Antifouling glycocalyx-mimetic peptoids

Article abstract of DOI:10.1021/ja404681x

The glycocalyx of the cell is composed of highly hydrated saccharidic groups conjugated to protein and lipid cores. Although components of the glycocalyx are important in cell-cell interactions and other specific biological recognition events, a fundamental role of the glycocalyx is the inhibition of nonspecific interactions at the cell surface. Inspired by glycoproteins present in the glycocalyx, we describe a new class of synthetic antifouling polymer composed of saccharide containing N-substituted polypeptide (glycopeptoid). Grafting of glycopeptoids to a solid surface resulted in a biomimetic shielding layer that dramatically reduced nonspecific protein, fibroblast, and bacterial cell attachment. All-atom molecular dynamics simulation of grafted glycopeptoids revealed an aqueous interface enriched in highly hydrated saccharide residues. In comparison to saccharide-free peptoids, the interfacial saccharide residues of glycopeptoids formed a higher number of hydrogen bonds with water molecules. Moreover, these hydrogen bonds displayed a longer persistence time, which we believe contributed to fouling resistance by impeding interactions with biomolecules. Our findings suggest that the fouling resistance of glycopeptoids can be explained by the presence of both a 'water barrier' effect associated with the hydrated saccharide residues as well as steric hindrance from the polymer backbone.

Full text of DOI:10.1021/ja404681x

Subscriber access provided by Penn State | University Libraries
Article
Antifouling Glycocalyx-Mimetic Peptoids
Hyun Ok Ham, Sung Hyun Park, Josh W. Kurutz, Igal G. Szleifer, and Phillip B. Messersmith
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja404681x • Publication Date (Web): 06 Aug 2013
Downloaded from http://pubs.acs.org on August 7, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Antifouling Glycocalyx-Mimetic Peptoids
Hyun Ok Ham1,5, †, Sung Hyun Park1,5, Josh W. Kurutz4,8, Igal G. Szleifer1,3,4,5,6,7, Phillip B. Messer-
smith1,2,3,5,6,7, *
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1Biomedical Engineering Department, 2Materials Science and Engineering Department, 3Chemical and Biological Engineer-
ing Department, 4Chemistry Department, 5Chemistry of Life Processes Institute, 6Institute for Bionanotechnology in Medi-
cine, 7Robert H. Lurie Comprehensive Cancer Center, and 8Integrated Molecular Structure Education and Research Center,
Northwestern University, Evanston, Illinois 60208, United States
Supporting Information Placeholder
ABSTRACT: The glycocalyx of the cell is composed of highly hydrated saccharidic groups conjugated to protein and lipid cores.
Although components of the glycocalyx are important in cell-cell interactions and other specific biological recognition events, a
fundamental role of the glycocalyx is the inhibition of nonspecific interactions at the cell surface. Inspired by glycoproteins present
in the glycocalyx, we describe a new class of synthetic antifouling polymer composed of saccharide containing N-substituted poly-
peptide (glycopeptoid). Grafting of glycopeptoids to a solid surface resulted in a biomimetic shielding layer that dramatically re-
duced nonspecific protein, fibroblast and bacterial cell attachment. All-atom molecular dynamics simulation of grafted glycopep-
toids revealed an aqueous interface enriched in highly hydrated saccharide residues. In comparison to saccharide-free peptoids, the
interfacial saccharide residues of glycopeptoids formed a higher number of hydrogen bonds with water molecules. Moreover, these
hydrogen bonds displayed a longer persistence time, which we believe contributed to fouling resistance by impeding interactions
with biomolecules. Our findings suggest that the fouling resistance of glycopeptoids can be explained by the presence of both a
‘water barrier’ effect associated with the hydrated saccharide residues, as well as steric hindrance from the polymer backbone.
INTRODUCTION
ed peptoids for prevention of surface biofouling, exploring a
number of alkyl, ether and hydroxyalkyl side chains.16-18
Poly-N-substituted glycines, or peptoids, are peptide-
mimetic macromolecules with a polyglycine backbone and
side chain derivatization at the amide nitrogen instead of the
α-carbon.1 In contrast to polypeptides, this structural substitu-
tion eliminates backbone chirality, prevents formation of β-
sheet stabilizing hydrogen bonds, and increases protease re-
sistance and stability in a wide range of salt, pH and solvent
conditions.2,3 An important characteristic of peptoids is that
they can be synthesized on automated peptide synthesizer in a
sequence-specific manner with precisely controlled length and
diverse natural or non-natural side chains. This feature permits
the design of sequence-specific molecules useful in fundamen-
tal studies of protein folding and in a variety of biomedical
applications.4,5 Biologically active peptoids have been investi-
gated as mimics of HIV-Tat proteins, lung surfactant protein,
and antibacterial peptides.6 Peptoids also have been explored
for gene therapy, genetic analysis,7 bioseparations,8 and as
ligands for cancer targeting.9
The glycocalyx is the outermost surface of the cell mem-
brane composed of a dense layer of highly glycosylated spe-
cies (e.g., glycoproteins, glycolipids, and other glycol-
conjugates). The idea of mimicking the carbohydrate-rich gly-
cocalyx of plasma cell membranes is an attractive one due to
the high natural resistance of the glycocalyx toward nonspecif-
ic interactions.19 Although carbohydrates have many charac-
teristics suitable for antifouling applications (e.g., high hydro-
philicity, neutral charge, flexible backbone), only a limited
number of antifouling carbohydrates, glycopolymers and car-
bohydrate containing monolayers have been investigated.
