DOI: 10.1002/cplu.
201500179 Full Papers
“On–Off” RGD Signaling Using Azobenzene Photoswitch-
Modified Surfaces**
Elisa Vaselli, Chiara Fedele, Silvia Cavalli,* and Paolo A. Netti*[a]
Photoresponsive surfaces were developed by modifying glass
slides with switchable Gly-Arg-Gly-Asp-Ser (GRGDS) peptides to
investigate light-controlled cell adhesion. The GRGDS peptide
sequence was attached to an azobenzene moiety and then
“clicked” to silanized glass substrates. The photoresponsive be-
havior of such cell-instructive materials (CIMs) was first checked
by contact angle technique and by recording local changes in
wettability owing to the isomerization of the azobenzene
Introduction
Material surfaces decoration with peptide or protein ligands
represents a powerful method to extend the functionality of
synthetic materials that presenting active signals to the cell
surface and are able to elicit specific cell functions.[1] This ap-
proach has been proved to be practical and effective in im-
proving cell recognition on synthetic materials.[2] Furthermore,
the degree of bioactivation can be controlled and modulated
by the orientation, the density, as well as the spatial distribu-
tion of the bioactive signal.[3] However, the native extracellular
matrix (ECM) guides cellular processes, thereby providing
a tight regulation of the temporal and spatial presentation of
cell-adhesive motifs, whereas most of the bioactivation strat-
egies lack the possibility to modulate the temporal presenta-
tion of the signal.[4] Therefore, novel classes of biomaterials
that better mimic the native ECM call for programmable plat-
forms that are able to regulate the presentation of matricellu-
lar cues in time and space to instruct the complex cellular pro-
cesses.[5] This advancement requires the implementation of
versatile systems in which external stimuli can tune the acces-
sibility of the cell ligands as desired. The sensitive response
can be triggered by altering pH[6] or temperature,[7] as well as
exposure to electric or magnetic fields;[8] in some cases irradia-
tion with light can also elicit a response.[9] Another important
feature of this new generation of biomaterials is the reversibili-
ty of their feedback to the external stimuli.[10] So far, many
[a] Dr. E. Vaselli,+ C. Fedele,+ Dr. S. Cavalli, Prof. Dr. P. A. Netti
Center for Advanced Biomaterials for Healthcare IIT@CRIB
Istituto Italiano di Tecnologia
Largo Barsanti e Matteucci, 53, 80125 Naples (Italy)
E-mail: silvia.cavalli@iit.it
paolo.netti@iit.it
[+] These authors contributed equally to this work.
[**] RGD = Arg-Gly-Asp
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201500179.
ChemPlusChem 2015, 80, 1547 – 1555
domain upon light stimulus. “On–off” switching was observed
for at least five cycles when illuminated consecutively with UV
and visible-light sources. Finally, cell-adhesion studies with
human umbilical vein endothelial cells (HUVECs) validated our
system as an effective light-responsive CIM platform and the
possibility of dynamically controlled cell adhesion in situ was
envisioned.
smart platforms have been already exploited to direct cell fate
by using light. In recent studies, the availability of biological
signals was modulated through photocleavable moieties,
thereby promoting either cell adhesion[11] or detachment.[12] In
this respect, azobenzene-modified surfaces[13] can provide fur-
ther improvement by introducing the concept of reversibility
through the isomerization mechanism.
Indeed, azobenzenes are excellent candidates as molecular
photoswitches,[14] since they can exist in two isomeric forms,
trans and cis, which can interconvert both photochemically
and thermally.[15] In addition, among several photoswitches,
azobenzene is one of the most resistant to optical fatigue.[16]
The isomerization process involved is reversible and can
induce a significant geometric change in the position of the
two phenyl rings. In general, the trans isomer is the most
stable and has a flat geometry because of the two aromatic
rings that lie on the same plane, and its strongest absorption
band is around 365 nm owing to the p!p* transition. Instead
the cis isomer presents an angular geometry and has a relative-
ly weak absorption around 445 nm related to the n!p* transi-
tion.[17] Because of the higher stability attributed to the trans
isomer, azobenzene derivatives are predominantly present in
the trans form and, when exposed to UV radiation, the trans-
to-cis isomerization occurs. This process is completely reversi-
ble, and the back-isomerization can take place either sponta-
neously (eventually accelerated by heating) or by visible-light
exposure. This reversible mechanism can lead to a variation in
the physical and chemical properties of the surrounding envi-
ronment. For example, azobenzene molecules that are linked
to a surface provoke a local alteration in wettability once they
modify their conformation, mainly due to a change in the
dipole moment of the molecules upon going from the trans to
cis isomer. Actually, such local variation in wettability can be
monitored by water contact angle technique, thereby provid-
ing useful information about the isomerization process.[18] Fur-
1547 Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

thermore, if the azobenzene molecule brings substituents on
the rings, during the isomerization the position of these
groups changes as well as their distance.[19]
Inspired by recent studies, which investigated a synthetic
matrix coated with azo derivatives that led to photocontrolled
cell[13, 20] or bacterial adhesion,[21] we designed a two-dimen-
sional platform based on an “on–off” photoswitching mecha-
nism that was useful for the dynamic exhibition of a well-
known peptide sequence (GRGDS) and exploitable for photo-
triggered cell-adhesion studies. The azobenzene moiety was
firstly linked to the RGD peptide, then grafted onto glass surfa-
ces with the chemoselective “click” chemistry approach.[22]
Here, the synthesis of our dynamic RGD platform is first dis-
cussed, followed by an extensive characterization of the photo-
responsive properties using UV/Vis spectroscopy and water
contact angle (WCA) techniques. Finally, cell-adhesion studies
on such substrates, conducted with human umbilical vein en-
dothelial cells (HUVECs), were shown to validate our system.
