Redox-switchable surface for controlling peptide structure

Article abstract of DOI:10.1021/ja203198y

A general surface chemistry strategy is described for the development of a new switchable material. The method modulates a surface-immobilized-molecules structure by using two orthogonal "click" reactions based on Huisgen cycloaddition and oxime chemistry, where the oxime linkage is redox active and switchable. We demonstrate this strategy by developing a noninvasive, biocompatible, in situ surface chemistry that is able to modulate the affinity of a cell-adhesive peptide to cell integrin receptors to study dynamic cell adhesion and cell migration in real time and as a new hide-and-reveal strategy for application in new types of smart biofouling biomaterials.

Full text of DOI:10.1021/ja203198y

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pubs.acs.org/JACS
Redox-Switchable Surface for Controlling Peptide Structure
Brian M. Lamb and Muhammad N. Yousaf*
Department of Chemistry, Carolina Center for Cancer Nanotechnology, Carolina Center for Genome Sciences, The University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
S Supporting Information
b
small-molecule structures on surfaces via a redox-responsive
ABSTRACT: A general surface chemistry strategy is
described for the development of a new switchable material.
The method modulates a surface-immobilized-molecules
structure by using two orthogonal “click” reactions based on
Huisgen cycloaddition and oxime chemistry, where the
oxime linkage is redox active and switchable. We demon-
strate this strategy by developing a noninvasive, biocompa-
tible, in situ surface chemistry that is able to modulate the
affinity of a cell-adhesive peptide to cell integrin receptors to
study dynamic cell adhesion and cell migration in real time
and as a new hide-and-reveal strategy for application in new
types of smart biofouling biomaterials.
orthogonal and biologically noninvasive electrochemical cue. The
dynamic and switchable covalent chemistry demonstrated herein
is general and may potentially be applied to hiding-and-revealing
pharmaceuticals or small molecules for cell biology assays.
Electroactive SAMs on gold have been extensively character-
ized and developed for a range of fundamental studies of
electrode processes and as a platform for biomaterial appli-
cations.11À17 We recently introduced an electrochemical “click”
methodology for immobilizing and releasing ligands to and from
a surface. The method is based on the conjugation of oxyamine
(OA)-terminated ligands on hydroquinone (HQ)-terminated
SAMs.18,19 The HQ group permits an electrochemical activation
and click reaction to occur with OA-terminated small molecules
that can be precisely monitored and actuated with cyclic voltam-
metry (CV). Although HQ lacks a carbonyl, it can be oxidized to
the benzoquinone (BQ) form (essentially a redox activation to
ketone groups) and reacted with OA to form a stable quino-
neÀoxime linkage (Qox).20 When Qox is reduced, cleavage of Qox
releases a primary alcohol and regenerates HQ. The methodol-
ogy has several inherent advantages: facile incorporation of OA
into biomolecules for subsequent conjugation, an ability to
conjugate and release ligands under biological conditions (pH
7.4, 37 °C), and precise electrochemical control of redox state for
switchable immobilization and release.
