chem soc rev · 4476 chem.soc.rev.,, 42,-- this journal is c the royal ociety of chemistry 2013...

4476 Chem. Soc. Rev., 2013, 42, 4476--4491 This journal is c The Royal Society of Chemistry 2013

Cite this: Chem. Soc. Rev.,2013,42, 4476

Glycopolymer probes of signal transduction†

Laura L. Kiessling*ab and Joseph C. Grima

Glycans are key participants in biological processes ranging from reproduction to cellular communication to

infection. Revealing glycan roles and the underlying molecular mechanisms by which glycans manifest their

function requires access to glycan derivatives that vary systematically. To this end, glycopolymers (polymers

bearing pendant carbohydrates) have emerged as valuable glycan analogs. Because glycopolymers can readily

be synthesized, their overall shape can be varied, and they can be altered systematically to dissect the structural

features that underpin their activities. This review provides examples in which glycopolymers have been used to

effect carbohydrate-mediated signal transduction. Our objective is to illustrate how these powerful tools can

reveal the molecular mechanisms that underlie carbohydrate-mediated signal transduction.

1. Introduction

All cells, from prokaryotes to eukaryotes, are cloaked in a glycancoat termed the glycocalyx.1,2 This coat is composed of a varietyof glycosylated proteins and lipids that report on cell type,environment, and the metabolic state of the cell. It was origin-ally thought that the primary role of the glycocalyx was to act asa physical barrier against the cell’s environment; however, the

a Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave,

Madison, WI 53706, USA. E-mail: [email protected] Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr.,

Madison, WI 53706, USA

† Part of the carbohydrate chemistry themed issue.

Laura L. Kiessling

Laura L. Kiessling holds theSteenbock Chair in Chemistryand is the Laurens AndersonProfessor of Biochemistry. Shereceived her BS in Chemistryfrom MIT and her PhD inChemistry from Yale Universitywith Professor Stuart Schreiber.She was an ACS postdoctoralfellow in Chemical Biology atthe California Institute of Techno-logy with Peter Dervan. In 1991,she joined the University ofWisconsin-Madison. She is the

founding and current Editor-In-Chief of ACS Chemical Biology. Sheis a Fellow of the American Academy of Arts and Sciences, theAmerican Society for Microbiology, and a Member of the NationalAcademy of Sciences. The Kiessling group is focused on elucidatingand exploiting the mechanisms of cell surface recognition processes,especially those involving protein–carbohydrate interactions. Hergroup interests include understanding how glycans are assembled,elucidating their biological roles, and using this information toco-opt or inhibit glycan interactions.

Joseph C. Grim

Joseph Grim was born in Chicago,Illinois. He received his BS inChemistry from the University ofWisconsin-Green Bay in 2008. Hethen joined the research group ofProfessor Laura Kiessling at theUniversity of Wisconsin-Madisonwhere he is currently a doctoralcandidate. He is designing andsynthesizing multivalent glyco-conjugates to study the functionof C-type lectins in immunity.

Received 8th March 2013

DOI: 10.1039/c3cs60097a

www.rsc.org/csr

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This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42, 4476--4491 4477

presence of carbohydrate-binding proteins on the cell surfaceaugurs the vital role of cell surface glycans in cell–cell recognition.The interaction of cell surface glycans with cell surface carbohydratereceptors is not only important for cell adhesion—it also can triggersignal transduction. This mode of information transfer is funda-mental for many biological processes, including fertilization andimplantation,3–6 pathogen invasion,7 immune system activation8,9

or attenuation,10–14 and cell proliferation.15 Recognition of thewide-ranging contributions of glycans to signaling is mounting.16,17

This growing appreciation of glycan function is providingimpetus to develop ligands to probe and perturb protein–carbohydrate interactions. While methods to isolate and char-acterize glycans from biological sources are advancing, it can bechallenging to elucidate the molecular features involved in glycanrecognition and function. Chemical synthesis is a powerful allyin addressing this challenge. It offers access to glycans whosestructures can be varied to dissect glycan function.18 Oneespecially valuable class of synthetic ligands for illuminatingcarbohydrate recognition is multivalent displays.

A hallmark of many protein–carbohydrate interactions ismultivalency. Most carbohydrate-binding proteins, whether onthe cell surface or secreted, are oligomers. They can exist as dimers,trimers, tetramers, or even higher order clusters.19–21 These oligo-meric proteins can bind either to multiple carbohydrate residueswithin a glycan or to multiple glycans on the surface of cells(Fig. 1).22–25 The advantages of multivalency have been revealedthrough chemical biology studies. While it is well-appreciated thatmultivalent binding can enhance the functional affinity (observedaffinity, also termed avidity) of cell surface protein–carbohydrateinteractions,22,26–29 it is often overlooked that multivalent bindingcan also improve specificity.28,30 If individual interactions at thecell surface were to occur with high functional affinity, it could beproblematic. During the encounter of two cells (one with carbo-hydrate ligands and the other with a protein-binding partner), for

example, the summation of multiple high affinity interactionswould render binding irreversible. In contrast, low affinitymultivalent interactions are kinetically labile;31 therefore, theyprovide the mean to capture a cell of interest while stillallowing for reversibility if the wrong cell type binds initially.19

The properties of multivalent carbohydrate derivatives (i.e., theirability to exhibit high functional affinity and increased specificity)have stimulated the development of methods to synthesize definedmultivalent carbohydrate derivatives, including polymers bearingpendant carbohydrates (glycopolymers). These agents can beemployed as potent inhibitors, but their ability to clustercarbohydrate receptors provides them with an additionalproperty—they can activate signaling.32 Accordingly, glyco-polymers have been used to mimic either polysaccharides(i.e., glycosaminoglycans), glycoproteins, mucins, or even largerentities such as viral particles or clustered glycans in cellsurface microdomains.29,33 Because they are synthetically tract-able, these surrogates can be altered to optimize a desiredactivity or to probe a specific biological process.34

Over the last two decades, many scaffolds have been developedfor displaying carbohydrates. These range from dendrimers,35,36

to oligomeric bioconjugates,37 to polymers,38–40 to quantumdots41–43 and nanoparticles.44–46 While each scaffold hasunique benefits, it is the polymers that can exhibit the greatestvariation in valency, individual binding group spacing, andoverall architecture. Access to this structural diversity has beenmade possible by advances in polymer synthesis, which haveprovided the means not only to synthesize polymers of differentstructures, but also to control the properties of the polymersthat result. By varying the structure of the monomer or thepolymerization conditions, a multitude of complex topologiescan be accessed (Fig. 2).47 Thus, polymers can be generated tocarry out systematic investigations into the effect of glycanstructure on biological function.