Dextran, which is a maltose polymer, exhibits very low non-
specific protein interactions, and is widely used as an alterna-
tive to poly(ethylene glycol).20-22 Dextran-oligomer modified
poly(vinyl amine) also showed good resistance against protein
and platelet adhesion on hydrophobic surfaces (graphite and
polycarbonate).23,24 It is also known that mannose, and other
oligosaccharide containing self-assembled monolayers
(SAMs) exhibit good short-term protein resistance.25-28 Gly-
cocalyx-mimetic peptoids, i.e. peptoids containing saccharide
or oligosaccharide side chains, have not been explored in an
antifouling context.
Biofouling involves the accumulation of biomolecules,
cells, micro- and macroorganisms on surfaces, and is often an
undesired and irreversible event of great practical importance
in industrial, military, consumer and medical settings. In the
healthcare area, biofouling of medical implants, surgical de-
vices, and biosensors can hinder biological performance, af-
fect device longevity, and increase healthcare costs. Common
strategies to prevent biofouling include surface grafting of
organic thin films including self-assembled monolayers
(SAMs)10 and antifouling polymer brushes such as
poly(ethylene glycol) (PEG),11 poly(2-oxazone),12 block co-
polymers containing PEG chains,13 polysaccharides,14 and
zwitterionic polymers.15 We as well as others have investigat-
Here we introduce a novel antifouling glycopeptide-mimetic
polymer composed of three distinct functional domains (Fig-
ure 1): a peptide anchor, a polypeptoid backbone, and oligo-
saccharide side chain. Glucose and β-D-maltose were chosen
as the oligosaccharide side chains because they provide physi-
cochemical properties that are known to be important in foul-
ing resistance, namely neutral charge and high
hydrophilicity.29 2’-azidoethyl-β-D maltopyranose (Mal(OH)-
ACS Paragon Plus Environment

Journal of the American Chemical Society
N3) was prepared from β-D-maltose-monohydrate as shown in
Page 2 of 9
1
2
3
4
5
6
7
8
Scheme 1. Then, the disaccharide was coupled to the peptoid
backbone via click chemistry as guided by previous reports of
glycopeptoid30 and glycomimetic polymer synthesis (Figure 1
and Figure 2).31,32 The resulting polymer was used for surface
modification of TiO2 substrates and the antifouling property
was evaluated by testing resistance against protein adsorption,
cell adhesion, and bacterial attachment. Insight into the role of
terminally linked saccharides in mediating antifouling perfor-
mance was provided by molecular dynamic simulations of
surface-grafted peptoids, revealing new features of the interac-
tion of water with glycopeptoids.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of 2’azidoethyl-β-D-maltopyranose. i)
excess Ac2O. ii) 2-bromoethanol and BF3-OEt2 in CH2Cl2.
iii) NaN3 in DMF:DCM (6:1). iv) CH3ONa in CH3OH.
Figure 1. Solid phase synthesis of M20Glu(OH) and
M20Mal(OH). First, the peptide anchor was synthesized by tradi-
tional solid phase peptide synthesis (Step 1), followed by peptoid
backbone synthesis by the submonomer approach (Step 2), after
which oligosaccharide was conjugated by CuAAC (Step 3).
Figure 2. Chemical structures of 20mer Nme peptoid (top),
M20Glu(OH) (middle), and M20Mal(OH) (bottom) (a). Analyti-
cal RP-HPLC spectra (214 nm, 2–50% acetonitrile in H2O with
0.1% v/v TFA 1.0 ml/min) (b), and MALDI-TOF mass spectra of
Na+ adducts (c) of purified M20Glu(OH) and M20Mal(OH).
RESULTS AND DISCUSSIONS
Characterization and evaluation of antifouling performance
of glycopeptoid-coated surfaces.
2
ACS Paragon Plus Environment

Page 3 of 9
Journal of the American Chemical Society
TiO2 was used in this study as a prototypical biomaterial
and aromatic carbons in the peptoid backbone and the DOPA-
Lys-DOPA-Lys-DOPA anchoring groups, as well as some
hydrocarbon contamination. Dramatic changes after polymer
coating occurred for binding energies in the range 286-287 eV
which correspond to the ether carbons (286.6 eV) of the Nme
side chains, to the carbons adjacent to amino groups (286.0 eV
[-(C)H2-NH2], 286.3 eV [-NH-(C)H-C=O]) of the peptoid
backbone36, the hydroxyl carbons (286.7 eV), and carbons
adjacent to the amine of the triazole linker (286.0 eV) and
saccharide. The peak at 288.0 eV represents the amide groups
of the peptoid backbone. The nitrogen signals originate from
the nitrogens in the triazole linkage, peptide/peptoid backbone,
and lysine side chains. Finally, the oxygen peaks are contrib-
uted by bulk TiO2 (529.9 eV), TiOH (531.1 eV), C-OH (532.5
eV), C-O (532.7 eV), and C=O (533.6 eV).36
1
2
3
4
5
6
7
8
that is representative of the surface composition present on
titanium-based alloys used extensively in orthopedic, cardio-
vascular and other medical devices. The DOPA-Lys-DOPA-
Lys-DOPA pentapeptide anchor is particularly well suited for
grafting peptoids to TiO2 surfaces because of the strong affini-
ty of the catechol OH groups of DOPA (pKa1 = 9.2; pKa2
=
13.0) to TiO2 (isoelectric point of TiO2 is ~ 6.2), which accord-
ing to Redfern et al.33 is expected to occur through bidentate
bridging bonds at pH 6.0.
