Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold
Gretchen PetersApril 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
O
ON
ZS
S
SNHR
O
ZS
S
S
O O
N
NHR
O174
1
2
3
0.5 mol%0.1 mol% ACVA
Dioxane, 90°C
10 eq N2H4
DMF, 0°C
HS
HN O
NH2
NHR
O174
4
acetate bufferpH=5.5, 50°C0.5% aniline
HS
HN O
NH
NHR
O174
5
O
O
OHZ= CH12H25; R=CH2CH2NH-biotinACVA= 4,4'-azobis(4-cyanovaleric acid)
NN
N
OH
ON
HO
O
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
NN
N
OH
ON
HO
O
N
HO
O O
ON
O
ON S
SZSNHR
O
O
ON
HO
OS
S
ZS
NHRO
S
SZSO
O
N
CN
O
HO
CN
CN
OHO
NHROO
ON
CN
O
HO
Reaction Scheme
O
ON
ZS
S
SNHR
O
ZS
S
S
O O
N
NHR
O174
1
2
3
0.5 mol%0.1 mol% ACVA
Dioxane, 90°C
10 eq N2H4
DMF, 0°C
HS
HN O
NH2
NHR
O174
4
acetate bufferpH=5.5, 50°C0.5% aniline
HS
HN O
NH
NHR
O174
5
O
O
OHZ= CH12H25; R=CH2CH2NH-biotinACVA= 4,4'-azobis(4-cyanovaleric acid)
NN
N
OH
ON
HO
O
Glycan Ligation
N
O
OH
H HH
O
NH
OH
NH
OH
NH2NH
O
R'
NH2
OH
NHNH
O
R'O H
NNH R'
O
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