Fig. 1 Carbohydrate-binding proteins often exist as oligomers and can interact with glycans through a variety of mechanisms. An oligomeric protein can interactwith an individual cell-surface glycan (A) or with multiple different cell-surface glycans simultaneously (B). Oligomeric proteins can also interact with soluble glycans orsoluble oligomeric lectins can engage cell surface glycans (C and D). Soluble proteins can cluster cell-surface glycoproteins to mediate signal transduction (E). Likewise,soluble glycans can cluster cell-surface receptors to mediate signal transduction (F).

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Despite the benefits of employing glycopolymers to studysignal transduction, to date this strategy has been surprisinglyunderutilized. Given the many new roles for glycans that arebeing revealed, we hope that this review will stimulate researchinto carbohydrate-mediated signal transduction. Because signaltransduction necessitates robust glycopolymer recognition byprotein receptors, we provide an overview of the structural featuresof glycopolymers that can influence their mechanism of proteinrecognition. We use these studies to offer some general parametersthat might influence the abilities of glycopolymers to affect cellsignaling. We also discuss examples of how glycopolymers havebeen employed to address specific problems in signal transduction.While there are many exciting examples where glycopolymers havebeen used to elicit an immune response,48–54 we highlight thosethat focus on a specific signal transduction pathway.

2. Maximizing protein recognitionof glycopolymers

In the 1970s, Schnaar and Lee55 and Horejsı et al.56 describedmethods for the synthesis of polyacrylamide-based glyco-polymers. Their demonstration that these synthetic conjugatescould bind lectins spurred investigations using glycopolymersas functional glycan mimics. These findings led manyresearchers to explore glycopolymers as potent inhibitors ofcarbohydrate-binding proteins.57,58 Accordingly, many poly-merization strategies have now been applied to the generationof glycopolymers (Fig. 3). The kind of radical polymerizationreactions carried out initially (free radical polymerization, FRP)have now been complemented by controlled polymerizationreactions, such as atom transfer radical polymerization (ATRP)

and reversible addition-fragmentation chain-transfer polymer-ization (RAFT). Non-radical polymerization strategies also haveemerged, including the ring-opening metathesis polymeriza-tion (ROMP), which has been used extensively in signalingstudies. The synthesis of glycopolymers is beyond the scopeof this review, but there many excellent resources on thetopic.39,40,48,59,60

For glycopolymers to affect signal transduction, they mustbind effectively to at least one but often multiple copies of theirprotein receptor. Using controlled polymerization methods,many aspects of the glycopolymer structure can be alteredsystematically to optimize its activity. Some key parametersinclude the length of the polymer, the density of the carbo-hydrate ligands, the flexibility of the polymer backbone, andthe overall structure of the glycopolymer. Thus, glycopolymers

Fig. 2 (left) A wide variety of monomers can be assembled in a controlled manner to generate polymers of a defined length and valency. (center) The polymers canbe generated with many different topologies. In addition, it is possible to generate polymers bearing multiple functionalities, such as biological ligands orfluorophores. (right) Finally, a key step in eliciting signal transduction is clustering of cell-surface receptors. Polymers bearing carbohydrates can cluster cell-surfaceproteins to elicit a signal. Figure adapted from ref. 47.

Fig. 3 Example polymer backbones generated from common polymerizationstrategies used in synthesizing glycopolymers. The R substituent represents a linkerbearing a carbohydrate ligand, but for many glycopolymers not every monomer unitbears a carbohydrate ligand. Abbreviations: FRP = free radical polymerization;ATRP = atom-transfer radical polymerization; RAFT = reversible addition fragmenta-tion chain transfer; ROMP = ring-opening metathesis polymerization.

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can be tailored to inhibit endogenous glycan interactions with acell surface receptor or to cluster receptors for signaling.39,61

Previously, we reported general principles for designing multi-valent ligands;62 subsequent studies have provided additionalinsight into how glycopolymer features influence activity. Someexamples follow that document the interplay between glyco-polymer structure and function.

2.1 Glycopolymer length and functional affinity

The advent of living polymerization reactions provided a meansto alter glycopolymer length. Living polymerizations, in whichthe rate of elongation is more rapid than termination, are idealprocesses for assembling glycopolymers of defined lengths. Theseparation between binding sites can vary for an oligomericcarbohydrate binding protein, and ligands that can occupymultiple binding sites will have increased functional affinity.63

Glycopolymers of defined lengths can serve as ‘‘measuringsticks’’; their length (and valency) can be optimized to spanmultiple binding sites within a carbohydrate-binding protein orbetween carbohydrate-binding proteins on a cell (Fig. 4). Forliving polymerization reactions with rapid initiation rates,the length of the polymer (degree of polymerization, DP) canbe easily predicted and controlled by varying the ratio ofinitiator to monomer.64–67 In this scenario, an initiator tomonomer ratio of 1 : 100 would afford a 100mer. This strategyof varying polymer length to bridge different lectin bindingsites was shown using glycopolymers generated by the ring-opening metathesis polymerization. Specifically, glycopolymerlength was optimized to facilitate binding to the tetramericlectin concanavalin A (Con A).66 The most active glycopolymerswere those that could bridge at least two binding sites withinthe Con A tetramer. Glycosylated dendrimers are anotherpopular scaffold for targeting oligomeric lectins,35,68–72 asdifferent generations or different linkers can be tested to findthose that span carbohydrate-binding sites within an oligo-meric lectin. To this end, Cloninger and coworkers haveemployed spin-labels and electron paramagnetic resonancespectroscopy to characterize the spatial distribution of carbo-hydrates on dendrimers.73,74

2.2 Density of the carbohydrate ligands

Another method to influence glycopolymer functional affinityinvolves altering the carbohydrate epitope density on a polymerbackbone. Studies employing glycopolymer ligands can com-plement those using natural glycans, whose variation in densitycan influence their activity.75 With controlled polymerizationreactions, the density of the carbohydrate ligand can bemanipulated in two ways—copolymerization of a monomerbearing the carbohydrate epitope with a biologically inertmonomer, or post-polymerization functionalization of a poly-mer with the carbohydrate ligand and a biologically inert ligand(Fig. 2).76 Using either approach, the overall length of thepolymer can be kept constant, while the level of carbohydratesubstitution can be varied.