Results and Discussion
The study presented here aimed to develop glass-modified
surfaces on which the exposure of the GRGDS peptide se-
quence could be triggered by light to tune cell adhesion in
a photocontrolled manner. To construct such photoresponsive
platforms, azobenzene was introduced into the chemical struc-
ture of our system and covalently immobilized on the sub-
strate. In general, to ensure the proper RGD signaling, it is im-
portant that the biomolecules maintain their bioactivity in
terms of conformation and chemical stability once immobilized
on a solid surface. For this reason, the target molecule was
grafted with a chemoselective method by exploiting the Huis-
gen 1,3-dipolar cycloaddition[23] between azides and terminal
alkynes, also known as a “click” reaction, which has been large-
ly established as a useful chemical handle for bioconjugation
in both non-copper-mediated[24] and copper(I)-catalyzed[22]
manners. The unreactive character of azides and alkynes to-
wards other functional groups present in the synthesized mol-
ecules, together with the thermal and hydrolytical stability of
their bonding, represent optimal features in our specific case,
as well as in the general field of bioconjugations.[25]
The density of packaging is another important parameter to
consider when grafting azobenzenes on a solid substrate. In
fact, a sufficient free volume for the isomerization to take
place has to be taken into account when dealing with azopoly-
mer coatings or unsubstituted azobenzene monolayers.[17–18a]
The degree of functionalization influences in turn the density
of biological signals over the surface, thus affecting cell re-
sponse. Indeed, cells need a certain architecture and number
of GRGDS ligands either to simply adhere onto the substrate
or to form integrin clusters called focal adhesions.[3a]
We designed our target molecule by taking into account
three important features: it contained a “clickable” moiety, pre-
sented a photoswitchable unit, and bore a cell-adhesive signal.
First, an alkyne that enabled the “click” reaction to occur was
introduced as the pendant group of a short oligo(ethylene
glycol) linker, thus ensuring an adequate degree of freedom
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Full Papers
for the isomerization to take place, even after confinement of
the azobenzene to the solid support.[3b, 9] Second, the selected
azobenzene guaranteed a cis stability of several hours, thereby
allowing cell-adhesion experiments. Third, the GRGDS peptide
was attached directly to the azobenzene as a biological signal
for cell adhesion. In this way, when the azobenzene was pres-
ent as a trans isomer the peptide was available for cell recogni-
tion, whereas, after trans-to-cis isomerization, the exposition of
GRGDS was compromised.[13]
In this study, the azobenzene unit was synthesized accord-
ing to a previously reported procedure[13b, 26, 27] to obtain 4,4’-di-
carboxylic acid and then the NHS derivative (AZO and 2 in
Scheme S1 in Supporting Information). In the synthesis of the
azobenzene unit the strategy involved a reductive coupling of
an aromatic nitro derivative, which occurred in situ in basic
aqueous solution in the presence of a large excess amount of
glucose as reducing agent. This reaction provided a symmetric
azobenzene with high yield; in addition the obtained byprod-
ucts were water soluble, so they were easily removed with
water washing steps. The NHS ester-activated azobenzene 2
was used for subsequent coupling reactions.
Afterward, the tert-butyloxycarbonyl (Boc) protecting group
of polyethylene glycol (PEG) linker 1 was cleaved with a solu-
tion of 50 % trifluoroacetic acid (TFA) in CH2Cl2. The obtained
amine salt was treated with triethylamine (TEA) to restore its
nucleophilic feature, and finally it was conjugated with com-
pound 2 to obtain the photoresponsive molecule 3. GRGDS
peptide was synthesized by standard solid-phase peptide syn-
thesis (SPPS) by means of a 9-fluorenylmethoxycarbonyl
(Fmoc) strategy. Then the conjugation between the azoben-
zene block 3 and the fully protected peptide 4 still anchored
onto the resin was performed as a final coupling to prevent
side reactions and simplify the workup of the target molecule
5 (Scheme 1). The final azo derivative 5 was checked with
mass spectrometry and nuclear magnetic resonance analyses
(see the Supporting Information), which confirmed the expect-
ed chemical structure for the designed compound.
Before use, the glass surfaces were washed, activated by
oxygen plasma treatment, and then immediately modified
with a short bromosilane. After silanization, the bromine head
group was displaced by sodium azide in N,N-dimethylforma-
mide (DMF; 6 A in Scheme 2).
