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Chemical Science

PERSPECTIVE

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Realizing the prom

LfOAdUIjooaww

inducted to the prestigious JohnsScholars in 2009. Dr Wang's reseabioorganic chemistry and glycobioglycoprotein synthesis, antibody glydesign.

aInstitute of Human Virology and Departme

University of Maryland School of Medicine,

21201, USA. E-mail: [email protected] of Chemistry, Chemistry Resea

Manseld Road, Oxford OX1 3TA, UK. E-ma

Cite this: Chem. Sci., 2013, 4, 3381

Received 1st April 2013Accepted 16th May 2013

DOI: 10.1039/c3sc50877c

www.rsc.org/chemicalscience

This journal is ª The Royal Society of

ise of chemical glycobiology

Lai-Xi Wang*a and Benjamin G. Davis*b

Chemical glycobiology is emerging as one of the most uniquely powerful sub-disciplines of chemical

biology. The previous scarcity of chemical strategies and the unparalleled structural diversity have

created a uniquely fertile ground that is both rich in challenges and potentially very profound

in implications. Glycans (oligosaccharides, polysaccharides, and glycoconjugates) are everywhere in

biological systems and yet remain disproportionately neglected—reviews highlighting this ‘Cinderella

status’ abound. Yet, the past two decades have witnessed tremendous progress, notably in chemical and

chemoenzymatic synthesis, ‘sequencing’ and arrays, metabolic engineering and imaging. These vital

steps serve to highlight not only the great potential but just how much more remains to be done. The

vast chemical and functional space of glycans remains to be truly explored. Top-down full-scale glycomic

and glycoproteomic studies coupled with hypothesis-driven, bottom-up innovative chemical strategies

will be required to properly realize the potential impact of glycoscience on human health, energy, and

economy. In this perspective, we cherry-pick far-sighted advances and use these to identify possible

challenges, opportunities and avenues in chemical glycobiology.

Introduction

25 years on from its rst concise denition,1 the eld of glyco-biology has continued to successfully highlight the myriad of

ai-Xi Wang received his PhDrom the Shanghai Institute ofrganic Chemistry, Chinesecademy of Sciences. Aer post-octoral studies at Johns Hopkinsniversity and Massachusettsnstitute of Technology (MIT), heoined the faculty of the Institutef Human Virology, Universityf Maryland as a tenure-trackssistant professor in 2000 andas promoted to full professorith tenure in 2009. He wasHopkins University Society of

rch interests are in the areas oflogy, including chemoenzymaticcoengineering, and HIV vaccine

nt of Biochemistry & Molecular Biology,

725 W. Lombard Street, Baltimore, MD

d.edu

rch Laboratory, University of Oxford, 12

il: [email protected]

Chemistry 2013

complex processes in which carbohydrates play a vital role. Itbecomes clear that glycans, in the form of oligosaccharides,polysaccharides, glycoproteins, glycolipids, proteoglycans andother glycoconjugates, can be key players in a number ofimportant biological recognition processes: intracellular traf-cking, cell adhesion, development, cancer progression, host–pathogen interaction, and immune response, to name a few.2–6

They may now be reasonably added to the ‘central dogmamolecules’ (nucleic acids, proteins) as playing a distinct role in

Benjamin Davis is a professor atthe University of Oxford. Hisresearch centres on chemicalbiology with an emphasis oncarbohydrates and proteins. Hisgroup's work has been recog-nized by several awardsincluding the RSC's Meldola(1999), Corday-Morgan (2005),Norman Heatley (2009) andBioorganic (2012) Medals; theWain Medal (2008); the ACSIsbell Award (2008); the Royal

Society Mullard (2005) and Wolfson Research Merit (2009) Awardsand the Tetrahedron Young Investigator Award (2012). He isEditor-in-Chief of Current Opinion in Chemical Biology and anAssociate Editor of Chemical Science. He is the Secretary of theEuropean Carbohydrate Organisation and President of the RSCChemical Biology Division.

Chem. Sci., 2013, 4, 3381–3394 | 3381

http://dx.doi.org/10.1039/c3sc50877c

http://pubs.rsc.org/en/journals/journal/SC

http://pubs.rsc.org/en/journals/journal/SC?issueid=SC004009

Chemical Science Perspective

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. View Article Online

information transfer7—they are an additional ‘language of life’(Fig. 1)—whose roles are constantly emerging.8 Yet decodingthis language has barely started. In comparison with proteinsand nucleic acids that contain linear sequences of buildingblocks, glycans frequently form branching structures and theinter-residue linkages (glycosidic) can exist in different cong-urations (anomeric isomers). The biosynthetic assembly ofglycans is also more complex—it is not template-driven, as islargely the case for proteins and nucleic acids—and involvescomplex sugar chain processing and trimming under the actionof a series of competing enzymes along secretory pathways. As aresult, glycans are generally more diverse in structure and areoen present as heterogeneous mixtures that are difficult toseparate for detailed structure–activity relationship studies.However, this structural complexity, which has oen beenblamed for the slow development of hypothesis testing withinthe eld, also offers powerful opportunities in the design ofmolecular experiments that will unpick the mechanism of thisbiology. In this way chemistry offers one unique, and as yetlargely unrealized, strategy for dissecting this complexity wherestrictly biological approaches may fail.

The increasing importance and realization of chemicalbiology as a eld have seen some successful analyses of itsintellectual thrust. We use here a concept of the study of bio-logical questions and problems through molecular insight.Current moves towards the cellular and organismal are vital butat the same time can create an imbalance in skills and a lack ofmolecular knowledge. This creates a current opportunity for thechemical biologist. The aesthetic of synthesis is a self-consis-tent goal that does not need to justify itself in disciplines. Andwithout genuine biological questions, the synthesis of struc-tural permutations (‘countless numbers of grapes’ [Bacon]) is astrategy that is unlikely to work. This is particularly pertinent toglycoscience, where permutations are too vast.9 Chemical biol-ogists, therefore, have an essential and powerful role in posingthe ‘right’ molecular questions in biology. In chemical glyco-biology, the exciting depth and breadth of the challenges that

Fig. 1 A modified central dogma of biological information flow.