Cairo et al. found that increases in binding epitope densityenhanced the ability of multivalent ligands to bind avidly to thelectin Con A.77 The authors used ROMP to generate glyco-polymers of various mannose densities by polymerization ofdifferent ratios of mannose-substituted monomers and bio-logically inert galactose-bearing monomers (Con A does notrecognize galactose). Their study revealed that as mannosecomposition increased, both the avidity and the ability of theglycopolymer to cluster Con A increased.

An examination of the role of ligand density at the cellular levelwas conducted by Rubinstein and coworkers.78 They sought toprepare glycopolymers that could exploit the observed upregulationof the galactose-binding lectin galectin-3 in metastatic tumorcells. They therefore prepared N-(2-hydroxypropyl)methacrylamide(HPMA) conjugates displaying different levels (10%, 20%, or 30%) ofa galactose, galactosamine, lactose, or trivalent galactose derivative.They tested the interaction of these glycopolymers with threedifferent colon cancer cell lines (Colo-205, SW-480, and SW-620).78

The level of galactose substitution influenced binding to somecell lines, but not others. For example, all three glycopolymersbound the SW-480 cell line, while the glycopolymers with thehighest substitution levels were the most effective ligandsfor the Colo-205 cells and SW-620 cells. While the molecularmechanisms underlying these differences have not been deter-mined fully, the results underscore that carbohydrate residuedensity can be an important parameter in devising effective celltargeting agents.

These aforementioned examples might lead one to concludethat increasing the density of carbohydrate ligands on a polymeralways leads to enhanced activity, but such a conclusion would beerroneous. One counter illustration is from Kiick and coworkers,who examined the ability of a series of glycopolymers to inhibitthe pentameric cholera toxin (CT),79 a member of the medicallyimportant class of AB5 toxins that have been targets for inhibitordesign.80–82 Glycopolymers were produced by coupling differentmole ratios of an amino galactoside to a poly-glutamic acidbackbone. Interestingly, the authors found that the glyco-polymer with the lowest level of galactose conjugation wasthe most effective CT inhibitor. A glycopolymer that consistedentirely of galactose interacted with CT much more weakly.The authors argue that when the density of the galactose

Fig. 4 Increasing polymer length (and valency) allows polymers to span multi-ple binding sites in oligomeric proteins, thereby increasing their functionalaffinity (avidity). When all accessible binding sites are occupied, further increasesin polymer length will not yield enhancements in functional affinity.


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moieties decreases, the spacing between the galactose moietiesincreases, thus approaching the approximate distance betweenbinding sites on CT. Because the activity is reported on agalactose residue basis, galactose residues that do not contri-bute to protein binding decrease the polymer’s activity. In asubsequent study, the authors engineered random coiled poly-glutamic acid polymers that were designed to contain galactosemoieties spaced apart at specific distances.83 The glycopolymerthat had the largest spacing between galactose moieties boundCT with the highest affinity, despite having the lowest valency.Although these glycoconjugates do not yet match the efficacy ofthe pentavalent glycan ligands devised by the Fan and Bundlegroups,80–82 the glycopolymers are quite potent, especiallywhen considering they are unable to occupy all five bindingsites within the toxin.

Like the glycopolymer strategy used by Kiick and coworkers,other methods have been developed that allow precise place-ment of saccharide residues on a polymer backbone, includingpolypeptides composed of repeating folded domains,84 poly-(amidoamines),85 and modified nucleic acids.86–91 Hartmanand coworkers have devised an iterative approach for assem-bling polymers with controlled carbohydrate ligand spacing.85

Their strategy entailed the sequential coupling of either aninert ethylenedioxy monomer or an alkynyl monomer thatcould be functionalized with an azide-bearing mannosideto generate mono-, di-, and tri-mannoside glycopolymers.The spacing between the mannose ligands of the polymerswas estimated to be 10 nm for di-mannoside and 7 nm fortri-mannoside. Interestingly, the glycopolymer containing onemannose moiety bound Con A with an IC50 of 8 mM, muchlower than that of other monovalent glycan ligands. Theauthors postulate that the enhanced affinity is due to the highlyhydrated ethylenedioxy units on the polymer backbone,92–94

but it also possible that the backbone itself contributes tothe higher affinity.95 In addition, the authors appended themannosides to the backbone via a triazole linker, which may alsohave contributed to the enhanced binding. The glycopolymer

that contains two mannose residues with an estimated 10 nmspacing exhibited only a moderate enhancement and was lesspotent than the monovalent ligand on a saccharide residuebasis (IC50 to 5 mM). The glycopolymer with the 7 nm spacingbetween saccharide residues was designed to be the mosteffective since it most closely matched the distance betweenbinding sites in Con A (B6.5 nm). Indeed, it was the best ligand(IC50 value of 1 mM) but the modest enhancement observed isnot indicative of multivalent binding. Nevertheless, this definedapproach could be used to prepare glycopolymers to examinewhether carbohydrate spacing affects signal transmission.

Another means of generating polymer constructs withdefined glycan spacing is to use peptide nucleic acid (PNA)backbones.89,96 For example, multivalent carbohydrate displaysof this type have been used to probe how the dimeric antibody2G12 interacts with mannosylated gp120 on HIV (Fig. 5).86

Because 2G12 can neutralize HIV, it is thought that compoundsthat bind tightly to 2G12 might serve as haptens for thedevelopment of effective anti-HIV antibodies. The displays wereproduced by appending mannose-containing oligosaccharidesto a single-stranded PNA and hydridizing the mannosylatedPNA to single-stranded DNA strands. The interactions of theseassemblies with 2G12 revealed the importance of carbohydratespacing. The authors used this strategy to generate a potentinhibitor of HIV infection.87 While the synthesis of PNA displaysis more labor intensive than assembling glycopolymers by poly-merization, the study highlights how the spacing of carbohydrateligands can be important for generating potent inhibitors.

