Scientific Report 2006

Chemistry

Bioorganic and Synthetic Chemistry

C.-H. Wong, C. Bennett, A. Brik, Y.-H. Chen, S. Dean, S. Ficht, M. Fujio, W. Greenberg, R. Guy, S. Hanson, Z. Hong, T.-L. Hsu, D.-R. Hwang, J.-C. Lee, P.-H. Liang, L. Liu, T. Polat, M. Sawa, M. Sugiyama, D. Thayer, S.-K. Wang, L. Whalen, C.-Y. Wu, D. Wu, M. Wuchrer, Y.-Y. Yang

Our research programs involve development of new chemical and enzymatic strategies and methods for the synthesis of biologically active compounds. We use the synthesized compounds as molecular probes to explore carbohydrate-mediated biological recognition events and enzymatic reactions.

Organic and Bioorganic Synthesis

Our work in organic and bioorganic synthesis includes the development of new chemical reactions and the exploitation of native and engineered enzymes for organic synthesis. In the past year, we developed new methods for making glycopeptides and glycoproteins via native chemical ligation methods. We will use these methods to synthesize homogenous glycoproteins that are important therapeutic agents in humans. We continue to develop covalent glycoarrays for high-throughput analysis of protein-carbohydrate interactions and the use of aldolases in the synthesis of glycosyltransfer enzyme inhibitors. Using directed evolution, we developed new aldolase variants capable of making both enantiomers of sugars.

Development of Inhibitors of Enzymes and Receptors

Our goals in the area of enzyme and receptor inhibitors are to develop new strategies to discover inhibitors and ligands with high selectivity as potential therapeutic agents. Current strategies involve the design and synthesis of structure- and mechanism-based inhibitors of enzymes associated with a variety of diseases. Targets for investigation include bacterial transglycosidase, sulfotransferases, retroviral proteases, lethal factor of Bacillus anthracis, and enzymes involved in the biosynthesis of carbohydrates essential for biological functions.

We have developed new iminocyclitols and derivatives as inhibitors of glycosidases and glycosyltransferases for potential treatment of inflammatory diseases. In addition, using a new strategy based on a rapid microscale synthesis coupled with in situ high-throughput screening, we developed new tight-binding inhibitors of anthrax lethal factor, a sulfotransferase, and drug-resistant HIV proteases. We also developed new reactions based on tetrabutylammonium fluoride–mediated N- and O-alkylation and epoxide opening in aqueous solution and used the reactions to identify potent enzyme inhibitors.

Carbohydrate Chemistry and Molecular Glycobiology

We continue to improve the programmable 1-pot oligosaccharide synthesis method for convenient and rapid preparation of oligosaccharides. So far, we have designed approximately 600 building blocks and measured the anomeric reactivity of each building block. Using the computer program OptiMer, developed in our laboratory, we rapidly assembled a number of oligosaccharides. We are using this method to define the specificity of interactions between carbohydrates and their receptors, with particular focus on optimization of the cancer antigen Globo H and HIV gp120 oligomannose as vaccine candidates. In collaboration with D.R. Burton, Department of Immunology, and I.A. Wilson, Department of Molecular Biology, we are evaluating a designed oligomannose-protein conjugate as an antigen to elicit antibodies for neutralizing HIV gp120 and variants.

We synthesized a series of bacterial glycolipids and analogs and found that they are active ligands for CD cell markers involved in activation of human natural killer T cells. These compounds may be useful as immunotherapeutic agents for the treatment of bacterial and viral infections, as well as cancer, and we are elucidating the structural basis for their activity. In collaboration with Dr. Wilson, we are determining the structures of the complexes formed by these glycolipids and CD1d; the results will assist in designing ligands with improved therapeutic potential. In collaboration with J.C. Paulson, Department of Molecular Biology, we developed new methods for microfabrication of saccharides on glass slides or microtiter plates for use in high-throughput analysis of sugar-protein interactions.

Publications

Brigl, M., van den Elzen, P., Chen, X., Meyers, J.H., Wu, D., Wong, C.-H., Reddington, F., Illarianov, P.A., Besra, G.S., Brenner, M.B., Gumperz, J.E. Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors. J. Immunol. 176:3625, 2006.

Brik, A., Ficht, S., Wong, C.-H. Strategies for the preparation of homogenous glycoproteins. Curr. Opin. Chem., in press.

Brik, A., Wu, C.-Y., Wong, C.-H. Microtiter plate based chemistry and in situ screening: a useful approach for enzymatic inhibitor discovery. Org. Biomol. Chem. 4:1446, 2006.

Brik, A., Yang, Y.-Y., Ficht, S., Wong, C.-H. Sugar-assisted glycopeptide ligation. J. Am. Chem. Soc. 128:5626, 2006.

