Glycopolymer Code: Synthesis of Glycopolymers and their Applications

by C. Remzi Becer (Editor), Laura Hartmann (Editor)

Overview

Glycopolymers are important for cell signalling, recognition pathways, and their role in the immune system. They are gaining attention for new applications in tissue engineering and drug delivery. Glycopolymer Code discusses the preparation, characterization and applications of glycopolymers providing a complete overview of the topic with examples from the latest research.

Specific chapters cover both basic and advanced synthesis techniques to prepare glycopolymers, the analytical techniques used to investigate lectin receptor glycopolymer interactions, the properties and types of lectins that are widely used to understand the multivalent interactions and various applications of synthetic glycopolymers.

With contributions from leading researchers in the field, the book is a unique source for graduates and academics new to the subject and already working in the area of glycopolymers.

Product Details

ISBN-13:	9781782624684
Publisher:	Royal Society of Chemistry
Publication date:	03/23/2015
Series:	ISSN
Sold by:	Barnes & Noble
Format:	eBook
Pages:	292
File size:	15 MB
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About the Author

Dr. Becer is currently a senior lecturer in Materials Science at Queen Mary, University of London. He has been working on developing new polymer synthesis techniques and functional materials such as smart polymers, glycopolymers. The latter has been in the focus of Dr. Becer’s research in the last five years and he has been published several research and review articles on this topic. Dr. Becer has several collaboration partners working on glycopolymers and has good links with other academics working on this field all around the World.

Dr. Hartmann is currently working as an independent Emmy Noether research groupleader at the MPI of Colloids and Interfaces in the Department of Biomolecular Systems (head: Prof. Peter H. Seeberger). Her research interests focus on the synthesis of biomimetic macromolecules and hydrogels combining synthetic scaffolds based on monodisperse, sequence-defined oligo/polymers with biological ligands such as sugars or peptides. She has published a number of papers related to her work on carbohydrate functionalized polymers and has several collaboration partners working in the area of carbohydrate and material research as well as good links with other academics working in this field around the World.

Read an Excerpt

Glycopolymer Code

Synthesis of Glycopolymers and Their Applications

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By C. Remzi Becer, Laura Hartmann

The Royal Society of Chemistry

Copyright © 2015 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-78262-468-4

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CHAPTER 1

Small Molecule Ligands for Bacterial Lectins: Letters of an Antiadhesive Glycopolymer Code

THISBE K. LINDHORST

1.1 Introduction

This chapter discusses how glycopolymers might function in the context of microbial adhesion. This is an important topic as attachment of viruses and bacteria to surfaces is a global problem and for host organisms it has fundamental implications for their vitality. This was considered when the human microbiome project was launched in 2008. Consequently, the human microbiome project is dedicated to research into how changes of microbial colonization influence human health and disease.

It has turned out that microbial colonization of the body is largely associated with the glycoconjugate decoration of the host cells, named the 'glycocalyx.' The glycocalyx of a cell is an extracellular compartment comprising a huge variety of different glycoconjugates. Strikingly, it forms an anchoring platform for invading microbes. It has been asked how carbohydrate recognition has evolved among microbes, how it is regulated and how it develops during the lifetime of an organism, in other words, how binding to cell surface carbohydrates is being 'spelled' (Figure 1.1). It has been suggested that the oligo- and polysaccharide structures that are expressed on cell surfaces function in the sense of a 'glycocode,' thus paralleling the biology of carbohydrates with the alphabet of a language, in order to decipher its meaning. Of course, it is sensible to consider the diversity of carbohydrate structures as a biologically meaningful concert corresponding to the whole of molecular interactions. Glycopolymers can be regarded as a means to interrogate a putative carbohydrate alphabet and, moreover, as a powerful tool to prevent microbial colonization of surfaces.

1.2 Lectin-Mediated Bacterial Adhesion

To colonize cell surfaces of the host, bacteria, for example, have to accomplish a process of adhesion in order to withstand natural defence mechanisms and mechanical shear stress. Stable adhesion can lead to the formation of bacterial biofilms, which is accompanied by vital advantages for the microbial colonies but disadvantages for the host. Finally, adhesion apparently is a prerequisite for bacterial infections that constitute a major global health problem, in particular in developing countries. Bacterial infections are especially dangerous for newborns and young children, with the most common serious neonatal infections involving bacteremia, meningitis and respiratory tract infections. Key pathogens in these infections are Escherichia coli, Klebsiella sp., Staphylococcus aureus and Streptococcus pyogenes.

