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Emerging Themes in Medicinal Glycoscience

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Emerging Themes in Medicinal Glycoscience © 2000 Nature America Inc. • http://biotech.nature.com REVIEW Emerging themes in medicinal glycoscience Kathryn M. Koeller and Chi-Huey Wong* Department of Chemistry, The Scripps Research Institute and Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037. *Corresponding author ([email protected]). Received 17 May 2000; accepted 5 July 2000 The recognition of complex carbohydrates and glycoconjugates as mediators of important biological processes has stimulated investigation into their therapeutic potential. New approaches for the simplifi- cation of glycoconjugate synthesis are overcoming the limitations of existing methods and providing a diverse array of these biomolecules. As the accessibility of glycoconjugates increases, carbohydrate- based constructs are becoming available for analysis as medicinal agents in a wide range of therapies. Keywords: glycoconjugate, synthesis, therapeutic, antibiotic, vaccine .com Numerous medicinally relevant physiological events rely on gly- difficult task, as carbohydrates are densely functionalized with h.nature coconjugates for their viability1–3. The recognition of glycopro- hydroxyl groups of similar reactivity. Synthetic carbohydrate cessing enzymes as targets for therapeutic intervention has chemists will attest to the many hurdles encountered in obtaining spurred the development of numerous drug candidates4,5, includ- even relatively simple oligosaccharides, including laborious protect- ing the well-known “flu” drugs that inhibit influenza virus neu- ing-group manipulations and long overall synthetic routes. In addi- http://biotec • raminidase. Likewise, the discovery of the selectins attracted tion, the degree of molecular diversity that can be generated from researchers to the study of carbohydrate–protein interactions in glycosidic linkage assembly is enormous: more than 10 million the inflammatory response6. The observation that cell surface gly- tetrasaccharides can be assembled from the 9 common monosac- coforms are altered in certain cancers has served as both a diag- charides, compared with 256 tetranucleotides and 16,000 tetrapep- nostic tool and the foundation of glycoconjugate-based vaccine tides from the corresponding 4 nucleotides and 20 amino acid development7. Glycans provide required recognition elements of building blocks, respectively. Consequently, advancing glycoconju- many antipathogenic agents8, and the glycopolymers heparin and gate synthetic technology to the same level as proteins and nucleic hyaluronic acid are employed clinically as anticoagulants9 or acids remains a daunting challenge. arthritis therapies, respectively. Furthermore, protein glycosyl On a macromolecular scale, various cellular expression systems phosphatidylinositol (GPI) anchors10 and polylactosamine have proven useful for protein production. Yet, obtaining homoge- 2000 Nature America Inc. 11 β © chains play structural roles on the cell surface, and a nuclear - neous glycoproteins is not a trivial matter. Glycosylation is a post- O-GlcNAc modification may function in signal transduction translational modification, rather than falling under direct tran- pathways12. Moreover, the glycoprotein hormone erythropoeitin, scriptional control, and glycan structure is therefore subject to sev- used for clinical treatment of anemia, requires sialyl oligosaccha- eral environmental factors. Enzyme competition for the same sub- rides for optimal activity in vivo13. strate, enzyme substrate specificity and availability, as well as variant Examination of the growing repertoire of glycoconjugate func- glycosylation patterns with cellular host and culture conditions all tion provides assurance that this field will be at the forefront of play a role in the form of the resultant glycan14. Glycoproteins research for years to come. Therapeutic agents based on carbohy- obtained from prokaryotic or eukaryotic expression systems there- drates or biological pathways in which they are involved have fore exhibit glycoform microheterogeneity15. This makes the contri- become important pharmaceutical targets. This review highlights bution of specific glycan structure to underlying protein structure areas in which glycoconstructs have been investigated for medicinal and function nearly impossible to assess. applications. Newly developed synthetic strategies Problems in the development of glycotherapeutics The ongoing investigation of carbohydrate-based therapeutic agents Many reliable options are available to protein and nucleic acid scien- is dependent on the development of reliable synthetic methods to tists for procuring a particular biomolecule. Automated synthesizers make glycoconjugates more readily available on a