Calarese, D.A., Lee, H.-K, Huang, C.-Y., Best, M.D., Astronomo, R.D., Stanfield, R.L., Katinger, H., Burton, D.R., Wong, C.-H., Wilson I.A. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc. Natl. Acad. Sci. U. S. A. 102:13372, 2005.

Cheng, Y.-S.E., Lo, K.-H., Hsu, H.-H., Shao, Y.-M., Yang, W.-B., Lin, C.-H., Wong, C.-H. Screening for HIV protease inhibitors by protection against activity-mediated cytotoxicity in Escherichia coli. J. Virol. Methods 137:82, 2006.

Chuang, M.-H., Wu, M.-S., Lo, W.-L., Lin, J.-T., Wong, C.-H., Chiou, S.-H. The antioxidant protein alkylhydroperoxide reductase of Helicobacter pylori switches from a peroxide reductase to a molecular chaperone function. Proc. Natl. Acad. Sci. U. S. A. 103:2552, 2006.

Fujio, M., Wu, D., Garcia-Navarro, R., Ho, D.D., Tsuji, M., Wong, C.-H. Structure-based discovery of glycolipids for CD1d-mediated NKT cell activation: tuning the adjuvant versus immunosuppression activity. J. Am. Chem. Soc. 128:9022, 2006.

Hong, Z.-Y., Liu, L., Hsu, C.-C., Wong, C.-H. Three-step synthesis of sialic acids and derivatives. Angew. Chem. Int. Ed., in press.

Hsu, H.-Y., Hua, K.-F., Su, Y.-C., Chu, L.-C., Su, S.-C., Chiu, H.-W., Wong, C.-H., Chen, S.-T., Shieh, C.-W., Yang, S.-S., Chen, Y.-M., Chao, L.K. Alkali-soluble polysaccharides of Rhizoclonium riparium alga induces IL-1 gene expression via protein kinase signaling pathways. J. Agric. Food Chem. 54:3558, 2006.

Huang, C.-Y., Thayer, D.A., Chang, A.Y., Best, M.D., Hoffmann, J., Head, S., Wong, C.-H. Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen. Proc. Natl. Acad. Sci. U. S. A. 103:15, 2006.

Huang, K.-T., Wu, B.-C., Lin, C.-C., Luo, S.-C., Chen, C., Wong, C.-H., Lin, C.-C. Multi-enzyme one-pot strategy for the synthesis of sialyl Lewis X-containing PSGL-1 glycopeptide. Carbohydr. Res. 341:2151, 2006.

Kinjo, Y., Tupin, E., Wu, D., Fujio, M., Garcia-Navarro, R., Benhnia, M.R.E.I., Zajonc, D.M., Ben-Menachem, G., Ainge, G.D., Painter, G.F., Khurana, A., Hoebe, K., Behar, S.M., Beutler, B., Wilson, I.A., Tsuji, M., Sellati, T.J., Wong, C-H., Kronenberg, M. Natural killer T cells recognize diacylglycerol antigens from pathogenic bacteria. Nat. Immunol. 7:978, 2006.

Lee, J.-C., Wu, C.-Y., Apon, J.V., Siuzdak, G., Wong, C.-H. Reactivity-based one-pot synthesis of the tumor-associated antigen N3 minor octasaccharide for the development of a photocleavable DIOS-MS sugar array. Angew. Chem. Int. Ed. 45:2753, 2006.

Liang, F.-S., Brik, A., Lin, Y.-C., Elder, J.H., Wong, C.-H. Epoxide opening in water and screening in situ for rapid discovery of enzyme inhibitors in microtiter plates. Bioorg. Med. Chem. 14:1058, 2006.

Liang, P.-H., Cheng, W.-C., Lee, Y.-L., Yu, H.-P. Wu, Y.-T., Lin, Y.-L., Wong, C.-H. Novel five-membered iminocyclitol derivatives as selective and potent glycosidase inhibitors: new structures for antivirals and osteoarthritis. Chembiochem 7:165, 2006.

Liang, F.-S., Greenberg, W.A., Hammond, J.A., Hoffmann, J., Head, S.R., Wong, C.-H. Evaluation of RNA-binding specificity of aminoglycosides with DNA microarrays. Proc. Natl. Acad. Sci. U. S. A. 103:12311, 2006.

Lin, K.-I., Kao, Y.-Y., Kuo, H.-K., Yang, W.-B., Chou, A., Lin, H.-H., Yu, A.L-T., Wong, C.-H. Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2-mediated induction of transcription factor Blimp-1. J. Biol. Chem. 281:24111, 2006.

