are assembled simultaneously in the course of the 

synthesis. The number of different sequences accessible in a split-and-mix synthesis is limited by the number of 

beads used in the SPPS synthesis batch. A typical synthesis batch of 500 mg of 90 micrometer diameter beads 

contains approximately one million beads, which imposes a limit of 100,000 sequences for the library size to ensure 

10-fold coverage of sequence space. 

Synthesis of Glycopeptide Dendrimers 

While the preparation of natural glycopeptides requires sophisticated chemical or enzymatic glycosylation 

chemistry, the decoration of synthetic macromolecules with carbohydrates is often more conveniently realized by 

reacting preassembled glycosidic building blocks by simpler coupling chemistries. Before its adaptation to 

combinatorial chemistry, we first investigated various glycosylation options for peptide dendrimers in a synthetic 

study aimed at the dendrimer mediated delivery of colchicine to cancer cells. 

Three different strategies were investigated to obtain glycopeptide dendrimers [37]. The first approach used oxime 

bond formation between a multivalent aldehyde-terminated peptide dendrimer and an oxyamine functionalized 

glycoside, as reported previously for the glycosylation of peptides [38, 39] and peptide dendrimers [40]. The second 

approach used reductive alkylation of the free N-terminus with lactose [41, 42], a method previously known for 

modifying whole proteins [43-45] and reported for the synthesis of glycodendrimers [46]. The third approach 

involved amide bond formation between an acetyl-protected glycosysidic carboxylic acid building block and the Ntermini 

of the peptide dendrimer directly on the solid support. Although this third approach required multigram 

quantities of carbohydrate building blocks, the synthesis was the most practical because it required only a single 

chromatographic purification and yielded products of excellent purity. For example the N-acetyl-glucosamine 

terminated peptide dendrimer colchicine conjugate A, which shows selective cytotoxicity to cancer cells, was 

obtained by this approach (Fig. 2). 

OH OH 

O 

HO 

AcNH 

O 

O 

O 

O 

HN 

H 

N 

O 

NH 

NH 

O 

N 

H 

N 

O 

OH 

O 

AcHN 

HO 

OH 

O 

S S 

N 

H 

O 

NH 

O 

MeO 

Figure 2: A cytotoxic colchicine glycopeptide dendrimer conjugate. 

NH 

OH OH 

O 

HO 

AcNH 

O 

HN 

H 

N 

N 

H 

NH 

O 

H 

N 

OMe 

O 

O 

OMe 

N 

O HN 

O 

O 

OH 

OMe 

A 

HN 

NH2 O 

NH 

O O 

HO 

AcNH 

O 

O O 

N 

H 

OH 

OH

Cyclopeptide-Based Glycoclusters 


Olivier Renaudet * , Didier Boturyn and Pascal Dumy 



CHAPTER 8 


Grenoble Cedex 9, France 

Abstract: The emergence of glycomics has deeply stimulated the design of new bioactive glycoclusters over the 

past decade. Among the increasing number of multivalent synthetic structures reported so far, cyclopeptide-based 

glycoclusters (CBGs) have recently shown promising interest for diverse biological applications. This chapter 

aims at describing the major advances in this field, with a special focus on the inhibition of carbohydrate-protein 

interactions and the synthetic vaccines. 

Keywords: Cyclopeptide, glycocluster, oxime, combinatorial chemistry, synthetic vaccine, lectin. 

INTRODUCTION 

Decoding carbohydrate/protein interactions involved in biological events [1, 2] remains crucial for the design of bioactive 

molecules. The “glycoside cluster effect” introduced by Lee et al. in 1995 describes that these complex recognition 

processes are regulated by simultaneous and cooperative contacts between cluster of glycans and multimeric proteins [3]. 

While their kinetic and thermodynamic parameters are still not fully understood [4-6], diverse mechanisms such as 

chelating, proximity/statistical or clustering effects were proved to promote high selectivity and strong binding 

improvement, depending on the structural features of both ligand and protein [7-9]. These findings have inspired bioorganic 

chemists to develop diverse molecules capable of mimicking the multivalent display of carbohydrates expressed in living 

systems, namely glycoclusters, with the aim to study multivalency effects or to inhibit biological processes [10-12]. 

Although impressive subnanomolar affinities were sometimes achieved [13], most of studies reported so far clearly 

highlight that the design of an optimal structure for an amplified biological effect remains extremely difficult to rationalise. 

The usual design to develop biorecognizable glycoclusters consists in grafting multiple copies of carbohydrates onto a 

carrier platform, so that they bind several, or ideally, every protein binding sites simultaneously. Since the biological 

potency of glycoclusters closely depends on the tridimensional protein structure, a large variety of synthetic structures 

exhibiting variable topology, flexibility, valency and sugar distribution were explored. In particular, linear (e.g. polymers 

[7, 14], oligonucleotides [15-17]), cyclic (e.g. calixarenes [18, 19], cyclodextrins [20], fullerenes [21, 22]) and branched 

platforms (peptide dendrimers [23-25], poly(amidoamine) dendrimers [26]), liposomes or nanoparticles [27, 28] have been 

intensively investigated in different biological contexts. The use of cyclopeptides as carrier platforms for carbohydrates 

was more rarely described until recent studies have considerably renewed their interests [29]. This chapter focuses on the 

synthesis of several cyclopeptide-based glycoclusters (CBGs) that revealed promising biological properties. 

CYCLOPEPTIDE PLATFORM 

Pioneering studies by Mutter et al. on protein mimics have introduced an original concept that addresses crucial 

questions on peptide assembly, protein folding and function [30, 31]. For this purpose, conformationally restricted 

cyclopeptides were utilized as topological platforms to assemble and stabilize the secondary structure of peptide building 

blocks in a native-like folding [32]. Following a similar concept, other sophisticated platforms, such as the 

Regioselectively Addressable Functionalized Templates (RAFT) [33], were further used for the construction of 

biorecognizable compounds [29, 34]. 

As illustrated in Fig. 1, a typical cyclopeptide platform is composed of amide bonds in the trans configuration and 

contains generally two -turn inducers i.e. L-proline-glycine (Pro-Gly in green, Fig. 1A) that constrained the 

structure into an antiparallel -sheet (yellow rubber, Fig. 1B). 

*Address correspondence to Olivier Renaudet: Département de Chimie Moléculaire, UMR-CNRS 5250 & ICMG FR 2607, Université Joseph 

Fourier, 38041 Grenoble Cedex 9, France; E-mail: olivier.renaudet@ujf-grenoble.fr

130 Synthesis and Biological Applications of Glycoconjugates Renaudet et al. 

A recognition domain 

B 

R 1 

R 1 

R 1 

R 1 

Gly Lys 

Lys Lys Pro 

turn turn 

Pro Lys 

Lys Lys Gly 

R 2 

R 2 

effector domain 

Figure 1: A) Chemical drawing and B) molecular model of an example of cyclopeptide scaffold. This platform is composed of 

ten amino acids, including two Gly-Pro β-turns and six Lys residues which define two addressable faces, namely “recognition 

domain” (upper, R 1 = carbohydrates, peptides) and “effector domain” (lower, R 2 = peptide, oligonucleotide, surface, drug). 

Alternating intramolecular hydrogen bonds stabilize the backbone into a locked and rigid cyclic conformation. The 

other amino acids into the peptide sequence may be selected to provide regioselective anchoring sites (e.g. lysine 

[Lys] as shown in Fig. 1, or aspartic acid [Asp], glutamic acid [Glu], aparagine [Asn], glutamine [Gln], threonine 

[Thr] in other cases) pointing to opposite faces of the template backbone. Thus these platforms display two 

multivalent addressable faces, defined as “recognition” and “effector” domains once decorated with binding and/or 

functional building blocks (Fig. 1B). 

Besides their topology and constrained conformation, the utilization of cyclopeptides offers other advantages. Their 

preparation indeed benefits from a plethora of technical improvements developed for peptide chemistry (e.g. solid 

supports, automation, non-racemizing coupling reagents…) that facilitate the rapid synthesis of their linear peptide 

precursor in high scale. More importantly, the large variety of commercial building blocks with orthogonal 

protecting groups provides both synthetic versatility and modularity. This ensures the stepwise construction and the 

structural integrity of complex macromolecules with tailor-made properties. Finally, it has to be mentioned that 

cyclic structures were proved to prevent proteolytic degradation in vivo compared to linear peptides [35], which 

strengthen their interest as addressable platforms for biological applications. 

CBGs AS CARBOHYDRATE-BINDING PROTEINS LIGANDS 

Selectins 

In 1996, the group of Kunz used for the first time a cyclopeptide for the multivalent presentation of biologically relevant 

carbohydrates [36]. Cycloheptapeptides displaying sialyl-Le x moiety were prepared using a convergent approach from 

the linear peptide sequence 1 which was synthesized by solid-phase peptide synthesis (Fig. 2). It contains a D-Ala 

residue as a -turn inducer and several protected Asp as anchoring site for the incorporation of sugars. After the 

intramolecular cyclization under high dilution then deprotection of the peptide by acidolysis with trifluoroacetic acid 

(TFA), a benzylated tetrasaccharide was conjugated to three Asp side chains in 48% yield using the Lansbury 

aspartylation reaction. The trivalent asparagine-linked glycoconjugate 2 was finally obtained after final deprotection. 

Selectins are transmembrane glycoproteins involved in cell-adhesion processes that are mediated by selective 

multivalent binding to Lewis-containing ligands [37]. To evaluate the inhibitory potency of the trivalent sialyl-Le X 

glycopeptide 2, cell adhesion assays were performed with recombinant human fusion proteins (E-selectinimmunoglobulin) 

in competition to tumor cells displaying the same carbohydrate moiety (cell line HL60). The 

synthetic compound 2 was found to inhibit the adhesion of tumor cells 3-fold more efficiently (0.35-0.6 mM) than 

the monomeric sialyl-Le X [36]. This modest binding has prompted the same group to design new ligands containing 

a flexible tetraethyleneglycol spacer between the sialyl-Le X moiety and the peptide platform [38]. However the 

biological evaluation of the divalent cyclopeptide 3 did not show a significant inhibition improvement on the 

adhesion of murine neutrophiles to E-selectin.