These examples all serve to illustrate that glycopolymerswith the appropriate spacing of carbohydrate ligands exhibitenhanced avidity. The relevance of carbohydrate residue densityand spacing in the context of signal transduction are stillunexplored. For signaling to occur, multivalent ligands mustorganize their protein targets into competent signaling com-plexes. This process typically requires that the multivalentligand can cluster multiple receptors. The ability to positioncarbohydrate ligands at defined distances provides the means

Fig. 5 Peptide nucleic acids (PNAs) were generated to control the spacing of carbohydrates. The library of PNAs was screened against the dimeric antibody2GI2, which binds to HIV gp120. As the distance between carbohydrates approached the distance between carbohydrate binding sites on 2GI2, the functionalaffinity increased.


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to test whether different receptor orientations alter signaling.Indeed, data is emerging that suggests it may be important(see, for example, the results of the Kiick Group described inSection 3.1). Still, it seems likely that glycopolymers that activatesignaling receptors need only to cluster them. If signalingcomplex assembly allows for different arrangements of receptors,glycopolymers with a variety of carbohydrate spacings should becapable of activating signaling. For randomly modified glyco-polymers, extremely high levels of carbohydrate substitutionmay sterically block protein binding; consequently, an inter-mediate density of carbohydrates offers a balance amongstenhancing functional affinity, avoiding unfavorable steric inter-actions, and presenting carbohydrate ligand arrangementscapable of facilitating receptor clustering.10,11,97

2.3 Glycopolymer length and receptor clustering

The length of a glycopolymer can influence its ability to clusterreceptors and how many receptors are in the cluster (Fig. 6). Both ofthese parameters can impact whether a signal is transmitted andhow effectively. It has been shown for some glycopolymer scaffolds,that as their length and valency increases, so does the number ofreceptor copies that they bind.77 This relationship between glyco-polymer length and the number of copies of lectin bound could beobserved directly using transmission electron microscopy.98

An example in which longer glycopolymers more effectivelytransmit signals than do shorter polymers has been described.Bacteria must move towards nutrients and away from toxins forsurvival.99–101 One attractant for Escherichia coli that promoteschemotaxis is galactose. Signals from galactose are transmittedthrough the transmembrane chemoreceptor Trg. The chemo-receptors cluster at the poles of the bacteria,102 suggesting that

they might communicate with each other. Indeed, galactose-sub-stituted polymers of different lengths uncovered the importance ofchemoreceptor–chemoreceptor interactions for E. coli responses toattractants. The attractant potency of the glycopolymers dependson their ability to alter the intrinsic organization of thechemoreceptors (e.g., cluster them) in the membrane. Specifi-cally, galactose-substituted polymers of sufficient lengths couldinduce chemoreceptor clustering. These glycopolymers weremore potent attractants than monovalent or oligomeric attrac-tants incapable of mediating receptor clustering.103 Moreover,the galactose-presenting polymers could potentiate responsesto other attractants, a result that highlights the ability of thechemoreceptor arrays to act as a kind of sensory organ todetect and integrate signals.104 These studies reveal that glyco-polymers can be used to explore the molecular mechanismscritical for signaling. They also highlight that glycopolymerlength can influence signal strength. A similar relationshipbetween glycopolymer length and signal transmission wasobserved in studies using glycopolymers as glycosaminoglycananalogs (vide infra).105,106

2.4 Flexibility of the polymer backbone

The flexibility of the polymer backbone can also impact aglycopolymer’s ability to bind to protein receptors.62 Initially,one might assume that a rigid polymer with the correct spacingmight interact with receptors more tightly, as any multivalentinteraction would avoid a conformational entropy penalty. Apotential downside of rigid polymers, however, is that theytypically are less capable of adapting to protein interfaces; there-fore, they will be less apt to adopt a spatial arrangement of glycanresidues that complements those of an oligomeric carbohydrate-binding protein (or cluster carbohydrate-binding proteins in amembrane). Indeed, Kobayashi and colleagues noted that rigidglycopolymers often bind weakly to lectins.107 For glycopolymersthat present their glycans in a variety of orientations, the back-bone rigidity might have little impact on binding.66

Immobilized glycopolymers also have been used to probethe role of polymer rigidity in protein recognition.108 For example,arrays of glycopolymers of varying flexibility were tested for bindingto Con A.109 When no cross-linker was added to the polymerization,little interaction with Con A was observed. When a low molefraction (0.5%) cross-linker was added, the lectin readily bound.When a higher level of cross-linker was employed (1.5%), bindingto Con A was reduced significantly. Similarly, Miura investigatedCon A binding to glycopolymeric hydrogels of different stiffness.110

They referred to these three states as follows: a flexible swollenstate, an intermediate transition state, or a stiff collapsed state. ConA most avidly bound to the transitional hydrogel with intermediateflexibility, the flexible swollen hydrogel was next, with binding tothe stiff hydrogel being the weakest.

The flexibility of the linker that connects the carbohydrate tothe polymer backbone may also contribute to protein recognition.For example, Stenzel and coworkers generated galactose glyco-polymers with either a stiff poly 2-hydroxyethyl methacrylate(pHEMA) linker or a flexible polyethylene glycol (PEG) linker.111

The authors investigated the polymers’ ability to inhibit theFig. 6 Polymers of sufficient length are capable of bridging multiple surfacereceptors, clustering them, and initiating signal transduction.


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plant lectin ricin and found the glycopolymers with the flexiblePEG linker were more effective inhibitors. Given the ability ofmore flexible backbones and linkers to adopt a conformation/orientation that leads to effective interactions, it seems likelythat the most active signaling agents will be glycopolymers thatmaintain this balance and can rapidly and effectively promoteprotein clustering.10,11,33,34,103,106

2.5 Glycopolymer architecture

Advances in controlled polymerization reactions have affordedaccess to diverse glycopolymer topologies (Fig. 2). The role ofglycopolymer shape in signal transduction, however, has yet tobe explored extensively. A number of studies do suggest thatthe shape of a multivalent carbohydrate ligand is a criticaldeterminant of its activity. If one considers a carbohydrate-modified surface as ‘‘an insoluble glycopolymer’’, it is apparentthat how glycans are displayed influences their functional affinitiesand protein binding specificities. One study showed that membersof the selectin family bind only weakly to surfaces displayingmonovalent sulfated galactose derivatives but avidly to surfacesthat present multivalent sulfated galactose glycopolymers.112 Thecarbohydrate residues were identical, but the multivalent displayshad different features. Thus, the manner in which the carbohydratemoiety is displayed can have a marked influence on binding.Differences in glycan presentation have been shown to beimportant both for implementing glycan array technologyand interpreting the data it affords.113 To this end, differentmethods to fabricate arrays are being examined, from usingglycolipids to immobilizing oligosaccharides via short linkersto presenting glycans on surface-linked protein or glycopolymerscaffolds.108,114–118 These data indicate that the architecture ofa glycopolymer will undoubtedly influence its functional affi-nity for its receptor. Still, functional affinity is just one factor toconsider in optimizing glycopolymer signals.

