Enhanced cell adhesion and mature intracellular structure promoted by squaramide-based RGD mimics on bioinert surfaces

Article abstract of DOI:10.1016/j.bmc.2013.02.032

Highly selective molecular binding and the subsequent dynamic protein assemblies control the adhesion of mammalian cells. Molecules that inhibit cell adhesion have the therapeutic potential for a wide range of diseases. Here, we report an efficient synthesis (2-4 steps) of a class of squaramide molecules that mimics the natural tripeptide ligand Arg-Gly-Asp (RGD) that mediates mammalian cell adhesion through binding with membrane protein integrin. In solution, this class of squaramides exhibits a higher potency at inhibiting mammalian cell adhesion than RGD tripeptides. When immobilized on a bio-inert background formed by self-assembled monolayers of alkanethiols on gold films, squaramide ligands mediate vastly different intracellular structures than RGD ligands. Immunostaining revealed that the focal adhesions are smaller, but with a larger quantity, for cells adhered on squaramides than that on RGD ligands. Furthermore, the actin filaments are also more fibrous and well distributed for cell adhesion mediated by squaramide than that by RGD ligands. Quantification reveal that squaramide ligands mediate about 1.5 times more total focal adhesion (measured by the summation of the area of all focal adhesions) than that by natural RGD ligands. This result suggests that cell adhesion inhibitors, while blocking the attachment of cells to surfaces, may induce more focal adhesion proteins. Finally, this work demonstrates that immobilizing new ligands on bioinert surfaces provide a powerful tool to study mammalian cell adhesion.

Full text of DOI:10.1016/j.bmc.2013.02.032

Bioorganic & Medicinal Chemistry 21 (2013) 2210–2216
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enhanced cell adhesion andmature intracellular structure promoted
by squaramide-based RGD mimics on bioinert surfaces
⇑
Sri Kamesh Narasimhan, Preeti Sejwal, Shifa Zhu, Yan-Yeung Luk
Department of Chemistry, 111 College Place, 1-014, Syracuse University, Syracuse, NY 13244-4100, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Highly selective molecular binding and the subsequent dynamic protein assemblies control the adhesion
of mammalian cells. Molecules that inhibit cell adhesion have the therapeutic potential for a wide range
of diseases. Here, we report an efficient synthesis (2–4 steps) of a class of squaramide molecules that
mimics the natural tripeptide ligand Arg-Gly-Asp (RGD) that mediates mammalian cell adhesion through
binding with membrane protein integrin. In solution, this class of squaramides exhibits a higher potency
at inhibiting mammalian cell adhesion than RGD tripeptides. When immobilized on a bio-inert back-
ground formed by self-assembled monolayers of alkanethiols on gold films, squaramide ligands mediate
vastly different intracellular structures than RGD ligands. Immunostaining revealed that the focal adhe-
sions are smaller, but with a larger quantity, for cells adhered on squaramides than that on RGD ligands.
Furthermore, the actin filaments are also more fibrous and well distributed for cell adhesion mediated by
squaramide than that by RGD ligands. Quantification reveal that squaramide ligands mediate about 1.5
times more total focal adhesion (measured by the summation of the area of all focal adhesions) than that
by natural RGD ligands. This result suggests that cell adhesion inhibitors, while blocking the attachment
of cells to surfaces, may induce more focal adhesion proteins. Finally, this work demonstrates that immo-
bilizing new ligands on bioinert surfaces provide a powerful tool to study mammalian cell adhesion.
Published by Elsevier Ltd.