Cyclopeptide-Based Glycoclusters Synthesis and Biological Applications of Glycoconjugates 131 

Figure 2: Synthesis of sialyl-Le x containing cycloheptapeptide conjugates. 

Plant Lectins 

To overcome the synthetic difficulties related to the synthesis of glycoconjugates, our group developed an oximebased 

chemoselective procedure that revealed extremely efficient for the construction of CBGs [39]. This strategy 

requires the prior incorporation of mutually reactive aldehyde and oxyamine groups into the molecules to be 

conjugated. Only few procedures (i.e. Mitsunobu coupling [40], stereoselective glycosylation using phase transfer 

catalysis [41]) were reported to introduce an aminooxy function to the anomeric position of glycans. We developed 

a synthetic alternative to afford alpha and beta aminooxy glycosyls starting from glycosyl fluoride donors and Nhydroxyphthalimide 

(PhthOH) in the presence of BF3.Et2O as promoter [42] (Fig. 3). This strategy was used 

successfully to synthesize biologically relevant carbohydrates (e.g. lactose 4, mannose 7 and fucose 8 [43]) or 

tumor-associated carbohydrate antigens (e.g. Tn 9 and Thomsen-Friedenreich (TF) 10 [44]). 

Figure 3: Synthesis of aminooxy glycosyls. 

With these aminooxy glycosyls in hands, glycoclusters with one or two addressable domains were next designed. 

Typically, linear peptides containing a various number of orthogonally protected Lys (e.g. Alloc, Boc, Dde) were 

prepared on solid-phase using acido labile resin (e.g. Sasrin TM ) then cyclised in solution through the C-terminal 

glycine to prevent epimerization. To design one domain-containing compounds, four Lys pointing above the 

platform were deprotected to incorporate serine residues. Successive treatment with sodium periodate generate 

glycoxyl aldehyde groups [45]. Fully deprotected aminooxy glycosyls were finally conjugated under mild aqueous


Oligonucleotide-Carbohydrate Conjugates 

Nicolas Spinelli * and Eric Defrancq 



CHAPTER 9 



Abstract: Nucleic acids have been widely investigated as biological tools for various applications including gene 

silencing, DNA chips and drug targets. Interactions involving nucleic acids are generally well known making 

their rationalization straightforward. However, they generally suffer from a lack of bioavailability. One of the 

main strategies to overcome these limitations is the conjugation of oligonucleotides with an appropriate reporter. 

As carbohydrates are involved in a huge variety of biological processes including molecular and cell surface 

recognitions, oligonucleotide glycoconjugates are perhaps one of the most promising approaches to improving 

cellular delivery of oligonucleotides. Other applications of these conjugates have emerged like the use of DNAdirected 

immobilization to prepare carbohydrate biochips or the construction of glycoclusters by self organization 

of oligonucleotides. This chapter summarizes the applications of these conjugates and approaches to prepare them 

through some recent examples. 

Keywords: Nucleic acids, conjugation, solid-supported synthesis, chemoselectivity. 

INTRODUCTION 

Nucleic acids have been widely investigated as biological tools especially for gene silencing. They can be used for 

targeting mRNA by either antisens [1-3] or RNAi [4] process or double strand DNA by antigen [5] strategy. 

However their applications are not just restricted to genetics [6]. They can be used as aptamers [7], for diagnostic 

applications [8] including DNA biosensors [9] and aptasensors [10], for designing of nanostructures [11-13] and for 

DNA mediated reactions or catalysis [14, 15]. However, their biological applications they suffer from a lack of 

bioavailability principally due to their susceptibility to nuclease degradation and their poor cellular uptake. One of 

the main strategy to overcome those limitations is the use of oligonucleotide-conjugates [16, 17]. 

Carbohydrates are involved in various biological recognition events [18-20] such as cell-cell adhesion, fertilization, 

immune defense and degradation of blood clots. In particular, study of carbohydrate-protein (lectin) interactions [21] 

has received a great interest toward the enhancement of targeting of drugs and gene delivery [22] via receptor 

mediated endocytosis. Lectin-carbohydrate interactions are generally very specific, but affinities are generally weak 

for monovalent interactions (millimolar range) [23]. These weak affinities are usually overcome by preparing 

glycoclusters on various scaffold (see the other chapters) for mimicking the “glycoside cluster effect” [24]. 

As a consequence, oligonucleotide glyconjugates are perhaps one of the most promising approaches for improving 

cellular delivery of oligonucleotides [25]. In addition, other applications of these conjugates involving the 

hybridization of oligonucleotides for studying carbohydrate-aromatic stacking, preparation of carbohydrate biochips 

or elaboration of glycoclusters have emerged. Preparation and applications of oligonucleotides-glyconjugates has 

been reviewed in 2004 [26], so this chapter deals with recent advancements concerning methodologies for synthesis 

of these compounds through some examples. The different synthetic approaches and corresponding applications are 

described in the following sections. 

ON-SUPPORT SYNTHESIS 

Since most of the protecting groups (e.g. acetyl) used for protection of carbohydrates are also compatible with the 

synthetic protocols of oligonucleotide (phosphoramidite or H-phosphonate), the approach consisting in the 

*Address correspondence to Nicolas Spinelli: Département de Chimie Moléculaire, UMR-CNRS 5250 & ICMG FR 2607, Université Joseph 

Fourier, 38041 Grenoble Cedex 9, France; E-mail: nicolas.spinelli@ujf-grenoble.fr

146 Synthesis and Biological Applications of Glycoconjugates Spinelli and Defrancq 

incorporation of phosphorus glycosides during oligonucleotide synthesis is perhaps the more straightforward. Solidphase 

conjugation of oligonucleotides and solid phase synthesis of glycoconjugates have been reviewed [27, 28]. 

Several carbohydrate derivatives have been prepared for 5’, in-sequence or 3’ conjugation depending on the purpose 

of the conjugate. 

Use of Carbohydrate Phosphoramites for 5’-Conjugation 

Carbohydrate phosphoramidites can be incorporated at the 5’-end of oligonucleotide. For example, some of them 

have been developed in order to elaborate a model system to conduct a systematic study of the carbohydratearomatic 

stacking [29]. Models were designed as follows: a self-complementary sequence ( 5’ Carbohydrate- 

XCGCGCG 3’ ) carrying benzene nucleotide (X) and a carbohydrate at 5’-end (Fig. 1). 

Figure 1: a) Use of DNA base pairing for studying carbohydrate-aromatic stacking. b) Carbohydrate phosphoramidites for the 

synthesis of 5’-glycoconjugates. 

Sugars were incorporated as the corresponding phosphoramidite synthon; phosphorus function is linked to the sugar 

through the anomeric position via an ethylene glycol linker. According to this strategy, several conjugates were 

synthesized including -glucose 1, -galactose 2, -2-deoxyglucose 3,-2-deoxyglucose 4 or -L-fucose 5. 

Sugar-benzene interactions were estimated measuring thermodynamic parameters of self-complementary duplexes 

and geometry of carbohydrate-benzene stacking was estimated by NMR. This study has shown that dispersion 

forces and non-conventional hydrogen bonding seems to take place between sugar and benzene ring. 

Synthesis of 5’-multivalent glycoconjugates, i.e. oligonucleotides bearing several carbohydrates motif has been 

performed with this strategy using branched phosphoramidites [30] (Fig. 2). In a recent work, the influence of the 

presentation of carbohydrates has been evaluated in terms of the cellular uptake and cell surface binding via glucose 

receptors [31]. Several conjugates were synthesized bearing monovalent glucose 6, maltose 7, maltotriose 8, 

branched divalent glucose 9 and tetravalent glucose 10. Monovalent conjugates were obtained using appropriate 

carbohydrate phosphoramidite (1, 11 or 12) and branched glycoclusters were obtained using a branching 

phosphoramidite 13 and glucose phosphoramidite 1. All these conjugates were labeled with Alexa 488 at their 3’ 

end in order to perform flow cytometry analysis with two types of cells (HeLa and U87) both in presence and in the 

absence of glucose in the culture media.

Oligonucleotide-Carbohydrate Conjugates Synthesis and Biological Applications of Glycoconjugates 147 

Figure 2: Monovalent, multivalent 5’-glycoconjugates and phosphoramidites for their preparation. 

Adsorption experiments highlighted slight difference of behavior of glycoconjugates in each cell type. For instance, 

the binding of tetravalent conjugate 10 was found to be less efficient with Hela cells than that with U87. The best 

cellular uptake was found for divalent 9 and maltose 7 conjugates. Conjugates linked through longer spacer (7 and 

9) showed better incorporation than these linked through shorter spacers (6 and 8). Surprisingly, tetravalent glucose 

conjugate 10 showed a bad incorporation presumably due to cell uptake hindering. 

Synthesis of heteroglycoconjugates [32], i.e. oligonucleotides bearing three different glycosyl groups (Fig. 3, 14) has 

also been achieved using appropriate orthogonally protected branching phosphoramidite 15 and carbohydratephosphoramidites 

(16-18). A derivative of bis(hydroxymethyl)-N,N’-bis(3-hydroxypropyl)malondiamide 15 was 

introduced during solid-phase synthesis. It contained a DMT protected hydroxyl and a phosphoramidite group 

allowing oligonucleotide elongation, and a levulinyl protected hydroxyl and a t-butyldiphenylsilyl protected 

hydroxyl for connecting glycosides residues. Conjugate syntheses were performed as follows: building block 15 was 

incorporated at the 5’ end of the oligonucleotide, DMT group was removed under acidic conditions and nucleotides 

were incorporated. At the end of the elongation, a carbohydrate-phosphoramidite was incorporated at the 5’ end. 