Water contact angles (WCA) were evaluated before the
plasma treatment and after each modification step to verify
the occurrence of the reactions. Collected values were in good
agreement with those reported in literature. For bromo-silan-
ized glasses in particular an average angle (qav) of 808 was
found, whereas after azide displacement the average value de-
creased to 768.[28]
In the final step, azo derivative 5 was attached to modified
glasses 6 A by means of “click” chemistry, as illustrated in
Scheme 2. In particular, we used a catalytic amount of
copper(II) salt (CuSO4·5 H2O) with a large excess amount of
a water-soluble reducing agent (sodium ascorbate) to produce
copper(I) in situ. A glass coverslip of 6 A treated without the
CuI catalyst (conditions under which the reaction did not
occur) was used as negative control, on one hand to further
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Full Papers
sorbing species (trans and cis) in
equilibrium with each other.[29]
The thermal back-isomerization
of the molecule was achieved
just after two days by keeping
the sample at room temperature
in the dark, whereas the photo-
induced back-isomerization was
reached using a common desk
lamp provided with a halo bulb.
The light source was placed far
enough away to avoid local
heating of the solution, and with
this simple tool we were able to
restore quite completely the
trans isomer just after 20 min of
exposure to white-light radia-
tion.
Moreover, these measure-
ments were able to give impor-
tant information on how the
substituents linked to the azo-
benzene affect the isomeriza-
tion.[19] As shown by our experi-
Scheme 1. Reagents and conditions for the synthesis of compound 5. (a) 50 % TFA in CH2Cl2, 2 h at RT; (b) 2
mental data, the substituents
(1 equiv) in DMF, 24 h at RT; (c) 4 (1 equiv), NHS (1 equiv), DIC (1 equiv), DIPEA (2 equiv) in DMF, overnight at RT.
did not hinder the isomerization;

verify the occurrence of the “click” reaction, and on the other

hand to ensure that changes in WCA were exclusively due to
the presence of the azobenzene covalently bound to the sur-
face.
To characterize our substrates in terms of topographical and
chemical distribution of the azobenzene molecules, atomic
force microscopy (AFM) and Raman measurements were per-
formed (for details, see the Supporting Information). On the
basis of these analyses, the presence of distinct areas of func-
tionalization emerged. In particular, according to the Raman
mapping spectra obtained by measuring all over the surface,
the N=N peak at 1466 cm¢1 was present with diverse intensi-
ties in different points of the sample.
As anticipated above, the isomerization mechanism of azo-
benzene molecules in solution can be monitored by means of
UV/Vis spectroscopy, thanks to the fact that each isomer pres-
ents a characteristic absorption spectrum.[17, 19b] The photores-
ponsive behavior was studied both for molecules 5 and AZO.
Even if slightly redshifted with respect to AZO (see the Sup-
porting Information), the spectra of azo derivative 5 showed
all the identifying features of an azobenzene. The spectra
indeed exhibited a p!p* band in the UV region (between 320
and 380 nm) related to the more stable trans conformation Figure 1. (A) UV/Vis absorption spectra of 5 in DMF. (Black line) before UV ir-
that decreased upon irradiation for 20 min with the UV lamp, radiation and (orange dashed line) after 20 min of UV exposure, (blue line)
thermal back-isomerization after two days in the dark, and (red line) photo-
as shown in Figure 1. controlled back-isomerization after 20 min of halo lamp illumination. (Inset)
Meanwhile, the redshifted broad band at 420 nm related to Magnification of the peak at 420 nm related to the cis isomer. (B) and (C) iso-
the n!p* transition of the cis isomer increased. In that time sbestic points of molecule 5 at 262 and 387 nm.
interval, a photostationary state was reached. The presence of
the isosbestic points at 262 and 387 nm ensured that all the in fact, the trans-to-cis conformational change was observed as
different peaks observed in each spectrum belong to both ab- well as the cis-to-trans back-isomerization. In addition, the

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 Full Papers
First of all, contact angle
values were collected before any
light treatment. Then, samples
were irradiated for 20 min at
365 nm with the same UV lamp
used in spectroscopic measure-
ments. After this illumination
step, the local contact angle was
measured as explained in detail
in the Experimental Section.
Thermal back-isomerization was
verified for each sample after
leaving it for three days in the
dark at room temperature,
whereas the photoinduced back-
isomerization was confirmed
after just 30 min of irradiation
with a desk lamp. Experimental
data collected for the two sam-
ples proved that both of them
underwent photodriven isomeri-
zation. Moreover, as expected,
they experienced a decrease in
water contact angle values after
UV illumination, even if some
differences were observed. In
the case of sample 7 B, in which
the azobenzene bore the NHS
group (Figure 2A), a lower varia-
Scheme 2. Reagents and conditions for the synthesis of sample 7 A. (a) CuSO4·5 H2O (0.5 equiv with respect to
tion in contact angle value that
compound 5), sodium ascorbate (10 equiv) in DMF, overnight at RT.
reached 48 was detected after
UV illumination, as reported in
thermal conversion of the cis isomer to the trans one was slow, Figure 2B, C.
thereby ensuring a sort of “frozen configuration” after UV irra-
diation, which was compatible with typical biological experi-
ment timing.