Received 10 December 2012
Revised 5 February 2013
Accepted 13 February 2013
Available online 27 February 2013
Keywords:
RGD mimics
Squaramide
Focal adhesion
Self-assembled monolayers
Bioinert surfaces
1. Introduction
into drugs have not addressed the cell signaling involving focal
adhesion and stress fiber in the living cells. Coincidentally, the pur-
Mammalian cell adhesion, central to many biological processes
and related diseases, is governed by highly selective molecular rec-
ognition between ligands in the extracellular matrix (ECM) and
membrane protein receptors on the cell surfaces.1,2 The most well
known ligand is the tripeptide Arg-Gly-Asp (RGD) in ECM proteins
such as fibronectin, that binds to a wide variety of integrins,
suit of using integrin antagonist as drugs has encountered numer-
ous setbacks in the pharmaceutical industry.29 Chew et al., judged
the development of oral
standstill after clinical reports on the high incidence of mortality
rates associated with IIbb3 antagonists, and that successful
therapeutic RGD mimics are still not in sight.30 Moreover, inhibi-
tors of integrins vb3 and vb5 that have entered clinical trials as
aIIbb3 antagonists had come to a complete
a
including
avb3,
a
IIbbIIIa and
a
5b1.3–8 The specific recognition be-
a
a
tween RGD and integrin receptors has been considered to be an
important target for developing therapeutic agents for a wide vari-
ety of diseases, including osteoporosis, cancer, diabetic retinopa-
thy, rheumatoid arthritis, and inflammatory diseases.2,9–12 To
develop potential therapeutic agents, molecules are synthesized
to mimic the natural RGD sequence to function as integrin antago-
nists that inhibit cell adhesion.6,9,13–28 Many reports on ‘integrin
antagonist’ showed inhibition for platelet aggregation or cell adhe-
sion, but did not show the attenuation of the cell signaling after
binding to integrin.6,9,13–23 The main focus for these RGD mimics
has been to achieve high potency at inhibiting cell adhesion and
to achieve selectivity between different types of integrins. Surpris-
ingly, many studies on developing these cell adhesion inhibitors
antiangiogenic agents for cancer treatment have been generally
unsuccessful.31,32 Interestingly, some studies demonstrated en-
hanced, instead of reduced, tumor growth and angiogenesis with
use of nanomolar concentrations of RGD mimics targeting
avb3
and
a
vb5 integrins.33–35
Mammalian cell adhesion is a complex multi-step process
including molecular recognition (integrin–RGD binding),36–40 cell
spreading on surface, integrin clustering,41 recruitment and
assembly of multiple cytosol proteins42,43 and linking them to
the dynamic process of stress fiber formation.44–47 Most of the
mechanistic studies of cell adhesion were conducted on RGD (lin-
ear or cyclic) ligands immobilized on different surfaces or polymer
scaffolds.48–54 Few studies compared cell adhesion mediated by
linear versus cyclic RGD peptides.39,54–56 One study reported the
strong binding of cyclic RGD to integrin caused small sizes of focal
adhesions, presumably by attenuating the lateral clustering of
⇑
Corresponding author. Tel.: +1 315 443 7440; fax: +1 315 440 4070.
E-mail address: yluk@syr.edu (Y.-Y. Luk).
0968-0896/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.02.032

S. K. Narasimhan et al. / Bioorg. Med. Chem. 21 (2013) 2210–2216
2211
Figure 1. General design and structures of squaramide-based RGD mimics.
integrin receptor proteins in the cell membrane.39 Cells response to
unnatural, strong binding ligands, and its consequent ramification
to the expression of proteins, however, is still an open question.
Understanding the response of mammalian cells to strong binding
ligands—especially in the context of pharmacoproteomics,57–62 will
provide guidelines for designing therapeutic agents for a wide
range of diseases, including osteoporosis, angiogenesis, cardiovas-
cular diseases and various cancers, as well as for designing materi-
als for tissue engineering.2,63,64
demonstrated substitution reactions that can be promoted by the
high dielectric property of water74 or driven chemoselectively by
the hydrogen bonds from water.75 Here, we used these reactions
to synthesize a new class of RGD mimics. The mimics, squara-
mides, are obtained by a two-step synthesis, which can be con-
ducted in one pot without using protecting groups (Fig. 2). The
first step was most efficient in water. Further derivatization with
4,40-ethylenedipiperidine in presence of triethylamine in water or
ethanol affords the desired squaramides.
In this work, we report the design and the efficient synthesis of
a series of unnatural, squaramide-based RGD mimics (Fig. 1). We
show that these squaramide ligands in culture medium inhibit
mammalian cell adhesion. When immobilized on a bioinert surface
resisting non-specific cell adhesion, the squaramides mediate
intracellular structures that represent more mature cell adhesion,
and induce more focal adhesion for promoting cell adhesion than
linear RGD ligands do.
To evaluate the efficacy of squaramides at inhibiting mamma-
lian cell adhesion, we used both commercially available cell adhe-
sion assays76,77 and a bio-inert surface assay.39,51,78 We found the
results to be most consistent by using the bio-inert surface assays.