Levulinyl group was removed by hydrazinolysis allowing the incorporation of another carbohydrate 

phosphoramidite and finally TBDPS was removed prior to incorporation of the last glycosyl-phosphoramidite. 

This method allows the synthesis of heteroglycoclusters such as 14 in a reasonable yield. Melting temperature 

measurements showed that the presence of the bulky glycocluster at the 5’ terminus destabilises the duplex. 

In Sequence-Conjugation 

In the last example, glycosides were incorporated at the 5’ terminus, but building block 15 should allow in-sequence 

incorporation. This can also be performed using modified nucleoside phosphoramidite. This strategy has been used


Glycoliposomes and Metallic Glyconanoparticles in Glycoscience 

Marco Marradi a,b,* , Fabrizio Chiodo a , Isabel García a,b and Soledad Penadés a,b 



CHAPTER 10 

a Laboratory of GlycoNanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE, Parque Tecnológico de 

San Sebastián, Pº de Miramón 182, 20009 San Sebastián, Spain and b Biomedical Research Networking Center in 

Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Parque Tecnológico de San Sebastián, Pº de 

Miramón 182, 20009 San Sebastián, Spain 

Abstract: Multivalent sugar-based materials have attracted attention since the functional role of carbohydrates in 

biology has been disclosed. The design of artificial systems that mimics the polyvalent carbohydrate organization 

at cell surface has been envisaged as a strategy to study and intervene in carbohydrate-mediated interactions. One 

of the first synthetic glycomaterials which appeared in the literature were glycoliposomes, dynamic systems that 

resemble the glycocalix in the phospholipidic bilayer of cell membranes. Glycoliposomes are non-covalent 

systems which have been used since the seventies as multivalent tools in carbohydrate-based interactions against 

pathogens, for enhancing immunity and as molecular carriers in drug delivery. In former years, the advent of 

nanotechnology has allowed the design and construction of new materials similar in size to biologically relevant 

molecules (proteins, nucleic acids, etc) and displaying unique physical properties. The bio-functionalization of 

metallic nanomaterials with carbohydrates generated a new class of glycomaterials, named glyconanoparticles, 

which present carbohydrates in a highly multivalent way and in high local concentrations. At the same time, the 

quantum size properties of metallic nanoclusters can be used for biosensing, diagnostics, and (in perspective) 

therapy. This review focuses on glycoliposomes and covalently-functionalized glyconanoparticles which make 

use of the “glyco-code” to address specifically pathogens or pathological-related problems. 

Keywords: Glycoliposomes, glyconanoparticles, glycocalix, multivalency, neoglycolipids. 

INTRODUCTION 

The Functional Role of Carbohydrates 

Once thought to be only energetic sources for living systems or structural molecules protecting the cell surface, 

carbohydrates are nowadays recognized as essential tools for cell adhesion and recognition processes [1]. The surface of 

mammalian cells is covered by a dense coating of carbohydrates named glycocalyx. In the glycocalyx, carbohydrates 

appear mainly conjugated to proteins and lipids (glycoproteins, glycolipids and proteoglycans) and it is as clustered 

glycoconjugates that they develop their biological function. The high variety of carbohydrates, which have a much 

higher information store-capacity than amino acids and nucleotides, gives rise to a complex “glycocode” which is used 

by living systems to stock and transmit biological information. The (oligo)saccharides of the glycocalix are involved in 

carbohydrate-protein [2] and carbohydrate-carbohydrate [3] interactions that mediate many physiological and 

pathological processes [4, 5]. Carbohydrate-mediated interactions are generally very weak and the low affinity has to be 

compensated by multivalent presentation of the ligands. Polyvalent ligand–receptor interactions are characterized by the 

simultaneous binding of multiple ligands on one biological entity to multiple receptors on another biological entity and 

can be cooperatively much stronger than corresponding monovalent interactions [6]. These interactions are ubiquitous in 

biology and include both cellular signalling and matching contacts of viral and bacterial surfaces to host cells. For 

example, the weak association constant of carbohydrate-protein interactions in living systems is overcome both by 

clustering of sugar binding sites in the protein partner and presenting the carbohydrate at cell surface in a multivalent and 

orientated way [2, 7]. The structural diversity, the variability of sugar–units sequence and linkage points, the anomeric 

configuration, the chemical modification and/or substitution at different positions and with different groups (phosphate, 

sulphate, N-acetyl, etc), and the interconversion of conformers are some characteristics that create a unique level of 

diversity in the “glycocode”. Another key feature of the “glycocode” relies on the potential of glycoconjugates to adopt 

various defined shapes, each with its own particular ligand properties (differential conformer selection) [8]. 

*Address correspondence to Marco Marradi: Laboratory of GlycoNanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE, 

Parque Tecnológico de San Sebastián, Pº de Miramón 182, 20009 San Sebastián, Spain: E-mail: mmarradi.ciber-bbn@cicbiomagune.es

Glycoliposomes and Metallic Glyconanoparticles in Glycoscience Synthesis and Biological Applications of Glycoconjugates 165 

Synthetic mimics of the glycocalix, as well as small molecules which intervene in carbohydrate biosynthetic and 

processing pathways can be used to interfere with cellular interactions [9]. The design and development of 

therapeutic glycomimetics is a great challenge of current glycoscience and the concept of carbohydrate-based drugs 

has thus spread out in the last years [10, 11]. The key role of polyvalency in many biological interactions has 

suggested new strategies for the design of artificial systems which mimic nature organization and help 

understanding cellular events at molecular scale. In particular, different types of scaffolds such as glycopeptides and 

glycoproteins [2, 12-14], glycopolymers [15-17], and glycodendrimers [18] have been developed to multimerise 

biologically relevant carbohydrates and address biological problems. A big challenge in this direction relies in 

combining the effects of multivalency and high local concentrations of the carbohydrates in such systems, with the 

possibility of tailoring other types of molecules (vectors, drugs, etc.) on the selected scaffold (multifunctionality) 

and/or using different imaging techniques (fluorescence, magnetic resonance imaging [MRI], etc.) to track them in 

vitro and in vivo (multimodality). Glycocalixarenes [19] and cyclopeptide-based glycoclusters [20] are presented in 

different chapters of this E-book. This chapter is focussed on glycoliposomes and glyconanoparticles (GNPs) as 

polyvalent systems for addressing biological problems. 

Glycoconjugation and Self-Assembly of Glycoconjugates 

The biosynthetic pathways of glycans are lead by enzymatic processes through the action of glycosidases and 

glycosyltransferases [21]. Although the isolation from natural sources can give access to biological relevant 

oligosaccharides, this method usually affords small amounts of the desired product and rarely provides high degree 

of purity. Chemical or chemoenzymatic syntheses still are the method of choice to obtain pure and well-defined 

oligosaccharides. The progress in glycoscience is undoubtedly related to the efficiency of routine synthetic methods 

of complex glycoconjugates. The developments in the set-up of general methods for the preparation of biologically 

relevant oligosaccharides, including multistep protecting-group manipulations and highly stereo- and 

chemoselective glycosylations, have recently been reviewed [13, 22, 23]. In addition to traditional solution phase 

synthesis, it is nowadays possible to use solid phase synthesis, even in automated fashion [24]. The preparation of 

neoglycoconjugates that mimic the functions of bioactive carbohydrates by using chemoselective approaches is of 

great importance for the development of new therapeutics and it is revised in two chapters of this E-book [25, 26]. 

The synthesis of suitable neoglycoconjugates is also essential for the construction of self-assembled glycosystems. 

There are two main strategies to construct multivalent carbohydrate-based systems. The first one is to create 

membrane-like systems by using carbohydrate-conjugates in the presence of self-assembly molecules, as it is the 

case of glycoliposomes (Fig. 1, left), in which a glycolipid is mixed with self-assembly phospholipids and/or 

cholesterol(Chol)-like molecules. The other way is to use a suitable scaffold to anchor the carbohydrates, as it is the 

case of GNPs (Fig. 1, right), in which a metallic nanocluster is used to covalently attach the glycoconjugates. Due to 

the advances in carbohydrates synthesis during the last 30 years, suitable glycoconjugates obtained by conventional 

coupling chemistry (amines with carboxy groups or isothiocyanates, thiols with maleimides, azides with alkynes, 

etc) have allowed the preparation of a great variety of glycoliposomes and glyconanoparticles. Carbohydrates have 

to be derivatised prior to the self-assembly process, although some approaches using unmodified sugars have been 

proposed. The main chemical methods used for immobilization and anchoring of oligosaccharides onto solid 

matrixes are also valid for surface-functionalization of liposomes and nanoparticles [27]. 

The self-assembly process of glycolipids in the presence of phospholipids or the self-assembled monolayers (SAMs) 

of thiol-armed glycoconjugates are some examples of construction of complex systems in a straightforward way. 

Glycoliposomes are non-covalent systems, while glyconanoparticles are based on covalent chemistry. In this 

chapter, we review these two types of multivalent systems bio-functionalized with conjugates of biologically 

interesting carbohydrates and their application in glycoscience. 

GLYCOLIPOSOMES 

Amphiphilic phospholipids are able to self-assembly and form closed bilayered structures in water. The resulting 

vesicles (liposomes) enclose an aqueous compartment and their structure resembles the phospholipid membranes of 

living cells [28]. Liposomes can easily incorporate hydrophilic molecules in their aqueous compartment and

166 Synthesis and Biological Applications of Glycoconjugates Marradi et al. 

hydrophobic compounds in their lipophilic bilayer, they can interact with cells by lipid bilayers fusion phenomena, 

and they are biocompatible. In general, liposomes are formed when a thin film of lipids containing phospholipids 

and Chol is hydrated. The hydrated lipid sheets detach during agitation and self-close to form large, multilamellar 

vesicles which prevent interactions of water with the hydrocarbon core of the bilayer. Reducing the size of the soformed 

particles requires energy inputs in the form of sonic energy (sonication) or mechanical energy (extrusion), 

among others [29]. 

