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Chemical Approaches to Glycobiology

Laura L. Kiessling1,2 and Rebecca A. Splain1

1Department of Chemistry, 2Department of Biochemistry, University of Wisconsin–Madison, Wisconsin 53706; email: [email protected]

Annu. Rev. Biochem. 2010. 79:619–53 Key Words First published online as a Review in Advance on array, glycan, glycomimetic, glycosylation, lectin, multivalency April 8, 2010

The Annual Review of Biochemistry is online at Abstract biochem.annualreviews.org Glycans are ubiquitous components of all organisms. Efforts to This article’s doi: elucidate glycan function and to understand how they are assembled 10.1146/annurev.biochem.77.070606.100917 and disassembled can reap benefits in fields ranging from bioenergy by University of Wisconsin - Madison on 07/29/10. For personal use only. Copyright c 2010 by Annual Reviews. to human medicine. Significant advances in our knowledge of glycan All rights reserved biosynthesis and function are emerging, and chemical biology ap- Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org 0066-4154/10/0707-0619$20.00 proaches are accelerating the pace of discovery. Novel strategies for assembling oligosaccharides, glycoproteins, and other glycoconjugates are providing access to critical materials for interrogating glycan function. Chemoselective reactions that facilitate the synthesis of glycan-substituted imaging agents, arrays, and materials are yielding compounds to interrogate and perturb glycan function and dysfunction. To complement these advances, small molecules are being generated that inhibit key glycan-binding proteins or biosynthetic enzymes. These examples illustrate how chemical glycobiology is providing new insight into the functional roles of glycans and new opportunities to interfere with or exploit these roles.

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that approximately 1% of each genome, Contents from eubacteria to archea and eukaryotes, is dedicated to sugar-processing enzymes (2). INTRODUCTION ...... 620 Moreover, these genes can be highly conserved, GLYCAN SYNTHESIS ...... 622 as the components of few other biochemical Chemical Synthesis pathways are so invariant as those responsible of Oligosaccharides ...... 623 for glycan biosynthesis (3). The importance of Engineering Enzymes this conservation is underscored by data indi- for Glycan Synthesis ...... 626 cating that defects in the glycan biosynthetic Glycoprotein and machinery in humans, known as congenital dis- Glycopeptide Synthesis ...... 628 orders of glycosylation, are rare and generally Chemical Glycobiology of have severe deleterious consequences (4). Glycolipids ...... 629 Genomic analysis is a powerful means to Chemoselective Reactions identify enzymes that generate or degrade gly- to Modify Glycans ...... 629 cans and the proteins that recognize the gly- INTERROGATION OF can products. Still, it does not reveal what GLYCAN RECOGNITION...... 632 glycans are present in a cell or organism be- Glycan Arrays ...... 632 cause the synthesis of glycans is not template Lectin Arrays ...... 634 directed. As a result, elucidating the molecu- PERTURBATION OF lar mechanisms that underlie glycan function GLYCAN FUNCTION ...... 634 has been a challenge. Nevertheless, researchers Perturbation of Protein-Glycan have uncovered numerous roles for glycans, Recognition with Monovalent including those in fertilization and develop- Ligands ...... 635 ment, hormone function, cell proliferation and Perturbation of Protein-Glycan organization, host-pathogen interactions, and Recognition with Multivalent the inflammatory and immune responses (3). Ligands ...... 637 These findings are providing additional impe- Perturbation of Glycan Assembly . . . 640 tus to devise new approaches that meet the chal- Exploiting Alternative Substrates lenges of elucidating and manipulating glycan in Glycan Biosynthesis ...... 643 function. Illuminating Glycan Biosynthesis . . . 644 The increased appreciation for the ubiquity CONCLUSION ...... 646 of glycans and their importance to human health has spawned the field of chemical glycobiology. Because of the complexities of by University of Wisconsin - Madison on 07/29/10. For personal use only. Glycan: a generic glycans, their study has compelled researchers term referring to a INTRODUCTION to pursue interdisciplinary approaches. Since Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org monosaccharide, oligosaccharide, Glycans, which are compounds that include the pioneering contributions of 1902 Nobel polysaccharide, or its monosaccharides, oligosaccharides, polysac- Laureate Emil Fischer, it has been apparent conjugate (e.g., charides, and their conjugates, are critical that our understanding of glycan function glycolipid, constituents of all organisms. Members of a can be advanced using approaches that span glycoprotein, or other glycan subset, the polysaccharides, are the most biology and chemistry. The nucleation of the glycoconjugate) abundant organic compounds on Earth. Gly- discipline of chemical biology is yielding new Glycoconjugate: one coconjugates (e.g., peptidoglycan, glycolipids, and innovative strategies to probe glycan func- or more saccharide units (glycone) glycoproteins) also are prevalent. In humans, tion (5). Indeed, there has been an explosion covalently linked to a for example, half of all proteins are glycosy- of research in this area. As a result, this review noncarbohydrate lated (1). Consistent with glycan abundance in cannot provide comprehensive coverage of the moiety (aglycone) nature, data from genomic sequencing indicate field but rather offers an overview of select

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advances that illustrate the unique contribu- agents can be used for purposes ranging from Glycobiology: the tions and exciting opportunities within the imaging glycans to cross-linking them to their study of sugars in field of chemical glycobiology. binding partners. Together, these chemical biological systems, The state of the art of chemical glycobiology strategies are illuminating the molecular including their is focused on key questions: How are glycans mechanisms that underlie glycan function. structures, made and degraded, what are their biological biosynthesis, and physiological roles roles once in place, and how can these roles be exploited? To address these questions, Interrogation researchers have employed the complementary a Glycan- b Cell strategies of interrogation and perturbation binding protein Lectin (Figure 1). The interrogation strategy strives to understand endogenous interactions between Antibody natural glycans and their cognate enzymes or Glycan binding partners. Access to naturally occurring array and novel glycans provides the means to examine protein-glycan or enzyme-glycan in- teractions. Arrays composed of glycoconjugates Surface (Figure 1a) or lectins (Figure 1b) are valuable tools for interrogating protein-binding speci- ficity or cellular glycosylation patterns. With Perturbation the complementary perturbation approach, in- c e hibitors, analogs, or other nonnatural substrates Lectin can serve as probes of both the biosynthesis and Inhibitor the biological roles of glycans. Indeed, novel nonnatural oligosaccharide mimics or syn- thetic glycoconjugates can inhibit or encourage specific biomolecular interactions within cells Glycosyl- transferase and organisms (Figure 1c,d ). Moreover, compounds have been identified that can block key steps within glycan biosynthetic pathways d Cell (Figure 1e). Finally, carbohydrate analogs can Normal Inhibitor sugar be incorporated into glycans using the cellular Lectin biosynthetic machinery (Figure 1f ). Such

by University of Wisconsin - Madison on 07/29/10. For personal use only. −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ f Nonnatural sugar Figure 1 Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org Interrogation and perturbation in chemical glycobiology. (a) Glycan arrays have been developed to interrogate the binding specificities of lectins (blue), antibodies (green), and other glycan-binding proteins (orange). (b) Lectin arrays can be used to fingerprint cell-surface or pathogen glycosylation Multivalent ligand patterns. Monovalent (c) and multivalent (d ) ligands for glycan-binding proteins can perturb protein- glycan interactions. (e) Inhibitors can prevent key steps in glycan biosynthesis, thereby reducing the production of specific glycan structures. ( f ) Nonnatural monosaccharides can serve as substrates for biosynthetic enzymes and thereby be incorporated into glycans.

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GLYCAN SYNTHESIS glycolipids, glycosylphosphatidylinositol an- Defined oligosaccharides and glycoconjugates chors, proteoglycans, and polysaccharides are are critical for unraveling the function of gly- influenced by accessibility of the nucleotide Lectin: a glycan- binding protein of cans. Obtaining these entities from natural donors, but the mechanisms governing the reg- nonimmune origin sources is difficult because their production ulation of these pathways are still being eluci- generally involves the participation of multiple dated. Thus, it is difficult to obtain sufficient transporters and enzymes (6). This complex- quantities of glycans for study from biological ity is illustrated by the pathway for eukaryotic sources. glycoprotein synthesis (Figure 2). The saccha- Chemical strategies are addressing this de- ride building blocks (typically nucleotide sug- ficiency by providing the means to generate ars) must be generated and then transported an ever-increasing diversity of glycans. Natu- to the appropriate cellular location, where they rally occurring glycans can be synthesized, as can be used by glycosyltransferases. The effi- can derivatives. In this way, critical structure- ciency of producing any particular glycan de- activity relationships can be elucidated. There pends on the concentration of building blocks, are two general approaches for the synthesis what glycosyltransferases and other biosyn- of oligosaccharides: chemical and enzymatic.

thetic enzymes are present, and the Km values Here, we outline some of the major advances of those building blocks for the glycosyltrans- that have occurred on both fronts. More de- ferases that use them. Pathways for the pro- tailed information can be found in several duction of N-glycoproteins, O-glycoproteins, excellent reviews (7–10).

Extracellular milieu

Monosaccharides Cell-surface glycoconjugates

Glycan- Metabolic specific receptor interconversions

ER/Golgi Cytosol by University of Wisconsin - Madison on 07/29/10. For personal use only. Monosaccharide donors Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org Glycoconjugate assembly in the secretory compartments

Figure 2 Schematic depiction of glycoconjugate biosynthesis and cell-surface recognition of glycans. Most exogenously supplied monosaccharides are taken up by cells and converted to monosaccharide donors in the cytosol. The donors are imported into the endoplasmic reticulum (ER) and Golgi compartments, where they are used by glycosyltransferases to assemble glycoconjugates. In the case of N-linked glycoproteins, a core oligosaccharide is assembled in the cytosol, transported into the ER where it is processed by glycosidases, and further elaborated by glycosyltransferases. Once displayed in fully mature forms on the cell surface, the glycoconjugates can serve as ligands for soluble lectins, cell-surface glycan-binding proteins, or glycan- binding proteins on other cells or pathogens. In principle, chemical glycobiology can yield molecules that can be used to inhibit or promote any stage of this process.