9
The dry thickness of the M20Glu(OH) and M20Mal(OH)
peptoid coatings adsorbed onto the TiO2 surfaces were meas-
ured by spectroscopic ellipsometry and the average thickness
values are reported in Table 1. Although the average thickness
values for M20Mal(OH)-modified surfaces appear to increase
slightly compared to M20Glu(OH)-modified surfaces, the
difference was not statistically significant, indicating that
grafted films of M20Glu(OH) and M20Mal(OH) were of simi-
lar thicknesses.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table
2.
Atomic
compositions
of
unmodified,
M20Glu(OH)-, and M20Mal(OH)- modified TiO2 sub-
strates as determined from high-resolution XPS spectra.
Atomic Composition (%)
Substrate
Advancing and receding contact angles for unmodified
TiO2, M20Glu(OH)-, and M20Mal(OH)-modified substrates
were measured and the results are summarized in Table 1.
Bare TiO2 immediately after oxygen plasma cleaning was
shown to be extremely hydrophilic (<10°) while both
M20Glu(OH)- and M20Mal(OH)- modified substrates demon-
strated advancing contact angles of 30-35°. M20Mal(OH) was
slightly more hydrophilic than M20Glu(OH), presumably due
to the influence of the additional hydroxyl groups present in
maltose versus glucose. The contact angle values for both gly-
copeptoid-modified surfaces fall near or between the reported
values in the literature for self-assembled monolayers with
hydroxyl ter al groups on gold (~10°)34 and the Nme peptoid
backbone with the same length but without oligosaccharide
(~39°).35
Ti
24.8
7.4
C
N
O
Bare TiO2
M20Glu(OH)
M20Mal(OH)
26.9
53.8
50.3
n/a
48.3
29.5
31.0
9.2
10.0
8.7
Table 1. Polymer thicknesses measured by spectroscopic
ellipsometry, and average advancing (θa) and receding (θr)
contact angles measured for bare TiO2 and polymer-
modified substrates.
Contact angle (°±SD)
Polymer Thickness
(Å)
Substrate
Adv. (θa)
7.8 ± 1.9
34.3 ± 0.1
31.5 ± 0.2
Rec. (θa)
7.2 ± 2.8
33.7 ± 0.1
29.5 ± 0.4
Bare TiO2
M20Glu(OH)
M20Mal(OH)
n/a
Figure 3. High-resolution XPS spectra of bare and glycopeptoid-
modified TiO2, substrates. Shown in each panel are the spectral
regions corresponding to Ti(2p) (a), C(1s) (b), N(1s) (c), and
O(1s) (d).
29.2 ± 1.0
31.4 ± 1.1
XPS survey scans reveal the adsorption of M20Glu(OH)
and M20Mal(OH) onto TiO2 surfaces after immersion of the
substrate into the polymer solution. Glycopeptoid modified
substrates exhibited Ti signal from the underlying TiO2 as well
as C, N and O signals from the chemical species found in gly-
copeptoids (see Figure S1). Compared to unmodified TiO2, the
decrease in Ti content and significant increase in C and N
signal intensity for glycopeptoid modified substrates indicate
successful modification. To calculate the atomic composition
of the polymer layer, detailed high-resolution spectra were
acquired at 455-467 eV for Ti(2p), 280-292 eV for C(1s), 394-
406 eV for N(1s), and 524-536 eV for O(1s); the results are
shown in Table 2 and Figure 3. Of particular note in the high-
resolution spectra were changes in the C1s and O1s spectra
upon modification with glycopeptoid. The peak at 284.6 eV
that emerges after peptoid modification is due to the aliphatic
As an initial antifouling performance test, resistance of the
modified surfaces against fibrinogen adsorption was investi-
gated. Both unmodified and modified TiO2 substrates were
treated with buffered fibrinogen solution for 20 minutes, 24
hours, or 72 hours and the amount of adsorbed fibrinogen
measured by ellipsometry. As shown in Figure 4, the fibrino-
gen adsorption values on both glycopeptoid-modified sub-
strates were significantly lower than unmodified substrates for
all time points. Whereas fibrinogen adsorption reached satura-
tion (with a mass of > 500 ng/cm2) on unmodified TiO2 within
20 minutes exposure time, fibrinogen adsorption onto
M20Glu(OH)- and M20Mal(OH)- modified surfaces under
identical conditions was less than 1 – 10 % of the controls at
all time points.
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 9
700
600
500
400
300
200
100
0
a)
4 hr
3 day
5 day
7 day
TiO2
M20Glu(OH)
M20Mal(OH)
1
2
3
4
5
6
7
8
TiO2
M20-
Glu(OH)
M20-
Mal(OH)
9
200 µm
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
b)
100
0.33
24
72
Time (hr)
TiO2
M20Glu(OH)
M20Mal(OH)
Figure 4. Glycopeptoid-modified surfaces exhibit resistance to
fibrinogen adsorption. Shown is the mass plot of fibrinogen ad-
sorption at 20 min, 24 h and 72 h on unmodified and glycopep-
toid-modified surfaces as measured by ellipsometry.
