1. Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) 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

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-glycosylated threonine or N-glycosylated amide of asparagine

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,

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

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,

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,

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,

17. 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

18. 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