Synthesis of functional polymer brushes containing carbohydrate residues in the pyranose form and their specific and nonspecific interactions with proteins

Article abstract of DOI:10.1021/bm100882q

Three novel N-substituted acrylamide monomers containing different carbohydrate residues, 2'-acrylamidoethyl- α-D-mannopyranoside, 2'-acrylamidoethyl-β-D-glucopyranoside, and 2'-acrylamidoethyl-β-D- galactopyranoside, in the pyranose form were synthesized. The corresponding glycopolymer brushes were prepared on silicon substrates by surface-initiated atom transfer radical polymerization (SI-ATRP) using unprotected glycomonomers. The formation of glycopolymer brushes was well-characterized using ellipsometry, ATR-FTIR, water contact angle analysis, atomic force microscopy analysis, and X-ray photoelectron spectroscopy. The effects of halogen, ligand, and solvent on the polymerization were thoroughly investigated. It was shown that CuCl/CuCl₂/tris(2- dimethylaminoethyl)amine (Me₆TREN) catalytic system with an optimized ratio of Cu(I)/Cu(II) produced glycopolymer with high molecular weight (M_n = 44-140 kDa) and relatively narrow molecular weight distribution (PDI = 1.4). The dry thickness of resulting glycopolymer brushes (10-36 nm) showed a proportional relationship with the molecular weight of free polymer generated in the solution. The grafting densities of obtained glycopolymer brushes were between 0.12 and 0.17 chains/nm². The grafting of glycopolymer resulted in highly hydrophilic surface layer with very low water contact angles (<10°). The glycopolymer brushes showed ultralow protein adsorption from bovine serum albumin (BSA) and fibrinogen (Fb) solutions. Glycopolymer brushes containing glucose units showed relatively better protection against BSA and Fb adsorption than those brushes containing mannose and galactose units. Synthesized glycopolymer brushes retained specific protein interactions, as evident from the interaction with Concanavalin A (Con A). The interaction of surface-grafted glycopolymer brushes with Con A depended on both the stereochemistry of carbohydrate units and the chemical structures present. In addition, the newly synthesized glycopolymer brushes performed significantly better in comparison with currently available structures in terms of specific protein interactions.

Full text of DOI:10.1021/bm100882q

Biomacromolecules 2010, 11, 3073–3085
3073
Synthesis of Functional Polymer Brushes Containing
Carbohydrate Residues in the Pyranose Form and Their
Specific and Nonspecific Interactions with Proteins
Kai Yu† and Jayachandran N. Kizhakkedathu*,†,‡
Centre for Blood Research and Department of Pathology & Laboratory Medicine, and Department of
Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
Received July 30, 2010; Revised Manuscript Received September 14, 2010
Three novel N-substituted acrylamide monomers containing different carbohydrate residues, 2′-acrylamidoethyl-
R-D-mannopyranoside, 2′-acrylamidoethyl-ꢀ-D-glucopyranoside, and 2′-acrylamidoethyl-ꢀ-D-galactopyranoside,
in the pyranose form were synthesized. The corresponding glycopolymer brushes were prepared on silicon substrates
by surface-initiated atom transfer radical polymerization (SI-ATRP) using unprotected glycomonomers. The
formation of glycopolymer brushes was well-characterized using ellipsometry, ATR-FTIR, water contact angle
analysis, atomic force microscopy analysis, and X-ray photoelectron spectroscopy. The effects of halogen, ligand,
and solvent on the polymerization were thoroughly investigated. It was shown that CuCl/CuCl2/tris(2-
dimethylaminoethyl)amine (Me6TREN) catalytic system with an optimized ratio of Cu(I)/Cu(II) produced
glycopolymer with high molecular weight (Mn ) 44-140 kDa) and relatively narrow molecular weight distribution
(PDI ) 1.4). The dry thickness of resulting glycopolymer brushes (10-36 nm) showed a proportional relationship
with the molecular weight of free polymer generated in the solution. The grafting densities of obtained glycopolymer
brushes were between 0.12 and 0.17 chains/nm2. The grafting of glycopolymer resulted in highly hydrophilic
surface layer with very low water contact angles (<10°). The glycopolymer brushes showed ultralow protein
adsorption from bovine serum albumin (BSA) and fibrinogen (Fb) solutions. Glycopolymer brushes containing
glucose units showed relatively better protection against BSA and Fb adsorption than those brushes containing
mannose and galactose units. Synthesized glycopolymer brushes retained specific protein interactions, as evident
from the interaction with Concanavalin A (Con A). The interaction of surface-grafted glycopolymer brushes with
Con A depended on both the stereochemistry of carbohydrate units and the chemical structures present. In addition,
the newly synthesized glycopolymer brushes performed significantly better in comparison with currently available
structures in terms of specific protein interactions.
efficiency of reorganization. Covalent grafting of monosaccha-
ride or oligosaccharide by direct grafting (“grafting to” or
“grafting from”) techniques, can potentially overcome these
shortcomings. These techniques include the formation of a self-
assembled monolayer (SAM),15,16 covalent immobilization of
modified carbohydrates directly onto functionalized glass or
silicon slides,17-19 activated ester-derivatized surface,20,21 and
the use of “click chemistry”.22,23 However, most of these
methods suffer from drawbacks such as stability under various
conditions, limitation of the range of immobilized carbohydrate,
and the substrate-dependent nature of specific covalent attach-
ment chemistry.24
Introduction
The glycocalyx is a “carbohydrate-rich coat” on the external
surface of the plasma membrane of cells. The interactions of
glycocalyx with proteins present in the surrounding environ-
ments are important recognition events that regulate a myriad
of biological and pathological processes, including cell
recognition,1,2 viral infection,3 and cancer metastasis.4 The
carbohydrate groups participate in these processes through the
specific interaction with their partner proteins with high
specificities. In addition, the glycocalyx also serves as a layer
that prevents the undesirable nonspecific adhesion of other
proteins and cells.5-8 Therefore, preparation of artificial cell
surface by mimicking of the glycocalyx may provide a promis-
ing route not only for comprehensive understanding of carbo-
hydrate-receptor interactions but also for the development of
highly biocompatible surfaces.
Despite the fact that carbohydrate-protein interaction has
shown high specificity, the affinity between the proteins and
simple sugar residues is low (Ka ) 103-104 L·mol-1) because
of the monovalent interaction.25,26 A multivalent presentation
of ligands is generally required to achieve physiologically
relevant association and affinities. Grafting glycopolymer chains
onto surface with high grafting density (keeping the glycopoly-
mer chains highly stretched state) may be a good scaffold to
mimic the glycocalyx and may provide a means to study the
carbohydrate-protein interactions. To achieve this objective,
Ejaz and Fukuda were the first to report the preparation of
glycopolymer brushes using surface-initiated atom transfer
radical polymerization (SI-ATRP) of a methacrylate-based
acetate-protected glucose-containing monomer,27 followed by
the deprotection to gain the sugar functionalities.28 Similar
The glycocalyx-mimetic surfaces prepared by the physical
adsorption showed efficient reorganization with receptor9,10 and
good nonfouling properties.11-14 Although this physical attach-
ment method gave useful properties, the low sugar density,
instability, and random orientation of sugars might not reflect
their natural presentation in living systems, which decreases the
* To whom correspondence should be addressed: E-mail: jay@
pathology.ubc.ca. Tel: (604) 822-7085.
† Centre for Blood Research and Department of Pathology & Laboratory
Medicine.
‡ Department of Chemistry.
10.1021/bm100882q
 2010 American Chemical Society
Published on Web 10/18/2010

