Synthesis and microcontact printing of dual end-functionalized mucin-like glycopolymers for microarray applications

Full text of DOI:10.1002/anie.200805756

Angewandte
Chemie
DOI: 10.1002/anie.200805756
Microarrays
Synthesis and Microcontact Printing of Dual End-Functionalized
Mucin-like Glycopolymers for Microarray Applications**
Kamil Godula, David Rabuka, Ki Tae Nam, and Carolyn R. Bertozzi*
Glycan arrays are increasingly popular tools for probing the
ligand specificities of glycan-binding receptors,[1] antibodies,[2]
and enzymes.[1c,d,f,3] In a typical architecture, the glycan
components are attached to the array surface using a linker
appended to the glycanꢀs reducing end. The multivalency of
surface display can mirror, to a limited extent, the environ-
ment of the cell surface. Therein, glycans are organized into
multivalent collections that are often critical for high-avidity
binding to cognate receptors.[4] However, the spatial arrange-
ment of glycans on a cell surface is surely different from that
on a synthetic glycoarray. On cells, many glycans are
displayed on glycoprotein scaffolds with three-dimensional
geometries that cannot be emulated on a two-dimensional
substrate. It is widely appreciated that the relative positioning
of glycans can profoundly influence recognition by oligomer-
ized receptors.[5] Additionally, changes in linker length,[1f]
glycan density,[1d,6] and mode of glycan immobilization can[7]
alter the avidity and specificity of glycan–protein interactions.
New modes of glycan display within array platforms should be
explored to better approximate native glycoconjugate struc-
tures.
lished that, as in native glycoconjugates, the shape and size
uniformity of these synthetic architectures[9] as well as the
density and relative positioning of their glycan appendages[10]
can significantly alter the outcome of their interactions with
protein receptors. Recently, surface-bound multivalent glycan
ligands were shown to exhibit higher avidity to protein
receptors compared to immobilized monomeric glycans,[11]
and glycodendrimers were integrated into a microarray
platform to study how multivalency affects lectin binding.[12]
Inspired by these examples, we have sought to develop
glycoconjugate mimetics that spatially position the pendant
glycans similarly to natural glycoproteins. Integration of such
constructs into arrays may create a more physiologically
authentic platform for probing glycan-binding proteins.
We were interested in generating glycopolymers that
mimic mucins, glycoproteins participating in numerous cell-
surface interactions.[13] Mucins possess dense clusters of serine
and threonine residues to which complex glycans are attached
using a core N-acetylgalactosamine (GalNAc) moiety (Fig-
ure 1a). We previously designed mucin mimetic glycopoly-
Natural glycoproteins can be more closely mimicked by
attachment of glycans to synthetic scaffolds with defined
structures. Glycopolymers have been skillfully employed as
soluble multivalent ligands that bind cell-surface receptors
and activate biological processes.[8] Previous studies estab-
[*] Prof. C. R. Bertozzi
Departments of Chemistry and Molecular and Cell Biology and
Howard Hughes Medical Institute, University of California and The
Molecular Foundry, Lawrence Berkeley National Laboratory
Berkeley, CA 94720 (USA)
Fax: (+1)510-643-2628
E-mail: crb@berkeley.edu
Figure 1. Design of a synthetic mucin mimic. a) Representative frag-
ment of a native mucin glycoprotein. R1, R2 =glycan extensions from
the core GalNAc residue. b) Schematic of a mucin mimic glycopolymer
containing oxime-linked a-GalNAc residues.