Peptides, small molecules, carbohydrates, and oligonucleo-
tides are routinely functionalized with both Huisgen cycloaddi-
tion and oxime conjugation chemistry (alkyne, azide, ketone,
OA), are readily incorporated into many biomolecules, and have
been shown to undergo mutually orthogonal chemoselective
reactions.21À26 In order to generate a switchable surface that
replicates dynamic ECM functions, such as revealing adhesive
proteins on connective tissue in response to injury, a bifunc-
tional redox-active HQ molecule was synthesized containing an
azide linkage for orthogonal Huisgen and oxime click ligations
(Scheme 1). Huisgen azide/alkyne cycloaddition chemistry was
chosen as an orthogonal and complementary chemoselective
strategy to the benzoquinoneÀoxime chemistry both for the
minimal steric size of the azide and alkyne moieties and for the
broad and facile applicability of the chemical coupling methodology.27
A synthetic scheme was designed to incorporate azide and HQ
functional groups in close proximity into an alkanethiol. The
synthesis is modular and provides a general framework for future
modification of the system for alternative applications (e.g.,
SAMs on indium tin oxide, replacement of azide). The synthesis
of 7, an alkanethiol containing a tetra(ethylene glycol) linker, a
witchable materials are important for a wide range of applica-
S
tions in polymer science, molecular electronics, nanotechnol-
ogy, and biology.1À5 These materials contain chemical groups or
linkages that can be altered with physical stimuli to produce
interesting properties and dynamic functions. Important exam-
ples of recently developed stimuli-responsive materials include
surfaces with electronically actuated hydrophobicity,6 switchable
hydrogels that selectively release therapeutic agents,7 self-as-
sembled monolayer (SAM) surfaces that employ ultraviolet light
to actuate small-molecule conformation,8 and polymers that
selectively bind and release cells due to changes in swelling
caused by mild temperature fluctuations.9
Cells live in a complex and dynamic environment and receive a
multitude of extracellular input cues ranging from physical stimuli to
small molecules and hormones that are then processed for a
range of output signaling and cell behaviors. In particular, the
extracellular matrix (ECM) environment cells occupy provides a
dynamic scaffold that is able to continually remodel itself and
elicits a range of cell functions. To study how cells respond to a
dynamic and evolving environment, model substrates based on
materials that can undergo molecular remodeling or conforma-
tional shifts in response to noninvasive triggers are required to
provide novel strategies for stimulating and controlling biological
functions both in vitro and in vivo.10
Herein a general strategy for switching an immobilized-mole-
cules structure on a surface is described based on two ortho-
gonal “click” reactions. We demonstrate this strategy by devel-
oping a redox-responsive chemistry that is able to switch the
activity of a cell-adhesive peptide (RGD) presented to cells.
The method relies on two orthogonal click reactions based on
Huisgen cycloaddition and benzoquinoneÀoxime chemistry
where the oxime linkage is redox active and switchable. This
is an innovative application of molecular switches to alter
Received: April 7, 2011
Published: May 19, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of a Redox-Active Bifunctional and
Orthoganol Molecule (7) for Huisgen and Oxime Click
Conjugationa
Figure 1. Illustration of a methodology switching small-molecule
conformations on surfaces. Small molecules containing both alkyne
and oxyamine functional groups are first immobilized onto the surface
via Huisgen 1,3-cycloaddition. Conversion of HQ to BQ occurs via a
mild oxidative potential. BQ then reacts with the OA group on the ligand
for an intramolecular cyclization via oxime chemistry. Upon application
of a reducing potential to the substrate, the oxime bond is cleaved,
regenerating the HQ and producing the linearized ligand. Each step is
monitored and controlled by electrochemistry since each molecule
(HQ, BQ, Qox) has a distinct and diagnostic CV signature.
a Reagents and conditions: (a) i, tert-butyllithium, THF, 0 °C; ii,
1-bromoheptene, HMPA, 3 h. (b) MCPBA, bicarbonate, dichloro-
methane (DCM), 3 h, 25 °C. (c) i, bicarbonate, phthalimide, 100 °C,
24 h; ii, mesityl chloride, 10 equiv of triethylamine, THF, 1 h 25 °C; iii,
DMF, sodium azide, 110 °C, 3 days. (d) i, hydrazine, methanol, 50 °C,
12 h; ii, THF, succinic anhydride, 30 min, 25 °C. (e) DMF, HBTU,
diisopropylethylamine, 9, 2 h, 25 °C. (f) DCM, trifluoroacetic acid,
triethylsilane, 30 min, 25 °C.
Scheme 2. Bifunctional Peptides and Small Molecules Used
for Characterizing Surfaces and for Generating Smart
Bio-interfacial Surfaces for Studies of Cell Behavior
secondary azide (for Huisgen cycloaddition), and a HQ is
described. A branching point for placing the two reactive func-
tional groups, HQ and azide, was introduced by ortho-lithiation
of 1 and reaction with 1-bromoheptene to produce olefin 2.
