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 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

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-glycosylated threonine or N-glycosylated amide of asparagine http://www.biology-online.org/dictionary

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

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.

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

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.

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

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.

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.

RAFT

Reaction Scheme

Glycan Ligation

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

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

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

Microarray: Lectin Specificity Microarrayed polymers 5a-r on streptavidin- coated glass Tested for binding of Cy5-labeled concanavalin A (ConA), Ricinus communis I (RCA I), Helix pomatia agglutinin (HPA), and Aleuria aurantea lectin (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 galactose epitopes 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

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