3074 Biomacromolecules, Vol. 11, No. 11, 2010
Yu and Kizhakkedathu
(Columbus, Ohio). Fb (from human plasma), Alex-fluor 594 conjugate
(product number: F13193), and tetramethylrhodamine conjugates of
concanavalin A (Con A, product number: C860) were purchased from
Invitrogen. (Eugene, Oregon). 2-Azidoethanol was prepared from
2-bromoethanol and sodium azide in the presence of sodium hydrox-
ide.39 1H NMR (300 MHz, CDCl3): 3.42-3.45 (t, 2H), 3.75-3.79 (t,
2H) ppm. Tris[2-(dimethylamino)ethyl]amine (Me6TREN) was prepared
by the previously reported procedure.40 1H NMR (300 MHz, CDCl3):
2.25 (s, 18H), 2.38-2.43 (t, 6H), 2.6-2.63 (t, 6H) ppm. Ester derivative
of surface ATRP initiator (11-(2-bromo-2-methyl)-propionyloxy)-
undecyl-trichlorosilane) was synthesized by the use of a similar
procedure reported in the literature.41 1H NMR (300 MHz, CDCl3):
1.28-1.42 (br m, 16H), 1.55-1.70 (m, 4H), 1.93 (s, 6H), 4.15-4.19
(t, 2H) ppm.
Characterization. NMR spectra were recorded on a Bruker Avance
300 MHz NMR spectrometer using deuterated solvents (Cambridge
Isotope Laboratories, 99.8% D) with the solvent peak as a reference.
Molecular weights and polydispersities of glycopolymer samples were
determined using gel permeation chromatography (GPC) on a Waters
2690 separation module fitted with a DAWN EOS multiangle laser
light scattering (MALLS) detector from Wyatt Technology. with 18
detectors placed at different angles and a refractive index detector
(Optilab DSP from Wyatt Technology). An Ultrahydrogel linear column
with bead size 6-13 µm (elution range 103 to 5 × 106 Da) and an
Ultrahydrogel 120 with bead size 6 µm (elution range 150 to 5× 103
Da) from Waters were used. The dn/dc value of glycopolymer carrying
mannose residues in the mobile phase was determined at λ ) 620 nm
to be 0.154 mL/g and was used for determining molecular weight
parameters. The dn/dc values are 0.158, 0.159, 0.148, and 0.15,
respectively, for poly(2′-acrylamidoethyl-ꢀ-D-galactopyranoside) (PAAE-
Gal), poly(2′-acrylamidoethyl-ꢀ-D-glucopyranoside) (PAAEGlc), PGA-
MA, and poly(3-O-methacryloyl-D-glucofuranose) (PMAGlc). Mass
spectra were recorded on a Bruker Esquire electrospray (ESI) ion-trap
instrument using samples dissolved in MeOH or water, with positive-
ion polarity.
ATR-FTIR absorption spectra were collected on a Nexus 670 FT-
IR ESP (Nicolet Instrument, Waltham, MA) with an MCT/A liquid
nitrogen cooled detector, a KBr beam splitter, and an MkII Auen Gate
single-reflection attenuated total reflectance (ATR) accessory (Specac,
Woodstock, GA). Spectra were recorded at 4 cm-1 resolution, and 128
scans were collected. Initiator-modified silicon wafer was chosen as
the background.
Water Contact Angle Measurements. A water droplet (6 uL) was
placed on the surface, and an image of the droplet was taken with a
digital camera (Retiga 1300, Q-imaging). The contact angle was
analyzed using Northern Eclipse software. Over three different sites
were tested for each sample.
XPS Measurement. X-ray photoelectron spectroscopy (XPS) was
performed using a Leybold LH Max 200 surface analysis system
(Leybold, Cologne, Germany) equipped with a Mg KR source at a
power of 200 W. Elements were identified from survey spectra. High-
resolution spectra were collected at 48 eV pass energy.
approaches were then reported by Mu¨ller and coworkers for
the preparation of glycopolymer brush on carbon nanotubes and
Si wafer.29,30 By using a sugar-containing monomer D-glucona-
midoethyl methacrylate (GAMA),31 Yang and Xu successfully
prepared linear and comb-like grafted glycopolymer layers on
polypropylene membranes using SI-ATRP without the depro-
tection step.32,33 It was shown that poly(D-gluconamidoethyl
methacrylate) (PGAMA) brushes exhibited promising resistance
to nonspecific adsorption of proteins, including BSA, lysozyme,
and fibrinogen (Fb).34,35 Although the glycopolymer surfaces
produced by these methods showed good reduction in nonspe-
cific protein adsorption, the specific protein interaction was
compromised. This is due to the fact that none of the glyco-
polymer brushes reported have sugar residues in the pyranose
form, and in some cases, the core carbohydrate structure is
modified or in the open form.28-35 It is important that sugar
residues should be in the pyranose form as found on the cell
surface36 and the differences in sugar structures and stereo-
chemistry may have influence in both specific and nonspecific
interactions with proteins.36,37 An exception to this is a recent
report by Mateescu et al., who used poly(2-lactobionamidoethyl
methacrylate) brushes that exhibited considerable binding
interactions with galactose-binding lectin via the “glycocluster”
effect.38 Unfortunately, only the galactose-functionalized poly-
mer layer can be prepared by this method, and variation of
carbohydrate structure is not possible.
Another important disadvantage in the synthesis of glyco-
polymer brushes using post-deprotection procedure is the
difficulty in quantitative removal of protecting groups on sugar
moieties. Incomplete deprotection of sugar residues might
impact the biological properties of sugar unit and may introduce
hydrophobic patches on the surface as well. These hydrophobic
binding sites may attract certain proteins to the surface,
especially in complex biological fluids. Therefore, it is critical
to develop methods for the synthesis of glyco-polymer surfaces
bearing carbohydrate units having similar stereochemistry to
that present on cell surface glycocalyx to investigate their role
in both specific interactions and nonspecific interactions in
biological environments. Therefore, in the present work, we
report the synthesis of three novel hydrolytically stable, N-
substituted acrylamide monomers carrying mannose, galactose,
and glucose in the pyranose form and the preparation of
glycopolymer brushes using these monomers without post-
deprotection procedure. A preliminary study on nonspecific and
specific interactions between glycopolymer brushes and proteins
was also carried out to illustrate the dependence of these
interactions on sugar structures and stereochemistry. We also
synthesized previously reported glycopolymer brushes and
compared them with the newly developed carbohydrate brushes.
Experimental Section
The variable-angle spectroscopic ellipsometry (VASE) spectra were
collected on an M-2000 V spectroscopic ellipsometer (J.A. Woollam,
Lincoln, NE) at 55, 65, and 75° at wavelengths from 480 to 700 nm
with an M-2000 50W quartz tungsten halogen light source. The VASE
spectra were then fitted with a multilayer model utilizing WVASE32
analysis software based on the optical properties of a generalized
Cauchy layer to obtain the “dry” thickness of the glycopolymer layers.
AFM Measurements. Atomic force microscopy measurements were
performed on a multimode, Nanoscope IIIa controller (Digital Instru-
ments, Santa Barbara, CA) equipped with an atomic head of 100 ×
100 µm2 scan range. The tapping mode was used to map the film
morphology under ambient conditions. Silicon tips with a spring
constant of 42 N/m and a frequency of 320 kHz were used. Root-
mean-square (rms) roughness was evaluated using the integrated
Materials. D-(+)-Mannose (99%) was purchased from Alfa Aesar
(Heysham, Lancashire, England) and used as received. ꢀ-D-Glucose
pentaacetate (98%), ꢀ-D-galactose pentaacetate (98%), boron trifluoride
diethyl etherate (purified by redistillation), 2-bromoethanol (95%),
palladium on carbon (10 wt % loading), acryloyl chloride (97%), sodium
methoxide solution (25 wt % in methanol), copper(I) chloride, copper(II)
chloride, tris(2-aminoethyl)amine, 2-chloropropionyl chloride (97%),
methyl 2-chloropropionate (97%), trichlorosilane (99%), and albumin-
fluorescein isothiocyanate conjugate (bovine serum albumin, product
number: A9771) were purchased from Sigma-Aldrich (Oakville, ON).
Karstedt catalyst (platinum-divinyltetramethyl-disiloxane complex in
xylene 2.1 to 2.4% Pt) was purchased from Gelest (Morrisville, PA).
1-Amino-10-undecene (97%) was purchased from GFS chemicals