Epoxidation of olefin 2 with m-chloroperoxybenzoic acid
(MCPBA) afforded 3 in high yield. Nucleophilic ring opening
of the epoxide with sodium phthalimide, transformation of the
resulting secondary alcohol to a mesylate, and substitution with
sodium azide provided 4 in a 90% yield. Reaction of 4 with excess
hydrazine in methanol yielded the free amine, which was
reacted with succinic anhydride 5 and then coupled to 9 with
HBTU and DIEA to yield 6. Subsequent deprotection in
DCM/TFA/TIPS yielded the target product 7 in near-quan-
titative yield. By creating a SAM containing two clickable
functional handles (7) via electrochemically controlled click
oxime chemistry and Huisgen cycloaddition, a general strategy
for immobilizing and switchably controlling ligand presenta-
tion on SAM surfaces was achieved (Figure 1).27
Small molecules and peptides were synthesized to contain
both OA and alkyne coupling groups to react with the dual
chemoselective HQ and azide functional groups presented on
the SAM surface. This strategy allows for the change of structure
of bound molecules on a surface by the general immobilization,
activation, conjugation, and release of the oxime bond.
Mixed SAMs composed of 7 and 8 were constructed by
immersing freshly evaporated gold surfaces in 1 mM total mixed
alkanethiol solution in ethanol (Scheme 2). To confirm the
availability of both the HQ and azide functionalities to react
selectively with their chemical partners, model alkyne- and
OA-terminated ligands were immobilized onto the SAM sur-
face. Ethynyl ferrocene was immobilized to a SAM surface
composed of 20 mol % 7 and 80 mol % 8 with standard ascorbic
acid/CuSO4 Huisgen cycloaddition conditions and character-
ized by CV (Supporting Information (SI) Figure S1).28
Further confirmation of ferrocene immobilization was obtained
by MALDI mass spectrometry of the surface (SI, Figure S3). In a
separate experiment, BQ was reacted with methoxylamine to form
Qox, which upon application of À100 mV reducing potential
regenerated the HQ with release of methanol (Figure S1). To
confirm the mutual compatibility of the azide and HQ, solution-
phase experiments demonstrating two-step immobilization, activa-
tion, conjugation, and release were performed and monitored by
UVÀvis spectroscopy. The addition of CuSO4 to the solution after
Huisgen cycloaddition oxidized the HQ group to BQ, promoting
cyclization via oxime bond formation (SI, Figure S2). Reduction of
Qox to HQ resulted in release of the alcohol product.
For cell adhesion studies we used the well-known cell-adhesive
RGD peptide as a dynamic ligand. The affinity and specificity of
RGD peptides for cell integrin receptors are strongly dependent
on the conformation of the peptide backbone, and many studies
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Figure 3. Switchable control of the RGDF peptide structure for cell-
adhesion and migration studies. (A,B) Representative images of stained
cells on the linear and cyclic RGDF switchable surfaces. (C,D) Cell
spreading and migration rates can be controlled by dynamic switching of
RGD peptide structure. On the more adhesive cyclic RGD surface
(blue), cells have larger diameters and slower migration rates. On the
less adhesive linear RGD surface (red), cells have smaller diameters and
migrate faster. The control of RGD peptide structure in situ in the
presence of adhered cells allows for real-time study of cell behavior.
Figure 2. Electrochemical monitoring and control of cyclization and
linearization of an immobilized cell-adhesive RGD peptide containing
terminal alkyne and OA functional groups. (A) The bifunctional SAM
contains a HQ group and an azide group. HQ-to-BQ interconversion
can be monitored by CV (À95 mV, 405 mV). (B) Upon reaction with
the bifunctional RGD peptide 10 followed by oxidation [O], a cyclic
RGD is formed. The cyclic RGD is redox active and shows diagnostic
linear RGDF. These observations are consistent with cell beha-
vior observed on separate linear and cyclic RGD surfaces in
previous work.31
Staining of cell cytoskeleton and focal adhesions with phalloi-
din and anti-vinculin showed that cells on cyclic RGDF peptide
exhibited a more intricately networked system of actin filaments
and more focal adhesions than cells on linear RGDF. As controls, a
scrambled GRFD peptide was immobilized, in which cells did not
adhere or bound very weakly and did not migrate on the surface due
to an inability of integrin receptors to recognize the peptide sequence.