The study described above provided a useful starting point
to set up water contact angle measurements using the same
parameters (such as time, light sources, and their distance
from the sample). In this study, the contact angle technique
was used as a powerful tool to prove the photoswitching pro-
cess of azo compounds once immobilized on glass surfaces
and to investigate local changes in wettability.[17] According to
the literature, the concept of wettability in azobenzene-modi-
fied surfaces is related to the change in the dipole moment of
the N=N double bond that occurs upon isomerization with
a concomitant switch in the geometry of the molecule it-
self.[19a] In a general case, when an unsubstituted azobenzene
is present as a trans isomer, the surface appears more hydro-
phobic; then, upon UV irradiation, the cis isomer is promoted
and an enhanced hydrophilic feature is exhibited, according to
an increase in the dipole moment of this isomer.[17] Two differ-
ent samples were investigated to understand the role of the
short peptide sequence linked to the azobenzene. The first Figure 2. (A) Chemical structure of sample 7 B. WCA images of a drop
(B) before UV illumination, (C) after UV illumination, and (D) after three days
sample was a glass surface modified with molecule 5 that bore
in the dark for thermal back-isomerization. For each drop, left and right con-
the GRGDS sequence (7 A), and the second one also contained tact angles were reported as raw data. WCA data were obtained analyzing
the azobenzene moiety but without the peptide (7 B). the same region of the sample.

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 Full Papers
mum 18 in few cases), thus proving once more that
wettability variations were strictly related to confor-
mational changes of the azo group. All the variations
in WCA values that occurred after UV stimulation
were completely recovered after three days in the
dark. In early experiments of photodriven back-iso-
merization we instead observed a partial recovery of
the initial value; here visible illumination time was
two hours and the intermediate washing step was in-
cluded (Figure 4C). Thus, we supposed that the wash-
ing step between the UV and visible-light exposure
affected the final value, and to overcome this prob-
lem we decided to skip the intermediate measure-
ment between the two irradiation steps by relying
on the fact that UV-induced variations had always
been observed. Therefore, WCA experiments were
performed just by collecting values before any light
treatment and after the final visible illumination step,
thus yielding reproducible data. The photocontrolled
Figure 3. (A) Chemical structure of sample 7 A. WCA images of a drop (B) before UV illu- back-isomerization was then also achieved in this
mination, (C) after UV illumination, and (D) after three days in the dark for thermal back- case with a reasonably short illumination time by
isomerization. For each drop, left and right contact angles were reported as raw data.
using a common desk lamp as trigger.
WCA data were obtained analyzing the same region of the sample.
Reversibility of photocontrolled isomerization was
confirmed irradiating the samples with UV and visible
However, sample 7 A (Figure 3A) revealed a decrease in con- light at least for five consecutive cycles (Figure 4D), where the
tact angle value up to 88 (Figure 3B, C) because of the pres- UV value reported as reference has been collected in previous
ence of charged polar amino acid residues (arginine, serine, measurements (“UV” in Figure 4C). As already mentioned, WCA
and aspartic acid). In fact, it is
known that not only the change
in dipole moment but also the
water affinity of the group pres-
ent at the outermost part of the
monolayer affects the change in
WCA.[30]
It is even possible to have an
opposite variation in wettability
(from a more hydrophilic mono-
layer in the trans state to a less
hydrophilic in the cis one), for
example, by placing a hydropho-
bic moiety in the meta position
of the azobenzene phenyl
ring.[31] In our specific case, how-
ever, the observed variations
were not uniform all over the
substrate. Each drop exhibited
its own change in WCA owing to
different degrees of local func-
tionalization of the surface. In
fact, some drops did not reveal
significant changes in WCA as
reported in Figure 4A, B.
In comparing these data with Figure 4. (A) WCA values for sample (red dots) 7 A and (blue rhombic shape) 7 B; (B) WCA variation for a different
those related to the negative area of samples 7 A and 7 B, together with negative control sample 6 A (green dots). (C) WCA values for sample
(red dots) 7 A and (blue rhombic shape) 7 B after visible illumination (VIS* refers to 2 h of illumination with visible
control 6 A, which did not pres-
light and includes an intermediate washing step). (D) Five cycles of consecutive UV/Vis irradiations for samples
ent any azobenzene, no contact (red dots) 7 A and (blue dots) 7 B. The WCA values after UV stimulation (black and gray dots) were reported in the
angle alteration occurred (maxi- graph as reference (“UV” data in 4 C). Each cycle is illustrated as “UV/Vis” with different colors.

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data reported were obtained by analyzing the same area of
the solid substrate.
For biological experiments, HUVECs were chosen as this cell
line is an important in vitro model in the field of tissue engi-
neering when studying adhesion and migration processes re-
lated to the regeneration of damaged tissues and vasculariza-
tion.