The assay employs gold-supported self-assembled monolayers
(SAMs) of alkanethiolates presenting a small percentage of cyclic
RGD ligand (cGRGDFK, alkanethiol 5) in a background of tri(ethyl-
ene glycol) (alkanethiols 8) (Fig. 3). This thiol-squaramate coupling
reaction, similar to thiol-maleimide coupling on surface,79 is highly
efficient in solution75 and on surfaces.78 We note that, for both sur-
face coupling reactions, no mass for unreacted disulfide of alkane-
thiol 8 and 9 (H adduct, 886.51; and Na adduct, 908.51) was
observed (see Supplementary data).
Self-assembled monolayers of alkanethiols on gold films pre-
senting tri(ethylene glycol) prevents non-ligand mediated cell
adhesion.53,80–86 On these surfaces presenting cell adhesion li-
gands, any observed reduction in cell adhesion to the surface-
bound cyclic RGD is attributed to the inhibition by the RGD mimic
that was added in the culture medium. Disulfides of alkanethiol 5
was synthesized and used directly to form mixed SAMs with
alkanethiol 8 to present cyclic RGD on the bioinert surfaces. Figure
4 shows the percentage of adhesion inhibition of 3T3 Swiss albino
fibroblasts by the RGD mimics, squaramide 1, 2, 3, or the control
ligand, linear peptide N0-GRGDS-C0 , on SAMs presenting cyclic
RGD ligand (alkanethiols 5 and 8). The percentage of adhesion is
2. Results and discussion
The design principle for molecular mimics of such RGD ligands
has been rather simple (Fig. 1); the molecule should have a positive
and a negative charge separated by a linker of about 14 atoms. The
tuning of the properties (rigidity and hydrophobicity) of the linker
for the potency and selectivity has been entirely empirical.20,28,65,66
This simple design strategy is consistent with the observation that,
based on the crystal structure of RGD-bound integrin
aVb3, the
positive and the negative charges on the bound RGD ligand are
spread out rather than folded in close proximity.6,67 However,
the organic synthesis of RGD mimics is not trivial, requiring 8–15
steps.18,20,28,68–72 Considering such
a simple design strategy,
squaramide moiety provides a promising structural element for
developing RGD mimics to function as potent ligand for binding
to integrin for the following reasons. First, the squaramide moiety
does not contain native amide bonds, and thus has a low propen-
sity to be degraded by enzymes. Second, the squaramide group is
rigid and exists in only four possible conformations due to the
p
NH
O
O
HN
H2N
COOH
O
O
conjugation. Compared to the glycine residue that bridges the argi-
nine and aspartic residues in the RGD tripeptide, the squaramide
significantly reduces the ensemble of conformations.
1
COOH
Et3N, H2O,
0 C to r.t., 80%
Et3N, EtOH,
reflux, 12 h, 66%
EtO
N
H
EtO
OEt
Because of the ring strain and partial aromaticity, the squarate
moiety exhibits unique reactivity in water.73–75 Recently, we have
Figure 2. Representative synthesis of squaramide-based RGD mimic.

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S. K. Narasimhan et al. / Bioorg. Med. Chem. 21 (2013) 2210–2216
HO
O
O
O
9
H
N
H
N
H
N
NH3
l
OPh
NH
OOC
S
o
O
N
H
N
H
i
N
H
h
R
SH
t
O
O
O
e
OOC
n
O
a
k
O
HN
l
Alkanethiol 6
A
NH
O
NH2
OH
H2N
OH
O
O
~
O
O
8
O
l
O
o
i
h
O
O
O
NH2
O
t
N
e
NH
O
n
a
O
O
k
O
l
O
H
S
A
O
H
N
O
O
H2N
H2N
HN
N
NH
O
O
O
HN
O
HN
O
H
N
~
HN
Alkanethiol 7
O
NH
O
S
Au
S
S
Alkanethiol 5
~
Figure 3. Surface presenting cyclic Arg-Gly-Asp-
D
-Phe-Lys consists of mixed SAMs of alkanethiols 5 (3%) and 8 (97%) in DMSO solution respectively; surfaces presenting
linear N0-AGRGDSC-C0 (alkanethiol 6) or the squaramide ligands 4 (alkanethiol 7) was prepared by surface reaction of N0-AGRGDSC-C0 or 4 with mixed SAMs of 9 and 8,
respectively.78
adhesion at concentrations at least 5-fold lower than the linear
peptide N0-GRGDS-C0.