Figure 1: Schematic representation of glycoliposomes (left) and calculated structure of a gold glyconanoparticle (right). The 

GNP is a model of a mannose-coated gold nanoparticle (the ligand being α-Man(CH 2) 5S) which was obtained using SYBYL suite 

program. Pictures are not in scale. 

It is nowadays widely accepted that liposomes belong to the first-generation of nanovectors, i.e. delivery systems 

which are governed by passive mechanisms of drug delivery [30]. Liposomal formulations of drugs have been 

approved and are currently in clinical use due to their relative safety and enhanced efficacy [31]. The enhanced 

permeation and retention effect of liposomes in vivo is the key factor of their success as drug carriers. In order to 

achieve selective targeting, strong efforts are being made to insert specific functionalities at liposome surface. 

Remote activation would be also desirable to trigger payloads release in a controlled way. New generations of 

liposomes have thus been designed to improve their potential in biomedical applications. From a simple 

phospholipids bilayer used for biophysical research, liposomes and analogous vesicular carriers which incorporate 

specific molecules for targeting purpose, fluorescent or magnetic dyes for diagnostic use, cell-penetrating peptides, 

pH-sensitive components or stimuli-responsive molecules for controlled release have been developed [32, 33]. A 

class of biomolecules which had an important role in liposome evolution are carbohydrates. Liposomes coated with 

multiple copies of carbohydrates (glycoliposomes) have been used since the early seventies to target protein 

receptors of pathogens, as we will account in the next section. Glycoliposomes are composed of phospho- and 

glycolipids and thus are good “dynamic scaffold” to mimic the glycocluster of cellular membranes. To understand 

the role that carbohydrates play in many biological processes is important to have in hand a dynamic cellular model 

in which multiple copies of sugar epitopes can diffuse in a membrane-like patch changing organization in front of 

external stimuli. Glycoliposomes have been proposed as potential systems for drug and gene targeting/delivery by 

exploiting the specificity of carbohydrates [34, 35]. 

In here, we describe the historical excursus of glycoliposomes as multivalent systems for carbohydrates presentation 

and their current biological applications. We will especially focus on glycoliposomes containing well-defined 

glycolipids. The presence of well-defined carbohydrates has been exploited first to understand their interactions with 

lectins in simple cellular models and then to selectively target lectins involved in physiological or pathological 

processes like inflammation and/or cellular immune response. Polysaccharide-anchored liposomes as synthetic 

models to study cellular adhesion and as potential therapeutics have been reviewed elsewhere [36]. 

The Seminal Works and Methods for Preparing Glycoliposomes 

Seminal works dealing with the construction of “model membranes” containing gangliosides, i.e. glycolipids 

containing N-acetyl neuraminic acid [5-(acetylamino)-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosonic 

acid; Neu5Ac], appeared in the early seventies [37-39]. Different gangliosides were inserted in bimolecular lipid




CHAPTER 11 

Chemoselective Glycosylation Techniques for the Synthesis of Bioactive 

Neoglycoconjugates, Glyconanoparticles and Glycoarrays 

Francesco Peri * 

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, 2; 20126 Milano, Italy 

Abstract: Chemoselective glycosylation (CG) methods have rapidly developed and diffused in glycochemist and 

glycobiologist’s community in the last fifteen years, and an increasing number of applications confirms the enormous 

potential of these techniques. CG has been successfully used for the synthesis of glycoproteins, glycopeptides, 

glycosylated natural compounds, carbohydrate-functionalized surfaces and nanoparticles. CG techniques can be 

considered as subset of click reactions that allow the efficient, high-yield conjugation of sugars with unprotected 

aglycons in aqueous media. In this review the CG methods have been divided into two main groups: “direct” CG that 

uses unmodified glycans, and “indirect” CG, in which the sugar has to be chemically modified before the glycosylation 

reaction. Recent applications of both CG techniques are reviewed, with special focus on the chemistry of CG reactions: 

mechanisms, stereochemistry issues, structure and stability of the glycoconjugates. 

Keywords: Chemoselective ligation, neoglycoconjugate, glyconanoparticle, microarray. 

DECONVOLUTING GLYCOME BY CHEMICAL TOOLS 

Like famous French and Italian sugar beads-covered cakes [1], cells from bacteria to mammals are covered by a 

sugar coating that presents the first information about the cell to the outside world. Increasing evidence indicates 

that carbohydrates have roles in a plethora of biological phenomena [2]. Carbohydrate-carbohydrate, carbohydrateprotein, 

and carbohydrate-nucleic acid interactions are the molecular basis of viral entry, signal transduction, 

inflammation, cell-cell interactions, bacteria-host interactions, fertility, and development, among other processes [3- 

5]. The discovery that a large part of the biological information is encoded in carbohydrate structures (or glycocode) 

rapidly became a fundamental concept in glycobiology, promoting the foundation of new biological disciplines first 

of all the specific branch of “omics” termed glycomics [6, 7]. In analogy with proteomics and genomics, glycomics 

explores the role of carbohydrates in biological processes. Carbohydrates, in the form of glycopeptides, glycolipids, 

glycosaminoglycans, proteoglycans, or other glycoconjugates have long been known to participate in all biological 

processes described above. Deconvoluting the importance of sugars in these biological events is challenging owing 

to many factors, including the complexity of the glycans (see below), the complex biosynthesis of the sugar part of 

glycoproteins (which is not under direct gene control), the multivalent nature of biological glycan recognition and 

the subtle phenotypes of glycan manipulation that often require multicellular environments to manifest. 

One of the principal obstacles to our study of carbohydrates comes from the complexity represented in the glycome 

itself, monosaccharides assemble into both linear and branched polymer structures, which are made more complex 

by the two possible stereochemical linkages between the units ( and ), resulting in a high degree of potential 

complexity. The complexity is further compounded by the different modes of attachment for cell-surface glycans, 

including glycolipids, and asparagine (-) and serine/threonine (O-) linkages to glycoproteins, and by further 

modifications of the monosaccharide units, such as methylation, sulfation, acetylation and phosphorylation. 

Organic chemists are therefore engaged in trying to reproduce synthetically the complexity of glycans. Synthesis 

provides access to relatively high quantities (mg) of glycans and glycoconjugates in a pure, chemically defined 

form. On the other and, milligram or sub-milligram amount of complex glycans (sugar epitopes, surface glycans, 

structural oligosaccharides) can be obtained by biological materials, and can be used as precious building blocks for 

the construction of chemically defined glycoconjugates. Despite how these complexes multivalent glycoconjugates 

have been obtained (by total synthesis or by chemical assembly of natural glycans) they are unique tools for basic 

structural glycobiology studies, and for use as therapeutics (Fig. 1). 

*Address correspondence to Francesco Peri: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della 

Scienza, 2; 20126 Milano, Italy; E-mail: francesco.peri@unimib.it

204 Synthesis and Biological Applications of Glycoconjugates Francesco Peri 

Figure 1: Complex glycoconjugates as valuable tools for glycobiology and medicinal chemistry: A) modified surfaces for 

microarrays and SPR, B) neoglycoproteins, neoglycopeptides, carbohydrate vaccines, C) glycosylated natural products and drugs, 

D) multivalent carbohydrate gold or magnetic nanoparticles. 

This review presents how the development of chemoselective glycosylation (CG) reactions, as well as the use of 

click conjugation techniques, has in the last decade provided access to highly complex, multivalent, 

polyglycosylated structures. This can be done by a full chemical approach or by assembling by efficient conjugation 

reactions natural sugar building blocks deriving from mammalian or bacterial cells. 

The first part of the review the CG techniques will be described in detail, in particular that based on the reactivity of 

reducing sugars with hydroxylamine-containing scaffolds. These techniques have enormous potential and wide 

applicability, because are based on the conjugation of unmodified sugars in water. CG presents several advantage on 

more classical glycosylation methods. Their use not limited to organic chemistry laboratories specially equipped to 

work in strictly anhydrous conditions, but can be extended to biochemistry or biology labs. The most recent 

applications of CG will be presented and discussed in the second part of this review, in particular, the 

neoglycosylation of pharmacologically active natural compounds, of surfaces (glycoarrays), and of nanoparticles. 

CHEMOSELECTIVE LIGATION OF UNMODIFIED AND MODIFIED GLYCANS 

The fundamental reaction to covalently connect two monosaccharide units or to link a sugar to other biomolecules 

and biopolymers (proteins, lipids, or other aglycons), is the glycosylation reaction [8-10]. Nature executes this 

reaction with exquisite stereo- and regioselectivity and high efficiency to yield linear and branched poly- and 

oligosaccharides, glycoproteins, glycolipids, and glycosylated secondary metabolites. However, the installation of 

the glycosidic linkage by chemical means remains cumbersome, even with the aid of modern technologies [11]. 

Owing to many recent breakthroughs in the field, the formation of most glycosidic bonds can be readily achieved. In 

the past three decades, much scientific effort has been dedicated to refining the glycosylation reaction through the 

development of new leaving groups, new promoters (activators), and through the optimization of the general 

reaction conditions. The “classical” glycosylation approaches for oligosaccharides, O- and N-linked glycopeptides 

and proteins and glycoconjugates (Scheme 1), require, regardless to the method used, protected sugar units, strictly 

anhydrous conditions, and the presence of glycosylation promoters or metal-based catalysts.

Chemoselective Glycosylation Techniques for the Synthesis Synthesis and Biological Applications of Glycoconjugates 205 

These techniques are often incompatible with aqueous conditions that are required when sugars should be linked to 

water-soluble molecules (such as proteins, peptides, lipids, polyphenols and other biological aglycones). On the 

contrary, strictly anhydrous conditions are required because water-labile glycosylation promoters are used. These 

glycosylation reactions therefore require specific equipment of organic chemistry lab and are not easily carried out 

in biochemistry or biology labs. Moreover, the extensive use of protecting groups and toxic and pollutant 

glycosylation promoters makes the classical glycosylation methods environmental unfriendly. 