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Chemical Synthesis The simplicity of this approach belies of Oligosaccharides its complexity. Glycosylation reactions are regulated by the reactivity-selectivity principle The chemical synthesis of oligosaccharides GAG: of organic chemistry. The anomeric group offers tremendous flexibility. It can give rise glycosaminoglycan must be sufficiently prone to leaving, such that to diverse glycans, including those available a relatively poor nucleophile like a hydroxyl in minute quantities from biological sources group can engage in bond formation; however, or those for which the biosynthetic enzymes the donor must not be so reactive that bond are unknown. Moreover, chemical synthesis formation occurs without stereocontrol. Thus, provides the means to test the importance of whether the reaction occurs via an S 2-like different functional groups because nonnatural N (inversion) or S 1-like (oxocarbenium inter- sugars can be introduced. The power of chem- N mediate) pathway has a critical influence on the ical synthesis is illustrated by the development stereochemical outcome. For this reason, the of potent and effective sulfated saccharides as dual problems of regiochemical control and anticoagulants (11). These defined compounds stereoselectivity of glycosylation reactions are were inspired by analysis of the properties of intertwined. Fraser-Reid and coworkers’ clas- heparin, an anionic glycosaminoglycan (GAG) sification of donors as “armed” (fast reacting) that has long been used as an anticoagulant. or “disarmed” (slow reacting) has led to major Heparin can bind antithrombin III, thereby advances in the field (14) because it offers producing a complex that blocks blood clot- insight into how the electronics of the glycosyl ting. Pharmaceutical-grade heparin is typically donor can be manipulated to control glyco- isolated from porcine intestinal mucosa as a sylation reaction outcomes. The reactivity of mixture of sulfated polysaccharides. Chemical the donor can be tuned by modifying several synthesis was important in verifying that factors, including the electron-withdrawing heparin’s activity resides in a critical pentasac- ability of the protecting groups, the lability charide recognition sequence. These findings of the anomeric leaving group, the method of indicate that proteins like antithrombin III leaving group activation, the conformation of can recognize specific sulfated oligosaccharide the donor or acceptor, and the nature of the sequences within GAGs. Additionally, they led solvent. These changes in reactivity impact to development of the defined anticoagulant glycosylation reaction stereochemistry because drug Arixtra®. The recent contamination of they influence the reaction mechanism (S 2- heparin isolated from biological sources, in N like versus S 1-like) (Figure 3a). It is possible which the presence of other sulfated polysac- N to tune a series of glycosylation reactions to charides led to over 100 deaths, highlights gain outstanding stereoselectivity.

by University of Wisconsin - Madison on 07/29/10. For personal use only. the utility of therapeutics that are based on A longstanding yet clever means to achiev- defined, synthetic glycans (12). ing stereocontrol is to exploit protecting

Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org The chemical synthesis of oligosaccharides groups that can influence glycosylation stereo- appears deceptively simple. It involves the chemistry via neighboring group participation formation of glycosyl bonds, a reaction first (Figure 3b). In the paradigmatic example, a 2- described in 1893 (13). The approach that was acyl group forms an acetoxonium ion by attack employed then is similar to that used by nature onto the anomeric carbon. With a protected and remains the preferred strategy for chemical glucose derivative, the 1,2-acetoxonium ion synthesis: A donor monosaccharide, equipped blocks nucleophilic attack of the acceptor from with a leaving group at the anomeric position, the α-face and results in the formation of β- undergoes reaction with a nucleophilic group glucosides; for mannose, the β-face is blocked, on an acceptor (Figure 3a). In chemical and α-mannosides are produced. This strategy synthesis, a promoter is added to the donor has been used extensively to generate β- monosaccharide to facilitate the departure of glucosides, β-galactosides, α-mannosides, and the leaving group.

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α-rhamnosides. More recently, novel variations glycosidic linkages, including α-glucosides and on the neighboring group participation strategy α-galactosides (7). Still, many key glycosidic have been employed to access a wider array of linkages cannot be formed via neighboring

OH a OH O HO O HO O HO OR OP OP' OH P = Ac OH O O 1. Promoter PO + HO or PO P'O OR 2. Protecting group OH PO OP' removal LG HO O Glycosyl donor Glycosyl acceptor HO OH HO P = Bn O P = Bn (armed) O HO OR or Ac (disarmed) OH

b OP OP OP PO O Promoter PO O ROH PO O PO LG PO PO OR NH HN+ NH O O O

c OP OP' PO O + HO O Promoter Promoter PO LG P'O LG' OP OP' OP" Most reactive donor Less reactive donor HO O "PO LG" OP" Least reactive donor OH 1. Promoter, ROH OH O OH HO O HO O O by University of Wisconsin - Madison on 07/29/10. For personal use only. 2. Protecting group HO O removal OH HO OR OH OH Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org

d 1. OP OP P1O O PO O LG LG PO OP PO OP OP Promoter O Promoter Linker HO HO PO O Linker 2. Selective protecting OP group removal Cleavage from OH OH support O HO O HO O HO OR Protecting group OH removal OH

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group participation strategies. Important ex- oligosaccharides are becoming more common. amples include β-mannosides, β-rhamnosides, Two notable examples include generation of and sialic acid derivatives. New insight into the the branched hexasaccharides GM1 and the interplay of stereoelectronic and conforma- tumor-associated carbohydrate antigen Globo- tional effects is providing strategies to assemble H (20). In the former, three components were these kinds of linkages (15). The synthesis joined in a single reaction; while in the latter, of sialic acid derivatives has been especially four building blocks were linked (21). challenging because the anomeric position Another strategy for oligosaccharide syn- is more hindered and possesses an electron- thesis that circumvents the need for multi- withdrawing group, but the use of protecting ple purification steps is solid-supported syn- groups to alter thiosialoside conformation in thesis (22). In this manifold, reactions can combination with novel conditions for donor be driven to completion by the addition of activation has led to dramatic improvements in an excess of one partner. In the typical con- glycosylation yields (16–18). The value of these figuration, a nucleophilic acceptor substrate new approaches is illustrated by the synthesis is appended onto a solid support and ex- of an α2,9-trisialic acid oligomer in a single posed to an excess of activated donor in so- reaction vessel (one-pot) (19). lution (Figure 3d ). Subsequent steps involve Efforts to streamline the chemical synthesis hydroxyl protecting group removal followed of oligosaccharides have focused on mini- by glycosylation. The multiple sites of re- mizing purification steps. One approach is activity and branching found in oligosaccha- to conduct the kind of one-pot glycosylation rides require monomers that possess orthogo- reactions described above, in which multiple nal protecting groups, which can be masked and glycosidic bonds are made without isolation unmasked at appropriate stages of glycan con- or purification of intermediates (Figure 3c). struction. Thus, the solid-supported synthesis There are three general strategies to achieve of oligosaccharides is complicated by the need this end. First, the relative reactivities of the for diverse building blocks. Nevertheless, the glycosyl donors can be varied by protecting potential of solid-supported synthesis has con- group selection, such that the addition of a pro- tinued to spark advances, including methods to moter triggers the most armed glycosyl donor automate the process. Automated synthesis now first, and the most disarmed donor eventually can be used to prepare even complex oligosac- engages in the final glycosylation reaction. charides (22), such as a branched β-glucan do- Second, glycosyl donors can be preactivated decasaccharide; blood group oligosaccharides before exposure to a glycosyl acceptor, and the Lewis x, Lewis y, and the Lewis x-Lewis y order and timing of their addition determine nonasaccharide; and tumor-associated carbo- by University of Wisconsin - Madison on 07/29/10. For personal use only. the reaction outcome. Third, orthogonal hydrate antigens Gb-3 and Globo-H. These anomeric leaving groups can be selected that latter examples are compounds with multiple Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org are activated by different promoters. Impres- types of glycoside linkages, and their success- sive one-pot syntheses of biologically relevant ful synthesis demonstrates that glycosylation ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 3 In its most versatile form, the chemical synthesis of oligosaccharides depends upon glycosylation reactions that involve activation of a glycosyl donor. (a) Treatment with a promoter facilitates the departure of the anomeric leaving group (LG) such that a glycosidic bond can be formed in a substitution reaction with a nucleophilic glycosyl acceptor. Depending on the electron-withdrawing potential of the protecting groups (P) on the glycosyl donor, the reaction can proceed through an SN2-like mechanism (P = Ac, acetate) or an SN1-like mechanism in which the stereochemical outcome is controlled by the anomeric effect (P = Bn, benzyl). (b) Neighboring group participation can dictate product stereochemistry. One-pot methods (c) and solid-phase synthesis (d ) are approaches that eliminate purification steps and thereby facilitate glycan assembly. Abbreviation: ROH, an alcohol, including a hydroxyl group of a protected sugar derivative.

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reactions in solid-supported synthesis can oc- glycone or aglycone acceptors. The use of these cur with excellent stereoselectivity. enzymes can facilitate the chemoenzymatic syn- There has yet to emerge a universal strat- thesis of glycans, and recent advances have in- egy to form glycosidic bonds with the requi- creased the utility of this approach. Historically, site regio- and stereocontrol. Suitable reaction nucleotide-sugar donors can only be generated conditions must be optimized for each glyco- with naturally occurring sugars, although ef- sidic variation. Thus, current methods are fo- forts in enzyme engineering indicate that this cused on developing sets of standard build- problem can be overcome (25). Indeed, the util- ing blocks that can be used to generate key ity of enzymes for generating oligosaccharides bioactive oligosaccharides. To this end, many has increased in the last decade (26, 27), owing, of the targets assembled to date possess link- in part, to the ability to identify glycosyltrans- ages that can be formed reliably, such as α- ferases from sequence data. mannosides, β-galactosides, and β-glucosides. Bacterial glycosyltransferases and related It is estimated that approximately 500 orthog- biosynthetic enzymes have proved especially onally protected monosaccharides would be useful for glycan assembly. These enzymes and needed to synthesize the bioactive oligosaccha- their variants can be produced and purified rides found in mammals (23), although a re- more readily than their eukaryotic counter- cent analysis suggests that 36 building blocks parts, and they often act on a broad array of could generate 75% of the known mammalian substrates. In a powerful example, Chen and oligosaccharides (24). The need for glycans that coworkers (28) used three classes of bacterial reflect the diversity of physiological systems sugar-processing enzymes (a sialic acid al- is driving efforts to develop methods to pre- dolase, a cytidine 5-monophosphate-sialic acid pare all the relevant glycosidic bonds, includ- synthetase, and a sialyltransferase) to produce ing those that have been challenging (e.g., β- a library of 72 biotinylated sialosides in an mannosides, sialic acid derivatives). Progress array format. By screening this array, detailed on this front has made accessible biologically information about the binding preferences important glycans, such as sulfated GAG se- of a key immunomodulatory protein, human quences and protein glycosylphosphatidylinos- CD22, could be gleaned. Another example itol anchors. Despite the rapid development in in which the broad substrate specificity of generating mammalian oligosaccharides, there bacterial glycosyltransferases was exploited is has been less emphasis on assembling glycans in the production of 70 glycoforms of the nat- found in microbes. Many of these contain non- ural products calicheamicin and vancomycin canonical sugars (e.g., deoxysugars and fura- (29). Because glycosylation can influence noses) that pose unique challenges. Methods to natural product biological activity, specificity, by University of Wisconsin - Madison on 07/29/10. For personal use only. assemble these glycans are needed to elucidate and pharmacology, the ability to introduce their roles in microbes, to probe host-pathogen different saccharide substituents is valuable. Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org interactions, and to investigate novel antimicro- The enzymes can be engineered to increase bial strategies. their substrate tolerance even further (30). Another chemoenzymatic approach to oligosaccharides is based upon glycosidases. Engineering Enzymes Nature’s antipode to the glycosyltransferase is for Glycan Synthesis the glycosidase, an enzyme that catalyzes the Enzymatic and chemoenzymatic methods for hydrolytic cleavage of glycosidic bonds. Re- glycan assembly complement those from chem- placement of the active-site water nucleophile ical synthesis. These approaches harness the with a glycosyl acceptor can result in trans- components used by physiological systems to glycosylation (26, 27). Because glycosidases can generate glycans. Specifically, glycosyltrans- readily be produced, often are highly soluble ferases transfer nucleotide-sugar donors onto and stable, and tend to be more promiscuous

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than glycosyltransferases, they are attractive as Glycosidases come in two varieties, retaining catalysts. Unfortunately, glycosidase-catalyzed and inverting (26, 27). In general, both pos- transglycosylation reactions suffer from low sess active sites in which catalytic carboxylic Glycosynthase: yields and product hydrolysis because the acid residues are proximal (Figure 4). Most gly- engineered glycosidase products are themselves substrates. A major cosynthases are based on retaining glycosidases, capable of catalyzing breakthrough in the field occurred with the which use a two-step, double-substitution transglycosylation invention of nonhydrolyzing glycosidases, or mechanism that proceeds through a covalent reactions glycosynthases. carbohydrate-protein adduct (Figure 4b). Sub- Glycosynthases were developed by exploit- stitution of the nucleophilic active site aspartate ing key features of glycosidase mechanisms. or glutamate with a small hydrophobic residue

a Inverting glycosidase mechanism Enz Enz

O O O O H H – ROH O O O H HO OH HO O O OR O O H Enz Enz

b Retaining glycosidase mechanism Enz Enz Enz

O O O O O O H H + H2O H O O O HO O – ROH O H HO OH R HO O O O O O O

Enz Enz Enz

c Glycosynthase mechanism

by University of Wisconsin - Madison on 07/29/10. For personal use only. Enz Enz

O O O O Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org H H – F– O O O R HO OR HO F CH 3 CH 3 Enz Enz

Figure 4 Catalytic mechanisms for glycosidases and glycosynthases. (a) Inverting glycosidases use two catalytic carboxylate residues positioned proximally. (b) Retaining glycosidases use a two-step, double-substitution mechanism, with a covalent carbohydrate-enzyme adduct. (c) Substitution of the nucleophilic active-site carboxylate of retaining glycosidases with a nonpolar side chain affords glycosynthases capable of transglycosylation. Abbreviations: Enz, enzyme; ROH, an alcohol, including a hydroxyl group of a protected sugar derivative.