1
2
To study the rate of cell adhesion and the cellular structure of
adhered cells mediated by non-natural ligands (squaramides) with
high binding constants, we prepared bio-inert surfaces presenting
either squaramide (alkanethiol 7) or linear RGD ligand (alkanethiol
6) with the same surface density, and compared the cell attach-
ment kinetics, focal adhesion and stress fiber formation on the
two surfaces. Self assembled monolayers presenting squaramide
(4) or linear RGD ligands (6) were prepared by surface reaction be-
tween 4 or N0-AGRGDSC-C0 and the squarate moiety on mixed
SAMs prepared from 3% of 9 and 97% of 8 in ethanol (Fig. 3).78
Two SAMs presenting either squaramide (alkanethiol 7) or linear
RGD ligand (alkanethiol 6) with a bioinert background (alkanethiol
8) were presented to a suspension of 3T3 fibroblasts (20,000 cells/
mL) in serum free media in the same culture flask and incubated at
37 °C. Cell numbers on each mixed SAMs were counted every
10 min on five replicates of gold films (ca. 1.0 ꢀ 1.0 cm2). During
the initial 30 min, cells assumed similar non-spread, spherical
shapes on both substrates, with more cells on SAMs presenting
squaramide 4 than that presenting linear RGD. The rates of cell
attachment during the first 30 min on the two surfaces is linear,
and that the rate of cell attachment on surface mediated by squara-
mide 4 was ꢂ1.6 times faster than on linear RGD ligand (about 152
cells per mm2 on squaramide, and 95 cells per mm2 on RGD
surface.
These results indicate that squaramide molecules in solution in-
hibit cell adhesion to cyclic RGD ligands on surface; but when
immobilized on surfaces, mediate cell adhesion. Together, we be-
lieve that these results suggest that the binding pocket for squara-
mide on integrin proteins is likely the same as that for RGD ligands.
Were it to be a different binding pocket for squaramide than that
for RGD, the faster cell attachment and stronger cell adhesion med-
iated by squaramide would likely imply a discovery of a new li-
gand–receptor interaction that is stronger than the known RGD-
integrin interaction.
N’-GRGDS-C’
3
Concentration (µM)
Figure 4. Percentage of cell adhesion inhibition of 3T3 fibroblasts by squaramides
1, 2, 3, and by the control peptide N0-GRGDS-C0 on bio-inert SAMs presenting
c(RGDFK)—prepared by soaking the gold film in 3% of 5 and 97% of 8 in DMSO (total
alkanethiol concentration: 1 mM). Data collected over six replicates.
computed by 100% ꢀ (nꢁn0)/n, where n0 and n are the number of
adherent cells with and without treatment of squaramide. Sus-
pended cells in medium (20,000 cells/mL) were incubated for
5 min with the squaramide derivatives, 1, 2, 3 or linear peptide
N0-GRGDS-C0 before the cells were introduced to the monolayers.
After cultured for 24 h, the adherent cell numbers were counted
for each of the SAMs. Six replicates were repeated for each
squaramide.
