Chemical Glycobiology Carolyn R

C ARBOHYDRATES AND G LYCOBIOLOGY REVIEW Chemical Glycobiology Carolyn R. Bertozzi1 and Laura L. Kiessling2 Chemical tools have proven indispensable for studies in glycobiology. provided substantial insight. For example, mice Synthetic oligosaccharides and glycoconjugates provide materials for cor- deficient in ␣ mannosidase II expressed an relating structure with function. Synthetic mimics of the complex assem- altered portfolio of N-linked glycans on their blies found on cell surfaces can modulate cellular interactions and are cell surface glycoproteins (2). The mice were under development as therapeutic agents. Small molecule inhibitors of prone to a systemic autoimmune response, sug- carbohydrate biosynthetic and processing enzymes can block the assembly gesting that abnormalities in N-glycosylation in of speciﬁc oligosaccharide structures. Inhibitors of carbohydrate recogni- humans may be a factor in the pathogenesis of tion and biosynthesis can reveal the biological functions of the carbohy- autoimmunity. Still, cell surface presentation of drate epitope and its cognate receptors. Carbohydrate biosynthetic path- simple as well as complex glycans requires ways are often amenable to interception with synthetic unnatural sub- many genes to be expressed in concert, which strates. Such metabolic interference can block the expression of oligo- complicates the analysis of single gene “knock- saccharides or alter the structures of the sugars presented on cells. outs” or “knockins.” Collectively, these chemical approaches are contributing great insight As outlined in this review, chemical ap- into the myriad biological functions of oligosaccharides. proaches are powerful allies to genetics and biochemistry in the study of glycobiology. Oligosaccharides and glycoconjugates (gly- geneous and chemically defined glycoconju- Chemical tools have been used to probe glycoproteins and glycolipids) have intrigued gates from biological sources. Without such cosylation at many levels. For example, cell biologists for decades as mediators of com- materials in hand, biological functions are surface carbohydrate-receptor binding events plex cellular events. With respect to structur- difficult to unravel. (Fig. 1) can be interrogated with synthetic al diversity, they have the capacity to far Genetic approaches have contributed con- oligosaccharides, glycoconjugates, and their exceed proteins and nucleic acids. This struc- siderably to our appreciation of oligosaccharide analogs. The biosynthesis of oligosaccharide tural variance allows them to encode infor- function. The availability of entire genome se- structures can be disrupted or modulated by mation for specific molecular recognition and quences has revealed the multiplicity of en- synthetic enzyme inhibitors. Unnatural sug- to serve as determinants of protein folding, zymes that contribute to glycoconjugate assem- ars that substitute for native monosaccharides stability, and pharmacokinetics. Given that bly. Their deletion in model organisms has or their downstream metabolic intermediates glycosylation is one of the most ubiquitious forms of posttranslational modification, the unexpectedly small number of genes identi- fied in the initial analyses of the human genome sequence provides even more impetus for understanding the biological roles of oligosaccharides. Oligosaccharide functions are now being elucidated in molecular detail, but advances in glycobiology have been slow to arrive compared with the pace of revelations in protein or nucleic acid biochemistry. The same structural diversity that has captivated biologists has also frustrated efforts to define oligosaccharide expression patterns on proteins and cells and to correlate structure with function. Some technical challenges are ana- lytical in nature; determination of the oligosaccharide sequence on a specific glycoconjugate is still far from routine. Others originate from glycoconjugate biosynthesis, which is neither template-driven nor under direct transcriptional control. Oligosaccha- rides are assembled in step-wise fashion pri- marily in the endoplasmic reticulum and Golgi apparatus (Fig. 1), a process that af- Fig. 1. Glycoconjugate biosynthesis and cell surface recognition. Exogenously supplied monosac- fords significant product microheterogeneity charides are taken up by cells and converted to monosaccharide “building blocks” (typically (1). As a result, it is difficult to obtain homo- nucleoside sugars) inside the cell. Several steps of metabolic transformation might take place en route from an exogenous sugar to a building block. The building blocks are imported into the secretory compartments where they are assembled by glycosyltransferases into oligosaccharides 1Departments of Chemistry and Molecular and Cell