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 ON

NHR

O174

1

2

3

0.5 mol%0.1 mol% ACVADioxane, 90°C

10 eq N2H4DMF, 0°C

HS

HN ONH2

NHR

O174

4

acetate bufferpH=5.5, 50°C0.5% aniline

HS

HN ONH

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 N

N

N

OH

ON

HO

O

N

HO

O O

ON

O

ON S

SZSNHR

O

O

ON

HO

OS

S

ZS

NHRO

S

SZSO

ON

CN

O

HO

CN

CN

OHO

NHROO

ON

CN

O

HO

Reaction Scheme

O

ON

ZS

S

SNHR

O

ZS

S

S

O ON

NHR

O174

1

2

3

0.5 mol%0.1 mol% ACVADioxane, 90°C

10 eq N2H4DMF, 0°C

HS

HN ONH2

NHR

O174

4

acetate bufferpH=5.5, 50°C0.5% aniline

HS

HN ONH

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

OHH HH

O

NH

OH

NH

OH

NH2NH

O

R'

NH2

OH

NHNH

O

R'O HN

NH 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