To evaluate the potency of inhibition by 1, 2 and 3, we com-
pared the concentrations at which 50% of cell adhesion inhibition
was observed. Whereas linear peptide N0-GRGDS-C0 inhibited 50%
of cell adhesion at about 500
able to inhibit 50% of cell adhesion at about 75
lM, the squaramides 1 and 2 were
l
M. The squara-
mide 3, however, did not inhibit cell adhesion effectively as com-
pared to N0-GRGDS-C0 peptide. This result suggests that
squaramide 1 and 2 are able to inhibit cyclic RGD mediated cell

S. K. Narasimhan et al. / Bioorg. Med. Chem. 21 (2013) 2210–2216
2213
Over the next 4 h of cell culture, there were more spread cells
on SAMs presenting squaramides than on those presenting the lin-
ear RGD ligands. Quantifying the cell number on the two SAMs
indicated that the ratio of the number of cells mediated by squara-
mide to that by linear RGD was about 1.8 before changing the ser-
um free culture medium with one containing 10% of fetal bovine
serum. After changing the medium, the total number of adherent
cells were reduced on both surfaces due to washing, but the ratio
of adherent cells on squaramide to linear RGD increased to about
2.6 (Fig. 5). Together with the faster attachment, these results sug-
gest that the squaramide derivatives are stronger adhesion ligands
than linear RGD ligands. We note that on the control surface pre-
senting only amino squarate moiety (alkanethiol 9) instead of
squaramide, there was no observable cell attachment. The reduc-
tion in adhered cells due to change of the culture medium also re-
flects the strength of ligand mediating cell adhesion. After the
change of culture medium at 5 h, about 38% of cells were retained
on by SAMs presenting linear RGD peptides (calculated by
100% ꢀ 109/288 cells per mm2) whereas 56% of cells were retained
on SAMs presenting squaramide ligands (calculated 100% ꢀ 295/
526 cells per mm2). This result suggests that the strength of cell
adhesion mediated by squaramide ligands is 1.5 times (56/38)
greater than those mediated by RGD ligands. As different cell types
have different strength of adhesion on surfaces, these squaramides
provide a class of strong capturing ligands for studying the adhe-
sion of other cell types.
integrin clustering, followed by the formation of focal adhesion
and three types of stress fibers.46,47 Here, we characterized and
compared the focal adhesion and stress fiber formation in cells
mediated by squaramide, 4, and by linear RGD ligands. Staining
the cells with anti-vinculin antibody followed by incubation with
fluorescein isothiocyanate (FITC)-tagged goat anti-mouse antibody
(green, for visualizing focal adhesion), and rhodamine–phalloidin
(red, for visualizing stress fibers) revealed the focal adhesions
and stress fibers formed during the early stage of cells mediated
by squaramide, 4, and by linear RGD ligands (Fig. 6). Different pat-
terns of focal adhesions and stress fibers were observed in cell
adhesion mediated by the two surfaces. Linear RGD ligands medi-
ated fewer FAs but with longer shape and larger sizes than those
mediated by squaramide ligands, 4 (Fig. 6). Measurement of the
size of fluorescent signals (Axiovision Imaging software) from anti-
bodies that label the focal adhesion revealed about 51 11 FA/cell
with an average size 5.57 1.45
adhesion, and about 81 18 FA/cell with an average size of
3.75 1.24
m2/FA for squaramide-mediated cell adhesion. This
l
m2/FA for RGD-mediated cell
l
result indicates that the FA mediated by linear RGD is about 1.5
times as large as that mediated by squaramide ligands, 4. We note
that the three quantities that characterize the changes in cell adhe-
sion (increase in the rate of cell attachment, increase in the
strength of cell adhesion and decrease in size of focal adhesion)
all coincide with a similar magnitude of 1.5. This result is also con-
sistent with the notion that higher binding affinity of squaramide,
4, and the faster capture of integrin on the surface causes more
nucleation for integrin clustering, which competes with the lateral
diffusion of integrin.39,54,87–89 Consequently, the average size of the
focal adhesion of cells mediated by squaramide, 4, is smaller than
that mediated by linear RGD ligands.
Mammalian cell adhesion is orchestrated by a sequence of spa-
tial-temporal resolved events, which includes both specific molec-
ular recognition between integrin and ligand in ECM and lateral
Recent studies on the mechanisms of stress fiber formation sug-
gested that transverse arcs were the precursors to ventral stress fi-
bers.46,47 Our results show that cell adhesion mediated by the
linear RGD ligands exhibit more transverse arcs in the form of cir-
cular bundles than the cells mediated by squaramide ligands. In
the cells mediated by squaramides, the stress fibers were relatively
separated and spanned across the whole area of the adhered cells (
Fig. 6). These fluorescent signals were consistent with the forma-
tion of dorsal and ventral stress fibers. As ventral stress fibers are
the more mature form of stress fibers, our results suggests that
stronger binding by squaramide 4 ligands promote more transfor-
mation of transverse arcs into ventral stress fibers46,47 and thus
more mature development of adhered cells.