bound to a protein (or lipid) scaffold. In the case of N-linked glycoproteins, a core oligosaccharide Biology and Howard Hughes Medical Institute, Uni- is assembled in the cytosol, then transported into the ER where it is processed by glycosidases, and versity of California, Berkeley, CA 94720, USA. 2De- then further elaborated by glycosyltransferases. Once expressed in fully mature form on the cell partments of Chemistry and Biochemistry, University surface, the glycoconjugates can serve as ligands for receptors on other cells or pathogens. of Wisconsin, Madison, WI 53706, USA. Chemical tools can be used to inhibit or control any stage of this process. www.sciencemag.org SCIENCE VOL 291 23 MARCH 2001 2357 C ARBOHYDRATES AND G LYCOBIOLOGY (Fig. 1) can intercept biosynthetic pathways, ate building blocks are produced and assem- metal activators are being replaced with mild- leading to changes in cell surface glycosyla- bled into oligosaccharides (Fig. 2B). In both er, more environmentally sound methods (9, tion. These chemical strategies allow one to approaches, the focus is on forming the crit- 10). Our understanding of how to obtain the perturb glycosylation and oligosaccharide-re- ical connection that links saccharide building desired configuration of a glycosidic bond is ceptor interactions in a cellular context. Fur- blocks: the glycosidic bond. also more sophisticated. Stereochemical con- thermore, chemically synthesized molecules Chemical synthesis and enzyme-based trol can be achieved by employing stereospe- that disrupt pathological carbohydrate-depen- routes are complementary. Enzymes can be cific activation methods, using protecting dent processes are emerging as important used to effect glycosylation with absolute groups that direct the orientation of the gly- therapeutic agents. regio- and stereo-control. If the necessary cosidic bond through intermolecular (neigh- enzyme is available, the desired bond can be boring group participation) or intramolecular Synthesis of Oligosaccharides and formed (Fig. 2A), often with high efficiency. (tethered aglycone delivery) participation, al- Glycoproteins In comparison, chemical synthesis offers ex- tering the steric environment around the ano- Access to structurally defined oligosaccha- ceptional flexibility. Natural and non-natural meric position to bias the desired outcome, or rides and glycoconjugates is a prerequisite for saccharide building blocks can be assembled exploiting the intrinsic stereoelectronic pref- unraveling their function. Chemical routes to with natural or non-natural linkages. Al- erences for reaction at the anomeric position. the production of oligosaccharides are, there- though some enzymes will act on alternative Two major advances are being applied to fore, essential. Advances on this front are substrates, chemical synthesis provides the streamline the chemical synthesis of oligo- providing materials for the assessment of gly- means to generate any oligosaccharide, oli- saccharides: one-pot reactions (11, 12) and can function, the establishment of the struc- gosaccharide analog, or glycoconjugate. polymer-supported synthesis (13, 14). In tural features important for function, the elu- The chemical synthesis of oligosaccha- one-pot processes, glycosylation reactions cidation of biosynthetic pathways, the cre- rides is formidable. It requires stereochemical occur sequentially in a single reaction vessel; ation of carbohydrate-based vaccines, the and regiochemical control in glycosidic link- the most reactive glycosyl donor is triggered production of non-natural glycosylated anti- age formation. The first viable method for first and the least is coerced to engage in the biotics, and the generation of inhibitors of controlled glycosidic bond formation, the final reaction. A key concept underlying glycoconjugate function. Koenigs-Knorr glycosylation, was reported progress on this front is that there are Two general strategies are used for in in 1901 (7). Although the search for alterna- “armed” (reactive) and “disarmed” (less re- vitro oligosaccharide production: enzymatic tive glycosylation reactions is ongoing (8), active) glycosyl donors (10, 15). A number of (including chemoenzymatic) synthesis and chemists have made remarkable strides in research groups have exploited this knowl- chemical synthesis. In enzymatic and che- carbohydrate synthesis. The problem of ob- edge to efficiently assemble oligosaccharides moenzymatic routes, saccharide intermedi- taining the proper regiochemistry of glyco- using one-pot reactions (12, 16–20). Solid- ates are elaborated with enzymes, typically sylation has been largely solved by the devel- phase synthesis of oligosaccharides similarly glycosyltransferases or glycosidases, to gen- opment

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