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Gretchen Peters April 14, 2011

Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly( acryloyl hydrazide ) Scaffold. Gretchen Peters April 14, 2011. Bertozzi Group. BS: Harvard; PhD: Berkeley; Post-Doc: UCSF Now faculty at UC-Berkeley

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Gretchen Peters April 14, 2011

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  1. Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloylhydrazide) Scaffold Gretchen Peters April 14, 2011

  2. Bertozzi Group • BS: Harvard; PhD: Berkeley; Post-Doc: UCSF • Now faculty at UC-Berkeley • Research interests: spans both chemistry and biology • Emphasis on changes in cell surface glycosylation pertinent to cancer, inflammation and bacterial infection • Nanoscience-based technologies for cell function probing and protein engineering methods http://www.cchem.berkeley.edu/crbgrp/bio.htm

  3. Definitions • Glycopolymer: a class of synthetic macromolecules that have mimic functions and structure to cell-surface glycoproteins • Glycoprotein: proteins covalently bonded to sugar units, via the OH group of serine, O-glycosylatedthreonine or N-glycosylated amide of asparagine http://www.biology-online.org/dictionary

  4. Glycopolymers: Why care? • Glycoproteins are vital for many biological processes (innate immunity, cellular communication, etc.) • Strength and specificity of glycoprotein/receptor interactions in these processes dependent on structure, valency, and spatial organization • Therefore, glycopolymers can be used to mimic these characteristics and probe the mechanisms of the biological processes

  5. Glycopolymers: Why care? • Another interest: Glycoproteins can be mucin mimics, which are used to control carbohydrate presentation in glycan microarrays • Important for interrogating ligand specificity of carbohydrate-binding proteins Godula, K.; Rabuka, D.; Nam, K.T.; Bertozzi, C. Angew. Chem. Int. Ed. 2009, 48, 4973-4976.

  6. Other Methodologies • Polymerization of glycan-containing molecules Okada, M. Prog. Polym. Sci. 2001, 26, 67-104.

  7. Other Methodologies • Attachment of prefunctionalized glycosides to polymer backbones containing complementary reactive groups Ladmiral, V.; Mantovani, G.; Clarkson, G. J.; Cauet, S.; Irwin, J.L.; Haddleton, D. M. J. Am. Chem. Soc. 2005, 128, 4830.

  8. New Synthesis • Benefits: eliminates carbohydrate prefunctionalization ; offers rapid access to glycopolymers with a broad scope of glycan structures

  9. RAFT • Reversible addition-fragmentation chain transfer • Radical polymerization; Thang, et al. 1998 • Done using thiocarbonylthio compounds as the monomer: R must be able to homolytically leave and initiate new chains • One of the most versatile methods: can be done with a wide range monomers with different functionalities and using many different solvents Chiefari, J.; Chong, Y. K.: Ercole, F.; Krstina; J.; Jeffery, J.; Le, T.; Mayadunne, R.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S.H. Macromolecules 1998, 31, 5559-5562.

  10. General RAFT • J & R are species that can initiate free-radical polymerization or they may be derived from radicals formed by the thiocompound or the initiator • Z should activate the C=S double bond for radical addition • R should be a good free-radical leaving group Chiefari, J.; Chong, Y. K.: Ercole, F.; Krstina; J.; Jeffery, J.; Le, T.; Mayadunne, R.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S.H. Macromolecules 1998, 31, 5559-5562.

  11. RAFT

  12. Reaction Scheme

  13. Glycan Ligation

  14. Ligation Efficiency • Ligation reversible; optimized conditions: 1.1 sugar eq., 2 eq. even better • Able to make mono-, di, and trisaccharides • Primarily b isomer • Diminished l.e. with lycans with N-acetylhexosamine

  15. Complex glycans • Used the new method to make polymers with complex glycans • Saw the expected trends in for ligation efficiency based on simpler cases

  16. Microarray: Lectin Specificity Godula, K.; Rabuka, D.; Nam, K.T.; Bertozzi, C. Angew. Chem. Int. Ed. 2009, 48, 4973-4976.

  17. Microarray: Lectin Specificity • Microarrayed polymers 5a-r on streptavidin-coated glass • Tested for binding of Cy5-labeled concanavalin A (ConA), Ricinuscommunis I (RCA I), Helix pomatiaagglutinin (HPA), and Aleuriaaurantealectin (AAL) (Figure 1B). • ConA: terminal R-mannose and R-glucose residues in polymers 5h and 5i, respectively • RCA I: polymers 5g and 5l, presenting terminal galactoseepitopes • HPA : N-acetylgalactosamine-containing polymer 5k and less strongly to polymer 5j, a much weaker HPA ligand • AAL bound to glycopolymers containing fucose (5d), (5o), (5q), and (5r), all of which contain the target residue

  18. Conclusions • New methodology for synthesizing biotinylatedglycopolymers • Can be used for glycan microarrays on streptavidin-coated glass slides. • These glycopolymers were recognized by lectins with high specificity

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