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we face preclude devotion to only one discipline or sub-disci-pline. The recently released US National Academy of Sciencesreport, entitled “Transforming the glycosciences: a roadmap forthe future”, offers a blueprint for future studies. It too empha-sizes the essence of a multi-disciplinary approach (http://www.nap.edu/catalog.php?record_id¼13446). This will forgesuperbly exciting ‘joined-up’ strategies and here we guess athow these might develop. With technical and conceptualdevelopment, the time is now ripe to bring glycoscience to themainstream of research.

Chemical glycobiology, as broadly dened, explores chem-ical approaches and concepts for deciphering and under-standing the function of entire glycomes and related partners.Remarkable progress has been achieved within the past twodecades, and this has been discussed in a number of excellentrecent reviews.10–16 While we should be proud of this, we mustbe also fully aware of the great structural and functional spacethat remains unexplored. The purpose of this speculative reviewis therefore not to recapitulate and list the many studies andreviews that comprehensively cover what has been tried in thepast, but instead to speculate on challenges that remain.

The generalities of sugar synthesis andglycosylation

The synthesis of glycans and related chemical probes is essen-tial if we are to precisely decipher structure, biosynthesis,metabolism and function in biological systems. The relativeimportance of synthetic glycans in glycobiology is exacerbatedsince homogeneous glycans (oen found as part of glyco-conjugates) are typically difficult to obtain from natural sourcesbecause of their structural micro-heterogeneity and, in manycases, their natural scarcity. Such synthetic glycans are alsobeginning to play essential roles in the development of carbo-hydrate-based therapeutics, such as vaccines.17 However, theissues involved in the construction of complex oligosaccharidesand glycoconjugates, including branching structures, anomericstereochemistry, regiochemistry (even in linear oligomers),and coupling efficiency, pose many synthetic challenges. Thecomplexity and diversity of oligosaccharides and glyco-conjugates demand the use of a range of the most efficientsynthetic methods with sufficient exibility and breadth of use,regardless of whether these are chemical, enzymatic, orchemoenzymatic.

The general aspects of chemical glycosylation and majorglycosylation methods have been well reviewed and discussedelsewhere13,18–21 but key aspects are worth reiterating. Carbohy-drate chemistry oen takes selective protection and depro-tection methods to their most comprehensive and elegantheights to achieve regioselectivity in glycosylation.13,18–22 Thislack of ‘atom efficiency’, of course, begs strategic questions butnonetheless it has proven to be a successful and reliableapproach, especially when coupled with novel glycoside-form-ing strategies. These include manipulations of stereoelectroniceffects, neighboring/remote protecting group participation, andconformational constraints—all usefully explored to controlstereoselectivity at the anomeric center. In addition, there are

This journal is ª The Royal Society of Chemistry 2013


Perspective Chemical Science

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continuous efforts to develop glycosyl donors with novelleaving groups and new promoters/catalysts in order to enhanceglycosylation efficiency, and to tune reactivity oen with usefulknock on consequences for the better control of stereochem-istry (Fig. 2). The progress in chemical synthesis has beenremarkable, as exemplied by the successful total chemicalsynthesis of complex heparin oligosaccharide-based drugs,23

highly complex carbohydrate-containing natural products,24

and even homogeneous natural glycoproteins.25–28 For thoselooking-in from outside the eld, such efforts spent on thecreation of what is in essence just a mixed acetal or ketal linkagemay seem a stunning self-indulgence for the carbohydratecommunity to still be so heavily involved in more than 100 yearsaer the rst realization of a real glycosylation reaction. Yet, wemust be clear, there is no general synthetic method availablethat can cover all of the diverse structures of glycans and gly-coconjugates and, in this era of oversimplied chemicalstrategy (“click” and “automated synthesis”), it is pertinent torealize that each glycan target may pose unique challenges of

Fig. 2 Chemical and enzymatic synthesis of glycosides and oligosaccharides. (a) Gglycoside construction.


reactivity and selectivity. More ‘programmable’ strategiesremain one option to create more general approaches, whilst onthe other hand ‘tricks’ in regioselectivity and stereoselectivityremain attractive as bespoke solutions to target structures. Eventoday, there remains an argument as to what active intermedi-ates may be involved in fundamental glycosylation reactionsand how the reactivity of the active species contributes to thecontrol of stereoselectivity.29 Innovations in achieving nearperfect stereocontrol in glycosylation remain urgently sought-aer goals30 and the mechanistic ambiguities of reactions atacetals ensures that there is still rich chemistry to be exploredin glycosidic bond formation.

Enzymatic and chemoenzymatic glycosidesynthesis and manipulation

The need to simultaneously control reactivity, regioselectivity,and stereoselectivity, some might argue, has created a double-bind for carbohydrate synthesis: such complexity requires the

eneral issues in glycoside synthesis; and (b) the common themes of biocatalytic

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greatest use of the available tools (e.g., extensive protectinggroup usage, arsenals of donors) and yet it is increasingly andstrikingly atom inefficient. In addition, much of the role of theprotecting group in glycan synthesis is to provide the ‘grease’ tosolubilize the substrate in a manner suitable for our traditionalorganic solvents. In contrast, nature has devised many thou-sands of biocatalysts (enzymes) to achieve oen perfect controlof stereo- and regioselectivity and usually high efficiency intransformations for glycan assembly in aqueous solution(arguably the ideal solvent for glycans), without the need ofprotecting groups. Thus, there seems no reason why we chem-ical biologists should not embrace such special and efficienttools in our synthetic business and we would argue thatsynthetic carbohydrate scientists have been forward-thinking intheir adoption of biocatalysis. In particular, combinedprocesses (incorporating enzymatic transformations as keysteps in a chemical synthesis, the so-called chemoenzymaticapproach), can rapidly simplify synthetic schemes.31