O 

PO LG 

PO 

glycosyl donor oxacarbenium ion 

Scheme 1: Classical glycosylation reaction for the formation of O-glycosides. 

O 

O 

HO 

glycosyl 

PO 

O 

O 

O 

acceptor glycoside (stereoselectivity?) 

In a typical glycosylation reaction, after detachment of the leaving group in anomeric position, the glycosyl donor is 

converted into an oxycarbenium ion (Scheme 1) whose stability and conformational preferences strongly influence 

the yield and the stereoselectivity of the reaction. In the case of the most common O-glycosylations, the nucleophilic 

attack of the oxycarbenium ion by the oxygen nucleophile of the glycosyl acceptor (in the case of oligosaccharides) 

is always in competition with the attack of water. For this reason strictly anhydrous conditions are needed to avoid, 

or at least minimize, the unproductive hydrolysis of glycosyl donor. 

In sharp contrast, the so-called chemoselective glycosylation (CG) and click methods (Scheme 2), allow the 

formation of a glycoconjugate in the presence of water (or even directly in aqueous buffers). Chemoselective 

ligation methods, first developed in peptide and protein chemistry for the assembly of large unprotected peptide 

fragment to have access of totally synthetic proteins [12], have been extended to sugar chemistry for the preparation 

of different types of glycoconjugates [13]. 

A 

B 

C 

HO 

O 

HO 

HO 

O OH 

Y aglycon 

O 

X + Y aglycon 

linker/peptide 

+ 

X 

+ 

Y aglycon 

direct 

chemoslective 

glycosylation 

indirect 

chemoslective 

glycosylation 

HO 

chemoselective HO 

ligation of 

glycosylated fragments 

HO 

O 

O 

Y aglycon 

O X Y aglycon 

linker/peptide 

X 

Y aglycon 

Scheme 2: Chemoselective glycosylation methods: A) Direct chemoselective glycosylation of unmodified glycans; B) indirect 

chemoselective glycosylation of glycans chemically modified in anomeric position; C) chemoselective ligation of glycosylated 

fragments. 

These methods have been designed so that a natural reducing sugar (mono or oligosaccharide) is reacted 

chemoselectively with a molecule bearing a nucleophile X group (Scheme 2A). Several copies of the reactive 

nucleophile X can be linked to the same scaffold when a multivalent glycoconjugate should be obtained. These 

methods, mainly based on the use of hydroxylamine and hydrazide nucleophiles, rely on the use of unmodified 

sugars, and will be treated here as “direct” CG methods because the glycosidic bond is formed in the ligation step. A 

second type of techniques has been developed based on the previous sugar modification, with introduction of a 

group X at the sugar’s anomeric position. Group X reacts selectively with a Y group on the aglycone moiety 

(protein, peptide, etc...) (Scheme 2B). These methods are somehow “indirect” because the real glycosidic bond 

(involving the anomeric C-1 carbon of the sugar) should be formed before the ligation step.




CHAPTER 12 

Glycosidases in Synthesis of Glycomimetics and Unnatural Carbohydrates 

Vladimír Křen * 

Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of the Czech 

Republic, Vídeňská 1083, CZ 142 20 Prague 4, Czech Republic 

Abstract: In recent years, carbohydrate-processing enzymes have become the enzymes of choice in many 

applications thanks to their stereoselectivity and efficiency. This chapter presents recent development in 

glycosidase-catalyzed synthesis of unnatural semisynthetic carbohydrate structures via two complementary 

approaches: the use of wild-type enzymes with engineered substrates and mutant glycosidases. Genetic 

engineering has recently produced glucuronyl synthases, an inverting xylosynthase and the first mutant endo-β-Nacetylglucosaminidase. 

A careful selection of enzyme producing strains and aptly modified substrates has resulted 

in rare glycostructures, such as N-acetyl--galactosaminuronates, 1,4-linked mannosides and 1,4-linked 

galactosides. The efficient selection of mutant enzymes is facilitated by high-throughput screening assays 

involving the co-expression of coupled enzymes or chemical complementation. Selective glycosidase inhibitors 

and highly specific glycosidases are finding attractive applications in biomedicine, biology and proteomics. 

Keywords: Glycosidase, glycosynthase, thioglycoligase, glycosyl azide, transglycosylation. 

INTRODUCTION 

In the past decades, the need for new carbohydrate materials and the development of glycomics have provided a 

boost to carbohydrate chemistry. Oligosaccharides can be prepared by classical organic chemistry methods [1] with 

all necessary tedious protection, deprotection, and activation strategies leading often to low yields and increased 

costs. As an alternative to the synthetic approach, enzymatic synthesis, ideally in a stereo- and regiospecific manner, 

is a valued option (Fig.1). Elegant glycosidic bond formation can be accomplished using two main enzyme groups – 

glycosyltransferases (EC 2.4.1.-) and glycosidases (EC 3.2.1.-) [2-5]. In this chapter, we will focus on recent 

advances in carbohydrate synthesis by glycosidases based on two approaches: the use of naturally occurring 

glycosidases with specially engineered substrates, and, use of glycosynthases leading to unnatural and modified 

carbohydrate structures, eventually to the glycomimetics. 

ENZYME ENGINEERING – MUTANT GLYCOSIDASES 

Glycosynthases, created by site-directed mutagenesis, represent a large group of mutant glycosidases. Since their 

introduction in 1998 [6, 7] they have caused a revolution in the high-yield enzymatic synthesis of carbohydrates and 

have been the topic of numerous reviews [8, 9]. 

The mutation of the active site nucleophile to a non-nucleophile, such as alanine, renders a retaining glycosidase 

hydrolytically inactive but the resulting mutant – glycosynthase – can transfer an activated sugar donor, such as a 

glycosyl fluoride, onto a suitable acceptor substrate (Fig. 2). All aspects of using glycosyl fluorides in enzymatic 

reactions were described in a classical review by Williams and Withers [10]. 

A number of new glycosynthases have been generated in recent years; for example, the glycosynthase derived from 

Thermus thermophilus -glycosidase, which can selectively synthesize the 1,3 glycosidic linkages in yields of up 

to 90 % [12], and the xylosynthase derived from Agrobacterium sp. -glucosidase [13]. Other xylosynthases 

followed, which were based on endo-1,4--xylanase from Cellulomonas fimi [14] and -xylosidase from 

Geobacillus stearothermophilus [15]. A noteworthy case of substrate engineering with hyperthermophilic 

*Address correspondence to Vladimír Křen: Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of 

the Czech Republic, Vídeňská 1083, CZ 142 20 Prague 4, Czech Republic; E-mail: kren@biomed.cas.cz

Glycosidases in Synthesis of Glycomimetics Synthesis and Biological Applications of Glycoconjugates 227 

glycosynthase from Sulfolobus solfataricus using glycosyl acceptors of varying anomeric configuration and 

lipophilicity was shown by Moracci et al. [16]. Glycosynthases have proved their potential in a number of 

applications involving the preparation of difficult-to-obtain glycosylated structures; for example glycosphingolipids, 

which are potential therapeutics for a range of pathologies including Alzheimer´s, Parkinson´s diseases and stroke 

[17], and, recently, glycosylated flavonoids, acting for example as immunosuppressants [18]. 

Figure 1: Hydrolysis mechanisms: A) of retaining and B) inverting glycosidase; C) of -N-acetylhexosaminidase, which uses a 

modified retaining mechanism.

228 Synthesis and Biological Applications of Glycoconjugates Vladimír Křen 

A 

B 

mutated acid/ base (Glu Ala) 

O 

O 

CH3 

O 

F 

O - 

- O O 

H 

HO 

O 

O 

nucleophile 

acid/ base 

CH3 

NO2 

HS 

HO 

OH 

O 

OH 

NO2 

OR 

mutated nucleophile (Glu Ala) 

C 

mutated acid/ base 

(Glu Ala) 

OH 

OR 

O 

OH 

O2N 

HO 

HF 

NO2 

O 

CH3 

O 

O 

O 

HO O 

- 

S 

HO 

HO 

O 

CH3 

CH3 

F 

O - 

S 

HO 

OH 

OR 

O 

O 

S 

HO 

OH 

OR 

O 

F 

H 

OH 

H 

OH 

- 

mutated nucleophile 

(Glu Gly) 

CH3 

OH 

O 

OH 

OH 

OR 

O 

OH 

OR 

O 

O 

CH3 

S 

HO 

O - 

OH 

OR 

O 

OH 

Figure 2: Reaction mechanisms of mutant glycosidases [11]: Transglycosylation mechanism: A) of a glycosynthase; B) of 

glycosyl thioglycoligase; C) of thioglycosynthase. A) Glycosynthase, in which the catalytic nucleophile Glu was mutated to Ala, 

uses fluoride of the opposite anomeric configuration as a donor. Its mechanism is common both for exo- and endoglycosynthases. 

R = aglycon, e. g., p-nitrophenyl; B) Thioglycoligase, in which the catalytic acid-base Glu was mutated to Ala, 

uses reactive 2,4-dinitrophenyl glycoside donors and nucleophilic thiosugar acceptors (R = aglycon); C) Double mutant 

thioglycosynthase, with the catalytic acid-base Glu mutated to Ala and the catalytic nucleophile Glu mutated to Gly, uses 

inverted glycosyl fluoride donors and thiosugar acceptors (R = aglycon). 

In 2006, the Withers group presented the first glycosynthase that was able to transfer glucuronyl residues [19]. 