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(i.e., alanine) (Figure 4c) renders the enzyme formed by engineered retaining glycosidases, incapable of hydrolysis. These variants can cat- inverting glycosidases have been generated alyze the formation of glycosidic bonds be- that afford α-linkages (36, 37). Thus, ad- tween an acceptor and an α-glycosyl fluoride vances in the identification and engineering donor. In the engineered enzyme, the product of both glycosyltransferases and glycosyn- is no longer susceptible to hydrolysis; there- thases are extending the range of accessible fore, transglycosylation reactions can occur in glycans. high yields and with high levels of regio- and stereoselectivity. Glycosynthases engineered to process al- Glycoprotein and ternative substrates can be generated by either Glycopeptide Synthesis rational mutagenesis or directed evolution. In parallel with the development of new meth- Because libraries of enzyme variants can be ods for oligosaccharide assembly, there have readily prepared, the major roadblock in the emerged new approaches for glycoconjugate discovery of novel glycosynthase enzymes has synthesis. The prevalence of glycosylated pro- been the development of high-throughput teins and the benefits of access to single-protein screens. The enzyme-catalyzed glycosylation glycoforms have inspired the development of reactions are not accompanied by the release of methods to generate N- and O-glycosylated a chromophore, so novel screens were needed. peptides and proteins. Protein glycosylation The approaches that have been devised fall into can influence the pharmacological properties three categories: a yeast three-hybrid chemical of therapeutic proteins, including their serum complementation assay (31), an assay based on half-lives, their ability to target specific cells or pH changes (32), and a fluorescence-activating organs, and their modes of clearance. Glyco- cell sorting (FACS) assay (33). In all three sylation can also exert an influence by playing approaches, glycosynthase activity is evaluated a direct role in recognition, such that whole in whole cells; therefore, protein isolation is glycosylated protein is more than the sum of not required. the parts. A notable example of the latter in- Glycosynthases have been identified (26, 27) volves P-selectin, a protein involved in the in- that act on a range of nucleophiles, including flammatory response, which binds to a highly glycone and aglycone acceptors. A notable fea- O-glycosylated protein (a mucin) bearing the ture of glycosynthases is that they can produce tetrasaccharide sialyl Lewis x. The tightest oligosaccharides that are difficult to obtain complexes between P-selectin and its ligand are using chemical synthesis (e.g., β-mannosides) formed when specific tyrosine residues adjacent (34). In addition, disaccharide fluoride donors to a sialyl Lewis x motif are sulfated (38). Thus, by University of Wisconsin - Madison on 07/29/10. For personal use only. and acceptors can be used in transglycosy- the identity of the glycoconjugate as a whole lation reactions, thereby enabling the rapid is important for recognition (39). Together, the Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org assembly of complex oligosaccharides (26). mode of P-selectin recognition and the require- Glycosynthases also can be used to generate ment for therapeutic glycoproteins with opti- oligosaccharides that contain β-glucuronic acid mized properties underscore the need to obtain or β-galacturonic acid residues, suggesting they defined glycoconjugates. can be used for GAG assembly (26). Notably, Several strategies have emerged that yield retaining endoglycosidases have also been de- defined glycoproteins and glycopeptides. One vised that catalyze the convergent assembly of approach is to employ engineered cell lines or N-glycans. Specifically, these modified enzymes recombinant enzymes to obtain glycoproteins. promote the reaction of oxazolines derived For instance, complete heterogeneous gly- from 2-deoxy N-acetyl glucosamine-containing coproteins can be expressed, isolated, and substrates with asparagine-containing peptides trimmed via glycosidases to bear individ- (35). To complement the linkages that can be ual monosaccharide moieties (40). These

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monosaccharides serve as starting points for synthesis is under investigation (44). Other embellishment by recombinant glycosyltrans- approaches have been described that capi- ferases or by transglycosylation using endogly- talize on removable or transient auxiliaries cosidases. The latter strategy is especially useful (45–47). One of these, sugar-assisted ligation for the rapid assembly of complex glycoproteins is particularly useful in the construction of because a large glycan motif can be added in a N-linked glycans (Figure 5b) (48). Thus, the single step (as described above). means to construct larger glycopeptides and Synthetic chemists also have taken on the glycoproteins are available and can be used challenge of glycopeptide and glycoprotein to examine the influence of glycosylation on preparation. In focusing on various complex protein function. glycopeptides and glycoproteins as targets for synthesis, the Danishefsky research group (41) has pushed the limits of existing synthetic meth- Chemical Glycobiology of Glycolipids ods. In pursuing their complex targets, includ- Glycolipids have been implicated in many criti- ing prostate-specific antigen, gp120 fragments, cal processes, but identifying their precise phys- and erythropoietin, they have developed new iological roles has been difficult. Recent discov- strategies for the assembly of multiple peptide eries have revealed that glycolipids can serve precursors. as critical immunomodulators. Natural killer T The chemical synthesis of glycoproteins is (NKT) cells are a class of T cells that play a fueled both by methods to construct complex central role regulating the immune response, glycopeptide fragments and the advent of and NKT cells can recognize glycolipids dis- chemoselective ligation reactions to join them. played by CD1d-positive antigen-presenting Solid-phase peptide synthesis is generally lim- cells. Both endogenous and exogenous glycol- ited to glycopeptides <50 residues. Chemical ipids can serve as CD1d ligands and thereby ligation reactions, such as native chemical lig- activate NKT cells. An endogenous glycolipid ation (NCL), alleviate this limitation because is presumably necessary for positive selection they can be used to link peptide fragments of NKT cells in the thymus, and NKT cells together (Figure 5a) (42). The NCL process can recognize exogenous lipopolysaccharides involves the transthioesterification reaction from bacterial pathogens (49). The synthetic of a C-terminal thioester with an N-terminal glycolipid antigen KRN7000 (Figure 6) and cysteine residue of a second peptide. The related compounds are illuminating the critical resulting thioester intermediate subsequently features of the glycolipid that result in NKT undergoes an intramolecular transacylation cell activation. This understanding can lead to reaction to produce a stable peptide bond. the development of new immunomodulators. by University of Wisconsin - Madison on 07/29/10. For personal use only. One valuable variation on NCL is expressed Additionally, because glycolipid trafficking and protein ligation (EPL), in which the peptide degradation are involved in several diseases, Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org component bearing the C-terminal thioester glycolipid analogs serve as probes and thera- is produced using recombinant DNA methods peutic leads (50). (43). Although both NCL and EPL increase the scope of glycopeptide synthesis, they require a cysteine residue, a relatively rare Chemoselective Reactions amino acid, at the ligation junction. to Modify Glycans The Staudinger ligation of peptide Glycoconjugates are critical tools in the ex- thioesters circumvents the need for cysteine at amination of glycan function. They can be the junction, as the two peptides couple when immobilized for affinity isolation of glycan- a C-terminal phosphinothioester undergoes binding proteins, used to generate glycan ar- reaction with an N-terminal azide. The utility rays, or converted to natural or nonnatural of the Staudinger ligation for glycopeptide probes. Such probes can be generated from

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a Expressed protein and native chemical ligation O HO

H2N Peptide

Recombinant DNA methods O HS or SPPS Peptide SR

O HO O HS HO H2N Peptide O

O Peptide N Peptide H Peptide S

b Sugar-assisted ligation O O HO O HO NH X NH X

HS Peptide S

O O O

Peptide SR H2N Peptide H2N Peptide

O O HO HO NH X NH X

HS Desulfurization

O O O O

Peptide N Peptide Peptide N Peptide H H

Figure 5 Ligation strategies for glycopeptide and glycoprotein synthesis. (a) In native chemical ligation (NCL), the by University of Wisconsin - Madison on 07/29/10. For personal use only. peptide components are obtained by solid-phase peptide synthesis (SPPS). For expressed protein ligation (EPL), recombinant DNA methods are used to produce a peptide or protein fragment with a C-terminal

Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org thioester. For both EPL and NCL, the thioester is captured by an N-terminal cysteine residue, and the incipient thioester conjugate rearranges to the amide. (b) In sugar-assisted ligation, a carbohydrate bearing a thiol substituent serves in the same capacity as the Cys side chain.

chemoselective reactions (i.e., reactions that HO OH O O occur among select functional groups in the HO NH OH presence of others) of natural and synthetic HO O oligosaccharides (51). One of the most common OH strategies is to use the intrinsic reactivity Figure 6 of oligosaccharides, which contain an elec- The structure of glycolipid antigen KRN7000, which functions as an trophile at the reducing end, most commonly, immunomodulator that leads to natural killer T cell activation. an aldehyde. This masked carbonyl group is

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susceptible to nucleophilic addition, which has been exploited for conjugate production. Nu- OH a R HO N cleophiles, such as alkoxylamine, hydrazine, or O O R H N acylhydrazine derivatives, can be employed to 2 afford glycoconjugates containing oxime, hy- O drazone, or hydrazide linkages, respectively HO OH (Figure 7a). These functional groups vary in H N their stability and in whether the reducing-end 2 NH O H HO N sugar exists in the open or closed form. Thus, R N R H the mode of conjugation can be chosen for a specific purpose. An alternative strategy is to generate a O R O glycan that bears a linker that possess a func- HO O HO O R tional group that can undergo reaction with a coupling partner in the presence of hydroxyl, acetamide, carboxylate, and other common car- bohydrate functional groups. Perhaps the most RR commonly used linker, and that used by the O Consortium for Functional Glycomics (CFG, b N NH2 http://www.functionalglycomics.org), is an H R R aminopropyl group appended to the anomeric position. The resulting amine-bearing O O NH oligosaccharides can undergo reaction with N O R R several types of partners, including those O bearing N-hydroxysuccinimidyl esters, alde- O hydes (followed by reductive amination), or H NH H CH3O N R N dimethyl squarate (Figure 7b) (52–55). Other R chemoselective linkage strategies rely on the O O O O unique reactivity of thiol-containing saccha- rides, which can undergo conjugate addition c N N N3 R N R −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ O O PPh2 NH CH3O by University of Wisconsin - Madison on 07/29/10. For personal use only. Figure 7

Chemoselective reactions used to generate PPh2 R O

Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org glycoconjugates. (a) Glycans with free reducing ends can undergo reaction with aminooxy- and O NH hydrazine-bearing linkers to form oxime and R Ph P S R hydrazone linkages. (b-e) Different reactant sets for 2 O the general reaction shown at the top of the table. ( ) Glycans bearing amino groups can attack O b d R N-hydroxysuccinimidyl esters, aldehydes, or R dimethyl squarate to generate adducts. (c) Glycans O O that possess azide functional groups can engage in O azide-alkyne cycloaddition [Cu(I)-catalyzed or strain-promoted reactions] and Staudinger ligation e reactions. Diels-Alder cycloadditions (d ) and olefin R metathesis reactions (e) are other examples of Grubbs’ catalyst R chemoselective methods for glycan attachment.