Lin, Y.-C., Brik, A., de Parseval, A., Tam, K., Torbett, B.E., Wong, C.-H., Elder, J.H. Altered gag polyprotein cleavage specificity of feline immunodeficiency virus/human immunodeficiency virus mutant proteases as demonstrated in a cell-based expression system. J. Virol. 80:7832, 2006.

Liu, L., Bennett, C.S., Wong, C.-H. Advances in glycoprotein synthesis.Chem. Commun. (Camb.) 21, 2006, Issue 1.

Liu, L., Hong, Z.-Y., Wong, C.-H. Convergent glycopeptide synthesis by traceless Staudinger ligation and enzymatic coupling. Chembiochem 7:429, 2006.

Liu, H., Wong, C.-H. Characterization of a transglycosylase domain of Streptococcus pneumoniae PBP1b. Bioorg. Med. Chem. 14:7187, 2006.

Qiul, H., Gabrielsen, A., Agardh, H.E., Wan, M., Wetterholm, A., Wong, C.-H., Hedin, U., Swedenborg, J., Hansson, G.K., Samuelsson, B., Paulsson-Berne, G., Haeggstrom, J.Z. Expression of 5-lipoxygenase and leukotriene A₄ hydrolase in human atherosclerotic lesions correlates with symptoms of plaque instability. Proc. Natl. Acad. Sci. U. S. A. 103:8161, 2006.

Sanna, M.G., Wang, S.-K., Gonzalez-Cabrera, P.J., Don, A., Marsolais, D., Matheu, M.P., Wei, S.H., Parker, I., Jo, E., Cheng, W.-C., Cahalan, M.D., Wong, C.-H., Rosen, H. Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P₁ antagonist in vivo. Nat. Chem. Biol. 2:434, 2006.

Sawa, M., Hsu, T.-L., Itoh, T., Sugiyama, M., Hanson, S.R., Vogt, P.K., Wong, C.-H. Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo. Proc. Natl. Acad. Sci. U. S. A. 103:12371, 2006.

Sawkar, A.R., Adamski-Werner, S.L., Cheng, W.-C., Wong, C.-H., Beutler, E., Zimmer, K.-P., Kelly, J.W. Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles. Chem. Biol. 12:1235, 2005.

Scanlan, C., Calarese, D., Lee, H.K., Blixt, O., Wong, C.-H., Wilson, I., Burton, D., Dwek, R., Rudd P. Antibody recognition of a carbohydrate epitope: a template for HIV vaccine design. Adv. Exp. Med. Biol. 564:7, 2005.

Shie, J.-J., Fang, J.-M., Kuo, T.-H., Kuo, C.-J., Liang, P.-H., Huang, H.-J., Wu, Y.-T., Jan, J.-T., Cheng, Y.-S.E., Wong, C.-H. Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic α,β-unsaturated esters. Bioorg. Med. Chem. 13:5240, 2005.

Thayer, D., Wong, C.-H. Vancomycin analogs with improved biological activity: a combined one-pot enzymatic glycosylation and chemical diversification strategy. Chem. Asian J., in press.

Wei, S.H., Rosen, H., Matheu, M.P., Sanna, M.G., Wang, S.-K., Jo, E., Wong, C.-H., Parker, I., Cahalan, M.D. Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses. Nat. Immunol. 6:1228, 2005.

Whalen, L.J., Wong, C.-H. Enzymes in organic synthesis: aldolase-mediated synthesis of iminocyclitols and novel heterocycles. Aldrichim. Acta 39:63, 2006.

Wu, C.-Y., Brik, A., Wang, S.-K., Chen, Y.-H., Wong, C.-H. Tetrabutylammonium fluoride-mediated rapid alkylation reaction in microtiter plates for the discovery of enzyme inhibitors in situ. Chembiochem 6:2176, 2005.

Wu, C.-Y., King, K.-Y., Kuo, C.-J., Fang, J.-M., Wu, Y.-T., Ho, M.-Y., Liao, C.-L., Shie, J.-J., Liang, P.-H., Wong, C.-H. Stable benzotriazole esters as mechanism-based inactivators of the severe acute respiratory syndrome 3CL protease. Chem. Biol. 13:261, 2006.

Wu, D., Zajonc, D.M., Fujio, M., Sullivan, B.A., Kinjo, Y., Kronenberg, M., Wilson, I.A., Wong, C.-H. Design of natural killer T cell activators: structure and function of a microbial glycosphingolipid bound to mouse CD1d. Proc. Natl. Acad. Sci. U. S. A. 103:3972, 2006.

Zajonc, D.M., Maricic, I., Wu, D., Halder, R., Roy, K., Wong, C.-H., Kumar, V., Wilson, I.A. Structural basis for CD1d presentation of a sulfatide derived from myelin and its implications for autoimmunity. J. Exp. Med. 202:1517, 2005.