80
60
40
20
0
To further test the antifouling properties of the glycopep-
toid-modified surfaces, cell adhesion studies were conducted
with fibroblasts. Substrates were modified following the same
procedure outlined above for protein adsorption, after which
the substrates were seeded with fresh 3T3 fibroblasts suspend-
ed in serum containing media. Cell attachment was quantified
from 4 hours to 7 days by live cell staining, fluorescent mi-
croscopy, and image analysis. At the initial 4 hour time point,
fibroblasts attached readily to the unmodified TiO2 substrates
while glycopeptoid modified substrates were all highly re-
sistant to adhesion. A plot of % surface area covered by cells
is shown in Figure 5. All glycopeptoid modified substrates
remained highly resistant to fibroblast adhesion throughout the
7-day experiment, despite being challenged with fresh cells at
each intermediate time point (4, 72 and 120 h). In contrast,
fibroblast adhesion on unmodified TiO2 reached nearly a con-
fluent monolayer by day 7. There was no significant difference
in cell fouling resistance between M20Glu(OH) and
M20Mal(OH) within the experimental time period. Low fibro-
blast adhesion suggests that serum protein adsorption on the
glycopeptoid modified substrates remained low throughout the
course of the in vitro experiment, thus indirectly confirming
the results of the protein adsorption experiments, but over a
longer time scale.
0.21
0.54
0.01
0.13
0.20
0.02
0.04 0.01
4
72
120
168
Time (hr)
Figure 5. Fibroblast adhesion on bare and glycopeptoid-modified
TiO2 substrates. Representative fluorescent images from each
time point measured (a) and quantified % surface area covered by
fibroblasts (b).
Unmodified, M20Glu(OH)-, and M20Mal(OH)-modified
TiO2 substrates were exposed to static suspensions of S. epi-
dermidis, P. aeruginosa, and E.coli for either 1 day or 4 days.
Adherent bacterial cells were stained with Syto 9 and propidi-
um iodide, and % surface area covered by bacterial cells was
calculated and normalized to control substrates. Glycopeptoid
modified substrates showed a 2-3 log reduction in bacterial
adhesion (> 99.8%) compared to unmodified TiO2 substrates
at both time points and against all three bacterial species as
shown in Figure 6. For S. epidermidis in particular, it is nota-
ble that both M20Glu(OH) and M20Mal(OH) glycopeptoids
demonstrated improved antifouling performance at 1 day and
4 days compared to previous findings with methoxyethyl
(Nme) peptoids,18 a finding with potentially important practi-
cal implications and which suggests a fundamental difference
in fouling resistance between glycopeptoids and 20mer Nme
peptoids.
Bacterial colonization of medical devices and implants can
severely impair their performance and increase infection rates
and health risks for patients.37 Three gram-positive and gram-
negative bacterial strains commonly associated with medical
device related infections were chosen for the static bacterial
adhesion test on unmodified and glycopeptoid modified TiO2
substrates. S. epidermidis is a gram positive bacterium that is
well known for its ability to colonize surfaces rapidly and is
closely linked to device-associated clinical infections of the
hip and urinary tract.38 E. coli is a gram negative strain of bac-
teria often found in surgically related infections;39 P. aeru-
ginosa is gram negative and often associated with infections in
patients with immune systems compromised by disease or
trauma. The release of powerful exotoxins and endotoxins by
P. aeruginosa can cause chronic and life-threatening condi-
tions which persist even after the bacteria have been killed off
by antibiotics.40
4
ACS Paragon Plus Environment

Page 5 of 9
Journal of the American Chemical Society
imentally. Therefore understanding the role of oligosaccharide
Day 1
1
2
3
4
5
6
7
8
residues in suppressing protein, cell and bacterial adsorption
was considered critical in understanding the antifouling mech-
anism of glycopeptoids.
TiO2
M20Glu(OH)
M20Mal(OH)
100.00
10.00
1.00
In order to test whether the “water barrier” mechanism
might play a significant role in the protein resistance of the
grafted glycopeptoids, we performed extensive atomistic mo-
lecular dynamics (MD) simulations of the peptoid layer. It
should be noted that experimental approaches to selectively
probe the hydration layers around local functional groups are
non-trivial. Therefore, all atom MD simulations on a system of
16 glycopeptoid (M20Glu(OH) or M20-Mal(OH)) chains
grafted onto TiO2 at a surface chain density of 0.65 nm-2 were
carried out in explicit water for 100ns. We focus on probing
the physical characteristics of the first hydration layer sur-
rounding the oligosaccharide residues in the surface-grafted
glycopeptoid chains.
0.10
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
0.01
Day 4
TiO2
100.00
10.00
1.00
M20Glu(OH)
M20Mal(OH)
The proximal radial distribution functions, pG(r)49,50 of wa-
ter oxygen atoms obtained from the simulations show that the
thickness of the first hydration layer surrounding maltose and
glucose residues is 4.4 and 4.7 Å, respectively (Figure 7a). For
comparison, we also determined the pG(r) of Nme residue, for
which the first hydration layer was found to be 4.7 Å thick.
Using the thickness information we can calculate, for the
chains grafted on TiO2 surface, the average distribution of the
number of water molecules in the first hydration layer around
the oligosaccharide functional groups. The results are summa-
rized in Figure 7b. The first hydration layer contains on aver-
age 32.7 water molecules on each maltose residue, and 26.1
and 15.7 water molecules around each of the glucose and Nme
residue, respectively. As the thickness of the first hydration
layer was more or less the same among the residues, it is rea-
sonable that the number of water molecules in the first hydra-
tion layer of each residue increases from Nme to glucose to
maltose as the van der Waals volume of the residue increases
in the same order.
0.10
0.01
S. epidermidis
P. aeruginosa
E. coli
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. S. epidermidis, P. aeruginosa, and E.coli adhesion to
unmodified and glycopeptoid-modified TiO2 substrates after 1 day
(top) and 4 day (bottom) exposure.
Molecular Dynamics (MD) of Water Interacting with Gly-
copeptoid Brush.
