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, 55595562.
General RAFT
• J & R are species that
can initiate freeradical
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, 55595562.
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 Nacetylhexosamine
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 streptavidincoated 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