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to maleimide-bearing glycans (56, 57) or form interactions for cell-cell recognition is that, disulfide-linked conjugates (58). Cycloaddition when each individual receptor-ligand interac- reactions, including Cu(I)-catalyzed azide- tion is weak, binding will be kinetically labile. CFG: Consortium for Functional Glycomics alkyne (Figure 7c) and Diels-Alder reactions In this way, only cells with the correct combina- (Figure 7d ), also have been used (59, 60). Al- tion of receptor-ligand pairs will interact stably. though olefin metathesis depends upon a metal The involvement of multivalent binding in carbene catalyst, remarkably, it has been shown many protein-glycan interactions complicates to be compatible with carbohydrate function- the identification of the relevant endogenous ality (Figure 7e), including sulfate groups (22, ligands. Standard receptor-ligand assays lack 61). The Staudinger ligation reactions of azides the necessary sensitivity to monitor low-affinity with phosphinoesters or phosphinothioesters binding. Accordingly, many methods to assess also are useful chemoselective reactions, and protein-glycan interactions depend upon mul- the phosphinoester version has been used in tivalent display of one or both binding part- diverse contexts (Figure 7c) (62, 63). Some ners. Such assays have higher sensitivity and examples of how the different aforementioned can have even higher specificity (67, 68). More- chemoselective reaction processes have been over, they can minimize the amount of ma- exploited to investigate glycan function are terial required, which is especially important described in subsequent sections. considering the challenges associated with the acquisition of glycans. Still, they are best used to compare compounds because determining INTERROGATION OF the true equilibrium constant for a multivalent GLYCAN RECOGNITION interaction is complicated. Indeed, many dis- Glycans are present both inside and outside of tinct types of binding modes can contribute to cells. Within cells, glycosylation is critical for the strength of a multivalent interaction (69, protein trafficking, and more recently, it has 70). Glycan arrays are a technology that in- been found to influence gene expression (64). vokes multivalent binding and allows many dif- Glycans on the surface of pathogens can serve ferent samples to be compared simultaneously. both as a protective shield and as a means for New tools for array fabrication and analysis recognizing and entering target cells. Similarly, have been advanced that depend on a combi- protein-glycoconjugate interactions are a nation of analytical, biochemical, and synthetic major line of communication between cells methods. The topic of glycan arrays has been and their environment. Lectins, proteins of reviewed extensively (71–75), and our goal is to nonimmune origin that bind to specific glycan highlight relevant contributions of chemistry to structural motifs, typically use solvent-exposed their development. by University of Wisconsin - Madison on 07/29/10. For personal use only. binding sites to interact with their target oligosaccharide ligands (65). As a result, they Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org bind weakly to single carbohydrate residues Glycan Arrays and even oligosaccharides. Indeed, monovalent Glycan arrays have been widely embraced as protein-glycan binding dissociation constants platforms suited to rapid screening of protein are often in the range of 10−4 to 10−3 M binding to carbohydrates. On the basis of prin- (66). These low affinities might suggest that ciples developed for DNA and protein microar- protein-glycan complexes are not important, rays, glycan arrays have emerged as tools to as- yet weak binding is ideal for mediating cell sess the specificities of lectins, antibodies, and adhesion. When cells interact, glycans on other glycan-binding proteins. There are many one cell surface can bind to multiple copies methods for fabricating glycan arrays, yet all of a lectin on another, thereby increasing have the same overall features. Specifically, nat- the apparent binding constant (functional ural or synthetic glycans are immobilized onto a affinity). The advantage of using low-affinity surface through either covalent or noncovalent

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attachment. The resulting glycan surfaces are of quantifying the immobilization efficiency. treated with whole cells, complex biological Reductive amination reactions of oligosaccha- samples such as sera, or isolated glycan-binding rides with the lysine side chains of proteins proteins. Binding can be assessed using fluores- also have been exploited to yield multivalent cence or another type of reporter. glycoconjugates that were subsequently immo- The first challenge to creating glycan arrays bilized; the presentation of these conjugates was to develop a method to spatially pattern on the surface can mimic that of glycosylated various oligosaccharides or glycoconjugates on proteins (86–88). a surface. To date, the methods implemented When glycans are generated by chemi- for array fabrication fall into three general cat- cal synthesis, tailored functional groups for egories: (a) immobilization by physical adsorp- immobilization can be introduced. As men- tion, (b) immobilization via high-affinity, spe- tioned previously, the mostly widely used strat- cific noncovalent interactions, or (c) covalent egy is to introduce a linker bearing an amine capture, in which complementary reactants are group and exploit its nucleophilicity with displayed on the glycan and the surface. Physi- N-hydroxysuccinimidyl ester-coated slides. A cal adsorption, which exploits the ability of gly- complementary approach is to build oligosac- cosylated proteins or glycolipids to adhere to charides directly onto the array surface (56). the surface, helped to establish the utility of gly- This general strategy provides not only a means can arrays as a multivalent assay platform (76– to construct known glycan structures, but also 78). Nonnatural glycolipids can be generated the opportunity to interrogate the selectivities from oligosaccharides that possess a free reduc- of glycosyltransferases (89). ing end (i.e., a masked aldehyde) using lipids Many relevant protein-glycan interactions that bear nucleophiles, such as amines (79) or can be uncovered using glycan arrays as exper- alkoxylamines (80). In an innovative variation iments focused on Tn antigen illustrate. The of the adsorption approach, fluorous lipid tags, Tn antigen is rarely expressed in normal tissues which can be used both for synthesis and im- but is associated with several cancers. Glycan mobilization, have been employed (81). An al- arrays were used to reveal that only a subset ternative approach is to immobilize a glycocon- of prostate tumors display the Tn antigen, a jugate through specific noncovalent complexes, finding that may have therapeutic implications such as biotin-streptavidin binding or DNA hy- (90). Another instructive feature of the Tn anti- bridization (82). In general, however, in most gen studies is that variations in the specificities arrays, the glycan is linked to the surface by co- determined by individual research groups were valent bond formation. different. This unexpected outcome highlights A common means for glycan array construc- the variability that can arise from glycan array by University of Wisconsin - Madison on 07/29/10. For personal use only. tion is to exploit the unique reactivity of the data and the need for standardization. anomeric position. For example, oligosaccha- Despite the challenges, glycan arrays are Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org rides can be appended to a surface that presents providing new insight into the protein-binding nucleophiles via reaction with the reducing end specificities in complex systems, including those (83). Similarly, the reducing end can undergo involving highly anionic sulfated GAG se- reductive amination with 2-aminobenzamine quences. GAGs such as heparin, heparan sul- derivatives in solution (84, 85), and these flu- fate, and chondroitin sulfate are involved in orescent saccharides can be subsequently at- processes ranging from development, angio- tached to the surface. In the latter approach, genesis, cancer metastasis, wound healing, and the fluorescent tag serves multiple purposes: It viral invasion (91). GAGs can be composed of provides a means to purify heterogeneous pools heterogeneous sequences, but the idea that spe- of natural glycans, it can react with an elec- cific sequences are recognized selectively has trophilic (e.g., succinimidyl ester- or epoxide- been controversial owing to a paucity of sup- functionalized) surface, and it provides a means porting data. To investigate this hypothesis,

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GAG arrays have been assembled from the mixtures, similar to that employed for DNA attachment of di- to hexasaccharides bearing arrays. The ratiometric data obtained from a amine- and aminooxy-terminated linkers to pair of dye-swapped arrays afford reproducible electrophilic surfaces (52, 53, 92). The resulting data. This method was used to support the arrays are bolstering the hypothesis that spe- hypothesis that the human immunodeficiency cific GAG sequences have defined physiological virus (HIV) co-opts the microvesicular exo- functions (93–95). cytic mechanism to exit T cells. Lectin array With all the advances in array fabrication, technology also has been used to elucidate the most significant barrier to the widespread differences in sialic acid expression between adoption of glycan arrays is the limited avail- nontumorigenic and adenocarcinoma cells (98). ability of oligosaccharide structures. The CFG Although lectin array technology is more provides arrays for the nonspecialist that focus nascent than that of glycan arrays, some of on human and mammalian glycans. The ver- the challenges are shared. Notably, just as gly- sion currently available to researchers (Version can arrays are limited by the availability of 4.0) displays 442 mammalian glycans, whereas oligosaccharides, lectin arrays are restricted by the pathogen array presents 96 glycans derived the recognition specificities of known lectins. from seven pathogen species. Researchers need Most commercially available lectins are isolated to continue to identify and generate a broad from plants, but pathogenic organisms often array of glycans to extend further the utility contain unique glycan structures that are not of the array platform. Additionally, although recognized by the available and characterized the array surface is suited to multivalent inter- lectins. As more carbohydrate-binding proteins actions, it is unclear how the mode of glycan become available, the value of these arrays and display influences protein recognition. New their ability to distinguish between different cell technologies to address the role of presentation types will increase. include the immobilization of multivalent Generally, glycan and lectin arrays use fluo- glycosylated scaffolds, such as proteins and rescence to detect protein-glycan interactions. peptides (87, 88) or polymer ligands (86). Methods of introducing probes include con- Additionally, fluidic arrays have been intro- jugation of fluorescent tags to cells or lectins, duced that are designed to mimic the mobility cell staining, and incubation with labeled anti- of glycoconjugates imbedded in a lipid bilayer bodies. Modification-free techniques for array (96). As glycan array technology continues to analysis are also under development, and these evolve, standard methods, from fabrication to include evanescent-field fluorescence detection interpretation, will undoubtedly emerge. (99, 100), surface plasmon resonance (101), and fluorescence interference contrast microscopy by University of Wisconsin - Madison on 07/29/10. For personal use only. (102). Lectin Arrays Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org Lectin arrays provide a means to assess lectin binding to individual glycoconjugates, patterns PERTURBATION OF of cell-surface glycosylation, and pathogen- GLYCAN FUNCTION lectin interactions (73). They can provide A traditional approach to perturb protein func- important structural information about un- tion is to delete a protein or proteins of inter- characterized glycans and serve as multivalent est within a cell or organism. The application and sensitive monitors of protein-glycan inter- of RNA interference or gene knockouts can actions. Lectin arrays are typically fabricated provide insight into the importance of a lectin using commercially available carbohydrate- or an enzyme involved in glycan biosynthesis binding proteins of defined specificity. Mahal (103). Drawing conclusions from single gene and coworkers (97) have pioneered a two-color deletions of enzymes involved in the biosyn- technique for the analysis of glycans from thesis or processing of glycans, however, can

