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![Page 1: Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold Gretchen Peters April 14,](https://reader038.fdocuments.us/reader038/viewer/2022103005/56649e585503460f94b518cc/html5/thumbnails/1.jpg)
Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold
Gretchen PetersApril 14, 2011
![Page 2: Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold Gretchen Peters April 14,](https://reader038.fdocuments.us/reader038/viewer/2022103005/56649e585503460f94b518cc/html5/thumbnails/2.jpg)
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
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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
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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
![Page 5: Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold Gretchen Peters April 14,](https://reader038.fdocuments.us/reader038/viewer/2022103005/56649e585503460f94b518cc/html5/thumbnails/5.jpg)
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.
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Other Methodologies•Polymerization of glycan-containing
molecules
Okada, M. Prog. Polym. Sci. 2001, 26, 67-104.
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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.
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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
![Page 9: Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold Gretchen Peters April 14,](https://reader038.fdocuments.us/reader038/viewer/2022103005/56649e585503460f94b518cc/html5/thumbnails/9.jpg)
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.
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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.
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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
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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
![Page 13: Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold Gretchen Peters April 14,](https://reader038.fdocuments.us/reader038/viewer/2022103005/56649e585503460f94b518cc/html5/thumbnails/13.jpg)
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
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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
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Complex glycans•Used the new method
to make polymers with complex glycans
•Saw the expected trends in for ligation efficiency based on simpler cases
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Microarray: Lectin Specificity
Godula, K.; Rabuka, D.; Nam, K.T.; Bertozzi, C. Angew. Chem. Int. Ed. 2009, 48, 4973-4976.
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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
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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