One important mechanism of bacterial adhesion is based on molecular interactions between cell surface carbohydrates of the host and specialized carbohydrate-specific bacterial proteins called adhesins or lectins. Lectins were first described at the end of the 19th century, when it was shown that plant lectins have the ability to agglutinate erythrocytes blood group specifically. As we know today, this is a result of a multivalent carbohydrate–lectin interaction. In 1954, Boyd and Shapleigh proposed the term lectin 'for these and other antibody-like substances' with blood group-specific agglutination properties. In the 1990s, Lis and Sharon suggested that 'lectin' should be used as a general name for all proteins of non-immune origin that possess the ability to agglutinate erythrocytes and other cell types. Early classification of lectins relied on their carbohydrate specificity. However, today lectins are grouped on the basis of their structural features and especially the relatedness of their carbohydrate binding sites, which are often called 'carbohydrate recognition domains,' or CRDs.

It is common knowledge today that lectins are ubiquitously spread in Nature, comprising many different functions in different organisms. Also, many bacteria, in particular those of the Enterobacteriaceae family, have the ability to agglutinate erythrocytes by their own lectins. This haemagglutination activity of bacteria is almost always associated with the presence of multiple filamentous protein appendages projecting from the surface of the bacteria. These are called fimbriae (from the Latin word for 'thread') and also, less correctly, pili (from the Latin word for 'hair') (Figure 1.2). Whereas pili are involved in gene transfer between bacteria ('sex pili') and flagellae have the role of sensory organelles used for moving, fimbriae serve as adhesive organelles. Fimbriae contain lectin subunits, which mediate carbohydrate-specific adhesion to cell surfaces (and also cell agglutination). Thus, bacteria utilize the sugar decoration of cells – the glycocalyx – to colonize the cell surface, wherever cells are in contact with the outside environment, as for example in the case of epithelial cells.

1.3 Carbohydrate Specificity of Type 1 Fimbriae

Type 1 fimbriae are particularly efficient adhesion tools of bacteria to mediate the colonization of various biotic and abiotic surfaces. They are uniformly distributed on the bacterial cell surface with their length varying between 0.1 and 2 µm and a width of ~7 nm. Since the 1970s, numerous studies have been carried out to elucidate the carbohydrate specificities of bacterial adhesion, in particular of type 1 fimbriae-mediated adhesion of E. coli. A key finding of this research was that the type 1 fimbrial lectin, called FimH, requires α-D-mannose and α-D-mannosides for binding. The other anomer, namely ß-mannosides, cannot be complexed within the carbohydrate binding site. This knowledge suggested that type 1 fimbriated bacteria can adhere to tissues expressing glycoproteins of the high-mannose type, exposing multiple terminal α-D-mannosyl units. For example, urinary tract infections are caused by uropathogenic E. coli (UPEC). Type 1 fimbriae are present in at least 90% of all known UPEC strains, where they are important pathogenicity factors. Today, it is known that bacterial adhesion to the surface of urothelial cells is mediated by FimH binding to oligomannoside residues of the glycoprotein uroplakin Ia. This interaction is a prerequisite for bacterial invasion. Consequently, much effort has been invested in the development of potent inhibitors of type 1 fimbriae-mediated bacterial adhesion in order to prevent bacterial adhesion to mucosa and thus treat bacterial infection in an approach that has been called antiadhesion therapy.

In this context, a second feature of type 1 fimbriae-mediated bacterial adhesion that was discovered already quite early is important. It was found that α-D-mannosides with an aromatic aglycone moiety exhibit an improved affinity to the bacterial lectin and an enhanced potency as inhibitors of type 1 fimbriae-mediated bacterial adhesion to surfaces. Today, this finding is well understood based on the X-ray studies of the structure of the type 1 fimbrial lectin FimH that have been published since 1999. Structural biology has shown that the entrance of the carbohydrate binding site of FimH is flanked by two tyrosine residues, Y48 and Y137, which make π–π interactions with an aromatic aglycone of an α-D-mannoside ligand that is complexed within the cavity of the FimH carbohydrate binding site (Figure 1.3).