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be difficult, as studies of CD22 illustrate. Mice Oligosaccharides based on endogenous gly- that lack CD22 are hypersensitive to B cell anti- cans are an obvious starting point (105), but gen receptor stimulation, but those that lack converting these polar molecules into potent Glycomimetic: the glycosyltransferase (ST6Gal-I) that gener- inhibitors has been difficult. The aim is to de- a small molecule ates the CD22 ligand have compromised B cell vise molecules with improved affinity and se- designed to mimic the responses to antigen (104). To determine that lectivity, reduced polarity, and greater stability function of a CD22-ligand interactions suppress B cell ac- than naturally occurring glycans. One strategy carbohydrate with tivation required further experimentation. An- to address these issues is to apply molecular de- improved pharmacological other complication of genetic experiments is sign principles. Although the rationale used to properties the masking of phenotypic changes because optimize a glycomimetic generally is tailored of compensation by other enzymes, which can to the specific lectin target, analysis of the suc- camouflage the function of the protein of in- cessful design efforts to date reveals some com- terest. Alternatively, a single protein may have mon strategies (105). First, either structure- multiple roles, but a null mutant lacks all of function relationship data or the structure of them. Perhaps most significantly, genetic meth- the complex is used to identify glycan func- ods were devised to examine protein function; tional groups that are critical for binding. even though they can be applied to a par- Second, nonessential polar functional groups ticular protein that binds or generates a gly- (e.g., hydroxyl and acetamido groups) are re- can, they do not report on the function of moved to increase lipophilicity. Third, confor- the glycan itself. Thus, although genetic meth- mational control elements are introduced to ods are powerful, complementary strategies are preorganize the oligosaccharide to adopt the needed. One alternative is to perturb glycan active, bound conformation. Fourth, the ob- function with compounds that disrupt or al- servation that many glycan-binding sites are ter specific protein-glycan interactions or the lined with aromatic residues can be exploited production of specific glycans. Such perturba- by introducing aromatic substituents at key po- tions can shed light on the physiological pro- sitions to enhance binding affinity. Some exam- cesses mediated by a glycan and also provide ples that illustrate successful implementation of therapeutic leads. In the following sections, we these design elements follow. outline recent advances in the use of synthetic The development of galectin inhibitors molecules to probe glycan function. highlights the value of the aforementioned strategies. Galectins are a class of glycan- binding proteins found in multicellular or- Perturbation of Protein-Glycan ganisms; humans possess 12 genes encod- Recognition with Monovalent Ligands ing galectin family members (106). Their by University of Wisconsin - Madison on 07/29/10. For personal use only. The recognition that protein-glycan complexes name comes from their propensity to bind are critical in physiological and therapeutically β-galactose-containing oligosaccharides, al- Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org important processes, which include inflamma- though individual galectins can exhibit distinct tion, immune system function, cancer, and selectivities. These proteins are involved in host-pathogen interactions, has fueled efforts a range of physiological processes, including to generate inhibitors. The aforementioned regulation of cell growth, differentiation and features of the proteins that bind glycans— apoptosis, cell adhesion, chemoattraction, and their low-affinity and solvent-exposed binding cell migration (107, 108). They also are impli- sites—render the generation of effective lig- cated in the inflammatory response and tumor ands a formidable challenge. Still, inhibitors progression. Unlike most mammalian lectins, with the requisite attributes are emerging from galectins are not membrane bound but rather an enhanced understanding of glycan-lectin are produced in the cytosol and then secreted. interactions coupled with advances in high- Consistent with their ability to occupy two dif- throughput methods for identifying them. ferent cellular locations, galectins appear to

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have important intracellular and extracellular bonds to the protein, whereas the remain- functions. Still, their functional roles have been ing hydroxyl groups do not make direct con- difficult to discern, and the relevance of glycan tact (109). These nonessential ligand hydroxyl binding is not always clear. Thus, inhibitors that groups serve as points for modification, and it could interact selectively with different mem- was postulated that aromatic substituents at the bers of the galectin family could serve as valu- 3-position would enhance binding. Inhibitors able biological probes. of this type possess dissociation constants that < Several of the galectins, including galectin- are 1000-fold more potent (Kd 50 nM) than 3, form complexes in which the 4- and 6- N-acetyllactosamine (110). They also show se- hydroxyl groups of galactose form hydrogen lectivity (∼100-fold) for galectin-3 over other galectins. A galectin-3 ligand of this type re- vealed that glycan binding by this lectin plays a HO a OH Sialyl Lewis x role in alternative macrophage activation (111). OH CH3 O Alternative macrophage activation is linked to OH NHAc HO OH HO processes ranging from asthma to wound re- HO C O O O O 2 O pair and fibrosis; therefore, these studies sug- O AcHN O OH OH gest that galectin inhibitors could have benefi- HO HO cial therapeutic effects. Many efforts to devise glycomimetic in- HO OH hibitors have focused on the C-type lectin fam- + b OH ily, whose members require Ca2 for binding. CH3 O HO OH c HO Three lectins from this group, E-, L-, and P- – O OH O2C O O selectin, have served as a major testing ground O OH CH O O OH OH 3 for glycomimetic design. The selectins have S been targets because of their participation in HO OH recruiting leukocytes to inflamed tissue and HN their putative roles in tumor cell migration. CO2H O Each selectin can bind to the structurally re- OH HO O lated tetrasaccharides sialyl Lewis x and sialyl

N Lewis a, and extensive structure-activity stud- CH3CH2O2C ies with oligosaccharide derivatives established Cl the key features that contribute to binding. For d E-selectin complexation, critical attributes in- clude the carboxylic acid group, the three hy- by University of Wisconsin - Madison on 07/29/10. For personal use only. e droxyl groups of fucose, and the 4- and 6- Cl hydroxyl groups of galactose. This motif was

Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org H N used to guide the design of a glycomimetic, N S in which the relevant groups were presented N N O F on a scaffold that is preorganized for binding H O (Figure 8b) (105). Ligands with even less re- Figure 8 semblance to the saccharide residues they were Monovalent ligands for perturbation of protein-glycan recognition. designed to mimic also were effective. For ex- Glycomimetics that present key functional groups in specific orientations have ample, peptide motifs appended to fucose bind been designed. The tetrasaccharide sialyl Lewis x (a) binds to the selectins, and to P-selectin. These also exhibit selectivity for compounds b–d have been designed to mimic critical attributes of the P- over E-selectin, which is consistent with the oligosaccharide. The naturally occurring oligosaccharide ligands are boxed, and important functional groups that have been incorporated into the glycomimetic ability of the former to recognize an epitope en- are highlighted in red. Compound e binds to another member of the C-type compassing a carbohydrate and a peptide back- (Ca2+-dependent) lectin family, DC-SIGN. Abbreviation: Ac, acetate. bone (Figure 8c) (112). As other examples of

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noncarbohydrate ligands, two small-molecule for effective inhibitors of other carbohydrate- inhibitors of P-selectin were generated from binding proteins. a quinoline salicylic acid scaffold (Figure 8d ). These compounds have progressed into clinical trials for rheumatoid arthritis and atherothrom- Perturbation of Protein-Glycan botic vascular events (113). Recognition with Multivalent Ligands To date, most glycomimetic design An alternative strategy to overcome low-affinity strategies have focused on individual protein- protein-glycan interactions is to employ multi- saccharide complexes. In contrast, pep- valent ligands. This approach can be especially tidomimetics often model structural elements effective for blocking protein-glycan engage- (e.g., a β-turn) known to be critical for protein- ment at the cell surface. Naturally occurring, protein contacts. There are common features multivalent glycan displays are widespread; of the C-type lectin complexes that might be representatives include glycosylated proteins, exploited in inhibitor design. Many C-type the glycan coats of bacteria, viruses, other lectin complexes use adjacent hydroxyl groups pathogens, and the surfaces of mammalian on fucose to coordinate the protein-bound cells. Many carbohydrate-binding proteins are calcium ion, which suggests that scaffolds that oligomeric and therefore are present in multi- possess key Ca2+-coordinating groups can be ple copies on the cell surface. In this way, both modified to enhance affinity or specificity. One cell-surface glycans and lectins are poised to such strategy has been described that employs engage in multivalent binding. focused libraries of glycomimetics using Multivalent carbohydrate derivatives can ex- shikimic acid as a building block (114). These ploit unique modes of recognition not available have yielded inhibitors of the prototypical to their monovalent counterparts. Many lectins lectin mannose-binding protein A. The identi- contain more than one saccharide-binding site fication of other approaches that can be applied or can oligomerize to form larger structures broadly to other lectin classes could accelerate with multiple binding sites. Multivalent ligands the pace of glycomimetic generation. that can span the distance between binding An alternative to the design approach is sites have an advantage over their monovalent to identify small-molecule inhibitors of lectins counterparts. This chelation mechanism is through screening. This strategy could be valu- advantageous because the translational entropy able if cell-permeable ligands could be found; cost is paid with the first receptor-ligand contact to date, however, a limited number of such (70, 117). Nevertheless, the apparent affinity compounds have been described. Studies of of a multivalent interaction often is less than the selectins have yielded some positive results might be expected, presumably because of the by University of Wisconsin - Madison on 07/29/10. For personal use only. (105), as have investigations focused on the C- conformational entropy restrictions incurred type lectin DC-SIGN. DC-SIGN facilitates by multipoint binding. Multivalent ligands also Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org several host-pathogen interactions, including can exhibit functional affinity enhancements dissemination of HIV (115). Inhibitors of by occupying secondary binding sites. Alterna- DC-SIGN were identified from a 35,000- tively, glycan-binding proteins may cluster in a compound small-molecule library using a high- membrane microdomain either in response to throughput fluorescence competition assay a multivalent ligand or in response to cellular (116). Seven compounds were identified that signals. Although multivalent ligands can be are ≥100-fold more potent than N-acetylman- potent inhibitors, their ability to cluster glycan- nosamine for DC-SIGN (Figure 8e). None binding receptors allows them also to serve as of the small molecules that bind the se- activators of signaling pathways (69). Thus, lectins or DC-SIGN resembles carbohy- depending on their binding modes, multivalent drates, an observation that provides impe- ligands can exhibit a wide range of different tus to use high-throughput screens to search activities.

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Chemical synthesis can provide architec- linker lengths (122). The activity of the mul- turally diverse multivalent ligands, including tivalent ligands against heat-labile enterotoxin low-molecular-weight displays, dendrimers, depended on the linker. The most potent ligand polymers, liposomes, and proteins (69, 118). possessed the longest linker and was 105-fold This diversity can be used to optimize a syn- more active than the corresponding monova- thetic ligand for a given application. For ex- lent galactose derivative. Dynamic light scatter- ample, unlike naturally occurring, multivalent ing experiments indicated a 1:1 protein-ligand glycan ligands, the valency of a synthetic lig- complex, suggesting the efficacy of the ligand and can be altered systematically by varying is the result of the chelate effect. In another the length or size of the scaffold. Polymers example, Bundle and coworkers (123) used glu- of defined lengths or dendrimers of different cose as a core structure to display two trisaccha- generations will possess different valencies and rides per glucose oxygen on long spacer arms therefore differing activities. Evaluating the im- (Figure 9a). This STARFISH ligand was de- pact of these changes on the biological re- signed to occupy a Shiga-like toxin through sponse can illuminate the mechanisms under- both the primary binding site and a subsite. It

lying the function of natural protein-glycan was a highly effective inhibitor (IC50 0.24 nM). interactions and lead to highly efficacious Unexpectedly, however, X-ray crystallographic inhibitors. analysis revealed that the designed multivalent Potent multivalent inhibitors for several ligand did not bind a single pentamer but rather medically relevant protein-glycan interactions it dimerized two copies by occupying all five

have been identified. One target that has been B subunits. Thus, for both AB5 toxin ligands, explored is influenza virus hemagglutinin, and the spacing between the binding elements was a number of multivalent sialic acid derivatives critical for activity, even though their modes of have been generated that block the interac- multivalent binding differ. tion of the virus with cells (119). Other host- Most applications of multivalency in gly- pathogen interactions also can be inhibited cobiology involve the use of multivalent lig- with multivalent ligands, and representative ex- ands as inhibitors, but multivalency also can amples include compounds that prevent Pseu- be used to activate particular cellular processes. domonas aeruginosa adhesion or the binding of An example involves blocking the action of uropathogenic Escherichia coli (120). Although L-selectin, which mediates leukocyte migra- the influence of scaffold structure is just begin- tion and recruitment from the blood to lym- ning to be explored (121), the inhibitors of the phatic tissues and sites of inflammation (124).