Interestingly, the alanine mutant (Glu383Ala) was the most efficient in the transfer of both glucuronyl and 

galacturonyl moieties, which is in contrast to previous observations with other glycosynthases, where generally 

serine [20] and glycine [21] mutants exhibited superior activity to those with alanine. Another glucuronyl synthase, 

Glu504Gly mutant of -glucuronidase from E. coli, was applied in the -glucuronylation of a range of alcohols [22]. 

Important advances have also been accomplished in the development of thioglycoligases – retaining glycosidases in 

which the acid–base catalytic residue is substituted by another amino acid, mainly Ala and Gln (Fig. 2B) [23]. These 

mutant enzymes use activated donor glycosides, such as dinitrophenyl glycosides with thiol acceptors. Müllegger et 

al. [24] discovered an improved mutant thioglycoligase (Glu170Gln) from Agrobacterium sp. by saturation 

mutagenesis, which can use donor sugars with a relatively poor leaving group such as -D-glucopyranosyl azide.


Diels-Alder Based Synthesis of Glycomimetics 

Cristina Nativi a,* , Elisa Dragoni b , Barbara Richichi a and Stefano Roelens c 



CHAPTER 13 

a Dipartimento di Chimica, Universita’ di Firenze, via della Lastruccia, 3,13 I-50019 Sesto F.no (FI), Italy; 

b ProtEra, Polo Scientifico Universita’ di Firenze, viale delle Idee, 22 I-50019 Sesto F.no (FI), Italy and c CNR, 

Istituto Metodologie Chimiche (IMC), Dipartimento di Chimica, Universita’ di Firenze, via della Lastruccia, 3,13 I- 

50019 Sesto F.no (FI), Italy. 

Abstract: [4+2] Cycloadditions between “in situ” generated ,’-dioxothiones and unprotected or variously 

protected glycals provide a powerful synthetic way to obtain glycomimetics chemo-, regio- and stereoselectively. 

Examples of glycopeptidomimetics and mimetic of tumor antigens are reported. 

Keywords: Hetero Diels-Alder, mimetics, tumor antigens, glycopeptides, matrix metalloproteinases. 

INTRODUCTION 

Diels-Alder reactions are a powerful tool to obtain regio- and stereoselectively complex molecules containing an 

unsaturated six member ring under mild reaction conditions. Owing to the simplicity of operation and to mildness of 

reaction conditions, [4+2] cycloadditions are largely preferred to other reactions when a suitable diene and 

dienophile can be envisioned. In addition, the variant in which either the diene or the dienophile can contain a 

heteroatom makes them attractive substrates for the synthesis of six-member ring heterocycles. 

Complex chiral molecules can effectively be prepared relying on the stereochemical features of [4+2] 

cycloadditions. Indeed, although the formation of more than one regioisomeric/stereoisomeric cycloadduct is 

possible in Diels-Alder reactions, the propensity for one regio- and/or stereoisomer is predictable. A representative 

example is the recently reported synthesis of the oxadecalin core of phomactin A 1 (Fig. 1), a selective antagonist of 

platelet activating factor (PAF) [1], via a stereoselective Diels-Alder reaction between the appropriately 

functionalized diene 2 and maleic anhydride (Fig. 1, transition state 3). 

Figure 1: Structure of phomactin 1, diene 2 and transition state 3. 

Me 

Me 

O 

Me 

1 

OTBS OTBDPS 

O 

Si 

O 

O 

iPr 

iPr 

Me 

2 

OH OH 

O 

Other literature examples concern the total synthesis of the natural product vernolepin 4 [2] or the Diels-Alder 

reactions between pyran-based alkoxy-dienes 5 and various dienophiles [3-5]. In these reactions, the endo addition is 

highly favored with a suitable dienophile like maleic anhydride [6] (Scheme 1). 

*Address correspondence to Cristina Nativi: Dipartimento di Chimica, Universita’ di Firenze, via della Lastruccia, 3,13 I-50019 Sesto F.no 

(FI), Italy; E-mail: cristina.nativi@unifi.it 

Me 

O 

TBSO 

O 

O 

Si 

O 

O 

O 

TBDPSO Me 

3

Diels-Alder Based Synthesis of Glycomimetics Synthesis and Biological Applications of Glycoconjugates 241 

Scheme 1: Structure of vemolepin 4 and Diels-Alder reaction between 5 and maleic anhydride. 

More recently, the synthesis of an interesting glycodendrimer showing both water-solubility and lipophilicity was 

accomplished employing Schimdt glycosylation and Diels-Alder cycloaddition recursively [7] (Scheme 2). 

In the last decade we successfully developed hetero Diels-Alder reactions between unusual heterodynes, i.e. ,’dioxothiones 

and 1-,4- and 5-glycals [8-10]. Glycals are versatile unsaturated sugar derivatives, widely used in 

carbohydrate chemistry as enantiomerically pure precursors [11-13] of saccharidic and non-saccharidic compounds. 

On the other hand, ,’-dioxothiones were a class of heterodienes little known until Capozzi and co-workers 

published the first applications in hetero Diels-Alder reactions [14, 15]. 

Scheme 2: Synthesis of aromatic glycodendrimers. 

O 

AcO 

O 

AcO 

AcO 

AcO 

AcO 

AcO 

AcO 

AcO 

AcO 

AcO 

AcO 

4 

O 

OAc 

5 

AcO 

AcO O 

O 

O 

OH 

O 

O 

NPht 

O 

PHtN 

Diels-Alder 

Reaction 

O 

PHtN 

O 

PHtN 

O 

O 

O 

O 

O 

PhH, reflux 

68% 

OMP 

NH 

CHl 3 

Schmidt 

Glycosylation 

AcO 

AcO 

AcO 

AcO 

AcO 

AcO 

O 

PHtN 

O 

PHtN 

AcO 

AcO 

AcO 

AcO 

O 

O 

OAc 

O 

O 

OAc 

O 

O 

O 

HO OH 

AcO 

AcO O 

O 

O 

O 

PHtN 

OR 

R=MP 

R = C(=NH)CCl3 

O 

O 

5:1 

O

242 Synthesis and Biological Applications of Glycoconjugates Nativi et al. 

’-Dioxothiones as Electron-Poor Dienes 

’-Dioxothiones are highly reactive conjugated thiocarbonyl derivatives formed in situ and trapped as dienes in 

[4+2] Diels-Alder reactions [16-18]. Some years ago we developed the efficient synthesis of ’-dioxothiones from 

sufenylphthalimides and their trapping as electron-poor heterodienes in highly selective Diels-Alder reactions. 

Phthalimidesulfenyl chloride 6, (PhthNSCl, Phth = phthaloyl), reacts smoothly with -diketones, -ketoester or ketotiolester 

7 or with silylderivatives 8 to afford -acyl-N-thiophthalimide derivatives 9, which easily and 

quantitatively generate the reactive thiones 10 in the presence of mild bases. The transient thiones 10 can be trapped 

in Diels-Alder reactions with electron rich alkenes thus forming 5,6-dihydro-1,4-oxathiin derivatives 11 [14, 19, 20] 

(Scheme 3). Phthalimidesulfenyl chloride as well as phthalimide derivatives, 9 are stable compounds which can be 

prepared in grams scale and obtained pure by crystallization. 

In these inverse electron-demand Diels-Alder reactions, the molecular orbitals involved in the cycloadditions are the 

HOMO of the dienophile and the LUMO of the diene [15], therefore the 1,4-oxathiins formed through this synthetic 

strategy are always single chemo- and regioisomers. Indeed, the shape of the molecular orbitals obtained by 

molecular calculations ¥ showed that the symmetry of the HOMO of dienophiles and of the LUMO of dienes (’dioxothiones) 

is optimal for superimposition and that the orbitals with the largest coefficients are found on the sulfur 

atom of the thione and on the vinyl methylene of the alkene (Scheme 3). 

6 

Scheme 3: Synthesis of phthalimide derivative 9 and oxathiin 11. 

O 

O 

N 

O O 

S + 

Cl 

R R' 

R=R'=Alk,Ph 

R = Alk, Ph; R' = OR, SR, CN, COR 

9 

6 + 

PhthN = 

Me 3SiO O 

R 

Py, CHCl3 

- PhthNH 

O 

N 

O 

R 

R' 

O 

7 

S 

10 

O 

CHCl 3 

22, CH 2Cl 2, -18 °C 

-Me3SiCl 

8 

R=R'=Alk,Ph 

R=Alk,Ph;R'=OR,SR,CN,COR 

R' OR'' 

H 

O O 

R R' 

SNPhth 

9 

Cycloaddition reactions of ’-dioxothiones with electron-rich alkenes are also characterized by high 

stereoselectivity. Retention of the dienophile Z/E geometry was always observed with cyclic or linear alkenes. 

Moreover, the use of chiral non-racemic ’-dioxothiones, obtained from -ketoesters bearing a chiral auxiliary in 

the ester and/or in the ketone residue, gave an appreciable discrimination between the enantiotopic faces of several 

dienophiles [21] (Scheme 4). 

¥ 

Molecular orbitals were obtained by geometry optimization through ab initio Hartree-Fock calculation, using a 3-21G* basis set implemented in 

the Spartan program for PC 

R' 

R 

O 

9 

O 

S 

11 

O R''


Analytical Tools for Protein-Carbohydrate Interaction Studies 

Cédric Goyer and Pierre Labbé * 



CHAPTER 14 



Abstract: A wide range of assays has been used to characterize protein-carbohydrates interactions allowing access to 

various quantitative information including thermodynamic ones such as stoichiometry of binding, binding constant, 

enthalpy of binding as well as kinetics and mechanistic ones. An understanding of each technique is essential in order 

to propose and validate appropriate interaction models that rationalize experimental data. Due to the complexity of 

carbohydrate-lectins interactions as an inherent consequence of multivalent interactions, a careful control of 

experimental conditions is necessary in order to avoid misinterpretation. This article will briefly present the various 

techniques that are commonly used to characterize sugar-lectin interaction: inhibition of hemagglutination assay 

(HIA), enzyme-linked lectin assays (ELLA), isothermal titration calorimetry (ITC) and surface plasmon resonance 

(SPR). Recently introduced, force spectroscopy by atomic force microscopy (AFM) is a new approach for measuring 

carbohydrate-protein interaction at the level of a single protein. 