Finally, to demonstrate the broad applicability of the metho-
dology as a dynamic biomaterial, an elongated RGDS peptide
(11) was immobilized onto the surface (Scheme 2). This strategy
allows for the substrate to become selectively adhesive and
nonadhesive to cell attachment via an electrochemical hide-
and-reveal. Spacer residues (Gly-Gly-Ser-Gly-Gly-Ser-Gly) were
included to separate the biospecific RGDS sequence from the
C-terminal end of the peptide, so that upon oxidation, the N-term-
inal OA functional group reacts to form Qox, thereby shielding the
RGDS sequence from cell integrin receptors (Figure 4). When
the oxime is cleaved, the RGDS sequence is released and
becomes available to bind cell integrin receptors. Cells did not
adhere to cyclized peptide on surfaces presenting 0.1% peptide
11 but did adhere to surfaces presenting the revealed linear
peptide, as shown in Figure 4. Importantly, the cell adhesion
profile is dependent on ligand density and shows that cells are
able to differentiate between hidden and revealed RGDS pep-
tides. These results indicate future potential of this method to
perform biomolecule hide-and-reveal for applications ranging
from fundamental studies of cell adhesion and dynamic micro-
arrays to wound healing and metastasis assays.
Q
ox redox peaks (55 mV, 310 mV). (C) The cyclic RGD can then be
linearized by cleavage of the Qox by application of a mild reductive [R]
potential at physiological conditions (pH 7.2, 37 °C). The cleaved linear
peptide regenerates HQ, which has distinct HQ-to-BQ CV peaks.
have examined and compared linear and cyclic RGD structures in
solution through competitive binding assays.29,30
Cyclic RGD peptides, in general, have a higher binding affinity
(nM) for cell integrins than linear RGD peptides (μM). To
demonstrate the immobilization of bifunctional dynamic RGD
peptides onto a SAM surface, a Pth-RGDF-Pg peptide was
synthesized by standard solid-phase peptide synthesis and in-
stalled via Huisgen cycloaddition to the surface. Although the
phthalimide protecting group for the OA is not necessary for
immobilization, it enables better control over oxidative activation
of the HQ. MALDI analysis of the surface showed complete
conversion of azide-terminated monolayer to peptide-functiona-
lized monolayer (SI, Figure 4). Phthalimide deprotection in
dilute hydrazine in methanol provided the free OA group. The
surface was then placed in PBS (pH 7.2) solution and oxidized at
650 mV to promote conjugation via oxime formation. Reduction at
À250 mV resulted in oxime release (Figure 2 and SI, Figure S2).
To determine whether on-surface in situ peptide conformation
changes would affect cell behavior, cells were seeded on 1%
dynamic peptide surfaces initially presenting cyclic RGDF. An
in situ electrochemical activation in the presence of cells switched
the peptide structure to the linear RGDF, and cell behavior was
observed via time-lapse microscopy (Figure 3 and Scheme 2). On
cyclized RGDF, cells were observed to bind more strongly to the
surface, as evidenced by a 25% greater surface adhesion diameter,
and showed a 35% slower rate of migration compared to cells on
We have designed a redox-switchable surface that is able
to alter small-molecule structure through a redox-responsive
linkage. The strategy is based on the immobilization of a
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’ AUTHOR INFORMATION
Corresponding Author
mnyousaf@email.unc.edu
’ ACKNOWLEDGMENT
We thank the Carolina Center for Cancer Nanotechnology
Excellence (National Cancer Institute), the NSF (CAREER
award), and the Burroughs Wellcome Foundation for funding.
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