Synthesis of Functional Polymer Brushes
Biomacromolecules, Vol. 11, No. 11, 2010 3075
software. The roughness was defined as the rms of height deviations
taken from the mean data plane.
(1H, d, J ) 1.5 Hz, H1); 5.25-5.38 (3H, m, H2, 3, 4). 13C NMR:
(75.5 mHz, CDCl3, δ): 20.82, 20.87, 21.01 (4C, 4CH3); 41.48 (1C,
CH2NH2); 62.63 (1C, CH2); 66.29 (1C, CH); 68.66 (1C, CH2O); 69.20,
69.62, 70.66 (3C, CH); 97.91 (1C, CH); 169.85, 170.11, 170.21, 170.80
(4C, CdO). LR-MS (ESI): Calcd for C16H24N3O10 [M+H]+, 392.37;
found, 392.3.
Deposition of ATRP Initiator on Si Wafer. We prepared the
initiator layer by placing the Si wafer (1 cm ×1 cm) cleaned with
Piranha solution (H2O2/H2SO4 3:7 (v/v) (Piranha solution is an
extremely strong oxidant and should be handled Very carefully!) in an
anhydrous initiator solution in toluene (10 µL in 10 mL). The deposition
time for amide derivatives of surface initiator was 20 min, whereas
the deposition time for ester derivatives of surface initiator was 20 h.
The initiator-modified Si wafer was then thoroughly rinsed sequentially
with toluene, acetone, and anhydrous ethanol, followed by drying in
nitrogen. The initiator-modified surface was characterized by contact
angle measurements, ellipsometry measurements, and XPS analysis.
Synthesis of Monomers. a. Synthesis of 1,2,3,4,6-penta-O-acetyl-D-
mannose (1). D-Mannose (25 g, 0.14 mol) and DMAP (2.5 g, 0.02 mol)
were dissolved in anhydrous pyridine (200 mL) and cooled to 0 °C in
an ice bath. Acetic anhydride (87.5 mL, 0.96 mol) was added dropwise
to the solution. The mixture was allowed to stir at room temperature
overnight. After completion of the reaction (followed by TLC), the
mixture was poured in ice water with vigorous stirring and extracted
with ethyl acetate. The extracts were subsequently washed with cold
water, saturated aqueous sodium bicarbonate, and water until the pH
reached ∼7. The organic layer was dried over anhydrous sodium sulfate
and evaporated in a rotary evaporator. The crude product was purified
by flash column chromatography using ethyl acetate/hexane (1:1, v/v)
to give compound 1 (44.6 g, 82.2% yield, R/ꢀ 4:1, Rf ) 0.39, a colorless
oil). 1H NMR: (300 mHz, CDCl3, δ): 2.01, 2.06, 2.10, 2.17, 2.18 (15H,
s, Me); 4.04-4.08 (1H, m, H5); 4.12-4.17 (1H, dd, J ) 2.3, 11.96
Hz); 4.26-4.31 (1H, dd, J ) 4.8, 12.32 Hz, H6); 5.25-5.36 (3H, m,
H2, 3, 4); 5.86 (1H, d, J ) 1.02 Hz, H1ꢀ); 6.06 (1H, d, J ) 1.8 Hz,
H1R). 13C NMR: (75.5 mHz, CDCl3, δ): 20.69, 20.7, 20.75, 20.81,
20.90 (5C, CH3); 62.15, 65.59, 68.40, 68.81, 70.67 (5C, C2, 3, 4, 5,
6); 90.49, 90.66 (1C, C1); 168.10, 169.57, 169.77, 170.01, 170.65 (5C,
CdO). LR-MS (ESI): Calcd for C16H22O11Na [M+Na]+, 413.33; found,
413.2.
b. Synthesis of 2′-Azidoethyl-2,3,4,6-tetra-O-acetyl-R-D-mannopyrano-
side (2). Compound 1 (20 g, 0.05 mol) was dissolved in 100 mL of
anhydrous dichloromethane, and azidoethanol (7.1 g, 0.08 mol) was
added via syringe. The resulting solution was stirred under argon and
cooled to 0 °C. BF3 ·Et2O (33 mL, 0.27 mmol) was then added
dropwise. The reaction was stirred at 0 °C for 1 h and an additional
48 h at RT. After dilution with dichloromethane, the reaction mixture
was washed with water, aqueous sodium carbonate, and cold water
until pH ∼7. The organic phase was dried over anhydrous sodium
sulfate and evaporated in a rotary evaporator. Flash chromatography
on silica gel (ethyl acetate/hexane 1:1) gives the title compound 2 as
a crystalline solid in 76% yield as pure R anomer. 1H NMR: (300 mHz,
CDCl3, δ): 2.0, 2.06, 2.11, 2.16 (12H, s, Me); 3.44-3.50 (2H, m,
OCH2CH2N3); 3.64-3.71 (1H, m, OCH2CH2N3); 3.84-3.91 (1H, m,
OCH2CH2N3); 4.02-4.08 (1H, m, H5); 4.11-4.15 (1H, dd, J ) 2.2,
12.2 Hz, H6); 4.26-4.32 (1H, dd, J ) 5.3, 12.2 Hz, H6); 4.87 (1H, d,
J ) 1.3 Hz, H1); 5.26-5.39 (3H, m, H2, 3, 4). 13C NMR: (75.5 mHz,
CDCl3, δ): 20.77, 20.83, 20.86, 20.99 (4C, CH3); 50.48 (1C, CH2N3);
62.58 (1C, CH2); 66.12 (1C, CH); 67.17 (1C, CH2O); 68.98 (2C, CH);
69.5 (1C, CH); 97.87 (1C, CH); 169.88, 169.91, 170.12, 170.72 (4C,
CdO). LR-MS (ESI): Calcd for C16H23N3O10Na [M+Na]+, 440.36;
found, 440.2.
d. Synthesis of 2′-Acrylamidoethyl-2,3,4,6-tetra-O-acetyl-R-D-mannopy-
ranoside (8). A solution of compound 5 (3 g) in anhydrous methylene
chloride (80 mL) cooled in an ice bath, TEA (1.28 mL), and acryloyl
chloride (0.69 mL) were added dropwise under an argon atmosphere.
The reaction mixture was allowed to come to room temperature slowly
and was stirred overnight. The solution was then washed with water
and 1 N HCl and then sodium bicarbonate solution and water. The
organic phase was dried over anhydrous sodium sulfate and concen-
trated in vacuum to yellow syrup (2.3 g). Flash column chromatography
on silica in EtOAc/hexanes (10:1, Rf 0.45) gave pure compound as
colorless syrup (1.1 g, yield 32.2%). 1H NMR: (300 mHz, CDCl3, δ):
2.01, 2.05, 2.10, 2.16 (12H, s, Me); 3.54-3.67 (3H, m, OCH2CH2NH-,
OCH2CH2NH-); 3.80-3.85 (1H, m, OCH2CH2NH-); 3.95-4.00 (1H,
m, H5); 4.09-4.14 (1H, dd, J ) 2.3, 12.2 Hz, H6); 4.22-4.28 (1H,
dd, J ) 5.7, 12.2 Hz, H6); 4.83 (1H, dd, J ) 1.5 Hz, H1); 5.22-5.36
(3H, m, H2, 3, 4); 5.66-5.70 (1H, dd, J ) 1.33, 10.1 Hz, -CHdCH2);
6.06-6.20 (2H, m, NH, -CH)CH2); 6.29-6.35 (1H, dd, J ) 1.5,
16.9 Hz, -CHdCH2). 13C NMR: (75.5 mHz, CDCl3, δ): 20.86, 20.88,
21.03 (4C, 4CH3); 39.29 (1C, CH2NH); 62.67 (1C, CH2); 66.32 (1C,
CH); 67.72 (1C, CH2O); 68.93, 69.15, 69.52 (3C, CH); 97.94 (1C, CH);
127.10 (1C, CH2dCH); 130.73 (1C, CHdCH2); 165.78 (1C, CONH);
169.85, 170.25, 170.82 (4C, CdO). LR-MS (ESI): Calcd for
C19H27NO11Na [M+Na]+, 468.41; found, 468.2.
e. Synthesis of 2′-Acrylamidoethyl-R-D-mannopyranoside (11). To
a solution of compound 8 (1 g, 23 mmol) in anhydrous methanol (20
mL) was added 25% sodium methoxide in methanol (468 µL, 23 mmol)
dropwise. The reaction mixture was stirred for 90 min at room
temperature. Dowex cation-exchange resin (H form) was added to adjust
the pH between 6 and 7 and filtered. The filtrate was concentrated in
a rotary evaporator. The residue was dissolved in water and filtered
again. After freeze-drying, the title compound 11 was obtained in 83%
1
yield as a white solid. H NMR: (300 mHz, D2O, δ): 3.45-3.65 (5H,
m, OCH2CH2NH-, OCH2CH2NH-, H5); 3.72-3.91 (5H, m, H2,3,4,6);
4.83-4.84 (1H, d, J ) 1.5 Hz, H1); 5.72-5.76 (1H, dd, J ) 1.9, 9.7
Hz, -CHdCH2); 6.13-6.19 (1H, dd, J ) 1.9, 17.1 Hz, -CHdCH2);
6.21-6.30 (1H, dd, J ) 9.7, 17.2 Hz, -CHdCH2). 13C NMR: (75.5
mHz, D2O, δ): 39.10 (1C, CH2NH); 60.91 (1C, CH2); 65.87 (1C, CH);
66.71 (1C, CH2O); 70.10, 70.20, 72.88 (3C, CH); 99.79 (1C, CH);
127.56 (1C, CH2dCH); 129.95 (1C, CHdCH2); 168.83 (1C, CONH).
LR-MS (ESI): Calcd for C11H19NO7Na [M+Na]+, 300.26; found, 300.2.
Details of the synthesis and characterization of glucose and galactose
monomers 2′-acrylamidoethyl-ꢀ-D-glucopyranoside (12) and 2′-acry-
lamidoethyl-ꢀ-D-galactopyranoside (13) are given in the Supporting
Information.
Synthesis of Amide Derivatives of Surface Initiator (11-(2′-
Chloro-propionamido)-undecyl-trichlorosilane (14). A solution of
1-amino-10-undecene (2 g) in anhydrous THF (20 mL) cooled in an
ice bath, pyridine (1.2 mL), and 2-chloropropionyl chloride (1.3 mL)
were then added dropwise via syringe under an argon atmosphere. The
reaction mixture was allowed to come to room temperature slowly and
was stirred overnight. The reaction solution was diluted by hexane and
washed with 2 N HCl and deionized water. The organic phase was
dried over anhydrous sodium sulfate and concentrated in vacuum to
light-yellow oil. Flash column chromatography on silica in EtOAc/
hexanes (1:4) gave the pure product (compound 13) as a colorless oil
(2.3 g, yield 75%). 1H NMR: (300 MHz, CDCl3, δ): 1.28-1.60 (14H,
c. Synthesis of 2′-Aminoethyl-2,3,4,6-tetra-O-acetyl-R-D-mannopyra-
noside (5). A suspension of compound 2 (0.8 g) and activated palladium
on charcoal 10% Pd/C (120 mg) in anhydrous MeOH (32 mL) was
stirred under H2. After 30 min, the reaction was stopped and the mixture
was filtered. The filtrate was concentrated in vacuum to give the title
1
compound 5 in 46.5% yield as a fluffy white solid. H NMR: (300
m,
7
×
CH2); 1.73-1.75 (3H, d, CH3); 2.0-2.04 (2H, q,
mHz, CDCl3, δ): 2.0, 2.04, 2.10, 2.16 (12H, s, Me); 2.90-2.93 (2H, t,
OCH2CH2NH2); 3.46-3.53 (1H, m, OCH2CH2NH2); 3.71-3.78 (1H,
m, OCH2CH2NH2); 3.99-4.04 (1H, m, H5); 4.08-4.13 (1H, dd, J )
2.1, 12.2 Hz, H6); 4.26-4.32 (1H, dd, J ) 5.3, 12.2 Hz, H6); 4.85
-CH2-CHdCH2); 3.23-3.30 (2H, q, -N-CH2-); 4.37-4.44 (H, q,
-CH-); 4.91-5.02 (2H, -CHdCH2); 5.74-5.88 (1H, m,
-CHdCH2); 6.56 (1H, s, -NH-).