It is well established that steric repulsions are responsible
for the ability of grafted polymer layers to reduce protein ad-
sorption.41-43 The term steric repulsion should be understood to
include two main contributions. They are the higher osmotic
pressure within the polymer layer due to the presence of the
polymers and the reduction in conformational entropy of the
polymer chains. Therefore increasing the polymer surface
coverage reduces protein adsorption and in general working in
the regime of stretched chains, so-called brush regime, pro-
vides optimal protein rejection. This concept should be applied
to relatively long chains and in the regime where the confor-
mational degrees of freedom of the polymers are accessible. In
the limit of short chains and very high surface density, as those
obtained in SAMs with short ethylene oxide chains or sugars,
the prevention of protein adsorption has been suggested to be
the result of the hydration layer around the terminal groups,
the so called “water barrier”. Examples of this class of coat-
ings include SAMs with a variety of functional groups25 in-
cluding oligo(ethylene glycol),10 and charged or zwitterionic
molecules.44,45 Saccharide-based SAMs also fall into this class
and exhibit excellent antifouling performance.26,27,46,47 It has
been speculated that the tightly bound hydration layer on sac-
charide containing SAMs creates a repulsive force on proteins,
reducing the interaction between the protein and the sur-
face.46,48
The stability of the interfacial water layer was probed by
measuring the average number and the lifetime of the hydro-
gen bonds between the oligosaccharide residues and water
molecules. The hydrogen bonds were identified by a widely-
accepted geometric criterion: a pair is considered to be hydro-
gen bonded if the oxygen-oxygen distance is no greater than
3.5 Å, and simultaneously, the O-H…O angle is less than
30°.51 The distributions of the number of oligosaccharide-
water and Nme-water hydrogen bonds are summarized in Fig-
ure 8a. The average number of hydrogen bonds is 11.8, 6.5
and 1.4 for the maltose-water, glucose-water, and Nme-water
pair, respectively. The results show that the maltose residues
in the surface-grafted glycopeptoids typically form the largest
number of hydrogen bonds with water, the glucose-water pair
about half as many, and each Nme residue in the surface-
grafted Nme-20mer chains forms less than 2 hydrogen bonds
with water on average. The results are consistent with the
number of hydrogen bond-eligible sites available in each resi-
due.
In our experiments, glycopeptoids were grafted on the TiO2
surface at surface chain densities that stretch the chains but are
not nearly the close packed densities achieved in SAMs.
Therefore, it may be expected that the ability of the peptoids to
prevent protein adsorption is associated with a combination of
both mechanisms. Namely, steric repulsion and hydration will
combine to provide the non-fouling properties observed exper-
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 9
for the oligosaccharide residues is reminiscent of the relaxa-
a)
3
2.5
2
1
2
3
4
5
6
7
8
tion process of disordered quenched systems such as glasses.
Therefore, the hydration layers around the oligosaccharide
residues are very long lived and possess glass-like dynamics
which can further enhance the protein resistance by causing
incoming proteins to slip and make the replacement of water
molecules by protein very difficult.
Maltose
Glucose
Nme
Nme
1.5
1
The autocorrelation function of the hydrogen bonds between
Nme and water, calculated from the simulation system of a
20mer Nme peptoid grafted on TiO2 and also shown in Figure
8b, decays in a similar way to those for maltose and glucose,
suggesting that the Nme -water hydrogen bond in Nme -20mer
system is as persistent as the maltose-water and glucose-water
hydrogen bonds in glycopeptoid systems. However, the num-
ber of hydrogen bonds between Nme and water is significantly
less than for the maltose-water and glucose-water pairs as
shown in Figure 8a resulting, as discussed below, in a less
effective protection.
0.5
0
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
0.25
0.5
0.75
1
1.25
1.5
r (nm)
b)
4000
3000
2000
1000
0
Nme
Maltose
Glucose
a)
8000
Nme
6000
Glucose
Maltose
4000
2000
0
10
15
20
25
30
35
40
Hydration OW per residue
Figure 7. (a) The proximal radial distribution, pG(r), of water
oxygen atoms surrounding maltose, glucose, and Nme resi-
dues, obtained from MD simulations. (b) The distribution of
the average number of water oxygen atoms within the first
hydration layer of maltose, glucose, and Nme, from MD simu-
lations.
0
5
10
15
HB per residue
b)
1
0.8
0.6
0.4
0.2
0
Perhaps the most interesting results involve the lifetime of
the hydrogen bonds, which is a crucial indicator of the hydro-
gen bond stability. The lifetime of the hydrogen bonds was
calculated from the autocorrelation function C(t) of the hydro-
gen bond operator h(t). The hydrogen bond autocorrelation has
been used to characterize the kinetic relaxation behavior of
hydrogen bonds52,53 and is defined as follows:54
Maltose
Glucose
NPmMeP1
Ac. Glucose
Water
h(0)h(t)
C(t) =
h
Here the hydrogen bond operator h(t) = 1 if a tagged pair is
hydrogen bonded at time t, and h(t) = 0 otherwise. The bracket
represents the ensemble average.
0
5
10
15
20
Time (ps)
Figure 8. (a) The distribution of the average number of hydrogen
bonds of maltose, glucose, and Nme with water, obtained from
MD simulations. (b) The autocorrelation functions of hydrogen
bonds for maltose-water, glucose-water, Nme-water, acetylated
glucose (Ac. Glucose)-water, and water-water pairs, calculated
from MD simulations.