K. Godula, D. Rabuka, K. T. Nam
Department of Chemistry, University of California and
The Molecular Foundry, Lawrence Berkeley National Laboratory
Berkeley, CA 94720 (USA)
mers comprising a poly(methylvinylketone) backbone to
which aminooxy glycans were conjugated using oxime link-
ages (shown schematically in Figure 1b).[14] Like natural
mucins, the synthetic glycopolymers possessed rigid extended
structures. These polymers were displayed on synthetic
materials[14a–d] and on live cells,[14e] where their pendant
glycans bound multivalent lectins. However, unlike natural
mucins, the synthetic polymers were highly polydisperse with
limited scope of end-functionalization, reflecting the classic
limitations of the free radical polymerization process that was
used to generate them.[15]
[**] This work was supported by the Director, Office of Energy Research,
Office of Basic Energy Sciences, Division of Materials Sciences, of
the U.S. Department of Energy under Contract No. DE-AC03-
76SF00098, within the Interfacing Nanostructures Initiative and
NIH (K99M080585-01). Portions of this work were performed at the
Molecular Foundry, Lawrence Berkeley National Laboratory, which is
supported by the Office of Science, Office of Basic Energy Sciences,
of the U.S. Department of Energy under Contract No. DE-AC02-
05CH11231. We thank Dr. Ramesh Jasti for stimulating discussions,
Dr. Marian Snauko for technical support, and Prof. Pil J. Yoo of
SKKU Advanced Institute of Nanotechnology for graciously pro-
viding PDMS stamps.
Herein, we report on a new class of mucin mimetic
glycopolymers that are suitable for integration with current
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200805756.
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4973

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microarray technology platforms. As shown in Figure 2a, the
polymers comprise three functional domains: a central mucin
mimetic domain on which glycans are displayed similarly to
of isolated P2 (see the Supporting Information). Glycopol-
ymers a- and b-P3 were then assembled by condensation of
the pendant ketones of P2 with a- and b-aminooxy-GalNAc,
respectively.[20] The ligation reaction proceeded efficiently
(>70%) to give glycopolymers that adopted mucin-like
extended structures, as evidenced by TEM analysis (see the
Supporting Information). Trapping of the newly released free
sulfhydryl group in P3 with a maleimide-conjugated Texas
Red fluorophore then afforded the target dual end-function-
alized glycopolymer P4 (Scheme 1).
The covalent printing of alkyne-terminated glycopoly-
mers by CuACC required the preparation of chips containing
surface azido groups. For this purpose we chose silicon wafers
with a thin layer of oxide (500 nm) treated with (3-azidopro-
pyl)trimethoxy silane (Figure 3a). Successful surface modifi-
cation was apparent from a significant change in contact
angle. We observed an increase in the contact angle of 808
after functionalization, indicative of a significantly more
Figure 2. Design of a synthetic mucin mimic. a) Modular approach for
the assembly of dual end-functionalized mucin mimetics. b) Schematic
of a key synthetic intermediate (P1) possessing orthogonal chemical
functionality in each domain.
native mucins, one terminal domain
bearing a surface attachment element,
and a second terminal domain outfitted
for conjugation of a reporter group. We
exploited the reversible addition-frag-
mentation chain transfer (RAFT) poly-
merization technique[16] to generate
polymers of low polydispersity, while
simultaneously introducing chemically
orthogonal capping groups onto each
terminus. The RAFT technique is
robust, tolerates the presence of myriad
functional groups, and accordingly, has
been employed previously in the syn-
thesis of glycopolymers.[17] We envi-
sioned using the RAFT process to gen-
erate key intermediate P1 (Figure 2b)
containing pendant ketones for the
attachment of aminooxy glycans, as
well as terminal pentafluorophenyl
(PFP) ester and trithiocarbonate
groups. The PFP ester offers a reactive
site for amine-functionalized surface
Scheme 1. Synthesis of dual-end-functionalized mucin mimics using polymer scaffold P1.
anchors, while the trithiocarbonate can
be cleaved to release a free sulfhydryl
group for conjugation of the probe.
The synthesis of intermediate P1 is shown in Scheme 1.
We found that (2-oxopropyl)acrylate (1) efficiently polymer-
ized in the presence of a PFP-ester and trithiocarbonate-
functionalized chain transfer agent (2) and a radical initiator,
4,4’-azobis(4-cyanovaleric acid) (ACVA), to give polymer
scaffold P1 of low polydispersity (PDI < 1.15). Reaction of P1
with propargylamine afforded alkyne-functionalized polymer
P2, which was primed for surface attachment by using a
copper-catalyzed alkyne-azide cycloaddition reaction
(CuAAC).[18] This highly selective reaction has been previ-
ously employed for surface attachment of individual gly-
cans.[19] The end-functionalization reaction was determined to
hydrophobic surface. X-ray photoelectron spectroscopy
(XPS) analysis of the functionalized wafers showed a new
emission at 401 eV corresponding to the 1 s transition of the
newly introduced nitrogen atoms. The presence of surface
azide groups was further corroborated by FTIR analysis. A
À
characteristic azide N N stretch absorption was clearly
evident at 2100 cmÀ1 (see the Supporting Information).