This and other structural features of the bacterial lectin FimH have been described elsewhere and are not further detailed in this account. Similarly, the biosynthesis of type 1 fimbriae has been elucidated and reviewed. Briefly, the fimbrial appendage is assembled in the outer membrane of Gram-negative bacteria in a process called the chaperone–usher pathway. To be able to judge the potential value of especially glycopolymers as inhibitors of type 1 fimbriae-mediated bacterial adhesion, it is important to know that FimH is a two-domain protein, terminating every type 1 fimbrial rod (Figure 1.4). The so-called pilin domain of FimH, FimH, is required to anchor the protein at the fimbrial tip, comprising also the subunits FimF and FimG. The lectin domain Fim[H.sub.L], on the other hand, accommodates the α-D-mannoside-specific carbohydrate-binding site. Both FimH domains are interconnected by a hinge region, permitting allosteric regulation of the carbohydrate binding site (see below).

1.4 Tailor-Made FimH Antagonists

Based on the information obtained in structural biology studies, molecular modelling was employed to design tailor-made ligands of FimH and FimH antagonists. Synthesis and testing of these non-natural α-D-mannosides have only recently led to a revival of the idea of an antiadhesion therapy against microbial infection. In the 1990s, Lindhorst et al. introduced the idea of using multivalent α-D-mannoside clusters to inhibit effectively type 1 fimbriae-mediated bacterial adhesion in a potential therapeutic context. Later, molecular docking studies led to promising new monovalent mannosides as potent FimH antagonists. Many groups have added various high-affinity ligands of FimH to the arsenal of synthetic FimH antagonists with even nanomolar affinities, which is particularly potent in the context of lectin binding. The literature on the design and testing of FimH antagonists and inhibitors of type 1 fimbriae-mediated bacterial adhesion was reviewed in 2011. Since then, Ernst's group has complemented the collection of promising FimH antagonists with new mannosides that were shown to be non-toxic and active in vivo. The best representatives have even entered clinical studies concerning their value in the treatment of cystitis.

Five key representatives of highly potent FimH ligands are illustrated in Figure 1.5. Heptyl α-D-mannoside (1) was found to possess a 440-times greater power as inhibitor of type 1 fimbriae-mediated bacterial adhesion than methyl α-D-mannoside (not shown). The squaric acid derivative 2 has approximately 10 times the inhibitory potency of its analogue o-chloro-p-nitrophenyl α-D-mannoside (not shown). The photosensitive azobenzene mannoside 3 has almost the same inhibitory power as p-nitrophenyl α-D-mannoside (not shown) but has little value as an inhibitor of bacterial adhesion in vivo owing to its limited water solubility. The respective mannobioside on the other hand, is beneficial as a water-soluble and photosensitive FimH ligand.

The biphenyl mannosides 4 and 5 are the result of recent systematic lead optimization. Ernst and co-workers determined the [KD values of 1 and 4, among others, by an in-solution affinity assay as KD (1) = 5.6 ± 1.6 nM and KD (4) = 0.71 ± 0.01 nM. Indolylphenyl and indolinylphenyl mannosides have also been added to the collection of potent FimH antagonists. Testing of the most potent indolinylphenyl mannoside 6 revealed that the administration of a low dosage such as 1 mg kg-1 (corresponding to approximately 25 µg per mouse) was sufficient to prevent urinary tract infection in mice for more than 8 h. Bacterial colonization of the bladder could be reduced by almost four orders of magnitude, comparable to a standard antibiotic treatment. Hence the vision of an antiadhesion therapy to complement antibiotic treatment, which currently struggles with antibiotic resistance, becomes realistic.

1.5 Multivalent Inhibitors of Bacterial Adhesion

Without doubt, multivalency of molecular interactions is an important factor in carbohydrate recognition. Numerous multivalency effects have been observed in the glycosciences and interpreted according to various models. Lee and Lee were the first to observe a multivalency effect in lectin binding employing relatively small di- and trivalent cluster glycosides and coined the term 'cluster effect' for the observations made. At the time, they concluded that multivalent carbohydrate binding sites occur in lectins, but when the multivalency of lectin CRDs became more and more obvious based on many crystal structures, researchers became especially interested in the design of multivalent glycomimetics to produce highly potent lectin ligands or inhibitors of lectin-mediated cellular adhesion. Among others, multivalent glycomimetics based on non-carbohydrate polymer scaffolds were launched and named 'glycopolymers' to distinguish them from natural polysaccharides. Roy and Kiessling and their colleagues were early pioneers in the field, but more recently modern polymer chemistry has further stimulated the synthesis and application of glycopolymers.