In theory, all types of enzymes involved in glycan biosyn-thesis, modication, and metabolism can be borrowed forsynthesis (Fig. 2b). The opportunities for catalysis are hugesince the numbers of enzymes involved in glycan assembly andmetabolism far exceed those involved in the transformations ofany other type of biomolecule. Glycosyltransferases are thenatural enzymes responsible for constructing glycosidic bonds.Their high selectivity in glycosidic bond formation and toler-ance of certain modications on the donor and acceptorsubstrates make glycosyltransferases unique biocatalysts forsynthesizing natural and selectively modied glycans.32 Sugarnucleotide regeneration systems have been used now for sometime to bypass the stoichiometric use of the expensive sugarnucleotides but this creation of ‘catalytic donors’ has not gainedthe prominence that it perhaps should in the syntheticcommunity. The perceived intolerance of some enzymes can, infact, sometimes be ‘forced’ in vitro using concentrations abovethe KM; this has actually allowed broad use of quite differentdonors in many cases. Moreover, mutational studies have led tomutant enzymes that demonstrate broader substrate tolerance,capable of accepting modied and altered substrates.31,33

Furthermore, enzymatic synthesis should not be limited topurely in vitro enzymatic transformations. For example, func-tional transfer of glycan biosynthetic genes or even gene clus-ters into the bacterium Escherichia coli (E. coli) has enabled thelarge-scale in situ production of heparin-like oligosaccharides,sialyloligosaccharides, ganglioside oligosaccharides andhuman milk oligosaccharides.34,35 Thus, E. coli, biotechnology's“work horse”, as well as other promising host systems (yeast,insect cells, and plant cells) could well be further explored asfactories for producing diverse glycans on a large scale.

Glycosidases, the natural function of which is to hydrolyzeglycosidic bonds in glycan metabolism, have strong potential intransglycosylation by virtue of their ability to manipulate bondformation at the anomeric center.36–38 One of the most strikingadvances in this area is the invention of glycosynthases:39,40

logically designed glycosidase mutants that are devoid ofproduct hydrolysis activity (a clear limitation in glycosidase-associated synthesis) but that are able to take activated donor

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substrates (e.g., glycosyl uorides or sugar oxazolines) fortransglycosylation.36–38 Since the report of the rst glycosynthasein 1998,39,40 various glycosynthases have been constructed fromglycosidases belonging to more than a dozen glycoside hydro-lase families. Yet, this reects only a small fraction of the manythousands of existing glycosidases; oen the principles of gly-cosynthase design do not translate. One reason for this could bethat observed reaction rates, in some cases, are simply belowthe threshold for detection. Even a little perturbation at thecatalytic site caused by mutation could result in a large impacton the reaction rates. It may also be that our assumptionsregarding modes of such catalysis may need rening; moredetailedmechanistic studies are undoubtedly required to unveilwhy some enzymes have more potent transglycosylation activi-ties than others (Fig. 2b).

In addition to the enzymes exploited in glycosidic bondformation, a number of glycan-modifying enzymes, such assulfotransferases, acetyltransferases, oxidases, and epimerases,to name a few, also bear high value for post-glycosylationmodications of oligosaccharides and glycoconjugates.31,41 Avery large space of enzyme capacity for synthesis remains to beexplored. Two notable examples showcase the power of thechemoenzymatic approach. One is the recently reported in vitrochemoenzymatic synthesis of homogeneous ultralow molecularweight heparins (heparin heptasaccharides) with well-denedsequence and sulfation patterns, which was fullled by acombined use of glycosyltransferases, sulfotransferases, andepimerase, starting from simple disaccharides.42 The synthesistook only 10 to 12 steps and resulted in a 45% overall yield. Thiswas achieved on the basis of an in depth understanding of thebiosynthetic pathways, together with the efficient cloning andexpression of those enzymes involved.42 In comparison, thepure chemical synthesis of the related anticoagulant drug,Arixtra (a heparin pentasaccharide), a landmark in targetchemical oligosaccharide synthesis, took more than 50 stepsleading to a low (1%) overall yield.23 The other example is theab initio glycan remodeling of glycoproteins via endoglycosi-dase-catalyzed deglycosylation and block oligosaccharidetransfer.43,44 This highly convergent approach enables a quickassembly of well-dened glycoforms, such as the homoge-neously Fc-glycosylated IgG antibodies, for structural andfunctional studies.

While oen specicity is an advantage in enzymatic trans-formations, the great diversity in the types of glycosidic linkagesalso potentially means that large numbers of enzymes must becollected to cover this diversity. However, the once popularnotion of ‘one linkage–one enzyme’ might not be quite accu-rate, as some enzymes such as glycosyltransferases, particularlythose from bacteria, can be, in fact, quite promiscuous insubstrate recognition, enabling “glycorandomization” to diver-sify the glycosylation patterns of complex natural products.45,46

Moreover, directed evolution, coupled with mechanistic studiesand protein engineering, provides a powerful method toimprove the catalytic efficiency and plasticity of enzymes.33,47–51

It is expected that studies along these lines will signicantlyexpand the repertoire of enzymes useful for synthesis. In ouropinion, the chemoenzymatic approach holds great promise,




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particularly in constructing those targets that are very large andcontain complex carbohydrates, such as proteoglycans, glyco-(sphingo)lipids, and glycoproteins.

The construction of homogeneous glycoproteins poses aspecial challenge (Fig. 3). One question to ask is: can we developroutine protocols to selectively alter the nature of glycans atspecic sites in a multiply glycosylated glycoprotein, in analogyto site-directed mutagenesis in recombinant technology?Theoretically, total and semi-chemical synthesis, via nativechemical ligation (NCL) and expressed protein ligation, offer apossibility to install different glycans at different sites in thecontext of a polypeptide (Fig. 3a).25–28 However, in reality,chemical technologies for total glycoprotein synthesis are farfrom maturity. The exploitation of various NCL strategies hassignicantly improved synthetic quality. Nevertheless, thechoice of ligation strategy, the design of ligation components,and the efficiency of tandem ligations should all rightly beconsidered on a case-by-case basis for individual targets.Convergence and efficiency of ligations are key to success andthe installation of glycan prior to protein backbone assemblynecessarily places a more linear emphasis on NCL routes.