As discussed previously, signal strength can depend uponhow many receptors a ligand can recruit to the signalingcomplex, the orientation of clustered receptors,119 and the rateat which a glycopolymer induces clustering. The link betweensignal transduction and receptor endocytosis120–122 offersanother means by which glycopolymer topology might alteractivity. There is growing evidence that the size of a ligand, itsshape, and perhaps even whether it is soluble or insoluble all arefactors that influence signaling, endocytosis, and trafficking.123,124

For instance, the dendritic cell lectin Dectin-1 is capable ofeliciting an immune response to particulate antigen; however,soluble antigen fails to elicit a response.125

While the field currently is lacking systematic investigationof how the shape of glycopolymers influences their signalingability, there has been an assessment of how different multi-valent carbohydrate ligands influences their mode of inter-action with a lectin.77 Gestwicki et al. generated a variety ofmultivalent mannosylated ligands, including bi- and tri-valentsmall molecules, globular protein conjugates, dendrimers,linear polymers of controlled lengths, and high molecularweight polydisperse polymers, and evaluated these conjugatesin a battery of assays that report on different aspects of their

ability to interact with Con A. Compounds were evaluated fortheir ability to block Con A binding to immobilized glycan, butalso many activities relevant for signal transduction: inductionof lectin clustering (a quantitative precipitation assay126 thatalso assessed the stoichiometry of lectin to ligand), the rate ofinduction of clustering (turbidity assay127), and the distancebetween receptors in a cluster (fluorescence quenching128). Theoligovalent small molecules and dendrimers were not potentinhibitors nor were they able to cluster Con A, suggesting thesetypes of structures might be less effective at eliciting signaling.Alternatively, ligands with high molecular weights, such as thecarrier protein conjugates and polydisperse polymers, couldbind many copies of Con A; however, fluorescence quenchingexperiments indicated the bound receptors were not in closeproximity to one another. These data suggest that the largerligands may not be as efficient at eliciting signal transduction.Well-defined glycopolymers efficiently clustered the lectin, andproteins within the cluster were proximal. In another studycomparing star polymer scaffolds, significant differences inlectin clustering also were observed. Medium-sized star-polymerswere more efficient at clustering the lectin than were large star-polymers.129 Linear glycopolymers, however, were more efficientat interacting with Con A than the star-shaped polymers.Together, the data indicate that ligand architecture can havea profound effect on the mechanism by which a glycopolymerengages its receptor.

Comparisons between linear glycopolymers, which typicallyexist as individual entities in solution, and diblock copolymers,which have the propensity to form micelles in solution,have revealed differences in their ability to cluster proteins.130

Contrasting linear glycopolymer 1 (Fig. 7) and micelles formedfrom the assembly of block copolymer 2 was instructive. Con Ainteracted more strongly with the glycopolymeric micelle thanwith the linear polymer. Subsequently, the potency of the micelleswas augmented by generating block copolymers that displaythe carbohydrate binding groups in clusters (Fig. 8).131,132 Aspredicted, the clustered glycopolymer-derived micelle was evenmore effective at clustering Con A.

The density of glycopolymer presented by the micelle can bealtered by doping in an inert polymer during self-assembly.This strategy was employed by Wooley and colleagues, whoutilized ATRP to generated a diblock glycopolymer containing amannoside moiety at one terminus and a diblock polymer withno functionality on its terminus.133 Mixing various ratios ofthe two polymers in water resulted in their assembly intomicelles with a mannose composition that ranged from 0%to 100%. Increasing the ratio of mannose glycopolymers in thecross-linked micelle increased its ability to block Con Amediated hemaglutinin.

The factors that govern protein recognition by the afore-mentioned conjugates, including glycopolymeric micellesare complex. The investigations carried out to date on glyco-polymeric micelles suggest that they are highly effective atclustering proteins. It seems reasonable, therefore, to postulatethat they can be used to deliver powerful signals. Theirutility for this purpose, however, has not been investigated.


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Indeed, many interesting glycopolymer structures have notbeen tested as signal activators.

3. Mimics of surface glycans

Since all cells present a glycan exterior, it is not surprising that signaltransduction can arise from interactions between cell-surface carbo-hydrate-binding proteins and cell surface-glycans. Glycopolymershave been used to mimic a variety of different types of cell surfaceglycans, including N-glycoproteins, mucins, and glycosaminoglycans.In this way, glycopolymers can be used to probe how cells commu-nicate with each other or how a pathogen facilitates infection.

3.1 Mimics of mucins

The ability of glycopolymers to mimic mucins53,134 has been exploredextensively in the context of selectin-mediated inflammation.

In the inflammatory response, leukocytes are recruited to a siteof injury or infection.1,135,136 The process involves multiplesteps: leukocytes roll across the endothelium; adhere tightlyto the endothelium wall; and migrate into the inflamed tissue.In certain disease states, aggressive leukocyte migration isdetrimental.137,138 A key mediator of leukocyte migration isthe cell-surface lectin, L-selectin, which interacts with glyco-proteins displayed on the endothelium of blood vessels.138,139

Physiological L-selectin ligands are highly glycosylated mucin-like glycoproteins capped with sialyl Lewis x (sLex) epitopes.140

Because its natural ligand is multivalent, it was postulated thatclustering of L-selectin could be important for its function.

A number of different types of glycoproteins were investigatedas inhibitors of L-selectin.141–143 Evidence that glycopolymerscould cluster this leukocyte surface receptor was obtained usingtailored ligands. Using ROMP, a series of different length glyco-polymers bearing a 3,6-disulfogalactose ligand and a fluorescenttag were screened for binding to L-selectin-positive cells.144

Because each polymer bore only one fluorescent tag, it waspossible to determine the ratio of L-selectin bound to polymer.As the valency of the polymer increased, so did the ratio ofL-selectin to polymer, providing evidence that the ligandscluster L-selectin on the cell surface.