Thus, glycopolymers may also be designed to inhibit fimbriae-mediated bacterial adhesion. In spite of the fact that the type 1 fimbrial lectin FimH is an explicitly monovalent lectin, multivalent mannosides have shown favourable effects as inhibitors of type 1 fimbriae-mediated bacterial adhesion. Such findings might be due to statistical effects on the one hand, taking advantage of the dense packing of mannoside ligands in close proximity to one FimH carbohydrate binding site. On the other hand, appropriately dimensioned multivalent mannosides could also lead to simultaneous binding of multiple FimH units on multiple (~100–400 per bacterial cell) copies of type 1 (Figure 1.6).

Certainly, multivalent binding of glycopolymers to fimbriated bacteria or other microbes, that adhere carbohydrate-specifically, is not per se effective as the entropic penalty that can occur upon fixation of the formerly flexible polymer on the bacterial surface might prevent high-affinity binding. However, based on modern polymer chemistry, this problem could be circumvented. Thus, appropriately 'encoded' glycopolymer inhibitors of microbial adhesion to surfaces could indeed be provided as powerful tools for various different applications.

1.6 Carbohydrate 'Letters' for Antiadhesive Glycopolymers

For the systematic fabrication of antiadhesive glycopolymers the principal design outlined in Figure 1.7 can be envisaged. According to this approach, a polymer backbone can be designed with desired hydrophobicity, steric and conformational properties and carrying appropriate functional groups to allow selective attachment of carbohydrate moieties. Ideally, the attachment chemistry should work according to the concept of 'click chemistry, but more classical ligation methods could also be employed. For the fabrication of antiadhesive glycoarrays, we and others have often used a collection of functionalized glycosides that can be easily ligated to prefunctionalized surfaces. Similar mannosides (cf.Figure 1.7) could be employed to produce glycopolymers that inhibit type 1 fimbriae-mediated adhesion, for example. Alkyne–azide cycloaddition, triol–ene reactions, peptide coupling and thiourea bridging are approved methods to ligate carbohydrates to molecular scaffolds, and even bioorthogonal chemistry is certainly amenable to the synthesis of glycopolymers.

A number of structural parameters are known to influence and direct multivalent carbohydrate–lectin interactions and cell adhesion. For example, the density of carbohydrate ligands can be influential. Moreover, the complexity of carbohydrate decoration and in particular clustering of glycosides of different nature have been found to improve ligand binding in many cases. The latter observation has been described as the 'heteroglycocluster effect'. All of these aspects of carbohydrate recognition could be studied and adjusted with the aid of glycopolymers, as outlined in Figure 1.8.

With regard to inhibition of type 1 fimbriae-mediated bacterial adhesion, the identified high-performance FimH antagonists 1–6 (Figure 1.5) can be easily ligated to functionalized polymers either directly or after the introduction of a suitable linker. Some practical ideas are depicted in Figure 1.9. The mercapto-functionalized azobenzene mannoside 9, for example, has recently been used for the fabrication of photoswitchable carbohydrate-decorated self-assembled monolayers on gold, so-called glyco-SAMs. The same molecule can be utilized to make photosensitive glycopolymers, in which reversible E ->Z ->E isomerization of the azobenzene N=N double bond might be used to modulate the antiadhesive power of the respective polymer.

Equally, the recently introduced 'dual click' approach for glyco-SAM production can enrich the field of glycopolymer synthesis (Figure 1.10). For biological adhesion studies, it is essential to use biorepulsive moieties to suppress the non-specific adsorption of proteins on a surface. Such protein-repelling properties are mediated by oligoethylene glycol (OEG) linkers, which can be introduced to a functionalized scaffold or surface by a first click reaction.' A second 'click reaction' allows the attachment of a carbohydrate head group, such as an α-D-mannoside, at the terminal end of the molecular construct. This 'dual click' concept has been demonstrated to be viable by systematic step-by-step assembly of glyco-SAMs and their testing as (anti)adhesive surfaces using fluorescent E. coli bacteria and fluorescence read-out.

(Continues...)

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Excerpted from Glycopolymer Code by C. Remzi Becer, Laura Hartmann. Copyright © 2015 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents

Synthesis of Glycopolymers by Direct Polymerisation; Glycopolymers used in Biosensor Applications; Supramolecular Glycopolymer Systems ; Synthesis and Applications of Glycoparticles; Hybrid Glyco Materials and their Expansive Applications; Glycopolypeptides; Small Molecule Ligands for Bacterial Lectins: 'Letters' for an Antiadhesive Glycopolymer Code; The Synthesis and Biological Properties of Calixsugars; Dendritic Glycopolymers and their Biomedical Applications;