Substrates were functionalized with molecule 5 covering
more than 30 % of the surface to guarantee enough GRGDS
sites and to ensure a specific adhesion.[32] UV-sterilized samples
7 A and 7 B were treated and studied in such a way that both
trans and cis configurations of the grafted azo derivatives
could be present at the same time in each experiment. In par-
ticular, the sample with azobenzene in trans configuration
(here in short 7 Atrans), which was obtained by leaving the
UV-treated surface one week in the dark, revealed a stronger
adhesion of HUVEC (Figure 5A) with respect to that one with
the molecule in the cis form (7 Acis, illuminated again with
a UV lamp right before cells seeding) on which less adherent
cells were observed (Figure 5B).
Figure 5. Phase contrast images (10 Õ magnification) of HUVEC on sample
7 A. (A) Left one week in the dark after UV treatment (7Atrans), (B) illuminat-
ed with UV lamp right before cells seeding (7Acis), and (C) illuminated with
UV and visible light one straight after the other, thus inducing the switch
from trans-to-cis and back to trans again (7Atrans’). After seeding, cells were
incubated overnight at 37 8C and 5 % CO2 atmosphere and then imaged in
fresh medium.
Moreover, to evaluate the real photocontrolled cell adhesion,
the experiment was repeated by inverting the two 7 A sam-
ples. That is, glass sample 7 Atrans became 7 Acis and vice
versa. As expected, cell-adhesion response was inverted, there-
by excluding any influence of possible variation in surface
functionalization on cell adhesion. Finally, in another experi-
ment, the photoinduced back-isomerization was tested as well
by switching first from trans-to-cis and then back to trans
again, applying UV and visible-light radiations one directly
after the other before seeding cells (7 Atrans’ in Figure 5C).
Here cell adhesion was comparable to that of sample 7 Atrans.
For control sample 7 B a low adhesion was observed in both
configurations because of the lack of the peptide. Further-
more, GRGDS and GRGES directly grafted onto silanized glasses
8 and 9, respectively (see the Supporting Information) were
treated under the same conditions as for 7 Atrans and 7 Acis.
As expected, cells adhered all over the GRGDS-modified surfa-
ces and negligible variations were observed before and after
light treatment. Moreover, we noticed a visible difference in
adhesion between samples 8 and 9 that becomes more evi-
dent after five days, which points to a slight proliferative capa-
bility of the cells seeded on the GRGES sample (see Figure S8
in the Supporting Information).
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Even if they are promising, these results require further im-
provements to achieve a truly dynamic photocontrolled cell
adhesion in situ. Thus, we are currently optimizing this plat-
form with a view to more engineered functionalization, such
as the use of UV masks to allow spatially controlled illumina-
tion before cell seeding and the introduction of non-fouling
surrounding layers (i.e., PEG or BSA).[33]
Conclusion
In summary, in this study a photoresponsive kind of cell-in-
structive material was developed by “clicking” azobenzene-
based photoswitches that bear the GRGDS peptide sequence
to silanized glass. The synthesis involved two key steps: firstly,
an optimized strategy to avoid side reactions by employing
solid-phase synthesis for the conjugation of the azobenzene
moiety to the GRGDS peptide, and secondly, the use of the
chemoselective “click” approach for the final linkage to the
glass. The photoswitching behavior of the synthesized mole-
cules was first verified in solution by using UV/Vis spectrosco-
py, thereby confirming that GRGDS peptide did not interfere
with the photoinduced trans-to-cis isomerization and its rever-
sibility. Moreover, photoreversibility of the switching molecules
linked to the glass surface was proven by using water contact
angle techniques. When no peptide was present (sample 7 B),
the decrease in WCA reached 48 after exposure to UV light.
This response has been mainly attributed to a change in
dipole moment of the N=N double bond. When GRGDS se-
quence was present (sample 7 A) water contact angle variation
reached 88, thus showing a further contribution to the change
in wettability from the peptide itself. Back-isomerization was
achieved either by thermal relaxation over time, or by illumina-
tion with white light by using a common desk lamp. The
switching was observed for at least five cycles when illuminat-
ed consecutively with UV and visible light, thus revealing the
possibility of tuning at will the availability of the peptide se-
quence with an “on–off” mechanism typical of switch regula-
tors. Finally, the platform was tested for its use in the photore-
versible control of cell adhesion with HUVECs. These prelimina-
ry results open up a wide range of possible investigation, rela-
tive to the field of in situ dynamic modulation of cell behavior.