Keywords: Biomolecular interaction, biosensors, surface plasmon resonance, isothermal calorimetric titration, 

atomic force spectroscopy. 

INTRODUCTION 

Lectins are carbohydrate binding proteins other than enzymes and antibodies [1] and exist in most living organisms 

ranging from viruses and bacteria to plants and animals [2]. They constitute cellular receptors that play a fundamental 

role in a wide range of biological processes including physiological and pathological events [3] such as adhesion of 

infectious agents to host cells, inflammatory processes, cell interactions in the immune system, growth and metastasis 

of tumour cells. Interactions between lectins and carbohydrates occur in the highly hydrated biological environment 

where hydration and solvation effects play a determinant role [4]. Carbohydrates primarily bind to polar groups of the 

lectin active site through hydrogen-bonding of their hydroxyl groups. Complementary hydrophobic interactions also 

occur between hydrophobic patches of the carbohydrate structure and the aromatic moieties that are present in the lectin 

constituents. It has been early recognized that an individual carbohydrate ligand binds to a lectin site weakly, with 

affinity constant generally in the range 1µm-1mM. The reason for this weakness lies in the solvent-exposed nature of 

the lectin binding-site, which are shallow pockets making few direct contacts with the ligands [5]. Considering the high 

specificity of biorecognition processes, such a weak binding is unlikely to be of any biological significance. However it 

is now well established that the weak binding affinities are efficiently overcome through so-called multivalent 

interactions, also known as “cluster glycoside effect”, that allow increased binding affinities and higher specificity 

required for a biologically relevant recognition process. Polyvalent associations that occur throughout biology present a 

number of characteristics that monovalent interactions do not exhibit. In most cases, polyvalent interactions rather than 

a strong single one are used in biological systems. Thus, the ability of lectins to distinguish between subtle variations of 

sugar structure makes them particularly efficient for decoding such carbohydrate-encoded information known as 

“glycocode”. Since lectins and carbohydrates are both commonly present at the cell surface and because sugars posses 

tremendous coding capacity, they constitute excellent cell recognition markers [6] that can be consequently exploited 

for therapeutic and diagnostic applications. 

A complete comprehension of the mechanism of protein-carbohydrate interaction at the molecular scale requires the 

elucidation of both structural and energetic aspects of the process. A large amount of structural data is now 

available, essentially from X-ray crystallography and NMR spectroscopy that have been used to elucidate the 

*Address correspondence to Pierre Labbé: Département de Chimie Moléculaire, UMR-CNRS 5250 & ICMG FR 2607, Université Joseph 

Fourier, 38041 Grenoble Cedex 9, France; E-mail: pierre.labbé@ujf-grenoble.fr

256 Synthesis and Biological Applications of Glycoconjugates Goyer and Labbé 

structures of lectins, sugars and their complexes. In addition an increasing number of experimental methods have 

been employed to determine the thermodynamics and kinetics of the binding process. Whereas the initial research’s 

were focusing on large glycoproteins and neoglycoconjugates, the recent progress in engineering new polymeric or 

small molecular weight sugar ligands provide new molecular tools for unravelling these complex ligand-receptor 

interactions. The main goal of this chapter is to give an overview of the various analytical tools that are classically 

used for studying sugar-lectin interactions. Since this topic was discussed in several reviews [4, 6-8] we will briefly 

present them and discuss their specificity and limitations. A special focus will be devoted to the new expanding tools 

and techniques that allow improving characterization at molecular level. 

A wide range of assays has been used to characterize protein-carbohydrates interactions allowing access to various 

quantitative information including thermodynamic ones such as stoichiometry of binding, binding constant, enthalpy 

of binding as well as kinetics and mechanistic ones. An understanding of each technique is essential in order to 

propose and validate appropriate interaction models that rationalize experimental data. Due to the complexity of 

carbohydrate-lectins interactions as an inherent consequence of multivalent interactions, a careful control of 

experimental conditions is necessary in order to avoid misinterpretation. Historically, the strength of sugar-lectin 

interaction has been quantified by inhibition of hemagglutination assay (HIA [9]), enzyme-linked lectin assays 

(ELLA [10]) although several spectroscopic techniques (NMR [11, 12], UV spectroscopy [13], fluorescence 

polarization [14-16]), have also been utilized. Finally among the more advanced methods, isothermal titration 

calorimetry (ITC) and surface plasmon resonance (SPR) constitutes two complementary and powerful techniques. 

Recently introduced, force spectroscopy by atomic force microscopy (AFM) has been applied for measuring 

carbohydrate-protein interaction at the level of a single protein. 

INHIBITION OF HEMAGGLUTINATION ASSAY (HIA) 

Hemagglutination is a specific form of agglutination that involves red blood cells. These erythrocytes present on 

their surface N-Acetylneuraminic acid, the predominant sialic acid found in mammalian cells. This negatively 

charged residue is found in complex glycan on mucins and glycoproteins found at the cell membrane. Chicken or 

porcine erythrocytes are most commonly used for this assay [9]. Starting from an anticoagulated erythrocytes 

suspension, addition of a soluble multimeric lectin cross-links erythrocytes and produces a gel-phase that does not 

settle down. Addition of soluble lectin in the presence of soluble carbohydrate ligands inhibits this hemagglutination 

process as a consequence of a competitive binding of the carbohydrate ligand of interest to the lectin active site. At 

high ligand concentration the hemagglutination reaction is completely inhibited and the anticoagulated erythrocytes 

settle down. The assay reports the minimum ligands concentration (IC50) that inhibits the hemagglutination process 

[7]. This historical assay suffers quite a lack of reproducibility and was used as a simple and qualitative method that 

allowed ordering soluble ligands in a rough order of binding affinity. 

ENZYME-LINKED LECTIN ASSAY (ELLA) 

Enzyme-linked lectin assays (ELLA) [17] assays are conceptually similar to the common ELISA (enzyme-linked 

immunosorbent assay) but are unique in that they rely upon non-immunologic reagents. ELLA assays constitutes a 

rapid method for evaluating lectin binding properties of soluble natural or synthetic lectin ligands in solution [18- 

21]. Microtiter plates wells are coated with a reference ligand that is often a (neo)glycoprotein or a polysaccharide. 

Lectin-enzyme conjugate and the soluble ligand are added to the wells and after an incubation period, the wells are 

evacuated, rinsed and refilled with a prodye substrate for the enzyme conjugate. The amount of color formed, 

monitored spectrophotometrically, is proportional to the concentration of lectin-enzyme conjugate immobilized on 

the wells and inversely proportional to the affinity of the soluble ligand. IC50 values for inhibition of the binding of 

an enzyme-labeled lectin to the immobilized reference ligand (the matrix) by the soluble ligands to be tested are thus 

determined. Generally the relative inhibitory powers are similar to those deduced from HIA assays although large 

differences in IC50 are often observed. One advantage of ELLA compared to solid-phase binding assays such as 

surface plasmon resonance (SPR) is that both, lectin and ligand are in solution. However, the use of glycoproteins 

from natural sources as reference ligands is limited because their varying complex carbohydrate composition due to 

microheterogeneity leads to reduced reproducibility of IC50 values. Furthermore, incomplete inhibition of lectin 

binding to the plate surface is often observed, that is, maximal inhibition occurs at considerably less than 100%. 

Finally ELLA provides inhibition constant and not a concentration binding constant.

Analytical Tools for Protein-Carbohydrate Interaction Studies Synthesis and Biological Applications of Glycoconjugates 257 

ISOTHERMAL TITRATION MICROCALORIMETRY (ITC) 

Isothermal Titration Calorimetry (ITC) is a well-established and powerful methodology for studying biomolecular 

interactions. Dam and Brewer [22] have published a very comprehensive review on the use of ITC to study lectincarbohydrates 

interactions whereas Lundquist and Toone [7] discussed the advantages and limitations of this technique. 

Titration calorimetry measures the heat evolved during ligand binding as a function of titrant concentration. Usually the 

lectin is placed in the calorimetric cell, the carbohydrate ligand is added in a series of 20 to 50 injections and the heat 

absorbed or produced during the binding is recorded. The shape of the resulting titration curve is a function of the 

number of lectin binding sites in the cell and of the binding constant. Analysis of the experimental data needs the use of 

a binding model. This model is then used to fit the experimental binding curve, which hopefully gives access to binding 

constant (Ka), free energy (G°), enthalpy (H°), entropy (S°) of binding and stoichiometry (n) of interaction for each 

class of binding sites that are assumed in the model. From temperature dependence of enthalpy and entropy changes, 

the change in heat capacity (Cp°) can be evaluated. Importantly, ITC is the only technique that allows the 

simultaneous determination of the thermodynamic binding parameters (Ka, G°, H°, S°) and stoichiometry (n) in a 

single experiment. Due to material availability, solubility limitations and sensitivity issues, ITC is practically effective 

for studying systems exhibiting binding constants ranging roughly from 10 3 to 10 7 M -1 . 

As previously discussed [7], ITC presents many advantages as compared to other methods. First ITC gives a 

concentration binding constant rather than an IC50, which can be related directly to free energy change of the 

interaction. It is also the only methods able to provide directly binding enthalpies in contrast to other methods that are 

based on the temperature dependence of binding constant and use of Gibbs-Helmholtz relationship. Thus ITC is unique 

in allowing direct measurement of the enthalpy of binding as a function of temperature. This in turn provides a direct 

measure of the change in molar heat capacity accompanying binding. This molar heat capacity change is of primary 

importance when studying the effect of solvent reorganization on the thermodynamic of ligand binding [23]. 