Further investigations of L-selectin targeted glycopolymerssuggested that they could promote L-selectin-mediated signaling.Specifically, the polymers not only bind to L-selectin but alsofacilitate its downregulation (Fig. 9).145,146 Treatment of lymphocyteswith glycopolymer 3 resulted in a dramatic decrease in cell-surface L-selectin levels. The data indicated that ligand bindingtriggered the proteolytic release (or shedding) of L-selectin.Consistent with this mechanism, after cells were exposed tothe glycopolymers, soluble L-selectin was detected. Monovalentligands were unable to mediate shedding of L-selectin. Theaccumulated data suggest that clustering of L-selectin leads to asignal transduction cascade that results in L-selectin shedding.147

Thus, glycopolymers are highly potent inhibitors of L-selectinfunction through multiple mechanisms: they block interactionsof L-selectin with endogenous ligands, promote L-selectin lossfrom the cell surface, and generate a soluble form of the proteinthat can inhibit cell surface interactions.

In a subsequent study, Kiick and coworkers investigated therole of carbohydrate-spacing in activating L-selectin shedding.148

Fig. 7 Stenzel and coworkers synthesized linear glycopolymers and diblock glycopolymers, which self assembled to form glycopolymeric micelles. The glycopolymericmicelle was more efficient at clustering Con A than the linear glycopolymer.

Fig. 8 An investigation of the role of ligand clustering. Glycopolymeric micellesdisplaying carbohydrate clusters were much more effective at clustering thelectin Con A.


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Using a polypeptide backbone, sialylated ligands were appendedto give a polymer with ligands separated by distances estimatedto range from 17–35 Å and a polymer with carbohydrate moietiesseparated by 35–50 Å. The polymer with shorter carbohydrate-spacing was more effective at eliciting L-selectin shedding thanthe polymer with the longer spacing. The diameter of L-selectinis thought to be about 22 Å, which falls in the range of thepolymer with shorter carbohydrate-spacing. Still, both polymersshould be capable of clustering L-selectin, so the origin of thedifferences is not obvious. The authors raise the possibility thatoptimal ligand spacing may be important in eliciting signaltransduction.

3.2 Mimics of pathogen glycans

The carbohydrates on the surfaces of pathogens can engage hostreceptors and activate signaling. Many pathogen recognitionreceptors (PRRs) of the innate immune system have evolved torecognized conserved carbohydrate epitopes on the pathogensurface. One key family of PRRs includes the C-type lectinreceptors (CLRs), whose members are named for their depen-dence on calcium ions to facilitate carbohydrate binding.149

Several CLRs are found on dendritic cells (DCs).150 DCs arethe major antigen-presenting cells of the immune system, andDC lectins function as antigen receptors, as regulators of DCmigration, and as facilitators that mediate binding to otherimmune cell types.151,152 The multiple functions of DC lectinscontribute to an appropriate immune response.

One CLR of particular interest, DC-specific ICAM-3-grabbingnon-integrin (DC-SIGN), is a lectin implicated in numerousfunctions.153,154 Through interactions with high mannose glycansor fucose-containing Lewis-type antigens on self-glycoproteinsICAM-3 and ICAM-2, DC-SIGN can mediate T cell interactionsand trans-endothelial migration.155–157 In addition, DC-SIGN isthought to play a role in pathogen recognition and processing,as anti-DC-SIGN antibodies are internalized, processed andpresented to T cells.158 Despite its putative role in healthyimmune function, DC-SIGN is exploited by a variety of patho-gens, which deploy pathogen-specific mechanisms.159,160

DC-SIGN binds to the mannosylated envelope glycoproteingp120 on HIV-1 to mediate infection of T cells in trans.7,156,161

Alternatively, binding of DC-SIGN to mannosylated glyco-proteins on the surface of Mycobacterium tuberculosis activates

signaling pathways that lead to immunosuppression.162 How eachpathogen exploits DC-SIGN to take advantage of distinct escaperoutes remains unclear. Glycopolymers may prove critical tools inunderstanding the role of antigen structure on DC-SIGN function.

Initial studies of DC-SIGN primarily focused on inhibitors(Fig. 10), as inhibitors could block its ability to disseminate viruses,such as HIV.163–166 Haddleton and coworkers, for instance, synthe-sized mannose-substituted glycopolymers using ATRP.167 Theycontrolled the ratio of a mannoside (DC-SIGN ligand) and galacto-side (non-binding) moiety incorporated into the polymers. Thisstrategy of using a non-binding carbohydrate group as a spacerligand is identical to that employed by Cairo et al. (Section 2.2).As their mannose density increased, so did the ability ofthe glycopolymers to disrupt the gp120–DC-SIGN interaction.Polymer scaffolds also served as vehicles to present non-carbohydrate glycomimetics (such as 4)168 to DC-SIGN.97 Thefunctional affinity of the glycomimetic was found to be enhancedsignificantly over that of the monovalent glycomimetic.

Increasing evidence suggests that the propensity of patho-gens to appropriate DC-SIGN arises from the lectin’s ability to

Fig. 9 Glycopolymers were used to promote L-selectin signal transduction. Upon clustering of the surface L-selectin by the glycopolymer, signal transduction occursthat leads to the proteolytic cleavage of L-selectin.

Fig. 10 Various inhibitors of DC-SIGN. For compound 4, R = H for a monovalentinhibitor; alternatively, R can be a linker appended to synthetic polymer orprotein backbone.


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participate in signal transduction complexes.169,170 DC-SIGNalso is involved in antigen uptake. It can internalize a variety ofsynthetic multivalent ligands, including mannose-functionalizedgold nanoparticles,171 glycoprotein surrogates,16 mannosylateddendrimers,172 and glycopolymeric nanoparticles.173 Whether thesesynthetic multivalent mannose derivatives promote signaling indendritic cells is largely unknown.