large scale. Toward allow the manufacture of short peptide and nucleic acid sequences, this end, the incorporation of enzymes into synthetic protocols has and expression systems and polymerase chain reaction (PCR) make the potential to drastically reduce the number of protecting-group obtaining large quantities of macromolecular material a trivial con- manipulations and synthetic steps required16–18. sideration. Unfortunately, this is not the case for oligosaccharides Glycosyltransferases, especially combined with regeneration of and other glycoconjugates. sugar nucleotides (Fig. 1A), have great synthetic utility in the for- This discrepancy largely results from the intrinsic structure of mation of glycosidic linkages16–18. Furthermore, genetic manipu- carbohydrates themselves. To synthesize peptides or oligonu- lation of the biosynthetic pathways in microorganisms has cleotides, a method is available for iterative formation of a single improved the production of sugar nucleotides19. The use of pro- type of bond (i.e., peptide or phosphodiester bond, respectively). teases as catalysts in peptide bond formation20 and lipases for the However, synthesis of specific glycosidic linkages is a much more mild removal of protecting groups21 have been extremely benefi- NATURE BIOTECHNOLOGY VOL 18 AUGUST 2000 http://biotech.nature.com 835 © 2000 Nature America Inc. • http://biotech.nature.com REVIEW Figure 1. Selected approaches for A B the synthesis of complex glyco- conjugates. (A) The combination of glycosyltransferase-catalyzed reactions and the recycling of sugar nucleotides for the synthesis of complex carbo- hydrates. (B) The regeneration of PAPS for the sulfation of oligosaccharides and other glycoconjugates. (C) Strategies for the construction of homo- geneous glycoproteins using endoglycosidases, proteases, and glycosyltransferases. (D) One-pot programmable method for the synthesis of oligosaccharides. C D .com h.nature http://biotec • cial in the area of glycopeptide synthesis18, as well as for site-spe- Although these unique one-pot and enzymatic synthetic meth- cific esterification22. Aldolases have provided access to numerous ods have proven utility in the generation of glycoconjugates, tradi- unnatural carbohydrate derivatives16,23, and directed evolution is tional chemical synthesis continues to be a backbone staple. The yielding novel catalysts with altered substrate specificity (S. Fong following sections outline various approaches to carbohydrate- & C.-H. Wong, unpublished data). New enzymatic protocols for based therapeutics that were possible through chemical and 2000 Nature America Inc. ′ ′ © the recycling of 3 -phospho adenosine 5 -phosphosulfate (PAPS) chemoenzymatic synthetic approaches. as a sulfotransferase donor substrate have been developed (Fig. 1B) and will further allow elucidation of the role of glycoconju- Antiviral agents gate sulfation24. Endoglycosidases, proteases, and glycosyltrans- The influenza virus is one of the best-known pathogens dependent on ferases can be sequentially employed to produce glycoproteins carbohydrate-recognition for its infectivity. The viral hemagglutinin with homogeneous glycoforms (Fig. 1C)20. As methods for the protein is responsible for initial recognition of host cell surface sialic construction of homogeneous glycoproteins are greatly needed, acid (NeuAc), which is subsequently cleaved by the viral neuraminidase, other synthetic avenues, such as native peptide ligation25, intein- allowing infection to proceed29,30. Anti-influenza drug design has catalyzed peptide bond formation26, and endoglycosidase-cat- focused on the inhibition of both hemagglutinin and neuraminidase, alyzed transglycosylation have also been explored18,27 (Fig. 1C). At though in markedly different strategies (Fig. 2). Hemagglutinin exists as present, enzymes specific for every desired linkage are not cur- a homotrimer present in multiple copies on the viral cell surface and, in rently available, and strategies combining chemical and enzymat- accordance, the most potent hemagglutinin inhibitors are polyvalent ic synthetic methods have been generally advantageous. constructs31,32. Inhibition of neuraminidase, in contrast, has relied on On the chemical forefront, many research groups have reported mimicking the transition state of the hydrolytic reaction by rational “one-pot” strategies for the synthesis of oligosaccharides18. These design. Several of these transition-state analogs are potent neu- methods involve the selection of glycosyl donors that will react in a raminidase inhibitors, and are now on the market33,34. predefined order, thus potentially simplifying the overall synthetic Galactosyl-ceramide (Gal-Cer) on host cells is employed as an procedure. The culmination of these efforts has resulted in the alternate receptor for HIV-1,
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