3076 Biomacromolecules, Vol. 11, No. 11, 2010
Yu and Kizhakkedathu
Scheme 1. Chemical Structures of 2′-Acrylamidoethyl-R-D-mannopyranoside (Compound 11), 2′-Acrylamidoethyl-ꢀ-D-glucopyranoside
(Compound 12), and 2′-Acrylamidoethyl-ꢀ-D-galactopyranoside (Compound 13)
To a 50 mL flask, 1 g of compound 13 and 5.3 g of trichlorosilane
were added, followed by the addition of 8 µL of Karstedt catalyst via
micropipette. The mixture was allowed to stir at room temperature for
5 h. The excess trichlorosilane was removed under reduced pressure.
The residue was diluted with 0.5 mL of anhydrous toluene. The solution
was quickly filtered through a plug of silica gel to remove the catalyst.
The filtrate was concentrated in vacuum to give the title compound 14
as light-yellow oil. 1H NMR: (300 MHz, CDCl3, δ): 1.28-1.61 (18H,
m, 9 × CH2); 1.72-1.75 (3H, d, CH3); 3.24-3.31 (2H, q, -N-CH2-);
4.38-4.45 (1H, q, -CH-); 6.56 (1H, s, -NH-).
Synthesis of Poly(2′-acrylamidoethyl-2,3,4,6-tetra-O-acetyl-r-D-man-
nopyranoside) (PAAETAM) Brushes from Hydroxyl Protected Mono-
mer. Copper(II) chloride (CuCl2, 3 mg, 0.02 mmol), copper(I) chloride
(CuCl, 7 mg, 0.07 mmol), and Me6TREN (52 µL, 0.18 mmol) were
added successively to a glass tube, followed by the addition of 12 mL
of DMSO. The solution was degassed with three freeze-pump-thaw
cycles. The solution was then transferred to the glovebox. The catalyst
solution (0.73 mL) was drawn and added to the vial, which contained
2′-acrylamidoethyl-2,3,4,6-tetra-O-acetyl-R-D-mannopyranoside (0.65
g, 1.46 mmol). After the monomer was completely dissolved, the surface
initiator (11-(2′-chloro-propionamido)-undecyl-trichlorosilane) deposited
silicon wafer was immersed in the polymerization mixture. Soluble-
free initiator, 22 µL of methyl 2-chloropropionate in DMSO (stock
solution 440 µL in 4 mL of DMSO), was added immediately to the
reaction mixture. The polymerization was allowed to proceed at RT
(22 °C) for 24 h. The substrate was then thoroughly rinsed with
methanol and sonicated in methanol for 30 min. The soluble polymer
formed along with the surface-grafted polymer was collected by passing
through a column packed with basic alumina and the polymer was
precipitated from diethyl ether.
PAAEGlc brushes and PAAEGal brushes were synthesized using a
similar procedure. All brush structures were characterized in terms of
thickness, graft density, molecular weight, contact angle measurements,
and AFM measurements.
Synthesis of Other Glucose-Containing Brushes. PGAMA and
PMAGlc brushes and brushes were synthesized following the polym-
erization procedures reported in the literature.28,35 and were character-
ized using ellipsometry and contact angle measurements.
Nonspecific Protein Adsorption Studies. Initially, the substrates
were equilibrated with phosphate-buffered saline (PBS) for 15 min;
then, the protein solutions were introduced. The protein solutions were
centrifuged at 10 000 rpm (9300 rcf) for 5 min. Supernatant was used
in the protein adsorption study. The final concentrations of BSA and
Fb were 1 and 0.25 mg/mL, respectively.
After incubation for 1 h at room temperature, the samples were
removed and washed with PBS buffer three times to remove loosely
adsorbed proteins. The images of the protein-adsorbed surfaces were
taken by a fluorescence microscope (NikonEclipse TE 2000-U with
an X-Cite 120 fluorescence illumination system, FITC and rhodamine
filters, and a DS-U1 suit digital camera). The fluorescence images were
transferred to gray scale by Adobe Photoshop 6.0, and intensity was
taken to be linearly related to the quantity of adsorbed proteins on the
surface.42,43 The initiator-modified substrate was chosen as the control
sample. The fluorescence intensity of the control sample was set as
100%, and the adsorption of proteins on various polymer grafted
surfaces was normalized on the basis of the calibration scale. After the
fluorescence measurement, the substrates were washed with water three
times. The dry thickness of the brushes after protein adsorption was
then determined by ellipsometry.
Specific Protein Interaction Studies. The studies followed the same
procedures described above except for the use of Con A and PBS buffer
containing 1 mM CaCl2 and 0.1 mM MnCl2. The pH of the buffer was
adjusted to 4.8. Con A is a lectin that specifically binds mannose and
glucose residues.25,26 The final concentration of Con A solution was
0.2 mg/mL.
Deprotection of PAAETAM. To a solution of PAAETAM (24 mg)
in anhydrous methanol (10 mL) was added 25% sodium methoxide in
methanol (11 µL, 23 mmol) dropwise. The reaction mixture was stirred
for 5 h at room temperature. Dowex cation-exchange resin (H form)
was added to adjust the pH to 6 to 7 and filtered. The filtrate was
concentrated in vacuum.
Deprotection of the Surface-Grafted PAAETAM. To a vial
containing PAAETAM grafted substrate, anhydrous methanol (5 mL)
and 25% sodium methoxide in methanol (5 µL) were added subse-
quently. The samples were removed from the vial after 20 h and
thoroughly washed with methanol and deionized water.
Results and Discussion
Synthesis of N-Substituted Acrylamide Derivatives of
Unprotected Glycomonomers. Chemical structures of synthe-
sized glycomonomers containing mannose, galactose, and
glucose residues are shown in Scheme 1. The synthesis of 2′-
acrylamidoethyl-R-D-mannopyranoside is outlined in Scheme
2. 1,2,3,4,6-Penta-O-acetyl-D-mannose was readily obtained by
peracetylation of D-mannose by using excess of acetic anhydride
in pyridine serving both as solvent and catalyst. Obtained
peracetylated mannose was a mixture of R- and ꢀ-anomer in a
Synthesis of Poly(2′-acrylamidoethyl-r-D-mannopyranoside)
(PAAEM) Brushes from Hydroxyl Free Monomer. Copper(II)
chloride (CuCl2, 1.35 mg, 0.01 mmol), copper(I) chloride (CuCl, 8 mg,
0.08 mmol), and Me6TREN (52 µL, 0.18 mmol) were added succes-
sively to a glass tube, followed by the addition of Milli Q water (12
mL). The solution was degassed with three freeze-pump-thaw cycles.
The solution was then transferred to the glovebox. The solution (1 mL)
was drawn and added to the vial, which contained 2′-acrylamidoethyl-
R-D-mannopyranoside (50 mg). After the monomer was completely
dissolved, surface-initiator-modified substrate (ester derivative) was
immersed in the solution. Soluble methyl 2-chloropropionate in water
with defined concentration was then added to the reaction solution.
The surface-initiated polymerization was allowed to proceed at RT (22
°C) for 24 h. The substrates were then thoroughly rinsed with water
and sonicated in water for 30 min, followed by drying with nitrogen
gas. The solution was dialyzed against deionized water and then freeze-
dried.
1
4:1 ratio (from H NMR analysis). Glycosylation of mannose
pentaacetate with 2-azidoethanol, which was easily accessible
from 2-bromoethanol and sodium azide, in the presence of
BF3 ·Et2O gave R glycosides as a sole product. The azide group
was then reduced to amine group via the Pd/C-catalyzed
hydrogenation. We found that a portion of the resulting primary
amine was self-condensed to generate symmetric secondary
amine (bis(2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyloxyethy-
l)amine) when the reaction time was extended beyond 30 min.
When the reaction time was changed from 30 min to 2 h, a