Data are emerging that implicate DC-SIGN in transducingsignals. Narasimhan and coworkers generated mannosylatednanoparticles that were capable of eliciting DC maturation,a phenomenon that occurs upon interaction with antigen;however, the outcome was not entirely DC-SIGN-dependent.173

Additionally, a study demonstrated that glycopeptidic dendrimerscan be internalized by DC-SIGN, activate DC-SIGN signal transduc-tion, and even deliver a peptide antigen for presentation to Tcells.70 In general, DC-SIGN signals in collaboration with anotherclass of PRRs, the Toll-like receptors (TLRs).174 TLRs bind to a widevariety of pathogenic epitopes and ultimately results in cytokineproduction. DC-SIGN stimulation in the presence of TLRs leads toan amplification of TLR-induced cytokine production. Onepotential means of regulating cytokine production is by controllingthe structural features of the DC-SIGN ligand, such as the typeof carbohydrate ligand, its valency, or its ability to present otherepitopes that promote cross-talk between receptors.170 Glyco-polymers and related displays might therefore not only inhibitDC-SIGN but also elicit tailored signals.

There are hints that glycopolymer-promoted DC-SIGN signalingmay lead to new insight into DC-SIGN function in immunity andpathogen evasion of the immune system. A recent study by Prostet al. suggests that glycopolymers that bind to DC-SIGN do elicitsignaling.16 Specifically, glycoprotein surrogates displaying glyco-mimetic 4 promote signal transduction. In contrast, Ribeiro-Vianaet al. produced mannosylated second-generation glycodendrimersthat were internalized by DC-SIGN, but no signaling wasdetected.172 While neither compound has been assessed for itsability to cluster DC-SIGN, the larger glycoprotein surrogates arelikely more capable of mediating DC-SIGN clustering. Thus, theextent of DC-SIGN clustering may determine the level of signaltransmission. Forthcoming systematic studies using glycopolymerswill undoubtedly aid in understanding how DC-SIGN mediatesdifferential responses to distinct pathogens.

4. Mimics of soluble polysaccharides

Glycopolymers share with glycoproteins (including mucins) ageneral arrangement in which the glycans emanate from a back-bone (either synthetic or proteinaceous) (Fig. 11). In contrast,glycosaminoglycans and other polysaccharides are linear carbo-hydrate chains. Proteins can assemble on these linear polysac-charide chains (e.g., heparan sulfate) to mediate signaling.175,176

Hsieh-Wilson and coworkers demonstrated that glycopolymerscould serve as glycosaminoglycan mimics. Their investigationwas prompted by their interest in the role of glycosaminoglycaninteractions in axon regrowth. A major barrier in functionalrecovery after injury to the central nervous system is theinhibitory environment encountered by regenerating axons.177

After recruitment of astrocytes to the injury site, these cells releasechondroitin sulfate proteoglycans (CSPGs).178–180 The highly sulfatedpolysaccharides are the principal inhibitory components of axonregeneration, but their mechanism of action was poorly understood.The consensus was that CSPGs primarily acted as an additionalphysical barrier to axon regrowth. The function of CSPGs inaxon regeneration was elusive because of the complexitiesassociated with GSPG structure. Though studies had suggesteddistinct roles for specific sulfation patterns in GSPGs, theseconclusions rested on experiments conducted using hetero-geneous polysaccharides.181–183

Hsieh-Wilson and colleagues used ROMP to synthesizeglycopolymers that served as CSPG mimics.105 They assembledpolymers bearing disaccharide and tetrasaccharide sequencesderived from the biologically active chondroitin sulfate-E (CS-E)epitope. The ability of the glycopolymers to promote outgrowthof hippocampal neurons was compared to that of a natural CS-Epolysaccharide. As expected, the natural CS-E polysaccharideinhibited 100% of neurite outgrowth. Intriguingly, the glyco-polymer bearing the tetrasaccharide also inhibited 100% ofneurite outgrowth. The activity of the disaccharide-substitutedglycopolymer depended on its length—as glycopolymer lengthincreased, neurite outgrowth decreased.

Access to CSPG surrogates allowed for definitive studies on therole of specific CSPG sulfation patterns in axon regeneration.Homoglycopolymers 5–7 were synthesized, and each contained akey disaccharide from chondroitin sulfate polysaccharides CS-A,CS-C, or CS-E (Fig. 12).106 The CSPG mimics were analyzed forinhibition of neurite outgrowth. For comparison, the natural poly-saccharides enriched in the three sulfation patterns were tested.Interestingly, only CS-E glycopolymer 7 and the CS-E enrichedpreparations blocked neurite outgrowth. These data suggest thatsulfation at both the 4- and 6-position of N-acetylgalactosamine inCS polysaccharides is required for inhibition.

The authors found that CS-E glycopolymer 7 could promotesignaling. CSPGs and myelin inhibitors activate Rho/Rho-kinase(ROCK) and epidermal growth factor receptor (EGFR) pathwaysto impede axon regeneration.184–186 Pharmacological inhibitionof ROCK and EGFR with small molecules reversed the inhibitoryeffects of CS-E glycopolymer 7 on the cells. Analogous effectswere observed on cells treated with natural polysaccharidesenriched with CS-E. The ability of CS-E surrogates to elicitdownstream signaling events suggests that they interact directlywith a cell-surface receptor. CSPGs can engage protein tyrosine

Fig. 11 The structure of mucins closely resembles that of glycopolymers.Proteoglycans, however, have a different binding epitope arrangement than thatfound in glycopolymers. It is a testament to glycopolymer utility that they canfunction as glycosaminoglycan mimics.


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phosphatase PTPs, a glycosaminoglycan binding cell-surfacereceptor,187,188 and this interaction may be involved in axonregeneration. PTPs was screened in a carbohydrate array andfound to interact only with the sulfation pattern present in CS-Epolysaccharides. In addition, deletion of PTPs attenuated theinhibitory effects of glycopolymer 7. Therefore, CS-E enrichedCSPGs may signal through PTPs to inhibit axon regeneration.

These investigations highlight the power of glycopolymers toact as functional mimics of chondroitin sulfate proteoglycans.They also underscore the remarkable ability of glycopolymers tomimic a wide range of glycans. Further highlighting that point,antibodies raised against CS-E glycopolymer were specific forthe CS-E epitope and had no cross-reactivity with other sulfatedglycans.106 These studies emphasize the utility of glyco-polymers for activating and probing signaling pathways thatinvolve proteoglycans or other polysaccharides.