Figure 8b shows the autocorrelation functions for the malt-
ose-water and glucose-water hydrogen bonds from the glyco-
peptoid chains grafted on the TiO2 substrate, and for the water-
water hydrogen bonds from a pure water system, calculated
from the MD simulations. The autocorrelations of the hydro-
gen bonds between maltose residues and water as well as be-
tween the glucose residues and water for the surface-grafted
glycopeptoid chains are found to decay much more slowly
than the autocorrelation of water-water hydrogen bonds. The
relaxation time of the hydrogen bonds, defined as the time
when the autocorrelation drops to e-1, was found to be 14.1 ps
for maltose-water, 14.4 ps for glucose-water, and 2.2 ps for
water-water pairs, showing the relaxation times being more
than 6 times larger for the maltose-water and glucose-water
hydrogen bonds than for the water-water hydrogen bonds.
Moreover, the long tail of the autocorrelation at longer times
In Figure 8b, we also present the autocorrelation function of
the hydrogen bonds between water and the acetylated glyco-
peptoid (abbreviated as “Ac. Glucose”) chains grafted on TiO2
at the same surface chain density. The molecular structure of
the acetylated glycopeptoid chain is identical to the
M20Glu(OH) chain, except that all five hydrogen atoms in the
alcohol groups in the glucose residue are substituted with ace-
tyl group (this is essentially the “protected” form of the glyco-
peptoid). With no alcohol functional groups available, the
acetylated glucose residue no longer has the ability to act as a
6
ACS Paragon Plus Environment

Page 7 of 9
Journal of the American Chemical Society
hydrogen bond donor and has only a limited capability to form
have previously been shown to exhibit excellent antifouling
properties,18,55 and predictions from the molecular theory men-
tioned above are in good agreement with the experimental
observation,35,56 suggesting that steric repulsion is one of the
main contributions to the antifouling mechanism provided by
the Nme peptoid linker of the glycopeptoids.
1
2
3
4
5
6
7
8
hydrogen bonds with water molecules as a weak acceptor via
the ester oxygens. As a result, the first hydration layer of the
acetylated glucose residue should be very weakly bound as
corroborated by the fast-decaying autocorrelation function
with the relaxation time of 3.9 ps, shown in Figure 8b. The
weakly bound hydration layer of the acetylated glucose resi-
due suggests a poor resistance to protein adsorption for the
acetylated glycopeptoid chains. Indeed, our experiments con-
firmed that the surfaces modified with acetylated glycopeptoid
chains displayed a high degree of fibrinogen adsorption at
short times (experimental data not shown). These results vali-
date the importance of hydration layers around surface-grafted
molecules in determining their effectiveness to protein re-
sistance. Further, our analysis illustrates a possibility that the
autocorrelation function of hydrogen bonds might be used as a
simple and effective predictor to scan for potential protein
resistance for systems where steric repulsions are suppressed
and hence the interfacial hydration layer is likely to make a
major contribution toward fouling resistance.
Thus, we find that the non-fouling behavior of the glycopep-
toids is the result of the combination of the steric repulsions
from the 20mer Nme peptoid linker of the glycopeptoid, as
well as the repulsion arising from the volume and bound water
surrounding the terminal oligosaccharide residues. Although
the results for the acetylated glycopeptoids indicate that the
bound water may be the major mechanism of protein re-
sistance for the glycopeptoid chains, it is not entirely clear
whether the substitution of the hydroxyl groups by acetyl
groups changes the bare interaction between the protein and
the peptoid, resulting in strong attractions that lead to the ob-
served adsorption.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finally, it is important to emphasize the differences between
our study and the previously proposed water barrier. The earli-
er simulation work describing the importance of the bound
water on the non-fouling capabilities of sugars has been done
on high density SAM’s and therefore the analysis is based on
complete water layers parallel to the SAM.57 In our work we
study the water molecules bound to the sugar functional group
in the polymer layer at finite, experimentally relevant, surface
coverage well below that of short oligomers SAMs. Therefore,
our study shows how bound water molecules contribute to the
non-fouling in addition to the steric repulsions arising from the
conformational degrees of freedom of the polymers and the
osmotic pressure contribution.
Antifouling Mechanism and the Role of Water.
The overall picture emerging from the above analysis of the
interfacial hydration layer surrounding the terminal saccharide
residues of the surface-grafted glycopeptoid chains is the fol-
lowing: the maltose and glucose residue carry a large number
of interfacial water molecules due to their large sizes. Many of
these interfacial water molecules are tightly bound to maltose
and glucose residues via hydrogen bonds that have a long du-
ration time and hence are likely to be much stronger than their
water-water counterparts. The tightly bound interfacial water
molecules surrounding the oligosaccharide residues mean that
a protein would need a longer time to displace the interfacial
water molecules surrounding the oligosaccharide residues
before it can penetrate the hydration layer and interact with the
grafted polymer chains, leading to a diminished possibility for
protein to adsorb on the surface. This could serve as one of the
major mechanisms that allow the glycopeptoid chains to effec-
tively resist protein adsorption as observed in our experiments.
CONCLUSION
We reported the synthesis and characterization of a novel
class of antifouling polymers that mimic the glycocalyx in
both composition and ability to inhibit nonspecific fouling
interactions. CuAAC and high-fidelity solid phase methods
allowed the incorporation of saccharides as N-substituted side
chains of a peptidomimetic polymer backbone, providing pre-
cise control of chemical composition, molecular weight, and
architecture of the polymers. The glycopeptoid polymers read-
ily adsorbed onto TiO2 substrates, conferring significant pro-
tein, cell and bacterial antifouling properties. All-atom molec-
ular dynamics simulations were conducted to provide insight
into the role of oligosaccharides in antifouling, demonstrating
the existence of strongly bound water molecules at the termi-
nal saccharide residues, which are likely to play an important
role in fouling resistance through inhibition of protein-surface
interactions in combination with polymer steric repulsions. In
the future, more detailed explorations of chemical composition
and architecture of glycopeptoids on fouling resistance may
provide further insight into antifouling mechanism and inform
the design of more effective glycomimetic antifouling poly-
mers.