Experimental Section
Materials
The majority of chemicals and solvents were purchased from
Sigma–Aldrich and used without further treatment. 9-(N-Boc-
amido)-4,7-dioxanonanoic acid was purchased by Cyanagen, amino
acids by Iris Biotech GmbH, and cell media from Gibco Life Tech-
nologies. Column chromatography was performed with granular
silica gel (60–100 mesh). Glass substrates were purchased from
Thermo-Scientific. Milli-Q water (18.2 MW cm) was obtained with
a Millipore system. Mass spectra were acquired with an Agilent
Technologies 6530 Accurate Mass (Q-TOF, LC/MS) instrument. 1H
and 13C NMR spectra were acquired with an Agilent Premium Com-
pact + instrument (600 MHz). Chemical shifts are reported in ppm;
calibration was carried out on the deuterated solvent used in the
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Full Papers
experiments by the related software function. Homonuclear and as follows: Fmoc-Ser-(tBu)-OH, Fmoc-Asp-(OtBu)-OH, Fmoc-Gly-OH,
heteronuclear (2D) experiments (TOCSY, 1H,13C HSQC) were per- Fmoc-Arg-(Pbf)-OH, Fmoc-Gly-OH. The steps performed for each
formed when required for peak assignments. amino acid coupling were: 1) Fmoc deprotection with a solution of
20 % of piperidine in DMF; 2) a coupling reaction of each amino
acid (4 equiv respect to resin) in the presence of HOBt·H2O
Synthesis of compound 1 (4 equiv), N,N’-diisopropylcarbodiimide (DIC; 4 equiv), and N,N’-di-
isopropylethylamine (DIPEA; 8 equiv) in 50 % of CH2Cl2 in DMF;
9-(N-Boc-amido)-4,7-dioxanonanoic acid (0.36 mmol, 100 mg), 1-hy-
and 3) after each coupling, a capping step for unreacted amine
droxybenzotriazole hydrate (HOBt·H2O; 1.2 equiv, 58.5 mg), N-ethyl-
groups was performed with a solution of acetic anhydride (0.5 m)
N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl;
and DIPEA (0.125 m) in DMF. Each reaction step was controlled and
1.2 equiv, 83 mg), and triethylamine (TEA; 2 equiv, 100 mL) were
confirmed using a Kaiser test. Final product 4 was kept linked to
dissolved in CH2Cl2 (10 mL) in a round-bottomed flask under mag-
the resin with Fmoc protection still present on the last amino acid,
netic stirring for 3 min of pre-activation. After that time, propargyl-
ready to use for the next synthetic step. The peptide sequence
amine (1.2 equiv, 25 mL) was added and the mixture was left under
was checked by cleavage of a testing sample (10 % in weight)
magnetic stirring overnight. The reaction was followed by thin-
using a solution of 2.5 % H2O and 2.5 % triisopropylsilane in TFA,
layer chromatography (TLC) on silica with 10 % of methanol
precipitated in diethyl ether, recollected by centrifugation, and fi-
(MeOH) in CH2Cl2 and stained with a solution of ninhydrin that
nally freeze-dried; MS (ESI): m/z calcd for C17H32N9O8 : 490.24
made the product visible after heating. Product 1 was purified by
[M++H] + ; found: 490.26 [M+
+H] + .
silica gel column chromatography and eluted with 3 % of MeOH in
CH2Cl2 as a white oil (90 % yield). 1H NMR (CDCl3, 600 MHz): d =
1.42 (s, 9 H; CH3), 2.20 (s, 1 H; Ž CH), 2.48 (t, 2 H; COCH2CH2), 3.30
(dd, 2 H; OCH2CH2NH), 3.53 (t, 2 H; OCH2CH2NH), 3.59 (s, 4 H; Synthesis of compound 5
OCH2CH2O), 3.71 (t, 2 H; COCH2CH2), 4.03 (m, 2 H; Ž CCH2NH), 5.00
Azo derivative 3 (32 mmol, 18 mg) was coupled to the anchored
(br s, 1 H; CH2CH2NHCO), 6.75 ppm (br s, 1 H; Ž CCH2NHCO);
13 peptide 4 (1 equiv with respect to compound 3 according to the
C NMR (CDCl3, 600 MHz): d = 28.61 (CCH3), 29.16 (CCH3, Ž
nominal loading of the resin). First, anchored peptide 4 was put in
CCH2NH), 36.88 (COCH2CH2), 40.52 (OCH2CH2NH), 67.16 (COCH2CH2),
a syringe with filter and washed several times using CH2Cl2 and
70.14, 70.46 (OCH2CH2OCH2), 71.51 (Ž CH), 80.02 (C Ž CH), 156.18,
DMF to swell the resin, and then Fmoc deprotection was carried
171.48 ppm (CO); MS (ESI): m/z calculated for C15H27N2O5 : 315.18
out using a solution of 20 % of piperidine in DMF. Then the cou-
[M+ +H] + ; found: 315.02 [M++H] + .
