The Skaggs Institute
for Chemical Biology

Scientific Report 2008

Bioorganic and Synthetic Chemistry

C.-H. Wong, C. Bennett, S. Dean, S. Ficht, Y. Fu, W. Greenberg, R. Guy, S. Hanson, Z. Hong, D.-R. Hwang, J.-C. Lee, P.-H. Liang, R. Payne, M. Schelwies, S.-K. Wang

We develop new chemical and enzymatic strategies for synthesis of bioactive small molecules and biomolecules. We use the methods to probe carbohydrate-mediated recognition events important in cancer, bacterial infections, and viral infections, including HIV disease and influenza.

Synthetic Methods

We have developed new methods for sugar-assisted ligation of glycopeptides for synthesis of homogenous glycoproteins. We have used the methods in conjunction with enzymatic glycosylation techniques to assemble complex glycopeptides by chemical synthesis, and we are optimizing the techniques to achieve the total synthesis of therapeutic glycoproteins. Glycoproteins are expressed in vivo as complex mixtures of glycoforms, a situation that hinders efforts to study the role of glycosylation in protein folding, stability, and function. By synthesizing pure glycoforms, we can characterize in molecular detail the effects of glycans on protein function.

Using chemical techniques such as programmable 1-pot oligosaccharide synthesis, as well as enzymatic synthesis, we create glycoarrays on glass slides for high-throughput quantitative analysis of protein-carbohydrate interactions. These arrays are being used to study the binding specificity of carbohydrate-binding receptors and antibodies. We have applied aldolases, glycosyltransferases, glycosidases, and other enzymes to develop practical new methods of synthesizing molecules such as iminocyclitols, which are inhibitors of glycosidases and other enzymes; glycopeptides; and other glycoconjugates. Using directed evolution, we are evolving these enzymes to catalyze new reactions and synthesize new molecules of pharmaceutical relevance.

Carbohydrate-Mediated Recognition In Disease

We are using our synthetic methods to discover inhibitors and therapeutic agents in several diseases related to carbohydrates. Current targets include bacterial transglycosidase, sulfatases, and glycoprocessing enzymes involved in the biosynthesis of carbohydrates that mediate cancer metastasis, inflammation, and viral infection. Enzymatically synthesized iminocyclitols are being investigated as treatments for osteoarthritis and Gaucher disease. Inspired by the broadly neutralizing anti-HIV antibody 2G12, which recognizes a dense array of oligomannose displayed on HIV gp120, we are designing dendrimeric oligomannose structures for development of an HIV vaccine. In collaboration with D.R. Burton, Scripps Research, we are testing the immunogenicity of these constructs. We have designed glycolipid ligands for CD1 that activate natural killer T cells and are a promising new immunotherapeutic approach for treatment of bacterial and viral infections and cancer. The ligands may also be useful as adjuvants in vaccine development.

Glycoproteomics And Molecular Glycobiology

Using metabolic oligosaccharide engineering, we have developed methods for incorporating tagged sugars into glycans expressed on mammalian cells. The engineered glycans can be labeled with a variety of molecules by using click chemistry. One application is glycan-specific fluorescent labeling, which is used for fluorescent imaging to compare glycosylation patterns of different cells, such as normal vs cancer cells or cancer cells vs cancer stem cells. We found that protein fucosylation and sialylation are both elevated in cancer cell lines.

A second application of this chemistry is GIDmap, a new method for glycoproteomic analysis (Fig. 1). Whole cells are fed with tagged sugars, and after biochemical incorporation into cellular glycoproteins, click chemistry is used to attach a handle for purification of tagged proteins. Mass spectrometric proteomic methods are then used to identify proteins that are differentially glycosylated. We are using GIDmap to identify proteins that are aberrantly glycosylated in different stages of cancer. These cancer-associated glycoproteins may be useful as biomarkers for diagnostics or as targets for therapeutic intervention.

Fig. 1. GIDmap glycoproteomic analysis via metabolic oligosaccharide engineering.

Publications

Astronomo, R.D., Lee, H.K., Scanlan, C.N., Pantophlet, R., Huang, C.Y., Wilson, I.A., Blixt, O., Dwek, R.A., Wong C.-H., Burton D.R. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120. J. Virology 82:6359, 2008.

Bennett, C.S., Dean, S.M., Payne, R.J., Ficht, S., Brik, A., Wong, C.-H. Sugar-assisted glycopeptide ligation with complex oligosaccharides: scope and limitations. J. Am. Chem. Soc. 130:11945, 2008.

Ficht, S., Payne, R.J., Guy, R.T., Wong, C.-H. Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies. Chem. Eur. J. 14:3620, 2008.

Giffin, M.J., Heaslet, H., Brik, A., Lin, Y.-C., Cauvi, G., Wong, C.-H., McRee, D.E., Elder, J.H., Stout, C.D., Torbett, B.E. A copper(I)-catalyzed 1,2,3-triazole azide-alkyne click compound is a potent inhibitor of a multidrug-resistant HIV-1 protease variant. J. Med. Chem. 51:6263, 2008.

Hanson, S.R., Greenberg, W.A., Wong C.-H. Probing glycans with the copper(I)-catalyzed [3+2] azide-alkyne cycloaddition. QSAR Comb. Sci. 26:1243, 2007.

Kinjo, Y., Pei, B., Bufali, S., Raju, R., Richardson, S.K., Imamura, M., Fujio, M., Wu, D., Khurana, A., Kawahara, K., Wong, C.-H., Howell, A.R., Seeberger, P.H., Kronenberg, M. Natural Sphingomonas glycolipids vary greatly in their ability to activate natural killer T cells. Chem. Biol. 15:654, 2008.

Liang, P.-H., Imamura, M., Li, X., Wu, D., Fujio, M., Guy, R.T., Wu, B.-C., Tsuji, M., Wong, C.-H. Quantitative microarray analysis of intact glycolipid-CD1d interaction and correlation with cell-based cytokine production. J. Am. Chem. Soc. 130:12348, 2008.

Liang, P.-H., Wu, C.-Y., Greenberg, W.A., Wong C.-H. Glycan arrays: biological and medical applications. Curr. Opin. Chem. Biol. 12:86, 2008.

Northen, T.R., Lee, J.-C., Hoang, L., Raymond, J., Hwang, D.-R., Yannone, S.M., Wong, C.-H., Siuzdak, G. A nanostructure-initiator mass spectrometry-based enzyme activity assay. Proc. Natl. Acad. Sci. U. S. A. 105:3678, 2008.

Payne, R.J., Ficht, S., Greenberg, W.A., Wong, C.-H. Cysteine-free peptide and glycopeptide ligation by direct aminolysis. Angew. Chem. Int. Ed. 47:4411, 2008.

Wang, S.-K., Liang, P.-H., Astronomo, R.D., Hsu, T.-L., Hsieh, S.-L., Burton, D.R., Wong, C.-H. Targeting the carbohydrates on HIV-1: interaction of oligomannose dendrons with human monoclonal antibody 2G12 and DC-SIGN. Proc. Natl. Acad. Sci. U. S. A. 105:3690, 2008.

Whalen, L.J., Greenberg, W.A., Mitchell, M.L., Wong, C.-H. Iminosugar-based glycosyltransferase inhibitors. In: Iminosugars: From Synthesis to Therapeutic Applications. Compain, P., Martin, O.R. (Eds.). Wiley-VCH, Hoboken, NJ, 2007, p. 153.

Wu, D., Fujio, M., Wong, C.-H. Glycolipids as immunostimulating agents. Bioorg. Med. Chem. 16:1073, 2008.