The importance of the bound water layer is clear, however,
it should not be considered the only component in the non-
fouling capabilities of polymer layers. The application of a
molecular theory to study protein resistance for end-grafted
PEG and polypetptoids has also shown the importance of the
polymer conformational degrees of freedom, the deformation
of the polymer layer, and the osmotic pressure repulsion to
quantitatively predict protein adsorption. Therefore, we be-
lieve that it is the combination of the steric repulsions and the
water layer that are responsible for protein resistance. Actual-
ly, the two effects complement each other since the long lived,
bound, water molecules make the effective volume of the pep-
toids units large and highly hydrophilic. Therefore, the steric
barrier is larger when water molecules are bound. This further
explains the differences between the peptoids with sugars as
compared to Nme. The latter has a smaller number of bound
water and therefore, for the same surface coverage it produces
a smaller steric barrier (recall that we refer to the steric barrier
as the combination of conformational entropy loss and osmotic
pressure).
ASSOCIATED CONTENT
Supporting Information.
Experimental details for glycopeptoid synthesis, surface modifica-
tion and characterization; additional NMR data including peak
assignments, and XPS data before and after glycopeptoid modifi-
cation. This material is available free of charge via the Internet at
http://pubs.acs.org
The glycopeptoids used in this study have a 20mer N-
methoxyethyl (Nme) peptoid linker that tethers the terminal
oligosaccharide residue to the TiO2 surface. Nme peptoids
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 9
(19)Reitsma, S.; Slaaf, D.; Vink, H.; van Zandvoort, M.; oude Egbrink,
M. Pflugers Arch. 2007, 454, 345.
(20)Massia, S. P.; Stark, J.; Letbetter, D. S. Biomaterials 2000, 21, 2253.
(21)Perrino, C.; Lee, S.; Choi, S. W.; Maruyama, A.; Spencer, N. D.
Langmuir 2008, 24, 8850.
AUTHOR INFORMATION
1
2
3
Corresponding Author
*philm@northwestern.edu
4
5
6
7
8
9
(22)Martwiset, S.; Koh, A. E.; Chen, W. Langmuir 2006, 22, 8192.
(23)Holland, N. B.; Qiu, Y.; Ruegsegger, M.; Marchant, R. E. Nature
1998, 392, 799.
(24)Sen Gupta, A.; Wang, S.; Link, E.; Anderson, E. H.; Hofmann, C.;
Lewandowski, J.; Kottke-Marchant, K.; Marchant, R. E. Biomaterials
2006, 27, 3084.
(25)Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.;
Whitesides, G. M. J. Am. Chem. Soc. 2000, 122, 8303.
(26)Fyrner, T.; Lee, H.-H.; Mangone, A.; Ekblad, T.; Pettitt, M. E.;
Callow, M. E.; Callow, J. A.; Conlan, S. L.; Mutton, R.; Clare, A. S.;
Konradsson, P.; Liedberg, B.; Ederth, T. Langmuir 2011, 27, 15034.
(27)Luk, Y.-Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 9604.
(28)Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am.
Chem. Soc. 2003, 125, 6140.
(29)Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.;
Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336.
(30)Ryle, A. P. Nature 1965, 206, 1256.
(31)Geng, J.; Mantovani, G.; Tao, L.; Nicolas, J.; Chen, G.; Wallis, R.;
Mitchell, D. A.; Johnson, B. R. G.; Evans, S. D.; Haddleton, D. M. J. Am.
Chem. Soc. 2007, 129, 15156.
(32)Slavin, S.; Burns, J.; Haddleton, D. M.; Becer, C. R. Eur. Polym. J.
2011, 47, 435.
(33)Redfern, P. C.; Zapol, P.; Curtiss, L. A.; Rajh, T.; Thurnauer, M. C. J.
Phys. Chem. B 2003, 107, 11419.
(34)Lee, S.-W.; Laibinis, P. E. Biomaterials 1998, 19, 1669.
(35)Lau, K. H. A.; Ren, C.; Park, S. H.; Szleifer, I.; Messersmith, P. B.
Langmuir 2011, 28, 2288.
(36)Huang, N.-P.; Michel, R.; Voros, J.; Textor, M.; Hofer, R.; Rossi, A.;
Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2000, 17, 489.
(37)Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Nat. Rev. Micro.
2004, 2, 95.
(38)Katsikogianni, M.; Missirlis, Y. F. Eur. Cell. Mater. 2004, 8, 37.
(39)Ruggieri, P.; Angelini, A.; Pala, E.; Mercuri, M. Spine 2012, 37, 420.
(40)Anwar, H.; Strap, J. L.; Chen, K.; Costerton, J. W. Antimicrob.
Agents Chemother. 1992, 36, 1208.
(41)De Gennes, P. G. Macromolecules 1981, 14, 1637.
(42)Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid
Interface Sci. 1991, 142, 149.
Present Addresses
†H.O.H.: Department of Surgery, Beth Israel Deaconess Medical
Center, Harvard Medical School, and Wyss Institute of Biologi-
cally Inspired Engineering at Harvard University, Boston, Massa-
chusetts, 02115, USA
Author Contributions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script.