In this sense, enzymatic glycan remodeling of natural andrecombinant glycoproteins (Fig. 3c) offers an exciting opportu-nity to convert heterogeneous glycoproteins to homogeneousglycoforms in a global and convergent glycosylation remodelingmanner,52 as it combines some advantages of recombinantDNA technology for protein synthesis and the selectivity ofin vitro enzymatic manipulation for attaching desired glycans.However, despite its power this method can struggle to controlthe site for convergent glycan attachment; methods are urgently

Fig. 3 Strategies for glycoprotein synthesis. (a) Linear chemical glycoprotein synthtag or sugar primer incorporation; and (c) chemoenzymatic glycosylation remodelin


needed to achieve differential site-selective glycan remodeling.One, yet to be demonstrated, solution would be novel endo-glycosidases that can distinguish the nature of glycans fordeglycosylation and/or the location (site) of themonosaccharideprimer needed for en bloc transfer of glycans.

Co-translational glycosylation to determine site selectivityprovides another broad strategy (Fig. 3b). In this regard, one ofthe ‘killer apps’ in the form of stop codon suppression by aglyco-amino acid has not yet been clearly demonstrated in auseable form,53–55 but hope remains. The bacterial PglB oligo-saccharyltransferase system has been nicely exploited in vivo tocreate valuable substrates using pathway recapitulation andrepositioning of the appropriate DNXS/T consensus motif.56 Inparticular, the relaxed specicity of the PglB toward someglycan structures has enabled the transfer of certain largebacterial oligosaccharides and polysaccharides (such as O-antigens) to proteins in a site-specic manner to form linkage-dened polysaccharide–protein conjugates, which points to apromising new avenue to making bacterial glycoconjugatevaccines in E. coli.57,58 Another way to determine the site ofglycosylation is to do this chemically by attaching a primerglycan through chemical site-selective protein chemistry. Inthis sense, the ‘tag-and-modify strategy’ has successfullyallowed conversion of recombinant proteins into homoge-neously glycosylated proteins, particularly when combinedwith in vitro enzymatic sugar chain elongation.59,60 Althoughthese systems are tolerated by endoglycosidases to allowtransglycosylation, one goal that remains to be addressed ishow to introduce tags with the fully native linkage found innatural glycoproteins.

esis; SPPS ¼ solid-phase peptide synthesis; (b) convergent approach via expressedg.

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Automated and programmable synthesis

Conventional oligosaccharide synthesis can oen involvetime-consuming manipulations of a similar reaction type:protection and deprotection steps to dene the regioselectivityand sometimes intensive chromatographic steps to purifythe desired anomer(s) aer each glycosylation. Automatedsynthesis is therefore a sensible and logical strategy to stream-line glycan assembly by minimizing the intermediate purica-tion steps and by minimizing the manual nature of anyrepetition.61 Major strategies that hold promise for automatedand programmable oligosaccharide synthesis are summarizedin Fig. 4. Solid-phase synthesis seems an appropriate adjunctand, indeed, di- or trisaccharides were targets rst addressed inthe 1970s soon aer the success of automated polymer-sup-ported synthesis of polypeptides.62 However, it was not until2001 that an automated oligosaccharide synthesis strategy(using glycosyl phosphates on a solid support) captured generalattention (Fig. 4a).63 This remarkable demonstration showed

Fig. 4 Strategies for automated oligosaccharide synthesis; RRV ¼ relative reactivit

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that an automated synthesis of complex oligosaccharides wasconceptually and technologically feasible. Some have correctlyhighlighted that this disclosure did not provide a generalsolution. However, its disclosure threw down a gauntlet to othersynthetic methods—there is no doubt it has prompted dramaticdevelopments in the synthetic sophistication of both automatedand non-automated methods.

Many exciting associated goals remain. Recent studies havestarted to effectively address initial drawbacks faced in this andother automated oligosaccharide syntheses, exemplied by thesynthesis of increasingly more complex targets.64–66 Neverthe-less, a number of problems still remain when diverse glycanstructures are concerned. In addition to the issues associatedwith choices of linkers and solid-supports, and the accessibilityof selectively protected monosaccharide building blocks, thegreatest problems surround the control of anomeric stereo-chemistry and the unpredictability of coupling efficiency ineach glycosylation step. Thus, more robust glycosylationmethods with perfect control of stereoselectivity (particularly

y values.




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for the 1,2-cis glycosidic linkages) with excellent associatedcoupling yields (>99%) are welcome in this regard30 and moreare urgently needed, if compounded inefficiencies are not tocontinue to prove strategically unwieldy. Although more rarelyconsidered, enzymatic transformations also hold great poten-tial for automation; in a recent report, an automated enzymaticsynthesis of sialyl Lewis X was fullled that applies sequentialenzymatic glycosylations on a globular protein-like dendrimeras the solid support67 albeit in a relatively low overall yield(mainly due to the low recovery efficiency of the polymer)(Fig. 4b). In another study, digital microuidic technology wasinterestingly employed for enzymatic modications of heparinsulfate chains immobilized on manipulable magnetic nano-particles.68 As more-and-more enzymes become available, bio-catalytic automated platforms may allow high-throughputaccess to diverse naturally occurring glycans and a range of non-natural derivatives as well. Such concepts deserve furtherexploration in order to develop articial systems that might, oneday, effectively mimic the natural Golgi apparatus for oligo-saccharide assembly and modication. Interestingly bothgroups cited here viewed their very different technologicalapproaches as possible ‘Golgi mimics’.