pling with 3 was performed in the presence of a mixture of NHS
(1 equiv, 4 mg), DIC (1 equiv, 5 mL), and DIPEA (2 equiv, 11 mL) in
DMF. As a final step, the cleavage from the resin as well as the re-
Synthesis of compound 2
moval of all side-chain protecting groups were performed using
Azobenzene 2 was synthesized according to an already published a solution of 10 % of H2O in TFA for 2 h. Azo derivative 5 was pre-
protocol;[13b] more details can be found in the Supporting Informa- cipitated in cold diethyl ether, recollected by centrifugation, and fi-
tion. nally freeze-dried. Crude was directly used for the next reaction
without any further purification (71 % yield). 1H NMR (DMSO,
600 MHz): d = 1.5–1.7 (m, 4 H; CbH2, CgH2 Arg), 2.34 (t, 2 H; COCH2),
Synthesis of compound 3 2.73 (m, 2 H; CbH2 Asp), 2.9 (s, 4 H; OCH2CH2O), 3.0–3.1 (m, 5 H; CdH2
Arg, Ž CH, CH2CH2NH), 3.4–3.9 (m, 13 H; CaH2 Gly, CaH2 Gly, CbH2
Compound 1 (64 mmol, 20 mg) was treated for 2 h with a solution
Ser, CH2NH, CH2OCH2, CH2CH2NH), 4.16 (m, 1 H; CaH Ser), 4.31 (m,
of 50 % trifluoroacetic acid (TFA) in CH2Cl2 to remove the Boc pro-
1 H; CaH Arg), 4.59 (m, 1 H; CaH Asp), 6.5 (t, 2 H; CH Ar), 7.15 (s, 2 H;
tecting group and treated by co-evaporation at rotavapor with tol-
CONH2), 7.4 (m, 1 H; CH2NH Arg), 7.5–7.6 (m, 2 H; CH Ar), 7.8 (t, 1 H;
uene. Then azobenzene 2 (1 equiv, 30 mg) was dissolved in dry
NH Ser), 7.9–8.1 (m, 5 H; NH Arg, CH Ar), 8.15–8.30 ppm (m, 3 H; NH
DMF (2 mL) at 60 8C for 1 h under stirring. After that, a solution of
Asp, NH Gly, NH Gly); 13C NMR (DMSO, 600 MHz): d = 28.29, 29.02
1 in CH2Cl2 was added dropwise, still at 60 8C. Once the reaction
(Cb, Cg Arg), 35.7 (COCH2), 36.01 (Cb Asp), 40.36 (Cd Arg), 40.03
started, the mixture was left at room temperature overnight. The
(OCH2CH2O), 40.36 (Ca Gly), 41.89 (Ž CCH2), 42.64 (Ca Gly1), 49.54
reaction was followed by TLC at 5 % of MeOH in CH2Cl2. The final
(Ca Asp), 55.27 (Ca Ser), 57.00 (Ca Arg), 66.55 (Cb Ser), 68.7
product 3 was purified by performing silica gel column chromatog-
(CH2CH2NH), 69.46 (CH2CH2NH), 72.89 (Ž CH), 81.12 (C Ž CH), 112.51,
raphy and eluted at 2 % of MeOH in CH2Cl2 (40 % yield). 1H NMR
122.53, 128.67 (CH Ar), 156.54, 165.77, 166.68, 168.83, 168.96,
(CDCl3, 600 MHz): d = 2.18 (s, 1 H; Ž CH), 2.44 (t, 2 H; COCH2CH2O),
169.79, 170.31, 171.77, 171.91 ppm (CO); MS (ESI): m/z calculated
2.91 (s, 4 H; COCH2CH2CO), 3.64–3.74 (m, 10 H;
+H] + ; found: 938.44 [M+
for C41H55N13O13 : 938.41 [M+ +H] + .
CH2OCH2CH2OCH2CH2), 4.05 (m, 2 H; Ž CCH2), 6.50 (s, 1 H; NH), 6.84
(s, 1 H; NH), 7.96–8.02 (m, 4 H; CH Ar), 8.28 ppm (d, 4 H; CH Ar);
13
C NMR (CDCl3, 600 MHz): d = 25.87 (COCH2CH2CO), 29.52 (Ž CCH2),
40.16 (OCH2CH2O), 67.07 COCH2CH2O), 69.99, 70.36 (OCH2CH2NH), Protocol of silanization with 3-(bromopropyl) trimethoxysi-
71.65 (Ž CH), 79.89 (C Ž CH), 123.40, 128.38, 131.97 (CH Ar), 154.16, lane (BPTMS) for the preparation of glass surface 6
156.12, 161.51, 166.89, 169.27 ppm (CO); MS (ESI): m/z calculated
Glass coverslips were washed in acetone under sonication and
+H] + ; found: 564.31 [M+
for C28H30N5O8 : 564.21 [M+ +H] + .
dried in an oven for about 30 min at 30 8C. Then oxygen plasma
treatment (electronic Diener plasma surface technology) was per-
formed for 3 min, and immediately after the activation step,
Solid-phase peptide synthesis of GRGDS sequence 4
glasses were put in a solution of BPTMS in toluene (4 % v/v) on an
GRGDS peptide was synthesized using standard Fmoc solid-phase oscillating plate overnight. After reaction, glasses were washed
peptide synthesis (SPPS). Fmoc-protected amino acids were loaded with toluene, ethanol, and finally acetone and dried under
onto Rink Amide AM resin (100–200 mesh, 0.48 mmol g¢1, 50 mmol) vacuum.

