REVIEW ARTICLE PUBLISHED ONLINE: 23 OCTOBER 2009 | DOI: 10.1038/NCHEM.399 Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research Thomas J. Boltje, Therese Buskas and Geert-Jan Boons* Synthetic oligosaccharides and glycoconjugates are increasingly used as probes for biological research and as lead compounds for drug and vaccine discovery. These endeavours are, however, complicated by a lack of general methods for the routine prepara- tion of these important compounds. Recent developments such as one-pot multi­step protecting-group manipulations, the use of unified monosaccharide building blocks, the introduction of stereoselective glycosylation protocols, and convergent strategies for oligosaccharide assembly, are beginning to address these problems. Furthermore, oligosaccharide synthesis can be facili- tated by chemo-enzymatic methods, which employ a range of glycosyl transferases to modify a synthetic oligosaccharide pre- cursor. Glycosynthases, which are mutant glycosidases, that can readily form glycosidic linkages are addressing a lack of a wide range of glycosyltransferases. The power of carbohydrate chemistry is highlighted by an ability to synthesize glycoproteins. here is a growing appreciation that post-translational modifi­ glycoproteins and glycolipids29,30. These compounds, which are cations, such as glycosylation, dramatically increase protein equipped with an artificial aminopropyl spacer, have been covalently Tcomplexity and function1–6. For example, almost all cell sur- attached to N‑hydroxysuccinimide-activated glass slides and the face and secreted proteins are modified by covalently linked carbo- resulting microrray has found wide use for profiling the specificity hydrate moieties, and the glycan structures on these glycoproteins of a diverse range of glycan-binding proteins, such as C‑type lectins, have been implicated as essential mediators in processes such as siglecs, galectins and anticarbohydrate antibodies. Glycan array tech- protein folding, cell signalling, fertilization, embryogenesis, neu- nology has also been employed for rapid assessment of influenzae ronal development, hormone activity and the proliferation of cells virus receptor specificity due to the species-specific nature of the and their organization into specific tissues. In addition, overwhelm- interaction between virus and host glycans31. ing data supports the relevance of glycans in pathogen recognition, inflammation, innate immune responses and the development of Synthetic heparin and heparan sulfate fragments. The power of autoimmune diseases and cancer7–10. The importance of protein gly- organic carbohydrate synthesis has also been demonstrated by the cosylation is also underscored by the developmental abnormalities development of a fully synthetic heparin fragment used for the treat- observed in a growing number of human disorders known as con- ment of deep vein thrombosis in humans32–34. Heparin and heparan genital disorders of glycosylation, caused by defects in the glyco­ sulfate are naturally occurring poly­disperse linear polysaccharides sylation machinery11. that are heavily O‑ and N‑sulfated5. For more than eighty years, Polysaccharides are major constituents of the microbial cell sur- heparin isolated from porcine mucosal tissue has been used as an faces and, for example, the bacterial cell wall can contain relatively anticoagulant drug. It exerts its activity by binding to the plasma large amounts of capsular polysaccharides (CPS) or lipopolysaccha- protein antithrombin III (ATIII) causing a conformational change rides (LPS)12. These components are important virulence factors in that results in its activation through an increase in the flexibility of that they promote bacterial colonization, block phagocytosis and its reactive site loop. The activated ATIII then inactivates thrombin interfere with leukocyte migration and adhesion. CPS and LPS can and other proteases involved in blood clotting, most notably factor be recognized by receptors of the innate immune system leading Xa. The ATIII-binding region of heparin consists of a unique penta- to the production of cytokines, chemokines and cellular adhesion saccharide domain. A fully synthetic analogue (Arixtra, Fig. 1a) molecules13–16. With a few exceptions, bacterial polysaccharides can of this domain has been developed, which is being produced on a induce an adaptive immune response and, not surprisingly, bacte- multikilogram scale for the treatment of deep vein thrombosis32,33. rial saccharides have been employed for the development of vac- Unlike heparin, the synthetic derivative is easy to characterize and cines for several pathogens17–20. has a much-improved subcutaneous bioavailability. Several other