In parallel to solid-phase synthesis, there are several solu-tion-based, one-pot, multi-step synthetic strategies that holdpromise for automation. These include: programmable one-potmulti-step oligosaccharide synthesis based on the relativereactivity of selected glycosyl donors (Fig. 4c),18,69 a one-potorthogonal activation strategy,70 and a one-pot pre-activationglycosylation strategy (Fig. 4d).71–73 Again, here, control is thename of the game—the success of these strategies for any givencomplex synthetic target relies on how one can efficiently ne-tune the many factors involved, even the stoichiometry andmode-of-use of the reactants and activators, as well as theiridentity. As for all the strategies highlighted in this section,these methods serve to illustrate that far from there being onegeneral solution, the answers will continue to lie in robust andrapid access to rapidly optimized bespoke synthesis.

Glycan (and associated) arrays

Glycan (micro)arrays are emerging as a popular and importanttool in deciphering glycan functions.14,16,74 Microarray tech-nology typically consists of immobilization on a surface (e.g.,microplates/glass slides) and subsequent probing of interac-tions between the printed analyte (glycan) and binding partners(lectins, antibodies, enzymes, etc.) either in an isolated form orpresent in a biological context (sera, cell surface, or cell lysates).This format can offer several important advantages for glycomicstudies, including high-throughput screening, sensitive andquantitative analysis, and the need for reduced amounts ofglycans. A major application of glycan microarrays has been todetect and quantify specic interactions between glycans anddiverse glycan-binding proteins in mammalian, microbial andplant systems.16 This has probed changes in affinity and speci-city of glycan–protein interactions aimed at identifying specicbiomarkers for the early diagnosis of diseases. Screening the serafrom patients or vaccinated individuals in a glycan microarray


format also allows quick assessment of immune responses,facilitating biomarker discovery and vaccine development.75,76 Incombination with sensitive mass spectrometric analysis, theglycan microarray format has also been successfully used forprobing enzyme substrate specicity77 and for discovering newglycosyltransferases.78

Despite these signicant advances, the technology is still farfrom reaching its full potential. Perhaps the most signicantlimitation of current glycan microarray formats is a lack ofsufficient number and structural diversity of glycans. Forexample, the most “comprehensive” glycan microarrays so faravailable from the Consortium of Functional Glycomics (CFG)(http://www.functionalglycomics.org) contain only 600 or soglycans. This covers only a very small fraction of basic naturalglycan structures, which are estimated to be in the tens ofthousands. Accelerated synthesis, including development ofmore efficient chemical and chemoenzymatic methods (likelyemployed for ‘on array’ synthesis), is urgently needed to ll thegap. But synthesis is not the only avenue to expanding thechemical space of glycans. In fact, a number of natural glycanscan be adequately prepared directly from natural sources ofglycoproteins, glycolipids, and glycosaminoglycans: over 200natural glycans from several rich sources such as human milk,chicken ovalbumin, egg yolks, and glycosaminoglycans can bereadily conjugated with a bifunctional uorescent linker tofacilitate their tagging, separation and printing in a microarrayformat.79,80 Here, the uorescent tag introduced in the glycanscan also serve as a means for quantifying the immobilizationdensity and efficiency—these particular glycan arrays weresuccessfully used for identifying the natural ligands for galec-tins.80 It should be noted that, in this pursuit, complete separa-tion may not even be necessarily required for initial screening;once hits are narrowed to subgroups, they can be identied byfurther separation and characterization. Thus, development ofmore robust methods for isolation, characterization, andtagging of glycans from diverse natural sources should be a highpriority for rapidly expanding the chemical space of glycans andas a way of accessing glycomes in a number of useful guises.

Another major limitation of current glycan array technologyis the lack of adequate control of glycan presentation (Fig. 5).Firstly, different array platforms clearly result in differences inthe density and orientation of glycan presentation; these maydramatically change the affinity (avidity) and even specicity inglycan–protein interactions. Continuing cross-comparisonamong different array platforms is necessary in order to eval-uate such factors in array fabrication.81 Secondly, the presen-tations of glycans printed on microplates or glass slides couldbe quite different from those of glycans present in the contextof glycoproteins and glycolipids. The synthesis of neo-glycoproteins and neoglycolipids for array presentation maypartially address this issue.82–84 However, future generations ofglycan microarrays should routinely include arrays of naturalglycoproteins (varied glycoforms also) and glycolipids in orderto understand the precise contributions of the lipid andprotein portions to glycan–protein recognitions. This will beparticularly important when micro- and macro-multivalency isinvolved (likely with variable mechanisms). Thirdly, future

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Fig. 5 General issues in glycan array design.


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generations of glycan microarrays should also consider thedynamic nature of glycans present on cell surfaces. Fluidicglycan arrays offer an intriguing avenue to mimicking themobility of glycoconjugates (ras) embedded in a lipid bilayer.85

With efforts in the development of new approaches and tools,glycan microarray technology is expected to continue to play keyroles in accelerating the decoding of the glycome. Nonetheless,we should not lose track of the vital importance of also delin-eating quantitative mechanistic information from glycan–protein interactions where we can—striking a balance betweenrapid, qualitative and semi-quantitative methods on the onehand, with a need to understand the subtleties of modes ofbinding, and how these inuence selectivity and kinetics on theother hand will prove vital. Moreover, whilst we perhaps natu-rally gravitate to arrays that contain glycans (since they looselymimic glycocalyx) we should also recognize that arrays ofcarbohydrate-binding proteins, as analytical tools for glyco-conjugate solutes, can also prove powerful.86

Glycan sequencing

Decoding the sequence and structure of glycans is of immediateimportance in elucidating their biological functions and asso-ciated structure–activity relationships. Whilst proteins andnucleic acids can be routinely sequenced using well-establishedand robust methods, the sequencing of glycans is more chal-lenging. In contrast to the well-dened unmodied buildingblocks for proteins (20 amino acids) and DNA (4 basic nucleo-tides) and their single backbone linkages (amide and phos-phodiester, respectively), the possible natural monosaccharidebuilding blocks for glycans far exceed the number for aminoacids or nucleotides. Moreover, these monosaccharide buildingblocks display more diversity in their context of glycans (D-/L-,furanosyl/pyranosyl rings, regioisomerism of linkage, linear/branching, a- or b-anomers) when forming glycosidic bonds.These structures can be further complicated by diverse post-glycosylation modications such as sulfation, acetylation,deoxygenation, amination, and phosphorylation. Moreover,natural glycans in many cases appear in the form of glyco-conjugates (glycoproteins, glycolipids, proteoglycans) and areusually present as mixtures.