AB5 bacterial toxin family highlight the benefits The natural ligands for L-selectin are mucins of multivalent ligand design. This toxin family that present a multivalent display of sialyl by University of Wisconsin - Madison on 07/29/10. For personal use only. is characterized by one active component (A) Lewis x derivatives. Experiments using mucin and a pentamer of subunits (B) that bind car- mimics highlight the importance of multiva- Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org bohydrates displayed on cellular surfaces. The lency for L-selectin recognition (Figure 9b).

AB5 toxins are responsible for diseases rang- In this study, synthetic multivalent ligands were ing from travelers’ diarrhea (heat-labile en- generated using the ring-opening metathesis terotoxin), to acute kidney failure in children polymerization (ROMP). This polymerization (Shiga-like toxins), to fatal cholera (Cholera is especially valuable for multivalent ligand syn- toxin). Efforts from several groups have high- thesis because the length, and therefore valency lighted the importance of distance between of the ligands, can be controlled. Interestingly, binding motifs (118), a parameter that appears the ROMP-derived polymers not only bind to be at least as important as the identity of L-selectin, but also promote its proteolytic re- the ligands themselves. For example, a penta- lease, or shedding, from the cell surface (125). cyclen core was used to display five galactose These results suggest that clustering L-selectin residues that were tethered using a range of may signal for its cleavage.

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Multivalent displays of oligosaccharides also A new but related aspect of multivalent are being tested as vaccines against bacteria, protein-glycan interactions involves exploiting parasites, and cancers (7, 9, 126, 127). Although noncovalent interactions to create functional oligosaccharides typically do not elicit robust supramolecular protein-glycan assemblies. Two immune responses, they are effective when ap- different general strategies have emerged; both pended to an immunogenic carrier protein. To boost immunity further, novel multivalent con- a jugates are being developed. An innovative ex- HO OH HO OH ample is a conjugate that simultaneously dis- O O plays multiple groups: a B cell epitope, a T HO HO HO HO O O helper epitope, and a toll receptor ligand (128). OH OH OH OH O OCH O These groups can synergistically augment im- HO 3 HO OCH3 HO O O HO O O mune responses by recruiting different aspects O O O OH O OH of the innate and adaptive immune responses. Multivalent ligands also can be used to O N N O H H recruit lectins to signaling complexes. Such O assemblies are useful in controlling cellular re- O NH R sponses because some glycan-binding proteins O (CH2)8 R enhance signaling and others diminish it. Al- O O though vaccine designs are necessarily focused HN O O O R O R on augmenting the immune response, com- H R R = N pounds that suppress autoimmune responses S N O STARFISH H also are needed. The aforementioned CD22, O which dampens immune activation, is a sialic acid–binding lectin from the Siglec family. A b Ph R' CD22 ligand [i.e., N-acetylneuraminic acid- n α(2,6)-galactose-β(1,4)-glucose] was attached O to a multivalent antigen, such that the result- ROMP HN ing polymer engaged both CD22 and the B cell R antigen receptor (Figure 9b) (129). This sialy- lated antigen inhibited B cell activation. These L-selectin ligand

results identify a mechanism by which antigen R = HO R' = H glycosylation can suppress immune activation. OH OH Moreover, they highlight how multivalent lig- CH3 O by University of Wisconsin - Madison on 07/29/10. For personal use only. HO OH OH HO OH – O ands can be used to perturb the assembly of O2C O O O O glycan-binding proteins on the cell surface. O Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org AcHN O OH OH HO HO −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→

Figure 9 CD22/B cell antigen receptor ligand O Selected examples of multivalent ligands that can be OH R = HO – R' = used to perturb protein-glycan recognition. (a) The O2C OH pentameric STARFISH core has been used to devise OH 6 AcHN O O O potent inhibitors of the AB5 toxin family. O HO HO HO OHO O (b) Polymers are useful scaffolds for producing OH OH multivalent ligands. The structures depicted were and NO generated using the ring-opening metathesis 2 H polymerization (ROMP), which differs from most N polymerization reactions in that it can afford defined CO2H ligands. Abbreviation: Ac, acetate. O2N

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A second approach is to employ compounds that are not polymeric yet possess two dif- ferent epitopes (54, 131). A bifunctional lig- and was used to selectively kill tumor cells via recruitment of a glycan-binding antibody, which subsequently led to complement activa- tion. The bifunctional ligand was designed such Target that, upon binding to a cell-surface receptor as- sociated with cancer, it could present the anti- genic epitope galactosyl-α(1,3)-galactose (α- Ligand Template Gal), which would allow cells to be recognized as foreign by the naturally occurring anti-α-Gal antibody (54). The α-Gal epitope is not found in humans (132), but human serum contains a significant level of anti-α-Gal antibody (about 3% of circulating antibody). Cells displaying α-Gal epitopes are subject to complement- mediated cell killing. Importantly, the recruit- ment of anti-α-Gal to the cell surface requires a multivalent presentation of glycan residues. The final conjugate consists of an α-Gal epi-

tope linked to an integrin αvβ3-binding ligand, as this integrin is upregulated on tumor cells. When cells are treated with the bifunctional lig- and, complement-mediated lysis occurs. Cells Supramolecular with low levels of the integrin receptor were complex spared, whereas tumor cells displaying high lev- Figure 10 els of integrin were killed selectively (54), and Polymeric ligands possessing a preorganized structure can form supramolecular this selectivity underscores the advantage of us- complexes that display increased functional binding affinity or increased serum half-life. ing multivalent interactions for cell targeting. Thus, ligands that promote macromolecular as- rely on bifunctional ligands. In the first, poly- semblies of carbohydrate binding-proteins can meric ligands that display multiple copies of co-opt the function of glycans and their recep- two distinct recognition elements, such as the tors for new purposes. by University of Wisconsin - Madison on 07/29/10. For personal use only. sialylated antigens described above, promote macromolecular protein assemblies. Multiva- Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org lent assemblies of this type were used to ad- Perturbation of Glycan Assembly dress the disappointing in vivo activity of the An emerging strategy to understand glycan aforementioned pentavalent inhibitors of Shiga function is to block selected steps in glycocon- toxin. Polymeric ligands that possess a preor- jugate assembly or disassembly. Tothis end, ef- ganized architecture promote the formation of forts have been launched to develop inhibitors complexes of Shiga toxin 1 and an endogenous of specific glycosyltransferases, glycosidases, circulating pentavalent receptor human serum and other sugar-processing enzymes. Inhibitors amyloid P (Figure 10) (130). When tested in can illuminate both the biological roles of gly- mice, a stable macromolecular complex was cans and the mechanisms behind their turnover generated with a prolonged half-life in circula- and assembly. Indeed, compounds that selec- tion. Most importantly, the animals were pro- tively target major pathways (including the pro- tected from the toxin. duction of N-glycoproteins and O-glycosylated

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mucins, as well as the attachment of O-GlcNAc a OH residues) could be used to probe the roles of HO O O different glycan classes. Moreover, inhibitors HO AcHN OH NH of the production of specific glycans in tu- H N O O N mors or pathogens could serve as therapeutic O (CH ) HO O leads. 2 9 OH O Nature has provided some design strate- HO OH gies for generating compounds that inhibit N- Tunicamycin glycosylation in the form of natural products. For example, tunicamycin (Figure 11a) blocks b c HO a crucial transphosphorylation reaction be- OH N tween UDP-GlcNAc and dolichol-phosphate HO NH HO that generates dolichol-PP-GlcNAc. This step HO H HO OH is required for the synthesis of N-glycoproteins, OH Deoxynojirimycin Castanospermine and tunicamycin has therefore been used to illu- minate the consequences of deficiencies in N- d e glycan production (133). Another strategy to OH inhibit N-glycosylation is to block the activity HO O CO2H of the oligosaccharyltransferase complex, and O CO2CH2CH3 AcHN O this mode of inhibition is exhibited by a cyclic HO AcHN HN + H3N peptide that adopts the conformation of the – NH H2PO4 peptide acceptor (134). Cell-permeable com- H2N pounds that block N-glycosylation selectively Zanamivir (Relenza®) Oseltamivir (Tamiflu®) in either bacteria or eukaryotes would be useful Figure 11 probes (135). Natural products (compounds 11a–11c) have inspired the generation of drugs Inhibitors of glycosyltransferases involved (compounds d and e) that act as transition-state analogs and thereby inhibit in O-glycan biosynthesis also have been sought influenza virus neuraminidase. Inhibitors of this type can be used to perturb (136). Given that O-glycans are involved in glycan assembly. processes from pathogen binding to cancer, inhibitors will be useful for investigating the been employed to identify compounds that tar- physiological and pathophysiological roles of get O-GlcNAc transferase (OGT) (Figure 12). this important class of glycans. For these tar- OGT is an essential enzyme that regulates sig- gets, there are no obvious starting points be- naling through its ability to mediate intracel- cause natural product inhibitors have not been lular O-glycosylation. Compounds that block by University of Wisconsin - Madison on 07/29/10. For personal use only. identified, and the peptide sequence require- this enzyme can serve as valuable probes that ments for glycosylation have not been fully de- complement those known for the correspond- Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org lineated. Thus, high-throughput screens were ing O-GlcNAc residue hydrolyzing glycosi- employed. One innovative assay exploits the ob- dase. These studies illustrate the value of high- servation that the rate of proteolysis of a glyco- throughput screens to identify inhibitors, and sylated peptide is diminished relative to that of a number of strategies have been implemented its unmodified counterpart (136). By append- including those that rely on glycan arrays (72, ing N- and C-terminal fluorophores that can 137), activity assays (138), and binding assays engage in Forster¨ resonance energy transfer, (139). a peptide’s susceptibility to proteolytic cleav- The enzymes responsible for glycan assem- age can be assessed. When a glycosyltransferase bly or modification can be critical for microbes UDP-GlcNAc: is present, cleavage is decreased, whereas the and therefore represent attractive targets. One uridine 5-diphosphate- presence of a glycosyltransferase inhibitor re- such target is the influenza viral coat protein N-acetylglucosamine stores rapid proteolysis. This assay format has neuraminidase, whose function is to catalyze

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O O wall biosynthesis: UDP-galactopyranose mu- tase (UGM), which is found in mycobacteria and catalyzes the interconversion of the iso- NH F 2 meric compounds UDP-galactopyranose and UDP-galactofuranose, and the glycosyltrans- ferase MurG, which is a bacterial glucosamino- S transferase involved in the biosynthesis of