(A)
Squaramide AGRGDSC
600
500
400
300
200
100
0
1.8/1
2.5/1
2.5/1
1.9/1
2.4/1
2.7/1
2.6/1
2
4
6
8
10
12
24
We characterized the amount of focal adhesion in different sizes
by the summation of the focal adhesion area (measured by fluores-
cence of vinculin protein) over nine cells mediated by linear RGD
ligands, and by squaramide ligands 4. The sizes of fluorescent sig-
nals measured using Axiovision digital imaging (Fig. 7). For the fo-
Time (h)
Serumadded
Figure 5. The number of adhered cells on monolayers presenting N0-AGRGDSC-C0
or squaramide at 2, 4, 6, 8, 10, 12 and 24 h of cell culture. Data were collected over
five separate experiments.
cal adhesions in the range 2–4 l
m2, cell adhesion mediated by
Figure 6. Fluorescent micrographs of fixed cells stained with anti-vinculin/FITC-tagged goat anti-mouse antibody, and rhodamine–phalloidin at 6 h of cell culture (30 min
after addition of serum) on surface presenting, (A) linear RGD ligand (mixed SAMs of 6 and 8) (B) squaramide 4 (mixed SAMs 7 and 8). Scale bar = 30 m.
l

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S. K. Narasimhan et al. / Bioorg. Med. Chem. 21 (2013) 2210–2216
i.e., 2.6/1.8), smaller size and more abundant focal adhesions, and
more spread out stress fibers than those supported by linear RGD
ligands. More importantly, using SAMs that afford the same surface
density of ligands, squaramide-mediated cell adhesion induces
about 1.4 fold as much focal adhesion as RGD-mediated cell adhe-
sion. Because the intracellular structure of adhered cells can be al-
tered by strong binding unnatural ligands, this class of molecules
can help exploring studies in tissue engineering and enhancing
the adhesion of endothelial cells, which adhere poorly on vascular
graft surfaces.90–93
Cell adhesion mediated by
4. Experimental procedures
4.1. Gold deposition on glass slides
Fisher’s finest premium microscope slides (Fisher Scientific,
Pittsburgh, PA) were cleaned in Piranha solution (3 parts of 35%
H2O2 in water and seven parts of concentrated H2SO4) at 75 °C for
45 min. After cooling, the piranha solution was poured off and the
slides were rinsed 20 times with water having a resistivity of
Focal adhesion size (µm2)
Figure 7. Number of FAs versus FA area (
l
m2) over 9 adhered cells mediated by
18 MX cm (Millipore, Billerica, MA), 10 times with ethanol and 10
squaramide through alkanethiol 7, and by linear peptide RGD through alkanethiol 6.
m2
m2 for RGD-mediated cell
times with methanol. The slides were then dried individually with
a stream of nitrogen and stored in an 80 °C oven overnight. Semi-
transparent gold films of approximately 280 Å thickness were
deposited onto the glass substrates with an electron beam evapora-
tor manufactured by Thermionics (Port Townsend, WA). A layer of
titanium (approximately 70 Å) was applied first to enhance the
adhesion of the gold films. The rates of deposition were set at
0.2 Å/s for both gold and titanium. Films were deposited at an obli-
que angle of 45° to the normal of the substrate. Pressure was main-
tained at or below 2 ꢀ 10ꢁ6 Torr throughout the deposition. The
slides were then removed, rinsed thoroughly with ethanol, and dried
with a stream of nitrogen prior to use in mammalian cell culture.
The total surface area under each curve (number of FAs ꢀ area of FAs) is 2686
l
for squaramide-mediated cell adhesion, and 1807
l
adhesion.
squaramide 4 exhibited four times more FAs than those mediated
by linear RGD ligands. For the focal adhesions in the range 1–2 and
4–5
times more FAs than those mediated by linear RGD ligands. Focal
adhesions with size larger than 6
m2 were predominantly seen
l
m2, cell adhesion mediated by squaramide exhibited 2.5
l
in cell adhesion mediated by RGD ligands. This data reveals how
cells respond to strong binding ligands at cell signaling level. For
nine cells, the total area of focal adhesion is 2686
l
m2 for squara-
mide-mediated cell adhesion, and 1807
l
m2 for RGD-mediated cell
4.2. Formation of self assembled monolayers (SAMs) on the gold
films
adhesion. The measurement indicates that the ratio of the total FA
area mediated by squaramide to that mediated by RGD is about
1.5, a value similar to the ratios obtained for other comparisons
such as rates of cell attachment and strength of cell adhesions. This
result suggests that strong integrin-binding ligands induce a higher
level of focal adhesion in mammalian cells.