ChemPlusChem 2015, 80, 1547 – 1555 www.chempluschem.org 1553 Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Bromine-azide nucleophilic substitution for the preparation
of glass surface 6 A
Bromo-silanized glasses 6 were put in a glass petri dish that con-
tained a saturated solution of sodium azide (50 mm in DMF) and
left overnight on an oscillating plate. The next day all glass cover-
slips were washed with DMF, ethanol, and finally acetone, then
dried under vacuum.
“Click” chemistry
Protocol followed in the case of sample preparation for water contact
angle experiments: In a petri dish with glass sample 6 A, a solution
that contained compound 5 (1.1 mmol, 1 mg), sodium ascorbate
(10 equiv respect to compound 5), and CuSO4·5 H2O (0.5 equiv) dis-
solved in DMF (3 mL) was added. The reaction was left on an oscil-
lating plate overnight at room temperature, and the next day sam-
ples were washed and finally dried under vacuum.
Protocol followed in the case of sample preparation for biological ex-
periments: Compound 5 (3 mol, 2.9 mg), sodium ascorbate
(10 equiv respect to compound 5), and CuSO4·5 H2O (0.5 equiv)
were dissolved in 10% H2O in DMF, and then the reaction was per-
formed as described above.
UV/Vis spectroscopy
UV/Vis measurements were performed using a Cary 100 UV/Vis
spectrometer and recording from 800 to 200 nm in double-beam
mode by using two quartz cuvettes (10 mm optical path) and DMF
as solvent. Previously, a zero-baseline correction was performed by
recording 100 and 0 % of the transmittance signal. A step of 1 nm
and an average time of 0.1 s were used for all measurements.
Water contact angle measurements
Water contact angles were acquired by the sessile drop technique
using a KSV-CAM 200 instrument. Measurements were performed
at room temperature deposing Milli-Q water drops of 4 mL of
volume. Contact angle measurements were executed after each
synthesis step on glass surfaces to verify the occurrence of each
chemical modification. Obtained values were determined as the
average of several frames for different drops. In the case of the ex-
periment to evaluate the wettability change upon azobenzene-
photoinduced isomerization, a specific protocol was developed. In
this case, samples were investigated by monitoring the local
change in wettability in the same area of the sample, before and
after illumination, with the help of some landmarks drawn on the
opposite side of the glass. The analysis was performed by collect-
ing five frames for each drop, and an average value was consid-
ered for every single drop.
Illumination experiments
For UV radiation a Spectroline E-Series UV lamp was used illuminat-
ing at 365 nm (after 20 min of warm up). The light source was
placed 5 cm away from the samples. The fluence rate[28]
(1.2 mW cm¢2) was measured with a power meter (Thorlabs). For
visible radiation a normal desk lamp equipped with a halo bulb
was used and placed 10 cm away from the samples. The fluence
rate (130 mW cm¢2) was measured providing the sensor of the
power meter instrument with a proper filter (435–443 nm).
ChemPlusChem 2015, 80, 1547 – 1555 www.chempluschem.org 1554
Full Papers
Cell culture and cell-adhesion experiments
Human umbilical vein endothelial cells (HUVECs) were purchased
from LONZA and cultured on gelatin-coated polystyrene flasks in
an incubator at 37 8C and a humidified atmosphere with 5 % of
CO2, using Medium 200 supplemented with LSGS Kit (Gibco Life
Technologies). For cell-adhesion experiments 3 Õ 104 cells were
seeded (8 to 12 passage). Gelatin-coated glass and GRGDS-modi-
fied glass were used as positive controls, while bromo-silanized
glass was used as the negative one (see the Supporting Informa-
tion). Each sample was present in triplicate in the same experiment
and all substrates were sterilized for one hour under UV light one
week before the test. The trans configuration of azo derivatives in
sterilized samples was achieved by keeping them seven days in
the dark before the experiment (thermal back-isomerization), while
the cis configuration was induced by illuminating again with UV
light for one hour, immediately before seeding cells. The photoin-
duced back-isomerization was achieved by leaving the sample
under the visible-light radiation of a normal desk lamp for one
hour, immediately after UV irradiation and just before seeding
cells. After the overnight incubation, all substrates were washed
twice with phosphate-buffered solution (PBS), moved to a new
petri dish, refreshed with new medium, and imaged with an invert-
ed phase contrast microscope at several magnifications.
Acknowledgements
We thank Dr. Anastasios Manikas for his precious help with
RAMAN spectroscopy, Dr. Luca Raiola for his valuable contribu-
tion in recording NMR spectroscopy data and Dr. Valeria Chia-
nese for her support in developing the silanization protocol. Dr.
Chiara Attanasio and Maria De Gregorio are acknowledged for
their important guidance during biological experiments with
HUVEC cells. Financial support was provided from IIT (Istituto Ital-
iano di Tecnologia).
Keywords: azo compounds · cell adhesion · click chemistry ·
peptides
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Manuscript received: April 23, 2015
Revised: June 9, 2015
Final Article published: July 1, 2015
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim