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Reactivity-Based One-Pot Synthesis of Oligosaccharides for the Development of a Photocleavable Sugar Array Jinq-Chyi Lee1, Chung-Yi Wu1,2, Junefredo V. Apon3, Gary Siuzdak3, and Chi-Huey Wong1,2* Abstract A major challenge in proteomics is to understand the functional impact of posttranslational modification, and protein glycosylation represents the most complex post-translational event. More than 50% of human proteins are glycosylated; however, the role of carbohydrates in glycoproteins is relatively unknown, due to the lack of tools for study. Carbohydrate arrays provide a solution to this long-standing problem. Using the programmable reactivity-based one-pot synthesis of oligosaccharides developed by Chi-Huey Wong allows a rapid access to a large number of oligosaccharides which have been very difficult to obtain by other means. These saccharides were then printed on silicon surface with a photocleavable linker for direct characterization by mass spectrometry and used for the high-throughput analysis of sugar-protein interaction. A major challenge in proteomics is to understand the functional impact of post-translational modification and of which protein glycosylation represents the most complex event. At the same time, increasing numbers of receptors are being characterized that operate through binding to specific oligosaccharide sequences on glycoproteins, glycolipids and polysaccharides. Such receptors are involved in the folding of nascent proteins, the subcellular targeting of enzymes, mechanisms of infection (microbe-host interactions), inflammation, cancer metathesis, differentiation and development, and immunity. More than 50% of human proteins are glycosylated; however, the role of carbohydrates in glycoproteins is relatively unknown, due to the lack of tools for study. The search for carbohydrate ligands or receptors and their functions remains, however, one of the most challenging areas in cell Figure 1. Schematic representation of (a) solid-phase and (b) programmable one-pot approaches to oligosaccharide synthesis. biology. The conventional approaches to carbohydrate ligand discovery are cumbersome, and there is a great need for sen- Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, CA, U.S.A. The Genomics Research Center, Academia Sinica, Taipei, Taiwan 3 Department of Molecular Biology and Chemistry and the Scripps Center for Mass Spectrometry, The Scripps Research Institute, CA, U.S.A. 1 2 65 ACADEMIA SINICA sitive, high-throughput technologies that allow rapid analyses of carbohydrate-protein interactions. Development of carbohydrate microarrays would provide a new tool for this study and for the identification of carbohydrate receptors or ligands associated with cancer metathesis, bacterial or viral infection, immune response, differentiation and many other intercellular recognition processes. Carbohydrate microarray technologies, analo- Figure 2. Programmed one-pot synthesis of Globo H oligosaccharide. gous to those developed for DNA and being developed for proteins, are new developments at the fron- hydrates but also elucidation of their ligands and tiers of glycomics and ideal for addressing this mechanisms. need. Only small amounts of product are required Unlike peptides and nucleic acids, oligosac- for fabricating microarrays, and many compounds charides are difficult to synthesize chemically. This can be screened in parallel in a single operation. is because some oligosaccharide chains are linear, Since the introduction of gene arrays, genomic others are branched, the glycosidic linkages are in research has progressed rapidly and various genetic α or β anomeric configurations, and adjacent variations related to diseases have been identified. monosaccharides are linked via different hydroxyl However, the result of gene array analysis may not groups in their sugar rings. The possibility of form- be directly correlated with the function at the pro- ing a tetrasaccharide from the nine common mono- tein level. Understanding the relationship between saccharides found in humans is in the order of 15 genetic changes and disease states represents a new millions, significantly higher than the correspon- challenge, and functional genomics or proteomics ding tetramers of peptides or nucleic acids, though has thus become an important subject for study. nature probably only uses a small fraction of these Results of 'proof of concept' experiments with car- tetramers. This degree of diversity makes the syn- bohydrate-binding proteins of the immune system – thesis of oligosaccharides very difficult, and multi- antibodies, selectins, cytokines and chemokines – ple selective protection and deprotection steps are and several plant lectins indicate that microarrays of required for the hydroxyl groups of monosaccha- carbohydrates (glycoconjugates, oligosaccharides rides during chemical synthesis of oligosaccharides. and monosaccharides) will greatly facilitate not An automated solid-phase method has been devel- only the discovery of proteins that recognize carbo- oped to facilitate the synthesis with elimination of intermediate isolation and purification, but the requirement of protecting group manipulation still exists. An alternative approach to the synthesis of oligosaccharides is a programmable reactivitybased ‘one-pot’ approach. The approach is a newly developed strategy which eliminates protecting group manipulation completely to further simplify the synthetic procedures. In this approach, an Figure 3. Methods for covalent immobilization of sugars on N-hydroxy succinimide ester-coated glass slides. ACADEMIA SINICA 66 oligosaccharide of interest is generated by the sequential addition of building blocks (thioglyco- sides) that are either fully protected or have one hydroxyl group exposed, and each building block has defined relative reactivity values (RRVs). It has been shown that the RRV of a thioglycoside building block in the glycosidation reaction can be tuned in the presence of protecting groups; more than 400 building blocks, with RRVs ranging from 1 to 105, have been designed and synthesized, and stored as a reactivity database. A computer programme called ‘Optimer’ has thus been developed to guide the selection of building blocks for the one-pot synthesis of a given oligosaccharide. Based on this database and simple chemistry logic, an appropriate Figure 4. Binding of monoclonal antibodies Mbr1 (b) and VK-9 (c) to Globo H and truncated sequences. combination of thioglycoside building blocks can be identified from the database for any given oligosaccharide structure and used in a sequential manner for the one-pot synthesis. If RRVs differ by more than 102, the desired glycosidic bonds can be formed by the sequential addition of building blocks in the order of the RRVs. Different from the qualitative one-pot synthesis, the reactivity of each thioglycoside building block is determined quantitatively. These RRVs form a reactivity database that, together with the “Optimer” computer program, provides an automatic methodology for the reactivity-based one-pot synthesis of oligosaccharides. Once the required building blocks with pro- Figure 5. Representative glycan structures on the array. tecting groups are prepared, oligosaccharides can be synthesized in a short period of time (in minutes or hours, instead of days or months using traditional methods) using this programmable one-pot approach. We have thus successfully prepared a small oligosaccharide library, the breast cancer antigenic determinant Globo H, the colon cancer antigen Lewis Y, and the lung cancer antigen fucosylGM1. Desorption/ionization on silicon mass spectrometry (DIOS-MS) is an ionization method that uses a porous silicon surface to generate gas-phase ions of small molecules (<3000 Da) without a matrix. With this technique, the process of sample Figure 6. Glycan microarray analyses of Viet 04 and DK 97 avian influen- manipulation is minimized and it only requires very za virus. 67 ACADEMIA SINICA biomedical interest become available for investigation, the evaluation of their carbohydrate binding specificity will be greatly helped by carbohydrate microarrays. In summary, the work reported by Wong and postdoctoral associates Chung-Yi Wu and others has solved a long-standing problem in carbohydrate array development. The one-pot synthesis allows a Figure 7. Preparation and analysis of a photocleavable rapid access to a large number of oligosaccharides DIOS-MS sugar array. which have been very difficult to obtain by other small volumes for analysis. The carbohydrates syn- means. In addition, with attachment of a photo- thesized are then printed on the surface of modified cleavable linker between the sugar moiety and glass porous silicon with a photocleavable linker, which surface, the array can be characterized directly by could be cleaved by a laser (=337 nm) in the mass mass spectrometry, as a mass spectrometer is spectrometric analysis to detect the carbohydrates equipped with a 337-nm laser which cleaves the on the porous silicon. sugar from the glass surface for direct characteriza- The characterizable carbohydrate microarrays tion. According to the method, one milligram of described in this report hold great promise as a saccharides can be used for 100-million spots on the high-throughput means for detecting the interac- surface and each glass slide contains thousands of tions of proteins with diverse oligosaccharide spots, each with different saccharides. This new sequences of glycoproteins, glycolipids and poly- breakthrough — the rapid synthesis of oligosaccha- saccharides. The development should find use in rides and attachment of them to glass slides through detailed characterizations of carbohydrate-protein a photosensitive linker — is expected to replace the interaction and would lead to or be accompanied by traditional ELISA method for diagnosis and to facil- structural and functional studies of such recogni- itate carbohydrate-based drug discovery. tions. As more and more recombinant proteins of The original paper was published in Angewandte Chemie International Edition 45 (2006): 2753-2757. References: 1. Z. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat T. Baasov, C.-H. Wong, (1999) J. Am. Chem. Soc. 121, 734-753. 2. P. Sears, C.-H. Wong, (2001) Science 291, 2344-2350. 3. K.-K.T. Mong, C.-H. Wong, (2002) Angew. Chem. Int. Ed. 41, 4087-4090. 4. T. K-K. Mong, H. K. Lee, S.G. Duron, C.-H. Wong, (2003) Proc. Natl. Acad. Sci. USA 100, 797-802. 5. T. Feizi, F. Fazio, W. Chai, C.-H. Wong, (2003) Curr. Opin. Struct. Biol. 13, 637-645 (2003). 6. H.-K. Lee, C. N. Scanlan, C.-Y. Huang, A. Y. Chang, D. A. Calarese, R. A. Dwek, P. M. Rudd, I. A. Wilson, D. R. Burton, C.-H. Wong, (2004) Angew. Chem. Int. Ed. 43, 1000-1003. 7. M. C. Bryan, F. Fazio, H.-K. Lee, C.-Y. Huang, A. Chang, M. D. Best, D. A. Calarese, O. Blixt, J. C. Paulson, D. Burton, I. A. Wilson, C.-H. Wong, (2004) J. Am. Chem. Soc. 126, 8640-8641 (2 8. O. Blixt, S. Head, T. Mondala, C. Scanlan, M. E. Huflejt, R. Alvarez, M. C. Bryan, F. Fazio, D. Calarese, J. Stevens, N. Razi, I. van Die, D. Burton, I. A. Wilson, R. Cummings, N. Bovin, C.-H. Wong, J. C. Paulson, (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 1703317038. 9. S. R. Hanson, M. D. Best, M. C. Bryan, C.-H. Wong, (2004) Trends Biochem. Sci. 29, 656-663. 10. C.-Y. Huang, D. A. Thayer, A. Y. Chang, M. D. Best, J. Hoffman, S. Head, C.-H. Wong, (2006) Proc.Natl. Acad. Sci. U.S.A. 103(1), 15-20. 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