Synthesis of Functional Polymer Brushes
Biomacromolecules, Vol. 11, No. 11, 2010 3077
Scheme 2. Synthesis of 2′-Acrylamidoethyl-R-D-mannopyranoside
new MS peak at 766.4 Da, which corresponds to the positively
charged symmetric secondary amine, appeared in the spectrum
of resulting product (Supporting Information, Figure S1). This
observation is consistent with the reports that primary amine
can undergo self-condensation and transform to a secondary
amine in the presence of palladium catalyst.44,45 By controlling
the reaction time, we can achieve the conversion of azide to
primary amine without the formation of side product, however,
the yield is sacrificed (Figure S2, Supporting Information). The
2′-aminoethyl-2,3,4,6-tetra-O-acetyl-R-D-mannopyranoside (com-
pound 5) was then treated with acryloyl chloride in the presence
of triethylamine to give 2′-acrylamidoethyl-2,3,4,6-tetra-O-
acetyl-R-D-mannopyranoside. It is important that the formation
of secondary amine should be prevented before this step. N,N-
Di(2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyloxyethyl)acryla-
mide (formed by the reaction between secondary amine and
acryloyl chloride) and main product (compound 8) have similar
polarity values, and it was difficult to purify this mixture by
column chromatography. The deacetylation of the 2′-acrylami-
doethyl-2,3,4,6-tetra-O-acetyl-R-D-mannopyranoside (compound
8) gave the final product (compound 11). Purity of the final
monomer has great influence on the polymerization. (See the
next section.) A similar route was followed for the synthesis of
2′-acrylamidoethyl-ꢀ-D-glucopyranoside (12) and 2′-acrylami-
doethyl-ꢀ-D-galactopyranoside (13).
Surface Initiator Functionalization. Besides the ester-
functionalized SI-ATRP initiator (11-(2-bromo-2-methyl)-pro-
pionyloxy)-undecyltrichlorosilane), a new amide derivative of
initiator was also synthesized. The ellipsometry measurements
gave thicknesses of 2.8 ( 0.4 and 2 ( 0.1 nm, respectively,
for the amide- and ester-functionalized initiator layers. The water
contact angle on amide-based initiator (62.9 ( 0.2°) layer was
smaller than that on an ester-based initiator (74.5 ( 1.1°) layer
because the amide group is relatively more hydrophilic than
the ester group. XPS data collected from the two initiator-
modified Si wafers were presented in Figure S4 of the
Supporting Information. The Cl (2p) and N (1s) appearing at
200.6 and 400.3 eV, respectively, confirmed the formation of
amide-based initiator layer on Si wafer. The Br (3d) signal
situated at 69.7 eV, confirmed the presence of ester-based
initiator layer on Si wafer.
monomer directly from the surface (Scheme 3B). The study was
designed to help us understand the polymerization properties
of both types of monomers and achieve the goal of producing
fully functional glycopolymer layer containing carbohydrate
residues.
We estimated the grafting density (σ) for glycopolymer
brushes by using the equation σ ) (hFNA)/Mn, where Mn is the
molecular weight of free polymer in the solution, NA is
Avogadro’s number, h is the polymer layer “dry” thickness
measured by elliposometer, and F is the density of glycopolymer.
(We assumed that the density of glycopolymer brushes is equal
to 1 g/cm3.) Assuming that the molecular weight of the
glycopolymer grown in solution is similar that of surface-grafted
glycopolymer,28,46 the grafting density of tethered glycopolymer
chains was estimated. Although this is not a very accurate
method for calculation of graft density of the surface-grafted
polymer layers, it is a regular practice, especially for polymer
chains grown on flat surfaces, and is widely accepted in the
literature.28,46
i. SI-ATRP of PAAEM Brushes Via Protected Monomer
Route. Initially, we explored the preparation of glycopolymer
brushes by using 2′-acrylamidoethyl-2,3,4,6-tetra-O-acetyl-R-
D-mannopyranoside, followed by the deprotection of acetate
groups. The concentration of sodium methoxide was critical in
the deprotection of acetate group in the grafted glycopolymer
layer. The amide-functionalized initiator layer was tested initially
under the deprotection conditions to ensure its stability. At
relatively high concentration (e.g., 0.5 wt % NaOMe in MeOH),
the amide-functionalized initiator layer was destroyed, as evident
from the decrease in thickness from 2.8 to 1.2 nm. Therefore,
a lower concentration of sodium methoxide was chosen for the
study (0.025 wt % NaOMe in MeOH). In the case of acetate-
protected glycopolymer brushes, upon treatment with NaOMe,
the thickness of the grafted layer decreased from 7.8 ( 0.4 to
5.5 ( 0.3 nm in 22 h. The water contact angle decreased from
53.8 ( 1.3 to 33.5 ( 0.8° (Table S1 of the Supporting
Information). The thickness and contact angle did not show
noticeable change by extending the hydrolysis time to several
days. In comparison with the water contact angles of the
glycopolymer brushes prepared directly from the deprotected
monomer (7.2 ( 1.3°) (see the next section), we confirmed that
the deprotection of acetate group in the grafted glycopolymer
layer was not complete. The incomplete deprotection may be
brought by limited access of reactant to highly dense glyco-
polymer grafted surface. In contrast, the acetate group of free
polymer generated in solution can be effectively removed by
Synthesis of Glycopolymer Brushes. We adopted two routes
for the synthesis of glycopolymer layer containing mannose units
by (i) polymerization of acetate-protected monomer, followed
by the deprotection of acetate groups in the surface grafted
polymer (Scheme 3A) and (ii) polymerization of unprotected