Main drawbacks concerns the large amount of needed material (typically milligram quantities of both lectin and 

ligand), which is often a strong practical limitation. As a matter of fact, an adequate model describing the binding 

process has to be elaborated and validated before using it for fitting experimental data in order to extract quantitative 

thermodynamic information. 

SURFACE PLASMON RESONANCE (SPR) 

Surface plasmon resonance (SPR) biosensor instruments are sensitive to the common property of refractive index or 

mass change that is induced by complex formation at the surface of a biospecific sensor [24-28]. This optical 

method can provide both equilibrium and dynamic characterization of the interaction process [29, 30]. The method 

is label-free since it is based on refractive index changes, and monitors the interactions in real time. Although SPR 

was first observed in 1902 [31], its use for biosensors was only developed in the mid 1980s. Today the technique is 

widely used for studying bio-interactions, in particular because of the commercial availability of several 

instruments. Assessment of carbohydrate-lectin interactions by SPR has been reviewed in several articles [4, 7, 32]. 

A very complete and detailed article by Wilson et al. [33] focusing on the use of SPR for quantitative analysis of 

small molecule- nucleic acid interactions provides fundamental and practical information of general interest. 

The physical basis of SPR has been presented in several reviews [34-35]. SPR phenomenon is observed under 

condition of total internal reflexion (incidence angle higher than critical angle, see Fig. 1) at an interface between a 

dielectric medium of high refractive index (usually a prism) that is covered by a thin film (2.5 to few tens of nm) of an 

adequate metal (aluminium, silver, gold), in contact with a dielectric medium of low refractive index (usually the liquid 

buffer of interest). 

Gold is usually preferred due to its inertness under biological conditions and to its easy and versatile 

functionalization using Au-S bond formation by reaction with thiolated derivatives. In absence of a metallic film, the 

fully reflected beam leaks an electrical field intensity (i.e. the evanescent field) into the medium of low refractive 

index. No photons exit the reflecting surface but the intensity of the corresponding evanescent electrical field 

decreases exponentially with distance from the interface, decaying over a distance of about ¼ wavelength beyond 

the surface in the low refractive index medium (Fig. 2a).

A 


Index 

β-N-acetylglucosaminidase 226 

β-N-acetylhexosaminidase 227, 229-234-236 

N-acetyllactosamine 93, 99 

Activated donor 228-230 

Acylhydrazide 206-207, 209, 218 

Adhesin 3-5, 7, 9, 12-16, 21, 23-24, 29-31, 186 

Agglutination 13, 15, 23, 49-50, 58-59, 167, 170, 256 

Agglutinin 

- Wheat Germ Agglutinin (WGA) 51, 94, 133, 169, 186 

- Helix pomatia agglutinin (HPA) 140, 263 

- peanut (Arachis hypogaea) agglutinin (PNA) 58, 94, 133-134, 140 

- Vicia Villosa Agglutinin (VVA) 50-51 

- Viscum album L. agglutinin (VAA) 52, 95, 136, 186, 246-248 

Alkyl mannoside 20 

Allosteric control 30 

1,2-alternate 40-41 

1,3-alternate 40-41, 46, 52-54, 105 

Amide bond formation 43, 45, 69, 78, 220 

Aminooxy 131-132, 134, 141, 153, 158, 179, 183, 208, 

216, 220 

Aminooxyacetyl 208 

Analytical ultracentrifugation (AUC) 86 

Anionic guest 57 

Anti-adhesion 12, 28, 29, 55, 58, 186 

Anti-adhesive 29, 31, 187, 188 

Antibody (Ab) 19, 56, 57, 139, 159, 185, 187, 220 

Antigen Presenting Cells (APC) 171-172, 176 

Anti-tumor effect 101, 139, 172, 174, 212, 244 

-L-arabinopyranoside 233 

Aromatic amino acid 57 

Asialoglycophorin 51 

Asialoglycoprotein receptor (ASGP-R) 94, 190 

Aspergillus oryzae 230-231, 234-236 

Atomic force microscopy (AFM) 29, 49, 59, 68, 86, 178, 184, 255-256, 260-263 

Azide 26-27, 47, 68, 71-72, 75, 82, 100, 153, 157, 

168, 212, 220, 226, 228-229, 233, 235 

B 

Bacillus circulans 230-231 

B-cell 137, 168 

-bromoalanine 214 

Betulinic acid (BA) 217 

Bingel reaction 69-73 

Biochips 145, 155-156 

Bioisosterism 207 

Biomaterials 61 

Biosensor 70, 145, 185-187, 255, 257 

BK virus 51 

Blood brain barrier 189 


All rights reserved - © 2011 Bentham Science Publishers

268 Synthesis and Biological Applications of Glycoconjugates Renaudet and Spinelli 

Bolaforms 58-59 

C 

(Glyco)Calixarene 36-61, 85, 105-106, 129, 165, 185 

Capillary tubes 141 

Carbohydrate mediated adhesion 60 

Carbohydrate phosphoramidite 146-147, 154, 160 

Carbohydrate recognition domain (CRD) 8-9, 15-17, 20-25, 27, 30, 64-66, 73, 92-93, 96, 

97 

Carbohydrate-carbohydrate interaction 169-170, 177-178, 184-185, 219 

Carbohydrate-processing enzyme 73, 226 

Carbohydrate-protein interaction 36, 129, 135, 140, 164, 169, 171, 184, 186, 

190, 220, 255-256, 264 

Carbopeptoid 150-151 

CD69 52, 138-139, 235 

C-glycoside 48, 82, 116, 212, 243 

Chaperone/usher pathway 13-14, 16 

Chemoselective 131-132, 151, 160, 165, 167-168, 177, 179, 

203-221 

Cholera toxin 6-7, 9, 44-45, 55, 65, 78-81, 87, 136, 167, 185, 

191, 200 

CH-π interaction 57, 146 

Cycloaddition 65-66, 73, 82, 242, 244 

- Azide-alkyne 46-47, 71, 86, 132, 153, 180 

- Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAc) 46-47, 71-73, 75, 132, 139, 153, 154, 155- 158 

- Diels-Alder 67, 213, 240-252 

- 1,3-dipolar 46-47, 66, 72, 99, 103, 105 

- Huisgen 46, 71-73, 153, 157, 180 

- [3+2] 68, 214 

- [4+2] 240, 243, 248 

Click chemistry 46-47, 64, 71, 73- 75, 151, 168, 214 

Cluster glycoside effect 92, 94, 104, 255, 264 

Cluster mannoside 12, 20-21 

Combinatorial 

- chemistry 95, 116, 118, 129, 133, 141 

- libraries 95, 116-117, 119-120, 123, 133 

Computer modeling 24 

Cone 40-41, 43-44, 46-47, 51-54, 56, 71, 105 

Conformational flexibility 17, 24 

(glyco) Conjugation 22, 156-157, 160, 165, 168, 176-177, 179-181, 

183, 190, 204, 207-208, 212-215, 219-220 

Cross-linking 27-28, 49-50, 55, 58-59, 68, 75, 137, 168, 178 

Cyclodextrin 37, 65, 102-103, 105, 129 

Cyclopeptide-based glycocluster (CBG) 129-141 

D 

Delivery 58, 79, 118, 123, 145, 151-152, 166-167, 171- 

172, 190 

- drug 36, 57, 60, 61, 68, 92, 116, 123, 125, 164, 166, 

172-173, 213 

- gene 36, 61, 94, 145, 175, 192 

Deltorphin 209

Index Synthesis and Biological Applications of Glycoconjugates 269 

(glyco)Dendrimer 9, 20, 37, 64, 69, 75, 78, 83-86, 102, 108-109, 

116-125, 129, 165, 170, 177, 184, 187, 219, 241 

Dendri-RAFT 134-136 

4-deoxy sugar 234 

N,N´-diacetylchitobiose 231-232, 234-235 

Diazirine 25-27 

Digitoxin 216-217 

Dithiogalactoside 100-102 

Dithiopyridyl 215 

DNA binder 60 

DNA encoded library 159 

DNA-Directed Immobilization (DDI) 155 

Docking 12, 22-24, 55, 234-235 

Donor Strand Complementation (DSC) 15 

Donor Strand Exchange (DSE) 13-15 

DOSY NMR 71, 184 

D-ribose-phosphate 59 

Dynamic Light Scattering (DLS) 49, 59, 68, 84, 86-87, 169 

E 

Electrode 140, 185 

Electrophoretic mobility 49 

ELISA (Enzyme-Linked Immunosorbent Assay) 19, 20-21, 23-24, 27, 85, 86, 94, 171-172, 174- 

175, 185, 187, 256 

ELLA (Enzyme-Linked Lectin Assay) 51, 105, 121-122, 133, 255, 256 

Enantioselectivity 57 

Enhancement factor 37-38, 54, 56 

Epimerization 131, 234 

Epitope orientation 52 

Erythropoietin (EPO) 215 

Escherichia coli (E. coli) 4, 6-7, 9, 12-16, 18-19, 23, 27-28, 31, 75, 78, 

81, 87, 181-182, 186-187, 190-191, 228-229, 

233 

ESI-MS 57 

F 

Fibrillum 13-14 

fim gene cluster 13, 18 

Fimbriae 3-4, 9, 12-21, 23, 27, 29, 31 

FimC/FimH chaperone-adhesin complex 16 

Flow cytometry 94, 146 

Fluorescence 20, 49, 56, 58, 82, 85, 105, 165, 186, 189, 190, 

219, 256 

- activated cell sorter scans 53 

- anisotropy 132, 140 

Frontal affinity chromatography (FAC) 94, 97 

Fucose 7-8, 95, 116, 119-122, 131, 146, 172-173, 189 

-D-fucopyranoside 233 

-D-fucosidase 229 

(glycol)Fullerene 64-75, 94, 129 

Fullerenol 68 

Functionalisation 40-42

chapter 2 - Bentham Science

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