HO2C O the crucial bacterial cell wall component peptidoglycan. NH In the case of MurG, a fluorescent probe S was designed based on the structure of the Figure 12 donor, UDP-GlcNAc, complexed to the en- Compounds identified in a high-throughput screen zyme (Figure 13) (139). A library of 64,000 as inhibitors of the essential glycosyltransferase compounds was screened, and subsequent O-GlcNAc transferase (OGT). analysis revealed many of the most potent binders possessed a common scaffold: a 1,3- the cleavage of sialic acids on the host cell disubstituted heterocyclic core. Because gly- surface to enable viral infection. The design of cosaminotransferases are present in all organ- these compounds was inspired by natural prod- isms, probes or lead compounds must bind the uct glycosidase inhibitors, such as deoxyno- bacterial enzyme selectively. Importantly, the jirimycin (Figure 11b) and castanospermine inhibitors are selective over other nucleotide- (Figure 11c). These compounds are amines sugar-processing enzymes (143). that are protonated at neutral pH, and they In another example, a UDP-fluorescein are thought to mimic an oxocarbenium tran- probe was used in a 16,000-compound, high- sition state (140). Design strategies inspired throughput inhibitor screen of the UGM from by transition state models have led to valuable Mycobacterium tuberculosis (144). The active drugs; these include zanamivir (marketed as compounds identified share structural features Relenza®)(Figure 11d ) and oseltamivir (mar- with those found to block MurG, including keted as Tamiflu®)(Figure 11e) for the treat- a five-membered thiazolidinone heterocycle, ment of influenza (141). These compounds, substituents at the 1- and 3-positions, and at which block viral replication and infection of least one aromatic group that might mimic the new host cells, underscore the value of target- uracil (Figure 13). This class of thiazolidinone ing enzymes that operate on glycans. More- derivatives is subject to reaction with nucle- over, this strategy has been applied to afford ophiles in a biological milieu (144), which lim- by University of Wisconsin - Madison on 07/29/10. For personal use only. inhibitors of other glycosyltransferases or gly- its their utility as biological probes or thera- cosidases (142). peutic leads. Still, their identification in two Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org Carbohydrate-modifying enzymes critical independent screens suggests that the shape for cell wall biosynthesis in microbes also of these compounds is well suited for target- have been the objects of inhibition studies. ing nucleotide-sugar-utilizing enzymes. Fur- Several investigations in this area have em- ther design and optimization based on this ployed ligand displacement assays that rely hypothesis resulted in the identification of upon fluorescence polarization (FP). FP as- compounds that block mycobacterial growth says serve as a general method to study en- (Figure 13) (145). These studies underscore zymes that use nucleotide sugar substrates, that high-throughput screens can be used as which are prevalent yet poorly understood. FP tools to aid in identifying new therapeutic tar- assays have been used to identify inhibitors gets and, more generally, as probes of glycan of two different enzymes essential for cell biosynthesis.

642 Kiessling · Splain ANRV413-BI79-22 ARI 27 April 2010 21:25

Enzyme Nucleotide-sugar FP probe Inhibitor target substrate O

O OH OH N O HO O MurG HO HO O N HO HO AcHN –OOC HN S O UDP O UDP S NH O O

CO2H O O N S S HO OH S Cl NH(CH2)6 O UDP O CO H UGM HO O NH 2 AcHN S O UDP COO– HN N Cl HO I Cl

Figure 13 Fluorescence polarization (FP) has been used to identify inhibitors for sugar-processing enzymes with essential roles in bacterial cell wall biosynthesis. These enzymes include the glycosyltransferase MurG and the isomerase UDP-galactopyranose mutase (UGM).

Exploiting Alternative Substrates bear sialic acid residues with N-propionamide in Glycan Biosynthesis groups (147). This general strategy has subse- quently been exploited for diverse applications. Modified glycans can be used to investigate gly- The incorporation of a modified building can recognition, biosynthesis, cellular or or- block can interfere with subsequent glycosyla- ganismal localization, and turnover. For exam- tion reactions, thereby blocking the production ple, primer saccharides can be introduced that of specific glycan structures. In this way, glycan compete with endogenous substrates for the functional roles can be interrogated. For glycosyltransferases (146). These compounds example, when cells are treated with N- serve as decoys by preventing the produc- butanoylmannosamine, they display truncated by University of Wisconsin - Madison on 07/29/10. For personal use only. tion of physiological glycoconjugates. This ap- polysialic acid chains and provide a means to proach can block the generation of specific cell- assess the influence of the length of polysialic Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org surface glycoconjugates, but the consequences acid on neuronal plasticity (148). Another of harboring the resulting glycan chains within example of altering glycan production employs cells have not been elucidated fully. Glycan 2-deoxygalactose, which precludes the gener- biosynthetic pathways also can be co-opted to ation of the fucosyl-α(1,2)-galactose epitope. incorporate monosaccharide analogs, thereby Investigations using this deoxysugar revealed generating modified glycoconjugates in cells that fucosylation prevents the proteolytic or organisms. The feasibility of this strat- degradation of synapsin, a critical regulator of egy was demonstrated by exposing cells to neuronal function (149). The incorporation N-propanoylmannosamine, an analog of N- of modified saccharide residues also has been acetylmannosamine, a key intermediate in the used to investigate the requirements for glycan biosynthesis of sialylated glycans. With this recognition. For example, the use of metabolic treatment, the cell-surface glycans generated engineering to generate N-glycosyl-substituted

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sialylglycoconjugates disrupts the interaction glycans causes self-assembly into large clus- of neural glycoproteins with myelin-associated tered cell aggregates, which may prove valuable glycoprotein (150). N-Acetylmannosamine for tissue engineering purposes (157). derivatives also have been used to decorate tumor cell surfaces with immunogenic glycans (151). Thus, altering glycan biosynthetic path- Illuminating Glycan Biosynthesis ways can provide insight into the functional Our understanding of glycans would be roles of specific carbohydrate epitopes. advanced by strategies to visualize and track Oligosaccharide biosynthesis also can their cellular and organismal distribution in be co-opted to introduce unique functional development or disease. The biosynthesis of groups into cellular glycans. The Bertozzi glycans containing specific residues can be group (152) has investigated different aspects visualized by the incorporation of functional of glycobiology using metabolic labeling com- groups that can be used to append reporter bined with chemoselective functionalization groups. Both the Staudinger ligation and the reactions. In one example, they showed that Huisgen 1,3-dipolar cycloaddition reaction of N-azidoacetylglucosamine (GlcNAz) could azides with alkynes (often referred to as click be incorporated at sites subject to O-GlcNAc chemistry) have been employed in biological modification. Azides are valuable handles be- environments (Figure 7c) (51). A major issue cause they are absent from biological systems in these transformations is the rate of reaction, and are relatively unreactive toward most bio- which determines the labeling time and sensi- logical function groups. As stated above, they tivity. Attempts to increase reaction efficiency can undergo transformations in the presence have focused on the azide-alkyne 1,3-dipolar of other nonphysiological function groups. cycloaddition. Although this reaction can be Specifically, an azide-bearing compound can catalyzed by copper(I), these conditions are be coupled using a Staudinger ligation reaction deleterious to cells. Using a more reactive, with a phosphinoester bearing a reporter strained difluorinated cyclooctyne partner (e.g., biotin) to generate a conjugate. This circumvents the requirement for copper catal- reaction scheme was utilized to profile proteins ysis (Figure 14a). A variety of sugar building modified with O-GlcNAc. Cells were treated blocks bearing azido groups can be incorpo- with GlcNAz to afford azide-substituted rated, including derivatives corresponding to glycoproteins that were subsequently modified sialic acid, N-acetylgalactosamine (GalNAc), for isolation and characterization (153, 154). GlcNAc, and fucose (152). The azide moiety A similar strategy has been used with N- can then undergo reaction with an alkyne bear- azidoacetylgalactosamine (GalNAz) to detect ing a fluorophore or other reporter group. The by University of Wisconsin - Madison on 07/29/10. For personal use only. mucins. Metabolic labeling also can be used advantages of this labeling strategy are illus- to introduce photoaffinity labels, such that trated by its use in visualizing glycans in whole Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org cross-linking to glycan-binding proteins can animals. Specifically, zebrafish embryos were be carried out. For example, aryl-azide (155) incubated with GalNAz to generate glycans or diazirine (156) moieties can be incorporated that bear azide groups. The GalNAz residues into sialic acid-bearing glycans at the C9 or could be visualized by their ability to undergo C5 positions of sialic acid, respectively. When a cycloaddition reaction with fluorophore- these photoactivable groups are displayed on labeled cyclooctynes. By using two different cell-surface glycans, they can be used to identify fluorescent tags, changes in O-glycosylation glycan-binding proteins by covalent trapping. during the course of zebrafish development The ability to modify glycans by metabolic could be observed. An alternative approach for incorporation also can be used to introduce glycan tagging in cells takes advantage of mild groups to alter cell adhesion. The display of oxidation, which occurs at endogenous sialic N-thioglycolylneuraminic acid in cell-surface acid residues, to afford an aldehyde at the C7

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a

HO OH N3 O HO Reporter F NH OH F O N3 N N F N F N3

Cell

N3 Glycosyltransferase

b

OH O OH O N AcHN AcHN AcHN HO HO HO O HO O O CO H CO2H CO2H

by University of Wisconsin - Madison on 07/29/10. For personal use only. 2

O Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org NaIO4 NH2

Figure 14 Strategies for visualizing glycans can be achieved by (a) metabolic incorporation of a sugar bearing a latent reactive group (e.g., azide) or (b) chemical modification of endogenous glycans to introduce a chemical handle that can be functionalized with fluorophores or other reporter molecules. Abbreviation: Ac, acetate.

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position (158). Aniline-catalyzed oxime ligation carbohydrates, stated in his Nobel Lecture with an aminooxy-modified molecular tag pro- (159), “... the chemical enigma of Life will vides a chemical handle that can be used for vi- not be solved until organic chemistry has mas- sualization or tracking of glycans (Figure 14b). tered another, even more difficult subject, the This method is complementary to metabolic proteins, in the same way as it has mas- labeling; not all systems will tolerate the mild tered the carbohydrates.” Though one can oxidation step, but the labeling can be done on a only admire Fischer, today his statement ap- faster timescale. Together, these results empha- pears ironic, especially given the formidable size the advances that have been made in strate- challenges of elucidating glycan function that gies to observe glycan production and turnover. remain. These challenges are being met by an assemblage of interdisciplinary approaches. Research in chemical glycobiology is driv- CONCLUSION ing discovery by offering new approaches and Emil Fischer, a pioneer in recognizing the tools to explore and exploit the functions of critical role of chemistry in understanding glycans.

SUMMARY POINTS 1. Glycans are involved in myriad specific molecular and cellular events from development to disease. 2. The field of glycobiology demands the development of new approaches to elucidate glycan function. Chemical biology approaches provide critical tools and strategies that can be used to probe or perturb glycan function. 3. The synthesis of glycans and the development of chemoselective reactions have greatly expanded the repertoire of oligosaccharides and glycoconjugates available. 4. Glycan and lectin arrays have emerged as valuable platforms to interrogate protein-glycan interactions. 5. The biosynthesis of glycans can be exploited to introduce new functionality that can be used for analysis or imaging. 6. Inhibitors, both rationally designed glycomimetics and small molecules, are promising new tools with which to investigate and interfere with the biological roles of glycans. 7. Synthetic multivalent ligands are valuable tools for investigating the low-affinity inter- by University of Wisconsin - Madison on 07/29/10. For personal use only. actions characteristic of glycan-binding events. Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org

FUTURE ISSUES 1. The continued development of new strategies and approaches in both chemical and chemoenzymatic syntheses will provide increased access to complex glycans. Methods are needed to assemble less explored glycans, including sulfated oligosaccharides and the unique glycans found in microbes and other pathogens. 2. Glycans lack functional group handles that can be used to install fluorophores or other reporters to directly assess binding or enzyme-catalyzed modification reactions. Thus, new strategies for devising high throughput are needed to identify and engineer enzymes for the synthesis of glycans and to promote the discovery of probes of glycan function.