synthetic heparin fragments are being devel- Synthetic oligosaccharides for glycobiology oped for anticoagulation therapy, and the most exciting example A major obstacle in glycobiology and glycomedicine is the lack of is Idraparinux (Fig. 1b)32. This compound, which contains methyl pure and structurally well-defined carbohydrates and glycoconju- ethers instead of hydroxyls and O‑sulfates instead of N‑sulfates, is gates21. These compounds are often found in low concentrations much easier to synthesize than Arixtra, has an increased half-life and in microheterogeneous forms, greatly complicating their isola- and a much improved potency. The latter property seems to be due tion and characterization. In many cases, well-defined oligosaccha- to the presence of the methyl ethers, which induce the biologically 22–28 2 rides can only be obtained by chemical or enzymatic synthesis active S0 conformation of the iduronic acid moiety. and such compounds are increasingly used to address important The importance of synthetic heparin for anticoagulation ther- problems in glycobiology research and for vaccine and drug discov- apy was recently highlighted by the discovery of batches of heparin ery. For example, the Consortium of Functional Glycomics (CFG) that were associated with anaphylactoid-type reactions, lead- has employed a chemoenzymatic approach for the preparation of a ing to hypotension and which resulted in nearly 100 deaths35,36. library of over 400 oligosaccharides derived from N‑ and O‑linked These adverse reactions were traced to contamination with a Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia, USA. *e-mail: [email protected] NATURE CHEMISTRY | VOL 1 | NOVEMBER 2009 | www.nature.com/naturechemistry 611 © 2009 Macmillan Publishers Limited. All rights reserved. REVIEW ARTICLE NAtUrE CHEMIstrY DOI: 10.1038/NCHEM.399 a b 2621D 2621D 2 2 2 2 +2 20H 1D262 2621D 2+ 2621D 2 1D26+1 2 1D262 1D2& 20H 1D2& 20H +2 2 0H2 2 0H2 +2 2 2621D 2 20H &21D 1D26+1 &21D 1D262 1D26+1 0H2 2 2 1D262 2 2 1D262 +2 2 2 0H2 2 2 +2 0H2 2621D 2621D c +2 2 2 2+ +2 2 2 2 2+ 2+ 2 1D2 3 2 2+ 2+ 2 2+ 6 + 2 3 2 2+ 1 1 2 2+ 2 1D2 2+ 2 2 Q a 2 +1 7HWDQXVWR[RLG FDUULHUSURWHLQ P d 66(W )PRF 4$//916643 2 66(W )PRF :(3/4/+9'.$96*/56/77//5$/*$4.($,633 2 66(W )PRF '$$6$$3 2 + /57,7$'7)5./)59<61)/5*././<7*($&57*'5 7&(3+&O+2%W',3($3LSHULGLQH 7&(3+&O+2%W',3($3LSHULGLQH 7&(3+&O+2%W',3($3LSHULGLQH 4$//916643:(3/4/+9'.$96*/56/77//5$/*$4.($,633'$$6$$3/57,7$'7)5./)59<61)/5*././<7*($&57*'5 Figure 1 | Examples of biologically active synthetic oligosaccharide and glycopeptide constructs. a, Structure of the anticoagulant drug Arixtra. b, Structure of the promising anticoagulant candidate drug Idraparinux, in which the methyl ethers are highlighted in blue. c, Synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b. d, Synthesis of the erythropoietin (EPO; from amino acids 78‑166) glycopeptide through sequential fragment condensation using auxiliary-based cysteine-free native chemical ligation. Glycans are shown in red and the ligation sites are shown in bold, amino acids are represented by their single-letter codes. DIPEA, N,N-diisopropylethylamine; HOBt, 1‑hydroxybenzotriazole; TCEP-HCl, tris(2-carboxyethyl)phosphine hydrochloride. semi-­synthetic over-sulfated chondroitin sulfate, which is a popu- synthetic analogues of heparin may find application in the treat- lar shellfish-derived oral supplement for arthritis. ment of several neurodiseases, cancer and infection33,34,37–42. There is a growing body of literature indicating that glycosyl­ aminoglycans (GAGs), such as heparin and heparan sulfate, can Carbohydrate-based prophylactic and therapeutic vaccines. have profound physiological effects on lipid transport and adsorp- Synthetic oligosaccharide epitopes offer promising possibilities tion, cell growth and migration and development5. Alterations in for the development of vaccines for the prevention of infectious GAG expression has been associated with cancer and, for example, diseases such as Haemophilus influenzae type b, HIV, Plasmodium significant changes in the structure of GAGs has been reported in falciparum, Vibrio cholerae, Cryptococcus neoformans, Streptococcus the stroma surrounding tumours, which is important for tumour pneumoniae, Shiga toxin, Neisseria meningitides, Bacillus anthracis growth and invasion. GAGs also have important neurobiological and Candida albicans17,18,43,44. Natural polysaccharides conju- functions and examples include neuroepithelial growth and differ- gated to carrier proteins have been successfully developed as entiation, neurite outgrowth, nerve regeneration, axonal guidance human vaccines, however, their use is associated with problems and branching, deposition of amyloidotic