3078 Biomacromolecules, Vol. 11, No. 11, 2010
Yu and Kizhakkedathu
Scheme 3. Synthesis of Poly(2′-acrylamidoethyl-R-D-mannopyranoside) Brushes by Polymerizing Protected Monomer
(2′-Acrylamido-2,3,4,6-tetra-O-acetyl-R-D-mannopyranoside), Followed by Deprotection Procedure (A) or by Polymerizing Deprotected
Monomer (2′-Acrylamidoethyl-R-D-mannopyranoside) Directly (B). The Brushes Prepared by Different Routes have Difference in the
Structure of Surface Initiator
Table 1. Effect of Halogen, Ligand and Solvents on the Polymerization of 2′-Acrylamidoethyl-R-D-mannopyranosidea
dry
grafting density
(chains/nm2)
static water contact
polymer
ligand
solvent
[CuCl2]/[CuCl]
Mn
PDI
thickness (nm)
angle (°)
1
2
3
4
5
6
7
8
H2O
H2O
H2O
H2O
H2O/DMF 1:1
H2O/DMSO 1:1
H2O
0
1/16
1/8
1/4
1.8
1/8
250 000
66 000
83 000
51 000
38 000
60 000
58 000b
55 000
2.1
1.7
1.4
1.5
1.4
3.6
4.5
3.2
25.2 ( 0.2
17.9 ( 0.8
24.1 ( 0.3
11.7 ( 0.4
6.6 ( 0.3
7.9 ( 0.5
14.3 ( 0.2
5.4 ( 0.1
0.06
0.16
0.17
0.14
0.10
0.08
0.15
0.06
5.3 ( 1.5
6.2 ( 1.7
4.4 ( 0.9
5.4 ( 1.3
5.6 ( 0.7
4.8 ( 1.7
4.1 ( 1.3
5.4 ( 1.9
Me6TREN
HMTETA
CuBr2/CuBr:1/8
1/8
H2O
a Polymerization conditions: M: 2′-acrylamidoethyl-R-D-mannopyranoside, 5% W/V. Initiator: methyl 2-chloropropionate, [I] ) 1.25 mM, reaction time:
24 h, RT. b Polymerization conditions: [M] ) 5% W/V. Initiator: methyl 2-bromoisobutyrate, [I] ) 1.25 mM, reaction time: 24 h, RT (22 °C).
using sodium methoxide in methanol. Figure S5 of the Sup-
porting Information shows the H NMR spectra of polymer
glycopolymer chains than the bromine system (polymer 7 in
Table 1). This is consistent with the report on polymerization
of acrylamide derivatives by using bromine system because Br
chain ends are more susceptible to nucleophilic substitution or
elimination reactions in protic solvents.47,48 The loss of terminal
C-Br groups will effectively “kill” the polymer chain ends,
and a relatively broad molecular weight distribution can be
expected. The use of chloride initiator in conjunction with CuCl/
CuCl2/Me6TREN resulted in better control of the polymerization
because chlorine chain ends are better preserved during the
polymerization process because of the decrease in nucleophilic
displacement.49 The effect of halogen in surface initiator on
polymerization was also investigated. The ester-based surface
initiator gave better surface-initiated polymerization, as noted
from the thickness of the grafted layer. Under similar polym-
erization conditions, glycopolymer brushes grown from ester
initiator gave a thickness of 17.9 ( 0.8 nm (Table 1, entry 2)
in comparison with 14.2 ( 1.3 nm thickness produced by amide-
based surface initiator (having a secondary chlorine).
1
before (I) and after deprotection (II). The disappearance of four
sharp peaks around 2 ppm, which were assigned to the acetate
groups, confirmed the completion of deprotection. The number-
average molecular weight of polymer after deprotection was
14 000, whereas the PDI value was 4.5. The broad polydispersity
indicated that the polymerization control was very poor.
ii. SI-ATRP of PAAEM Brushes Via Unprotected Mono-
mer Route. Because of the inefficient removal of acetate groups
in the grafted glycopolymer layer and other complications, we
prepared the mannose-containing polymer graft layers using
unprotected 2′-acrylamidoethyl-R-D-mannopyranoside by SI-
ATRP. Silicon wafer modified with ester-based ATRP initiator
was used as a substrate for this study. Several experimental
conditions were investigated to improve the control of polym-
erization in solution and from surface.
Effect of Halogen. The polymerization behaviors of the
bromine-containing system (alkyl bromide as the initiator, CuBr/
CuBr2/Me6TREN as the catalyst) and the chlorine-containing
system (alkyl chloride, CuCl/CuCl2/Me6TREN) were studied and
compared. The chlorine system gave a narrower distribution of
The effect of molar ratio of Cu(I)/Cu(II) on polymerization
was also investigated in detail. Without the addition of deactiva-
tor, CuCl2, the polydispersity of resulting glycopolymer (poly-

Synthesis of Functional Polymer Brushes
Biomacromolecules, Vol. 11, No. 11, 2010 3079
mer 1) was broad (PDI ) 2.1). The use of a mixture of CuCl/
Me6TREN and CuCl2/Me6TREN complex (molar ratio 8:1)
resulted in a decrease in polydispersity to 1.4 (polymer 3). The
addition of more CuCl2 (molar ratio of Cu(I)/Cu(II) 4:1)
complex did not show any large change in polydispersity. The
thickness of brushes also showed dependence on the molar ratio
of Cu(I)/Cu(II). At a molar ratio of 8:1, the highest thickness
of the grafted layer was achieved while the polydispersity of
free polymer was maintained as low as 1.4.
Effect of Ligands. 1,1,4,7,10,10-Hexamethyltriethylenetet-
ramine (HMTETA), which is considered to be a good ligand
for the acrylamide polymerizations,50,51 was used for the
polymerization of mannose-containing monomer. The polydis-
persity of resulting PAAEM (polymer 8 in Table 1) was high
(>3.2), indicating a poor control of the polymerization. The
thickness for the PAAEM brushes was also low (5.4 nm) with
a grafting density 0.06 chains/nm2. When Me6TREN was
employed as a ligand, substantial improvement in polymerization
was observed, unlike other commonly used ATRP ligands.52,53
The resulting glycopolymers showed lower polydispersity (PDI
1.4 to 1.7).
Figure 1. Relationship between grafted PAAEM layer thickness and
molecular weight of free polymer (9). Polydispersity of free polymer
(2) formed along with surface initiated polymer. Polymerization
condition: [M] ) 5% W/V. Initiator: methyl 2-chloropropionate, [CuCl]/
[CuCl2]/[Me6TREN] ) 8/1/18 mM, RT. Ester-based surface initiator
was used.
Effect of Solvents. Polymerization was carried out in different
solvent systems, and the results are shown in Table 1. The
thickness of PAAEM brushes prepared in the mixed solvent
DMF/H2O was much lower than those prepared in H2O.
Polymerization in mixed solvent DMF/H2O produced low
molecular weight with similar polydispersity (PDI ) 1.4),
whereas H2O/DMSO gave broader dispersity of chains (PDI )
3.6).
Effect of Addition of Soluble ATRP Initiator. The thickness
of PAAEM can be controlled by the addition of free soluble
initiator in the polymerization medium. By increasing the
concentration of free initiator from 0.55 to 3.75 mM, the
thickness of grafted polymer layer proportionally decreased from
34.7 to 10.3 nm, whereas the calculated grafting density of
brushes maintained relatively constant at 0.17 chains/nm2. There
was also proportional decrease in the thickness of the brush
with decrease in molecular weight of free polymer (Figure 1).
When unpurified monomer mixture (previously described)
containing 7% N,N-di(R-D-mannopyranosyloxyethyl)acrylamide
(Figure S3 of the Supporting Information) was used for
polymerization, both thickness and grafting density were
decreased. For instance, under similar conditions, the purified
AAEM monomer gave a layer thickness of 34.7 nm with a graft
density (0.17 chains/nm2), whereas the mixture of monomers
(93 mol % AAEM and 7 mol % N,N-di(R-D-mannopyranosy-
loxyethyl)acrylamide) gave a polymer layer thickness and
grafting density 19.6 nm and 0.09 chains/nm2, respectively. This
large difference showed the importance of the previously
mentioned purification step.
Figure 2. Thicknesses (9) and grafting densities (2) of PAAEM
brushes with polymerization time. Polymerization condition: [M] ) 5%
W/V, Initiator: methyl 2 chloropropionate, [I] ) 0.75 mM, [CuCl]/
[CuCl2]/[Me6TREN] ) 8/1/18 mM, RT, ester-based surface initiator
was used.
brushes, whereas the grafting densities were 0.12 chains/nm2,
respectively.
Kinetics of Polymerization of 2′-Acrylamidoethyl-r-D-man-
nopyranoside. We monitored the growth of PAAEM brushes
during SI-ATRP by measuring grafted polymer layer thickness.
Figure 2 shows the increase in polymer layer thickness measured
by ellipsometry as a function of polymerization time. In the
first 10 min, thickness increased rapidly to 22.9 ( 1.4 nm,
indicating a very fast initial polymerization. After the reaction
time was extended to 2 h, thickness increased to 33.9 ( 1.7
nm. Further extending the reaction time to 24 h resulted in only
a small change in the thickness. Two possible reasons may be
accounted for the arrest of the growth of brushes: catalyst
deactivation and chain termination by bimolecular coupling or
Effect of Different Carbohydrates in the Monomer. We
prepared PAAEGlc brushes and PAAEGal brushes from their
respective unprotected monomer (Scheme 1) by adopting a
similar polymerization condition as that used in the polymeri-
zation kinetics study (see the caption of Figure 3), and the
polymerization time was kept constant at 24 h. The results are
summarized in Table 2. The thickness for the PAAEGlc and
PAAEGal brushes was slightly higher than that for PAAEM
Table 2. Characteristics Results for Poly(2′-acrylamidoethyl-ꢀ-D-galactopyranoside) and Poly(2′-acrylamidoethyl-ꢀ-D-glucopyranoside)
Brushes and Corresponding Free Polymer in Solution
polymer
Mn
PDI brush dry thickness (nm) graft density (chains/nm2) water contact angle (°)
poly(2′-acrylamidoethyl-ꢀ-D-galactopyranoside) 220 000 1.4
poly(2′-acrylamidoethyl-ꢀ-D-glucopyranoside) 209 000 1.5
42.7 ( 0.82
42.8 ( 0.61
0.12
0.12
6.9 ( 1.2
5.6 ( 0.7