This diversity and heterogeneity of glycans therefore pres-ents a unique challenge in sequencing and structural determi-nation. Yet the level of difficulty and complexity in structural

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analysis varies depending on the nature and source of theglycans, which also dictate the choice of analytical strategies.Conventional and longstanding monosaccharide and per-methylation analysis can provide useful initial information onmonosaccharide composition and site of linkages on mono-saccharide building blocks, but it has not typically been able toprovide information on linkage types, linkage sequence, andanomeric conguration. MS and NMR are two most importanttools for glycan sequencing and structural analysis and bothtechnologies have now begun to take on greater challenges inthe structural characterization of complex oligosaccharides andglycoconjugates. For example, advances in mass spectrometry(nanoLC-ESI-MS, tandemMS, electron-capture dissociation andelectron-transfer dissociation) have made it possible to reliablycharacterize N- and O-glycosylation of glycoproteins.87 However,using MS it has been hitherto difficult to characterize regio-and stereoisomers of glycans. Recent work onMS assignment ofthe stereochemistry and anomeric conguration of mono-saccharides in oligosaccharides has shown promise in thisdirection.88,89 MS coupled with specic enzymatic trans-formations also provides a useful tool for sequencing glycans,yet one current limitation is the availability of sufficientlydiverse linkage-specic glycosidases.

Linear oligomers allow some simplication; for glycosami-noglycans (GAGs), recently, a top-down Fourier transform massspectrometric sequencing technology was employed forsequencing bikunin, one of the simplest proteoglycans.Surprisingly, the GAG chain of proteoglycan bikunin was shownto have a “homogeneous”, dened sequence for activity.90 ‘Top-down’ strategies preserve whole sequences that conventionalbottom-up strategies might miss. This example also emphasizesthe importance of the choice of method—for samples consist-ing of mixtures of numerous different glycosaminoglycans,such a top-down strategy might prove tedious and difficult.

NMR has been particularly useful for polysaccharidesequencing through the application of various homo- and het-eronuclear coupling techniques, which allow reliable assignmentof the ring forms of monosaccharides, anomeric congurations,linkage types, and monosaccharide sequential connections.91 Inaddition, it can also provide detailed three-dimensional struc-tures of glycans in the context of glycoconjugates.92 However,NMR usually requires relatively large quantities of samples, andthe sensitivity of natural isotope abundance is an issue. Thus,development of NMR techniques with greater sensitivity forpolysaccharides and glycoconjugates of natural isotope abun-dance is a highpriority, whilst at the same timenewmethods thatallow easy isotope labeling and enrichment of glycans withspecic isotopes (13C, 15N, and/or 18O, by chemical, enzymatic, orbiosynthetic means) would prove powerful.

Novel approaches to glycan functions

For chemical biologists, function should be a major goal. Onecriticism leveled at chemical glycobiology is that this aspect ofour eld is in its infancy as compared withmethods for buildingand characterizing glycans. Key methods may offer incisiveoptions as follows (Fig. 6).




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Glycochemical genetics

An important approach in chemical biology is to explore specicexogenous (small molecule) ligands to probe gene-productfunctions in a cellular or organismal context.93,94 In this so-called ‘chemical genetics’ approach, small molecule ligandsthat bind directly to proteins with high affinity and specicityare used to perturb protein functions in cellular processes,resembling effects of genetic mutations. By analogy, novel smallmolecule probes that can specically perturb the functions of

Fig. 6 Some chemical biology approaches to glycan functions.


glycans should be particularly useful in functional glycomicstudies. In fact, glycochemical genetics is not new to our eld,and many specic (natural or synthetic) inhibitors of glycan-processing enzymes, including glycosidases and glycosyl-transferases, have been discovered and implemented formanipulating functions in cells or in vivo (Fig. 6a).95 Tunica-mycin, a uridine analogue isolated from Streptomyces thatblocks the early steps of N-glycan assembly thus deleting N-glycosylation globally, has played a key role in elucidatingthe functions of protein N-glycosylation in protein folding,

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intracellular trafficking, and cellular communication. Inhibi-tors against polypeptide N-acetylgalactosaminyltransferases(ppGalNAcTs), a class of enzymes responsible for the attach-ment of the rst sugar in mucin O-glycosylation have beendiscovered by screening a synthetic uridine-based small mole-cule library;96 these inhibitors were shown to abrogate mucin-type O-linked glycosylation and to induce apoptosis in culturedcells. Recently, potent inhibitors against the two key enzymes(OGA and OGT) responsible for a dynamic O-GlcNAc glycosyla-tion97 were discovered.98,99 As demonstrated in a mouse modelof Alzheimer's disease, direct use of a sugar thiazoline-based O-GlcNAcase (OGA) inhibitor led to an increase of O-GlcNAcglycosylation level, resulting in the slowing of neuro-degeneration without apparent adverse effects.100 Precursors/prodrugs may also prove powerful (Fig. 6b); the acetylated 5-thioanalog of GlcNAc could be taken into the biosynthetic pathwayof UDP-GlcNAc in cells, leading to the in situ formation of UDP-5SGlcNAc, which inhibited the OGT (O-GlcNAc transferase)activity99 that uses UDP-GlcNAc as a donor. Similarly, it wasmore recently demonstrated that selectively uorinated sialicacids and L-fucose analogues could be taken up and trans-formed into respective sugar nucleotides that can also act assubstrate-based inhibitors of enzymes that process them insidethe cell.101 Their application resulted in global down-regulationof sialylation and fucosylation with clear consequent changes tothe glycome. The resulting functional consequence in myeloidcells was a loss of selectin binding and impaired leukocyterolling101 due to a reduced display of the associated sialylatedand fucosylated glycoconjugates that act as selectin ligands.