5. Assembling multireceptor complexes

CD22 is a member of the Siglec (sialic acid binding immuno-globulin-like lectins) family. Siglecs are prevalent on the surfacesof immune cells, where they play key roles in innate and adaptiveimmunity.189,190 Glycopolymers have been used to probe howone member of the Siglec class, CD22, leads to attenuation of Bcell responses.

Initiation of an immune response and the prevention ofautoimmunity is influenced by the ability of the B cell antigenreceptor (BCR) to transmit signals that both positively andnegatively regulate B lymphocyte survival, proliferation, anddifferentiation.191 Co-receptors that modulate BCR signalinghelp ensure that distinctions between self and non-self aremade. CD22 is an inhibitory coreceptor that can attenuate BCRsignaling.190 Studies in CD22-deficient mice suggest that CD22

acts to increase the threshold for B cell activation.192–194 CD22recognizes a-(2,6)-linked sialylated glycans,195 which are presenton some antigens but also abundant on the surface of B cells.195

Cis interactions between CD22 and proximal surface glycans canmask the coreceptor to exogenous (trans) ligands. No binding ofeven multivalent trisaccharide CD22 ligands was observed, untilthe cells were treated to remove or disable cell surface sialic acidresidues.14,196–199 Paulson and coworkers, however, generatedmodified oligosaccharide derivatives with higher affinity forCD22, and multivalent displays of these ligands could out-compete the cis interactions to bind CD22 in trans.14 Dataindicate that the functional role of the cis interactions betweenCD22 and surface glycoproteins is to sequester CD22 from theBCR prior to antigen stimulation.13,200

In addition to the importance of cis interactions, evidencehad been mounting that trans interactions are relevant.12,201

A molecular mechanism by which trans interactions altersignaling can be formulated. CD22 possesses cytoplasmicmotifs that can recruit a phosphatase to the BCR signalingcomplex to counteract kinases that lead to B cell activation. Iftrans interactions are relevant, antigens that can co-clusterCD22 and the BCR should give rise to attenuated immuneactivation. Mixed glycopolymers provided an ideal vehicle withwhich to test this model, both at the level of signal transductionin cells11 and immune suppression in vivo.202

Glycopolymers were used to elucidate a role for trans inter-actions with CD22 in modulating B cell signaling (Fig. 12).198

Glycopolymers were synthesized that display glycan ligandsfor CD22 (Fig. 12, R2 = H) and the 2,4 dinitrophenyl (DNP,R1 = �NO2) hapten that could engage the BCR. DNP-displayingpolymers had been shown to elicit activation in a B cell linewith a DNP-binding BCR,10 and this cell line was used to testwhether trans interactions could influence signaling throughCD22 (Fig. 13).11 Specifically, if only cis CD22 interactions arerelevant, CD22 would be masked, and the polymers would interactsolely with the BCR to promote B cell activation. In contrast, ifthe polymers engage both the BCR and CD22 through transinteractions, B-cell activation would be dampened.

To determine the outcome, a DNP homopolymer, CD22Lhomopolymer, and CD22L–DNP copolymer were employed. Ahallmark of B cell signaling involves the influx of intracellularCa2+. If BCR signaling is activated, as with DNP homo-polymer,10 an influx of Ca2+ should be observed. The CD22Lhomopolymer did not elicit any change in intracellular Ca2+,consistent with its inability to bind the BCR. If the copolymercould recruit CD22 to the BCR complex, B cell activation shouldbe suppressed and Ca2+ influx should be modulated. It is thatoutcome that was observed. These data indicate that co-clusteringof CD22 and the BCR results in signal attenuation. These glyco-polymers were used further to identify which proteins involved inBCR signaling were activated and which were deactivated uponco-clustering of CD22 and the BCR.

Paulson and coworkers carried out in vivo studies thathighlight the role of trans interactions with CD22 for attenuatingB cell activation.202 Using a polyacrylamide polymer function-alized with a nitrophenyl hapten (R1 = H) and a high affinity

Fig. 12 Glycopolymers were synthesized by Hsieh-Wilson and coworkers bear-ing carbohydrate epitopes for CS-A, CS-C, and CS-E. The CS-E glycopolymers werefound to inhibit axon regrowth through signaling.


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CD22 ligand (R2 = biphenyl), mice were treated with the glyco-polymers and their B cell response was measured. The glyco-polymers were non-immunogenic, and they promoted long-livedtolerance, preventing B cell responses when the mice were chal-lenged with an immunogen. When mice deficient in CD22 weretreated with these polymers, no tolerogenic response was observed.Together, the cell-based and in vivo investigations lead to theimportance of trans CD22 interactions. They support a role forantigen glycosylation as an innate form of self-recognition.201

These experiments also demonstrate the utility of using glyco-polymers to address mechanistic questions in signaling. Polymerscaffolds can present multiple ligands for multiple receptors. Thus,they are ideal tools to dissect cross-talk between cell surfacereceptors. Additionally, the glycopolymers described in this sectioncan serve as therapeutic leads for the design of agents that inhibitor suppress autoimmune responses.

6. Conclusions and future outlook

Cell surface protein receptors are tasked with the essential roleof sensing the cell’s environment and initiating a rapidresponse to ensure survival. These receptors rarely act asindividual entities, but instead work in concert to form highlysensitive, macromolecular signaling assemblies. Glycopolymersthat can promote and stabilize these complexes are powerfulagents for understanding and exploiting mechanisms of signaltransduction. The advent of new polymerization methodsthat can yield glycopolymers of diverse architecture providesthe means to optimize glycopolymers to elicit or inhibitsignal transduction. Thus, they can elucidate critical aspectsof signal transduction that elude traditional approaches.

How glycopolymer topology influences signaling is largelyunexplored, and this arena remains an exciting frontier.125,203–205

Glycopolymers may also prove useful in dissecting the role ofoligosaccharides in the assembly of multiprotein supramolecularcomplexes in signal transduction. In addition to their utility inunderstanding signal transduction, glycopolymers have possibletherapeutic uses.206 Their ability to modulate immune responsesmay lead to new therapeutic strategies.48–54,207 For example, theBundle and Paulson groups described heterobifunctional glyco-polymers that template IgM onto the surface of lymphoma cells toelicit humoral cytotoxicity.199 Glycopolymers are also being exploredin vaccine development208 and drug delivery.209–212 We hope thatthis overview will spur new applications of synthetic glycopolymersto explore and exploit diverse signal transduction processes.

Notes and References

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