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3. Methods for monitoring glycan degradation are needed, as this process is critical for human health and has ramifications for harvesting energy from plant cell walls. 4. Although great strides have been made in the development of small-molecule ligands for glycan-binding proteins, inhibitors exist for only a handful of lectins. Selective inhibitors for a wide range of lectins are needed. 5. Small molecules that interfere with glycan processing are valuable, as the drugs that block influenza virus neuraminidase illustrate. Recent studies indicate that small-molecule in- hibitors of key enzymes in glycan biosynthesis can be found, but more probes could expedite the elucidation of glycan function. 6. Great strides are being made in devising glycoconjugates that can elicit or inhibit immune responses. Promising glycolipids and synthetic glycoconjugates are being designed as vaccines, adjuvants, and immunomodulators. 7. With an increased understanding of how glycans function, chemistry can provide new tools to co-opt these functions for new purposes. From the metabolic incorporation of unique functional groups to the use of ligands that recruit anticarbohydrate antibodies to kill tumor cells, glycan function can be used to elicit new and valuable biological responses in cells and organisms.

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We apologize to authors whose contributions were omitted from this review owing to limitations of space and limitations in the number of references. This research was supported by the National Institutes of Health (GM49974, GM55984, AI063596, and AI055258) to L.L.K. R.A.S. acknowl- edges the American Chemical Society Division of Medicinal Chemistry for a fellowship. We thank C.D. Brown, S.L. Mangold, L. Li, and M.R. Levengood for their comments on the manuscript. by University of Wisconsin - Madison on 07/29/10. For personal use only. LITERATURE CITED

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138. Rose NL, Zheng RB, Pearcey J, Zhou R, Completo GC, Lowary TL. 2008. Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase. Carbohydr. Res. 343:2130–39 139. Helm JS, Hu Y, Chen L, Gross B, Walker S. 2003. Identification of active-site inhibitors of MurG using a generalizable, high-throughput glycosyltransferase screen. J. Am. Chem. Soc. 125:11168–69 140. Lillelund VH, Jensen HH, Liang X, Bols M. 2002. Recent developments of transition-state analogue glycosidase inhibitors of non-natural product origin. Chem. Rev. 102:515–53 141. Asano N. 2003. Glycosidase inhibitors: update and perspectives on practical use. Glycobiology 13:R93–104 142. Compain P, Martin OR. 2003. Design, synthesis and biological evaluation of iminosugar-based glyco- syltransferase inhibitors. Curr. Top. Med. Chem. 3:541–60 143. Hu Y, Helm JS, Chen L, Ginsberg C, Gross B, et al. 2004. Identification of selective inhibitors for the glycosyltransferase MurG via high-throughput screening. Chem. Biol. 11:703–11 144. Carlson EE, May JF, Kiessling LL. 2006. Chemical probes of UDP-galactopyranose mutase. Chem. Biol. 13:825–37 145. Dykhuizen EC, May JF, TongpenyaiA, Kiessling LL. 2008. Inhibitors of UDP-galactopyranose mutase thwart mycobacterial growth. J. Am. Chem. Soc. 130:6706–7 146. Brown JR, Crawford BE, Esko JD. 2007. Glycan antagonists and inhibitors: a fount for drug discovery. Crit. Rev. Biochem. Mol. Biol. 42:481–515 147. Kayser H, Zeitler R, Kannicht C, Grunow D, Nuck R, Reutter W. 1992. Biosynthesis of a nonphysio- logical sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors. J. Biol. Chem. 267:16934–38 148. Mahal LK, Charter NW, Angata K, Fukuda M, Koshland DE, Bertozzi CR. 2001. A small-molecule modulator of poly-α2,8-sialic acid expression on cultured neurons and tumor cells. Science 294:380–82 149. Murrey H, Gama C, Kalovidouris S, Luo W, Driggers E, et al. 2006. Protein fucosylation regulates synapsin Ia/Ib expression and neuronal morphology in primary hippocampal neurons. Proc. Natl. Acad. Sci. USA 103:21–26 150. Collins B, Yang L, Mukhopadhyay G, Filbin M, Kiso M, et al. 1997. Sialic acid specificity of myelin- associated glycoprotein binding. J. Biol. Chem. 272:1248–55 151. Liu T, Guo Z, Yang Q, Sad S, Jennings H. 2000. Biochemical engineering of surface α2–8 polysialic acid for immunotargeting tumor cells. J. Biol. Chem. 275:32832–36 152. Agard NJ, Bertozzi CR. 2009. Chemical approaches to perturb, profile, and perceive glycans. Acc. Chem. Res. 42:788–97 153. Vocadlo DJ, Hang HC, Kim E-J, Hanover JA, Bertozzi CR. 2003. A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc. Natl. Acad. Sci. USA 100:9116–21 154. Hang H, Yu C, Kato D, Bertozzi C. 2003. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation. Proc. Natl. Acad. Sci. USA 100:14846–51 155. Han S, Collins BE, Bengtson P, Paulson JC. 2005. Homomultimeric complexes of CD22 in B cells by University of Wisconsin - Madison on 07/29/10. For personal use only. revealed by protein-glycan cross-linking. Nat. Chem. Biol. 1:93–97 156. Tanaka Y, Kohler JJ. 2008. Photoactivatable crosslinking sugars for capturing glycoprotein interactions. Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org J. Am. Chem. Soc. 130:3278–79 157. Sampathkumar S-G, Li AV, Jones MB, Sun Z, Yarema KJ. 2006. Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nat. Chem. Biol. 2:149–52 158. Zeng Y, Ramya TNC, Dirksen A, Dawson PE, Paulson JC. 2009. High-efficiency labeling of sialylated glycoproteins on living cells. Nat. Methods 6:207–9 159. Fisher E. 1902. Syntheses in the purine and sugar group. Nobel lect. http://nobelprize.org/nobel prizes/chemistry/laureates/1902/fischer-lecture.pdf

www.annualreviews.org • Chemical Approaches to Glycobiology 653 AR413-FM ARI 28 April 2010 16:43

Annual Review of Biochemistry

Contents Volume 79, 2010

Preface

The Power of One James E. Rothman ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppv

Prefatory Article Frontispiece Aaron Klug pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppxiv From Virus Structure to Chromatin: X-ray Diffraction to Three-Dimensional Electron Microscopy Aaron Klug ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1

Recent Advances in Biochemistry Genomic Screening with RNAi: Results and Challenges Stephanie Mohr, Chris Bakal, and Norbert Perrimon pppppppppppppppppppppppppppppppppppppp37 Nanomaterials Based on DNA Nadrian C. Seeman pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp65 Eukaryotic Chromosome DNA Replication: Where, When, and How? Hisao Masai, Seiji Matsumoto, Zhiying You, Naoko Yoshizawa-Sugata, by University of Wisconsin - Madison on 07/29/10. For personal use only. and Masako Oda pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp89

Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org Regulators of the Cohesin Network Bo Xiong and Jennifer L. Gerton pppppppppppppppppppppppppppppppppppppppppppppppppppppppppp131 Reversal of Histone Methylation: Biochemical and Molecular Mechanisms of Histone Demethylases Nima Mosammaparast and Yang Shi ppppppppppppppppppppppppppppppppppppppppppppppppppppp155 The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway Michael R. Lieber ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp181 The Discovery of Zinc Fingers and Their Applications in Gene Regulation and Genome Manipulation Aaron Klug pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp213

vii AR413-FM ARI 28 April 2010 16:43

Origins of Specificity in Protein-DNA Recognition Remo Rohs, Xiangshu Jin, Sean M. West, Rohit Joshi, Barry Honig, and Richard S. Mann pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp233 Transcript Elongation by RNA Polymerase II Luke A. Selth, Stefan Sigurdsson, and Jesper Q. Svejstrup pppppppppppppppppppppppppppppp271 Biochemical Principles of Small RNA Pathways Qinghua Liu and Zain Paroo pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp295 Functions and Regulation of RNA Editing by ADAR Deaminases Kazuko Nishikura pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp321 Regulation of mRNA Translation and Stability by microRNAs Marc Robert Fabian, Nahum Sonenberg, and Witold Filipowicz pppppppppppppppppppppppp351 Structure and Dynamics of a Processive Brownian Motor: The Translating Ribosome Joachim Frank and Ruben L. Gonzalez, Jr. pppppppppppppppppppppppppppppppppppppppppppppp381 Adding New Chemistries to the Genetic Code Chang C. Liu and Peter G. Schultz ppppppppppppppppppppppppppppppppppppppppppppppppppppppp413 Bacterial Nitric Oxide Synthases Brian R. Crane, Jawahar Sudhamsu, and Bhumit A. Patel ppppppppppppppppppppppppppppp445 Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective Olga Khersonsky and Dan S. Tawfik pppppppppppppppppppppppppppppppppppppppppppppppppppppp471 Hydrogenases from Methanogenic Archaea, Nickel, a Novel Cofactor, and H2 Storage Rudolf K. Thauer, Anne-Kristin Kaster, Meike Goenrich, Michael Schick, Takeshi Hiromoto, and Seigo Shima ppppppppppppppppppppppppppppppppppppppppppppppppppppp507 Copper Metallochaperones pppppppppppppppppppppppppppppppppppppppppppppppppp537 by University of Wisconsin - Madison on 07/29/10. For personal use only. Nigel J. Robinson and Dennis R. Winge High-Throughput Metabolic Engineering: Advances in Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org Small-Molecule Screening and Selection Jeffrey A. Dietrich, Adrienne E. McKee, and Jay D. Keasling pppppppppppppppppppppppppp563 Botulinum Neurotoxin: A Marvel of Protein Design Mauricio Montal pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp591 Chemical Approaches to Glycobiology Laura L. Kiessling and Rebecca A. Splain ppppppppppppppppppppppppppppppppppppppppppppppppp619 Cellulosomes: Highly Efficient Nanomachines Designed to Deconstruct Plant Cell Wall Complex Carbohydrates Carlos M.G.A. Fontes and Harry J. Gilbert pppppppppppppppppppppppppppppppppppppppppppppp655

viii Contents AR413-FM ARI 28 April 2010 16:43

Somatic Mitochondrial DNA Mutations in Mammalian Aging Nils-G¨oran Larsson ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp683 Physical Mechanisms of Signal Integration by WASP Family Proteins Shae B. Padrick and Michael K. Rosen pppppppppppppppppppppppppppppppppppppppppppppppppppp707 Amphipols, Nanodiscs, and Fluorinated Surfactants: Three Nonconventional Approaches to Studying Membrane Proteins in Aqueous Solutions Jean-Luc Popot pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp737 Protein Sorting Receptors in the Early Secretory Pathway Julia Dancourt and Charles Barlowe pppppppppppppppppppppppppppppppppppppppppppppppppppppp777 Virus Entry by Endocytosis Jason Mercer, Mario Schelhaas, and Ari Helenius pppppppppppppppppppppppppppppppppppppppp803

Indexes Cumulative Index of Contributing Authors, Volumes 75–79 ppppppppppppppppppppppppppp835 Cumulative Index of Chapter Titles, Volumes 75–79 pppppppppppppppppppppppppppppppppppp839

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found at http://biochem.annualreviews.org by University of Wisconsin - Madison on 07/29/10. For personal use only. Annu. Rev. Biochem. 2010.79:619-653. Downloaded from arjournals.annualreviews.org

Contents ix