In addition to global inhibition of glycosylation, individualglycan functions can be also dissected through the use of morespecic inhibitors or molecular probes. These include the use ofchemical primers serving as competing acceptor substrates forglycosyltransferases, the use of inhibitors against specicglycan-processing enzymes such as distinct a-mannosidaseinhibitors, and the metabolic incorporation of sugar chainterminators to generate truncated glycoforms.11 We know thatstructure-based design can lead to the discovery of potentinhibitors, as powerfully and now archetypally exemplied bythe class of inuenza sialidases—these were successfullydeveloped into two widely used anti-inuenza drugs, Relenza(zanamivir) and Tamiu (oseltamivir).102,103 These inhibitorswere designed tomimic the transition state of a sialoside duringenzymatic hydrolysis, with the installation of a guanidinium orammonium group at the C-4 position for ion pairing with thepositively charged residues in the binding pocket, thusenhancing the affinity and specicity for viral sialidases overhuman enzymes. We are therefore aware as a eld that mech-anism can prove uniquely important in designs that generatepotent small molecules as probes and drugs. More recently,novel mechanism-based inuenza sialidase inhibitors weredesigned based on the structures of Relenza and Tamiu inwhich uorine atoms were introduced at the C-2 and C-3 posi-tions.104 These new inhibitors react specically with inuenzasialidases and inactivate the viral enzymes via the formationof stable, covalently linked inhibitor–enzyme intermediates.Notably, these mechanism-based inhibitors demonstrate

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broad-spectrum antiviral activity against those drug-resistantinuenza strains, making them attractive for further drugdevelopment.

As well as targeting catalytic function, synthetic glycan-basedligands are emerging as highly valuable tools to probe specicglycan–protein interactions in cellular processes. An example isthe discovery of multivalent sialylated glycan ligands for CD22,a sialic acid-binding lectin from the Siglec family that attenu-ates B cell receptor signaling, for suppressing B cell activa-tion.105,106 It was found that exposure of B cells to sialylatedantigens led to inhibition of key steps in B cell receptorsignaling and induced B cell tolerance. These studies showcasethe value of the glycochemical genetic approach to dissectingcellular functions that would be otherwise difficult to unveil bytraditional genetic methods. The power of chemistry thereforeallows us to consider the creation of even quite large hybridmolecules in pure form to act as such perturbing probes ormolecules. A so-called glycodendrinanoparticle (with a valencyof up to 1620) was constructed in homogeneous form recentlythrough the use of a nested multivalency in both glycoden-drimer attachments and the self-assembly of many proteinsinto a virus-like particle.107 The resulting constructs proved to bepowerful binders of dendritic cell (DC) lectin DC-SIGN andpicomolar inhibitors showing large multivalency effects inmodels of DC infection by Ebola virus.

The unique advantage of the glycochemical genetic approachusing molecular ligands or inhibitors as probes is to perturbfunctions of a cellular process in a dynamic, reversible andtunable manner. This complements well the traditional geneticapproaches that make “permanent” alterations of functions.However, the success of the glycochemical genetic approachrelies on the discovery of high-quality and specic carbohy-drate-based or noncarbohydrate-based probes. Future studiesshould be directed not only to systematic, high-throughputscreening of small-molecule compounds targeting specic sitesor stages of glycan functions but also to the rational design ofhigh-affinity ligands or inhibitors specic for glycan-recogni-tion receptors and enzymes and the design of novel probes thatare suitable for in vivo (not just in vitro) perturbation of glycanfunctions.

Glycan imaging

The ability to directly detect the changes of glycans in biologicalsystems offers an exciting opportunity to decipher their rolesand functions in normal physiological and disease states. Whilelectins and glycan-specic antibodies have been widely used formany years for detecting cell-surface glycans and for glycanvisualization on tissue sections, they are not widely suitable forall in vivo imaging (e.g., due to cytotoxicity and/or membraneimpermeability) and the truly characterized selectivities ofmany ‘anti-sugar’ antibodies can be unclear or even absent. Oneemerging technology for glycan imaging is the metabolic engi-neering of glycans with so-called bioorthogonal chemicalreporters (Fig. 6c).108,109 In this approach, a specic chemicalreporter is incorporated into the glycan via biosynthetic incor-poration of a sugar precursor modied with the chemical




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reporter. The reporter group is then visualized by covalentreaction with an imaging probe that can specically detect andreact with the chemical reporter. The currently most commonchemical reporters are azide or alkyne groups, since they aresmall and relatively biologically inert and they have the poten-tial to react specically under physiological conditions. Forexample, an azide reporter has been detected by reaction with abiotin-tagged phosphine via Staudinger reaction or with a u-orophore-tagged alkyne via copper-catalyzed azide–alkynecycloaddition reaction.110 To avoid perceived cytotoxicity of thecopper catalyst in living systems, a strain-promoted azide–alkyne cycloaddition has also been devised that uses cyclo-octyne derivatives as reactants, enabling reaction withoutcopper mediation.111–114 Early focus was dominated by imagingof cell surface sialylated glycans using azide- or alkyne-taggedManNAc variants (ManNAz or ManNAl) as precursors. Sincethen the metabolic glycan imaging approach has beenexpanded to the imaging of other types of glycans includingmucin-type O-glycans (using azide-tagged GalNAc as theprecursor), O-GlcNAc glycoproteins (using azide-tagged GlcNAcas the precursor), and fucosylated glycans (using 6-azido-fucoseor alkynyl fucose as the precursor). In addition, the imagingtechnology has also expanded from its use in cultured cellsystems to living organisms including zebrash and mice.110