Funding Sources
This work was supported by NIH grant R01 EB005772.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We acknowledge Dr. Xinqi Chen for his assistance with XPS
studies performed at the Keck-II/NIFTI facilities of NUANCE
Center at Northwestern University. We thank Yufei Tian for tech-
nical support. NMR and Mass spectroscopy analysis were per-
formed in the IMSERC facility at Northwestern University.
REFERENCES
(1)Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell,
D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.
Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367.
(2)Kirshenbaum, K.; Barron, A. E.; Goldsmith, R. A.; Armand, P.;
Bradley, E. K.; Truong, K. T.; Dill, K. A.; Cohen, F. E.; Zuckermann, R.
N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303.
(3)Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am.
Chem. Soc. 1992, 114, 10646.
(4)Nam, K. T.; Shelby, S. A.; Choi, P. H.; Marciel, A. B.; Chen, R.; Tan,
L.; Chu, T. K.; Mesch, R. A.; Lee, B. C.; Connolly, M. D.; Kisielowski,
C.; Zuckermann, R. N. Nat. Mater. 2010, 9, 454.
(5)Shin, S. B.; Yoo, B.; Todaro, L. J.; Kirshenbaum, K. J. Am. Chem. Soc.
2007, 129, 3218.
(6)Patch, J. A.; Barron, A. E. Curr. Opin. Chem. Biol. 2002, 6, 872.
(7)Vreeland, W. N.; Meagher, R. J.; Barron, A. E. Anal. Chem. 2002, 74,
4328.
(8)Vreeland, W. N.; Slater, G. W.; Barron, A. E. Bioconjugate Chem.
2002, 13, 663.
(9)Hooks, J. C.; Matharage, J. P.; Udugamasooriya, D. G. Peptide Science
2011, 96, 567.
(10)Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E.
J. Phys. Chem. B 1998, 102, 426.
(11)Lu, H. B.; Campbell, C. T.; Castner, D. G. Langmuir 2000, 16, 1711.
(12)Konradi, R.; Pidhatika, B.; Muhlebach, A.; Textor, M. Langmuir
2008, 24, 613.
(13)Bearinger, J. P.; Terrettaz, S.; Michel, R.; Tirelli, N.; Vogel, H.;
Textor, M.; Hubbell, J. A. Nat. Mater. 2003, 2, 259.
(14)Osterberg, E.; Bergstrom, K.; Holmberg, K.; Schuman, T. P.; Riggs,
J. A.; Burns, N. L.; Van Alstine, J. M.; Harris, J. M. J. Biomed. Mater.
Res. 1995, 29, 741.
(15)Huang, C.-J.; Li, Y.; Jiang, S. Anal. Chem. 2012, 84, 3440.
(16)Crowley, M. M.; Zhang, F.; Koleng, J. J.; McGinity, J. W.
Biomaterials 2002, 23, 4241.
(17)Lin, S.; Zhang, B.; Skoumal, M. J.; Ramunno, B.; Li, X.;
Wesdemiotis, C.; Liu, L.; Jia, L. Biomacromolecules 2011, 12, 2573.
(18)Statz, A. R.; Barron, A. E.; Messersmith, P. B. Soft Matter 2008, 4,
131.
(43)Szleifer, I. Biophys. J. 1997, 72, 595.
(44)Chen, S.; Yu, F.; Yu, Q.; He, Y.; Jiang, S. Langmuir 2006, 22, 8186.
(45)Cho, W. K.; Kong, B.; Choi, I. S. Langmuir 2007, 23, 5678.
(46)Ederth, T.; Ekblad, T.; Pettitt, M. E.; Conlan, S. L.; Du, C.-X.;
Callow, M. E.; Callow, J. A.; Mutton, R.; Clare, A. S.; D’Souza, F.;
Donnelly, G.; Bruin, A.; Willemsen, P. R.; Su, X. J.; Wang, S.; Zhao, Q.;
Hederos, M.; Konradsson, P.; Liedberg, B. ACS Appl. Mater. Interfaces
2011, 3, 3890.
(47)Prime, K.; Whitesides, G. Science 1991, 252, 1164.
(48)Szleifer, I. Physica A 1997, 244, 370.
(49)Wei, T.; Carignano, M. A.; Szleifer, I. Langmuir 2011, 27, 12074.
(50)Merzel, F.; Smith, J. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5378.
(51)Luzar, A.; Chandler, D. J. Chem. Phys. 1993, 98, 8160.
(52)Ebbinghaus, S.; Kim, S. J.; Heyden, M.; Yu, X.; Heugen, U.;
Gruebele, M.; Leitner, D. M.; Havenith, M. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 20749.
(53)Starr, F. W.; Nielsen, J. K.; Stanley, H. E. Phys. Rev. E: Stat.,
Nonlinear, Soft Matter Phys. 2000, 62, 579.
(54)Luzar, A.; Chandler, D. Nature 1996, 379, 55.
(55)Statz, A. R.; Meagher, R. J.; Barron, A. E.; Messersmith, P. B. J. Am.
Chem. Soc. 2005, 127, 7972.
(56)Statz, A.; Kuang, J.; Ren, C.; Barron, A.; Szleifer, I.; Messersmith, P.
Biointerphases 2009, 4, FA22.
(57)Hower, J. C.; He, Y.; Bernards, M. T.; Jiang, S. J. Chem. Phys. 2006,
125, 214704.
8
ACS Paragon Plus Environment

Page 9 of 9
Journal of the American Chemical Society
1
Antifouling Glycopeptoids!
2
3
4
Cell Fouling of TiO2!
5
MD
6
Simulation!
7
8
TiO2!
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
ACS Paragon Plus Environment