We explored the microcontact printing (mCP) technique[21]
as a means to array the glycopolymers on azide-functionalized
chips in well-defined patterns. In a typical experiment, a
poly(dimethylsiloxane) stamp with approximately 2 mm cir-
cular features was inked with a solution of a glycopolymer
(0.2 mgmLÀ1), copper sulfate (50 mm), tris[(1-benzyl-1H-
1
be quantitative from 19F and H NMR spectroscopy analysis
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Chemie
microcontact-printed glycopolymers
on silicon wafers can distinguish
glycan-binding proteins based on
their ligand specificity.
In summary, we have developed a
modular approach for the assembly of
a new class of orthogonally end-func-
tionalized mucin-mimetic glycopoly-
mers. Micropatterns of these polymers
can be generated by microcontact
printing followed by CuACC, and the
surface-displayed glycopolymers bind
lectins specific for their pendant gly-
cans. A key element of this approach
is that the densities and orientations of
the glycan ligands are determined by
the polymer structure rather than by
poorly understood features of the
underlying surface. These parameters
should therefore be more controllable
than is the case with conventional
glycan arrays. The utility of glycopoly-
mer microarrays as a platform for
profiling glycan-binding proteins is a
matter of considerable future interest.
Figure 3. Glycopolymer microarrays for probing specific glycan–protein interactions. a) Schematic
of the preparation of azide-functionalized silicon oxide wafers and covalent microcontact printing
(m-CP) of glycopolymer a-P4. b) Fluorescence micrograph of wafers printed with a-P4 in the
presence (top) and in the absence (bottom) of a copper catalyst. c) Binding of Texas Red-labeled
HPA lectin (TR-HPA) to microarrayed patterns of nonfluorescent glycopolymers a- and b-P3 (all
scale bars are 10 mm).
Received: November 26, 2008
Published online: May 28, 2009
1,2,3-triazol-4-yl)methyl]amine (TBTA; 50 mm), and sodium
Keywords: click chemistry · glycopolymers · micropatterning ·
mucin · polymerization
ascorbate (250 mm) in a mixture of water and DMSO (1:1).
The stamp was dried in a stream of nitrogen, brought to
contact with the chip surface, and pressure was applied (see
the Supporting Information). In the presence of the copper
catalyst, the terminal alkyne of the glycopolymers reacted
with the surface azides to form stable triazol linkages and,
thus, the resulting patterns became covalently attached to the
surface. After 30 minutes, the stamps were removed; the chips
were extensively washed with water and incubated for one
hour in a solution of bovine serum albumin (BSA) in
phosphate-buffered saline (PBS; 50 mgmLÀ1). The resulting
patterns of fluorescent glycopolymer a-P4 could be directly
observed using fluorescent microscopy (Figure 3b, top).
Notably, fluorescence was not detected when the printing
was performed in the absence of copper catalyst (Figure 3b,
bottom).
For microarray applications, it is important that the
printed glycopolymers interact in a ligand-specific manner
with glycan-binding proteins. To ascertain whether our plat-
form fulfills this requirement, we tested the binding of Helix
pomatia agglutinin (HPA), a lectin that recognizes a-GalNAc,
to the printed glycopolymers (Figure 3c, top). We incubated
separate silicon wafers displaying nonfluorescent a- and b-P3
polymers with Texas Red-HPA (0.25 mgmLÀ1, TRIS buffer,
pH 7.2) for 20 minutes. Fluorescence microscopy analysis of
the surfaces revealed specific binding of HPA only to the a-
GalNAc-modified polymer a-P3 (Figure 3c, bottom). HPA
binding to printed a-P3 was inhibited in the presence of free
GalNAc (200 mm), further confirming that this process is
indeed glycan specific. These observations establish that
.
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