The mixed SAMs of 8 and 9, were prepared by immersing the
gold film (1 ꢀ 1 cm) in a DMSO solution containing 3% squarate
based alkanethiol, and 97% 8 (1 mM total thiol) for 10 h. For the cell
adhesion inhibition assay, mixed SAMs of 8 and 5 were prepared
analogously. The substrates were rinsed with absolute ethanol
and dried under a stream of nitrogen.
Considering inhibition of different types of mammalian cell
adhesion has the potential for treating a wide range of diseases,
including osteoporosis, cancer, diabetic retinopathy, rheumatoid
arthritis, and inflammatory diseases, this result reveals that block-
ing the integrin receptors can induce undesired cell signaling re-
sponses, by which cells may become more adhesive via increased
expression of receptors. However, our result suggests that blocking
cell adhesion alone is likely not an indicator sufficient for thera-
peutic development. For all RGD mimics that bind to integrin,
the structure of focal adhesion and stress fiber, and cell signal
events should also be characterized. As the more focal adhesions
in small sizes were observed, our result suggests that squaramides,
when immobilized on surfaces, likely induce more adhesion points
within an adhered cell, and thus should be considered as an agonist
for integrin rather than an antagonist. Our work also suggests that
SAMs-based cell adhesion study will useful in complementing
studies in pharmacoproteomics.60–62
4.2.1. Surface reactions
The linear peptide N0-AGRGDSC-C0 and the squaramide 4 were
immobilized onto the SAMs by immersing the SAMs into a phos-
phate buffer solution (pH 7.0) containing 2 mM of the N0-
AGRGDSC-C0 or 4 for 8–10 h. The slides were then rinsed with copi-
ous amounts of water, dried with a stream of nitrogen and used for
cell culture without delay.
4.2.2. Mass spectrometry (MS)
All SAMs were characterized by matrix-assisted laser desorp-
tion/ionization time-of-flight (MALDI-TOF) mass spectrometry.94,95
Briefly, monolayers were treated with acetonitrile solution con-
taining matrix 2,4,6-trihydroxyacetophenone (THAP, 10 mg/mL),
dried, and loaded in a Waters MALDI Micro MX spectrometer. A
300 nm nitrogen laser was used as desorption/ionization source
with accelerating voltage of 19 kV and extraction delay time of
100 ns. All spectra were acquired using reflector mode for positive
ions. 100–300 laser shots were added to obtain each spectrum.
Spectra were obtained by moving the laser beam to at least three
different locations. For mixed SAMs presenting alkanethiol 5 and
8, MALDI revealed a peak at m/z 1396.34, corresponding to the
monoprotonated mixed disulfide and a peak at m/z 694.04, corre-
3. Conclusion
We have developed a class of potent RGD mimics based on the
squaramide structure. The synthesis of this class of squaramide
molecule is highly efficient. When immobilized on the surface,
the squaramide 4, facilitated a faster rate of cell attachment (1.6
times faster), stronger cell adhesion (about 1.4 times stronger,

S. K. Narasimhan et al. / Bioorg. Med. Chem. 21 (2013) 2210–2216
2215
sponding to the sodium adduct of the symmetrical disulfide 8. For
mixed SAMs presenting alkanethiol 7 and 8, MALDI revealed the
peaks for the sodium adduct of the mixed disulfide ([M+Na]+, m/z
1187.39), and the monoprotonated alkanethiol 7 ([M+H]+ m/z
853.25). For mixed SAMs presenting alkanethiol 6 and 8, MALDI re-
vealed the peaks for the monoprotonated adduct of the mixed
disulfide ([M+H]+, m/z 1456.33), and the monoprotonated alkane-
thiol 6 ([M+H]+ m/z 1122.17). See supporting materials for spectra
and synthesis details.
found, in the online version, at http://dx.doi.org/10.1016/j.bmc.
2013.02.032.
References and notes
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The authors thank Professor Dacheng Ren for the use of fluores-
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of the manuscript, and Professor Milan Mrksich for the use of MAL-
DI-TOF. This work was supported by NSF-CAREER (#0845686) and
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