3080 Biomacromolecules, Vol. 11, No. 11, 2010
Yu and Kizhakkedathu
Figure 3. (A) First-order kinetic plot for surface-initiated ATRP of 2′-acrylamidoethyl-R-D-mannopyranoside in H2O. (B) Dependence of molecular
weight (Mn) and molecular weight distribution (Mw/Mn) on monomer conversion. Polymerization condition: [M] ) 5% W/V. Initiator: methyl
2-chloropropionate, [I] ) 0.75 mM, [CuCl]/[CuCl2]/[Me6TREN] ) 8/1/18 mM, RT. Ester-based surface initiator was used.
Table 3. Characteristics Results of Brushes Containing Glucose with Different Structures and Corresponding Free Polymer in Solution
glucose-containing brushes
dry thickness (nm)
grafting density (chains/nm2)
Mn
PDI
water contact angle (°)
PAAEGlc
PGAMA
PMAGlc
42.8 ( 0.6
37.2 ( 0.2
29.9 ( 0.1
0.12
0.3
0.25
209 000
73 600
72 000
1.5
1.6
1.2
5.6 ( 0.7
19.8 ( 1
39.8 ( 1.3
disproportionation. To examine whether the catalyst was
deactivating during the reaction, we carried out a control
experiment in which fresh catalyst54,55 was added after 24 h of
polymerization. This fresh catalyst addition did not lead to any
significant change in polymer growth, indicating that catalyst
deactivation was not the primary reason for cessation of layer
growth. Therefore, we attribute the chain arrest to either
bimolecular coupling or disproportionation.
and, subsequently, a high polymerization rate in the initial stage.
In aqueous ATRP, water acts as both an accelerator to increase
the activity of the catalyst and a diluent.
Surface Characterization. The PAAEM grafted layers with
thicknesses ranging from 10 to 40 nm gave highly hydrophilic
surface layers, as evident from the very low water contact angles
(<10°) (Table 1). PAAEGlc and PAAEGla brushes also behaved
similarly (Table 2). The water contact angles of newly synthe-
sized brushes are much lower than the reported glycopolymer
brushes having similar thickness. A comparison of glycopolymer
brushes containing glucose units is given in Table 3. The
pronounced hydrophilic character of PAAEGlc grafted surface
maybe attributed to the unprotected carbohydrate unit within
the grafted layer. Figure 4 shows the ATR-FITR spectra of
PAAEM, PAAEGal, and PAAEGlc brushes. The characteristic
peaks at 1647 and 1550 cm-1 correspond to the CdO stretching
vibration and the N-H bending vibrations of the amide group.
The molecular weights of free polymer in solution and total
conversion were also monitored during the polymerization.
Figure 3 shows the first-order kinetic plot (A) and the evolution
of Mn with conversion for ATRP of AAEM in water (B). The
initial rate of the polymerization was very fast (the Mn rapidly
increased to 100 000), whereas the total monomer conversion
(contributions from both solution polymerization and surface
polymerization) reached 4.8% in 10 min. After the reaction time
was extended to 2 h, the Mn increased to 135 000, whereas the
total conversion increased to 16%. After 24 h, the Mn increased
to 140 000 with the monomer conversion to 22.3%. The
polydispersity of the chains decreased with monomer conversion
and remained relatively constant above 10.7% conversion.
Although the unusual nonlinear nature of the first-order kinetic
plot and the evolution of Mn with conversion are indicative of
termination reactions in the early stage, the symmetrical
unimodal feature of GPC traces of the glycopolymer at high
conversions with relatively narrow molecular weight distribution
is telling a different story (Figure S7, Supporting Information).
Similar unusual polymerization behavior has been observed by our
group56,57 as well as other investigators on surface initiated
polymerizations in aqueous solutions.58-62 Messersmith observed
similar rapid initial growing rate during the SI-ATRP of methyl
methacrylate macromonomers with oligo(ethylene glycol) side
chains.58 Xu also reported that there was a rapid increase in the
degree of polymerization of GAMA during the initial stage of
polymerization.32 The high dielectric constant, especially water,
increased the activity of the catalyst.63,64 The high activity of Cu(I)
complex brings a high concentration of active species (or radicals)
Figure 4. FTIR spectra of glycopolymer brushes containing different
carbohydrate units: (A) PAAEGlc brush (38.4 nm), (B) PAAEGal brush
(36.8 nm), and (C) PAAEM brush (27 nm).

Synthesis of Functional Polymer Brushes
Biomacromolecules, Vol. 11, No. 11, 2010 3081
Figure 5. AFM topographic images (scan area: 2 × 2 µm2) and the corresponding cross section of glycopolymer brushes containing different
carbohydrate units: (A) PAAEM brush (38.4 nm), (B) PAAEGal brush (39.5 nm), and (C) PAAEGlc brush (38.7 nm).
The broad peak centered at 3334 cm-1 is assigned to the OH
stretching vibration. The peak at 2929 cm-1 is due to the C-H
asymmetric vibration mode of the -CH2- groups. AFM
topographical images (Figure 5) showed that the glycopolymer
grafted surface is relatively smooth. The surface roughness
values for the PAAEM, PAAEGal, and PAAEGlc brushes are
about 0.72, 0.9, and 1.4 nm, respectively.
Nonfouling Properties: A Proof-of-Concept Study. We
performed a preliminary study to understand the nonbiofouling
properties of new glycopolymer brushes containing different
carbohydrate units. The ability to resist nonspecific protein
adsorption by these surfaces was measured by fluorescence
microscopy and ellipsometry. Samples having similar dry
thickness and graft densities were used for the measurements.
Two proteins, FITC labeled-albumin and Alex Fluor594 labeled
Fb, were used in our experiments. Albumin is the most abundant
protein in the circulatory system (∼65%) and contributes 80%
to colloid osmotic blood pressure. Fb is a large, blood plasma
protein that strongly adsorbs to hydrophobic surfaces and is
commonly used as a model for sticky serum proteins.
a. Effect of Carbohydrate Units. The thicknesses of PAAEM,
PAAEGal, and PAAEGlc brushes did not show any noticeable
change after incubation with BSA and Fb solution for 1 h
(Figures 6A and 7A). The fluorescence intensities of the
glycopolymer brushes after protein incubation were also com-
pared with that of initiator-modified silicon wafer. The glyco-
polymer brushes containing mannose, galactose, and glucose
significantly reduced the BSA adsorption, as evident from 149-,
172-, and 500-fold reduction in fluorescence intensity compared
with the initiator-modified surface (Figure 6C). The Fb adsorp-
tions were reduced by 52, 115 and 135-fold for the PAAEM,
PAAEGal, and PAAEGlc brushes, respectively, (Figure 7C)
compared with the initiator-modified surface. In the case of
glycopolymer layer (PAAEM*) prepared from acetate-protected
monomer, followed by deprotection, the reduction in the
fluorescence intensity was only 2.4-fold. The large difference
in reduction of protein adsorption again highlights the impor-
tance of preparation of glycopolymer brushes from unprotected
monomer directly. The large reduction in protein adsorption
brought by PAAEM, PAAEGla, and PAAEGlc brushes prepared
from deprotected monomer directly may be due to (i) highly
hydrophilic nature of brushes originated from carbohydrate
residues possessing hydrogen-donating abilities,65-68 (ii) en-
tropic repulsion brought by the highly stretched chains in
brushes,69-71 and (iii) the chemical nature of carbohydrate
units.72,73 It is also shown from the nonspecific protein adsorp-
tion study that the PAAEGlc brushes (containing glucose units)
showed better performance against BSA and Fb adsorption than
the PAAEM and PAAEGla brushes (containing mannose and
galactose units, respectively). The reason for the difference in
nonbiofouling property is not clear at this time and is under
investigation. The differences in the 3D arrangement of the
hydroxyl groups of the carbohydrate units in the glycopolymer
layer may disrupt the structural and dynamic properties of water
layer bond to this layer through the hydrogen-bonding interac-
tion.73 Because protein-resistant properties of the hydrated brush
surface are originated from the repulsive forces between the
hydration layer and protein, any differences in the structural
and dynamic properties of the water layer will bring a difference
in the protein adsorption characteristics.65,73
b. Effect of Carbohydrate Structure. We compared the
protein-resistant properties of PAAEGlc brushes with other
glucose contain polymer brushes reported in the literature28,35
(Figures 6B,D and 7B,D). PGAMA brushes, containing glucose
in a linear structure (Scheme 4), showed similar abilities to that
of PAAEGlc brushes against BSA and Fb adsorption, that is,
200- and 172-fold reduction, respectively, in comparison with
initiator-modified layer. PMAGlc brushes, in which hydroxyl