Glycan imaging provides an important tool for decipheringthe localization and trafficking of glycans in cellular processes.Moreover, noninvasive glycan imaging in living animals offers aunique opportunity to detect, monitor, and quantify thedynamic changes of glycans associated with development anddisease states, which may unveil novel biomarkers for diagnosisand important targets for drug discovery. However, a potentiallimitation of metabolic glycan imaging is the lack of cell type ortissue specicity, as the injected sugar precursors would beincorporated into all the shared glycan biosynthetic pathways. Arecently reported cell-specic metabolic labeling strategy thatuses ligand-targeted liposomes to encapsulate and selectivelydeliver an azidosugar precursor to target cells provides apromising solution to circumvent this problem.115 Futurestudies should be directed to the exploration of novel biosyn-thetic pathways to cover additional types of glycans in the gly-come, and to understanding their limitations, when sugarprecursors are biosynthetically used for more than onepurpose;116 new unnatural sugar precursors can be envisagedthat possess better biocompatibility and favorable pharmaco-kinetic properties and new orthogonal chemical reporters areneeded that permit simultaneous imaging of different types ofglycans in real time.

Selective cell-surface glycan remodeling

Diverse subtypes of glycans are present on cell surfaces. Site-specic remodeling of this glycocalyx provides an attractiveapproach to altering and understanding cell surface functionsas well as gaining novel ones. One notable example is the ex vivoglycan engineering of CD44 on human mesenchymal stem cells(hMSCs) that enabled redirection of cell migration to bone.117

Thus, the native CD44 glycoform on MSCs that lacks the key a-


1,3-fucosyl residue was converted into a hematopoietic cellE-selectin/L-selectin ligand via enzymatic a-1,3-fucosylationunder mild physiological conditions; this conferred potent E-selectin binding and tropism to bone, without drastic effects oncell viability or multipotency. This and a subsequent study118

unveil a great potential to program cellular trafficking viachemical engineering of glycans on a distinct membraneglycoprotein for directing cellular migration.

Another example is the development of universal red bloodcells through enzymatic removal of blood group ABO antigenson the cell surface.119 While this idea had been consideredpreviously, a practical approach to remodeling the ABO anti-gens had not been fullled because of the lack of enzymes toefficiently remove the GalNAc and galactose residues present inthe group A and group B antigens, respectively, withoutaffecting red blood cells' integrity. The discovery of two specicbacterial glycosidases, an a-GalNAcase and an a-galactosidase,provided new hope; these are efficient and specic for cleavageof the A and B immunodominant monosaccharides under themild conditions suitable for maintaining the integrity andfunctions of red blood cells.120 This study suggests a practicalapproach to transforming group A, B, and AB red blood cells tothe universal group O type for transfusion. The study alsoemphasized the importance of discovering new, more efficientmethods for glycan-specic cell-surface engineering for basicand clinical applications. Whilst many of these methods haverelied to date on alterations using naturally derived enzymeactivities, other approaches can be envisaged. The directedevolution of novel glycose oxidases offers tantalizing opportu-nities in this regard.51 Catalytic chemical methods may also beconsidered; the Pd-catalyzed remodeling of cell surfacesthrough direct C–C bond formation121 has recently been appliedto the remodeling of bacterial glycocalyx leading to a ‘chemicaldifferentiation’ that alters cellular interactions as determinedby appropriate, different chemical glycosylating agents.122

Conclusion

As a nal general comment, we suggest that in this burgeoningdiscipline we may need to be more clear-minded than in manyother elds. Whilst an unnecessary (and oen false) oppositionis sometimes created between “blue skies” research and appliedscience there is, nonetheless, a clear and useful distinctionbetween the creation of knowledge (science) and its use (tech-nology). This is particularly pertinent to glycoscience since onetempting way that we might consider solving some problemsthat we face is through the use of ‘bigger-and-better’ and ‘more-and-more’. In the eld of arrays there is an argument that oncewe have a big enough array with enough sugars then we will beable to ‘probe’ every interaction. In the eld of ‘glyco-proteomics’ there is an argument that if we perform largeenough, comprehensive ‘top-down’ proteomic studies then wewill see sufficient examples to get a better handle on thepatterns that are relevant to glycobiology.

Both of us would like to nish by highlighting that whilstsuch ‘powerful’ resource-driven analyses will create greatquantities of very useful data, the strength of many academic

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institutions is best found in asking the right questions andcreating the experiments to answer them. The size of the manychallenges in our eld means that this may end up being evenmore vital for our community than for others; it may be thatchemical glycobiology is one of the rst to be tested by thetemptations of large amounts of correlative data that lack causallinks. Sometimes we decide as a community that where wecurrently do not yet understand or see the direct causal linksthat we wish for, this might simply be because we haven't yetlooked widely enough or deeply enough. This argument to ‘domore’ is one approach. Yet another approach, perhaps, is topause and think about new mechanisms and roles for sugars inbiology. There is the temptation for us to imbue sugars with‘meaning’ that might not be there. Why does nature preservesuch a diversity and complexity of glycans aer so many years ofevolution? Is the heterogeneity of glycoforms a requirement forfunction or is it simply the result of a less perfect control ofbiosynthesis? Why might an additional GlcNAc on a proteinhave function cf. an additional hydroxyl? Although we know thathydroxylation and GlcNAc-ylation have specic functionaleffects it may also be that sometimes they provide simply aregister or ‘read-out’ of a background organismal state. Therewill be a long list of questions and we have just begun to decodethe function and dynamics of the glycome of any givenorganism. It becomes clear that chemical biology, whichprovides unique molecular tools and approaches, will continueto play increasingly important roles in deciphering the diversefunctions of glycans in biological systems.

Acknowledgements

This work was supported in part by the US National Institutes ofHealth (NIH grants R01 GM080374 and R01 GM096973 to LXW)and the Bill and Melinda Gates Foundation, Cancer ResearchUK and the UK research councils (EPSRC, BBSRC, MRC, toBGD). BGD is a Royal Society Wolfson Merit Award recipient.

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