3082 Biomacromolecules, Vol. 11, No. 11, 2010
Yu and Kizhakkedathu
Figure 6. Comparison of nonspecific interaction of BSA: The increase in thicknesses of glycopolymer brushes containing (A) different carbohydrate
units and (B) glucose units with different structures after BSA adsorption. (C) Fluorescence intensity reduction brought by glycopolymer brushes
containing (A) different carbohydrate units and (D) glucose units with different structures after incubating with fluorescently labeled BSA. Initiator-
modified silicon wafer was set as the control sample. PAAEM*: poly(2′-acrylamidoethyl-R-D-mannopyranoside) brushes prepared by polymerizing
acetate protected monomer and followed by hydrolysis for 22 h. The original thicknesses for PAAEM, PAAEGla, PAAEGlc, PAAEM*, PGAMA,
and PMAGlc before BSA adsorption are 38.4 ( 0.4, 39.6 ( 0.3, 38.8 ( 0.8, 5.5 ( 0.1, 37.2 ( 0.2, and 29.9 ( 0.2 nm, respectively. The grafting
densities for PAAEM, PAAEGla, PAAEGlc, PAAEM*, PGAMA, and PMAGlc brushes are 0.16, 0.12, 0.12, 0.24, 0.3, and 0.25 chains/nm2,
respectively.
Scheme 4. Chemical Structures of (A) Poly(2′-acrylamidoethyl-ꢀ-D-glucopyranoside) brushes, (B) Poly(D-gluconamidoethyl methacrylate)
(PGAMA),35 and (C) Poly(3-O-methacryloyl-D-glucofuranose) (PMAGlc) Brushes28
groups at position 3 of the pyranose ring in the glucose structure
(Scheme 4) have been modified, gave a reduction of 77- and
3-fold against BSA and Fb adsorption, respectively. The
relatively small reduction brought by the PMAGlc brushes
maybe related to the incomplete deprotection within the brushes.
The large difference in Fb adsorption between the PMAGlc and
other brushes again highlights the importance of preparation of
glycopolymer brushes from unprotected monomer directly.
Specific Interaction with Protein: a Proof-of-Concept
Study. Con A, is a 26 000 MW monomer that exists as a dimer
at low (<5.5) pH and as a tetramer at high (>7) pH.74 Each

Synthesis of Functional Polymer Brushes
Biomacromolecules, Vol. 11, No. 11, 2010 3083
Figure 7. Comparison of nonspecific interaction of fibrinogen: The increase in thicknesses of glycopolymer brushes containing (A) different
carbohydrate units and (B) glucose units with different structures after fibrinogen adsorption. (C) Fluorescence intensity reduction brought by
glycopolymer brushes containing (A) different carbohydrate units and (D) glucose units with different structures after incubating with fluorescently
labeled fibrinogen. Initiator-modified silicon wafer was set as the control sample. The original thicknesses for PAAEM, PAAEGla, PAAEGlc,
PGAMA, and PMAGlc before BSA adsorption are 39.3 ( 0.2, 40.1 ( 0.2, 39.2 ( 0.5, 37.2 ( 0.2, and 29.9 ( 0.2 nm, respectively. The grafting
densities for PAAEM, PAAEGla, PAAEGlc, PGAMA, and PMAGlc brushes are 0.16, 0.12, 0.12, 0.3, and 0.25 chains/nm2, respectively.
Figure 8. Comparison of specific interaction of Con A: (A) Increase in thicknesses of glycopolymer after Con A adsorption and (B) fluorescence
intensity reduction brought by glycopolymer brushes containing different carbohydrate units after incubating with fluorescently labeled Con A.
Initiator-modified silicon wafer was set as the control sample. The original thicknesses for PAAEM, PAAEGla, PAAEGlc, PGAMA, and PMAGlc
before Con A adsorption are 41.5 ( 0.8, 42.1 ( 0.5, 42.5 ( 0.3, 37.6 ( 0.4, and 30.5 ( 0.5 nm, respectively. The grafting densities for PAAEM,
PAAEGla, PAAEGlc, PGAMA, and PMAGlc brushes are 0.16, 0.12, 0.12, 0.3, and 0.25 chains/nm2, respectively.
subunit contains a binding site that binds ligands with unmodi-
fied hydroxyls of the carbohydrate units.
a. Effect of Different Carbohydrate Units. Figure 8 shows
that Con A has a specific interaction with glycopolymer brushes
containing mannose and glucose units, not galactose units. After
incubation with Con A, the thickness of brush layer increased
by 7.2 and 0.6 nm, respectively, for PAAEM brushes and
PAAEGlc brushes. The fluorescence intensities increased by
20.5- and 2.9-fold compared with control samples, respectively.
It has been reported that D-mannopyrannoside shows ap-
proximately four times affinity to Con A compared with
D-glucopyrannoside in the case of monovalent binding.75 The

3084 Biomacromolecules, Vol. 11, No. 11, 2010
Yu and Kizhakkedathu
enhanced difference in specific interaction with glycopolymer
brushes may also be brought by the multivalent binding due to
the “glyco-cluster” effect. In summary, our results show that
PAAEM and PAAEGlc brushes prevented nonspecific interac-
tions and preserved the specific interaction with Con A as well.
b. Effect of Modification of Carbohydrate Structure. To
illustrate the effect of carbohydrate structure on the specific
interaction with lectins, we compared the ConA interaction
profiles of PAAEGlc with PGAMA35 and PMAGlc28 brushes
(Figure 8). In comparison with PAAEGlc brushes, PGAMA and
PMAGlc showed very weak interaction with Con A. As
previously discussed, the glucose unit in the PGAMA has a
linear glucose structure, which is quite different from its natural
pyranose form, not able to interact with Con A specifically. In
the case of PMAGlc brushes, although the carbohydrate units
in PMAGlc brushes are in the pyranose form, the hydroxyl
group at position 3 has been modified. The modification
hindered the specific protein interaction. Because PAAEGlc
brushes have sugar units in pyranose form and the sugar unit is
not modified, they gave much better interaction with Con A
compared with PGAMA or PMAGlc. The residual adsorption
shown by these surfaces may be due to the nonspecific
interactions. This is consistent with the report by Goldstein et
al. that a monosaccharide is required to have D-manno- or
D-glucopyranose configuration with unmodified hydroxyl groups
at the C-3, C-4, and C-6 positions to bind strongly to ConA.76
Our results highlight the importance of natural sugar structure
for retaining the specific protein interactions.
Supporting Information Available. Experimental details and
characterization of compounds containing galactose and glucose
units, XPS data of surface initiator, 1H NMR spectra of poly(2′-
acrylamido-2,3,4,6-tetra-O-acetyl-R-D-mannopyranoside) before
and after deprotection, details of deprotection of poly(2′-
acrylamido-2,3,4,6-tetra-O-acetyl-R-D-mannopyranoside) brush,
GPC traces of glycopolymers, and so on. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Acknowledgment. We acknowledge the funding provided
by the Canadian Diabetes Association, NSERC, and CIHR. The
LMB Macromolecular Hub at the UBC Centre for Blood
Research was funded by CFI and MSFHR. K.Y. is a recipient
of a CIHR/Canadian Blood Services (CBS) postdoctoral fel-
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postdoctoral fellowship from the Strategic Training Program
in Transfusion Science at the Centre for Blood Research
supported by CIHR and Heart and Stroke Foundation of Canada.
J.N.K. is a recipient of a CIHR/CBS new investigator in
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