Synthesis and alignment of discrete polydiacetylene-peptide nanostructures

Article abstract of DOI:10.1021/ja211539j

Oligopeptides bearing internal diacetylene units are shown to self-assemble in water into one-dimensional nanostructures and aligned macroscopic hydrogels. The diacetylene units can be photopolymerized into polydiacetylenes that run coincident to the nanostructure and noodle long axes, and the resulting nanostructures show evidence for ambipolar charge transport. This self-assembly, alignment and polymerization technique provides a rapid way to produce globally aligned collections of conjugated polymer chains.

Full text of DOI:10.1021/ja211539j

Communication
pubs.acs.org/JACS
Synthesis and Alignment of Discrete Polydiacetylene-Peptide
Nanostructures
Stephen R. Diegelmann,†,‡,§,⊥ Nikolaus Hartman,§,∥ Nina Markovic,§,∥ and John D. Tovar*,†,‡,§
†Department of Chemistry, ‡Department of Materials Science and Engineering, §Institute for NanoBioTechnology, and ∥Department
of Physics and Astronomy, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
S
* Supporting Information
directly within the backbones of water-soluble oligopeptides
ABSTRACT: Oligopeptides bearing internal diacetylene
units are shown to self-assemble in water into one-
dimensional nanostructures and aligned macroscopic
hydrogels. The diacetylene units can be photopolymerized
into polydiacetylenes that run coincident to the nano-
structure and noodle long axes, and the resulting
nanostructures show evidence for ambipolar charge
transport. This self-assembly, alignment and polymer-
ization technique provides a rapid way to produce globally
aligned collections of conjugated polymer chains.
and directs the formation of electronically delocalized 1D
amyloid-like nanostructures, including those that are comprised
of single PDA conjugated chains. Biologically compatible 1D
“wires” would provide important advances for bioelectronics.
We prepared several peptide−DA−peptide molecules to
explore their self-assembly, topochemical polymerization, and
alignment. We subjected α,ω-diacid DAs 115 and 216 to solid-
phase dimerization reactions developed in our lab for the
incorporation of π-electron units into short oligopeptide
chains.4 Using the aliphatic diacid 1, we prepared peptide 3,
and the aromatic diacid 2 led to peptides 4 and 5 (Figure 1).17
niformly aligned macroscopic assemblies of individual π-
U
conjugated polymers or nanoscale fibrils thereof, as
opposed to the microscale domains commonly found in thin
films of crystalline organic semiconductive polymers, would
offer many opportunities for nanotechnology. We report how
short oligopeptides bearing internal diacetylenes (DA) self-
assemble into amyloid-like nanomaterials that can be aligned
via a simple solution dispensing process and subsequently
photopolymerized into polydiacetylene (PDA) nano- and
macrostructures. These PDA−peptide structures are water
processable, manipulatable, and (for the macrostructures)
consist of globally aligned individual bundles of isolated
conjugated polymers. Unique to this work are demonstrations
of chemically driven chromic alteration and gate-induced carrier
formation within nanostructures arising from PDAs derived
from unusual topochemical polymerizations of diarylbuta-
diynes. Oligomeric π-electron materials have been incorporated
directly into the backbones of organo-1 and water-soluble2−5
peptides leading to intermolecular electronic delocalization
within self-assembled one-dimensional (1D) nanostructures,
but this is the first report to use simple water-soluble peptides
embedded with reactive chromophores to prepare aligned
intramolecular electronic conduits of conjugated polymers.
PDAs have received considerable attention following the
pioneering demonstration by Wegner that crystalline DAs with
specific intermolecular geometric constraints can undergo
topochemical polymerization to form PDAs.6 Several material
systems have encouraged PDA formation, from single crystals7,8
to self-assembled monolayers and vesicles.9,10 There have only
been a few examples of PDA formation within 1D
nanostructures under organic11 or aqueous environments,12−14
but the PDAs thus formed (e.g., those within peptide
amphiphile nanofiber matrices) are bundled collections of
multiple polymer chains. Our design incorporates the DA units
Figure 1. Molecular structures of DA diacids and peptides.
Peptide 3 was soluble at basic pH and formed a self-
supporting hydrogel under more acidic pH, a common
indicator for these types of materials that suggests molecular
self-assembly into 1D nanostructures. Unfortunately, UV
irradiation of the peptide nanostructures formed from 3 did
not produce PDA. Analysis of the light irradiated product
indicated the formation of a hydrolyzed yne-one [Figure S11,
Supporting Information (SI)]. This suggests that the DA units
are either exposed to water during irradiation or that the
flexibility of the butyl spacers prevents polymerization.15
Nanostructures comprised of diphenyl DAs 4 and 5 placed
the reactive diynes in proper position to allow for successful
topochemical polymerizations (Figure S18, SI). This is
remarkable because there are very few reports of successful
diphenyl DA polymerizations.18−21 The peptide assembly
energetics thus override unfavorable steric or quadrupole
stacking within the nanostructures while affording a dynamic
environment to promote cross-linking. The nanostructures
obtained after assembly of 4 were dispersed in water and
Received: December 9, 2011
Published: January 10, 2012
© 2012 American Chemical Society
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irradiated with UV light (254 nm) and the formation of PDA
monitored by absorption spectroscopy (Figure 2b). The low-
Figure 3. (a) UV−vis spectra of polymerized 5 in acidic (pH ∼ 2, solid
blue), basic (pH ∼ 8, red), and reacidified (pH ∼ 2, dashed blue)
aqueous solution. (b) CD spectra of polymerized 4 in acidic (solid
blue), basic (red), and reacidified (dashed blue) 1:1 water/ethanol
solution.
our future work will address more biologically relevant
detection scenarios23 (e.g., with biotin-mediated binding, SI).
The nanostructures formed from 4 and 5 were examined
with AFM. Peptide 4 was dissolved in basic water (pH 7−8) to
form a clear solution (up to 10 mg/mL or 9.3 mM). The
acidification of that solution resulted in an opaque gel that
slowly formed macroscopic aggregates over approximately 5
min. Although we could observe 1D nanomaterials by diluting
this gel and imaging by atomic force microscopy (AFM),
visualization was difficult due to the presence of larger
aggregates (Figure S12, SI). Despite their water solubility, we
found that the addition of ethanol (1:1 v/v with water) prior to
the assembly led to better dispersion of the nanomaterials
presumably through better wetting of the substrate. In all cases,
the assembled solutions were polymerized with a hand-held UV
lamp prior to sample deposition and imaging by AFM. Peptide
4 assembled into nanostructures with diameters ranging from
larger 30−40 nm bundles down to smaller branched 3−5 nm
structures (Figure 4). The hydrophobicity of the DFAG
Figure 2. (a) Stacked DA peptides (right) and resulting PDA. (b)
UV−vis spectra taken after continual UV irradiation of 4 in acidic
solution (at 0, 0.5, 5, and 20 min intervals). The spectrum recorded at
basic pH is indicated on the graph.17 Inset: Film of an assembled
solution of peptide 4 cast onto a glass slide (∼ 3 cm2) followed by
irradiated with UV light for 30 min to form blue PDA.
energy absorption bands (λabs = 663, 606 nm) and visible blue
color after irradiation indicated PDA formation. This polymer-
ization occurred in solution and in the solid state as a drop cast
film (inset in Figure 2b). The absorption bands indicate a very
high degree of planarity and conjugation throughout the PDA
backbone relative to other reported diphenyl DA polymer-
izations. Irradiation of 5 was accompanied by the formation of a
yellow solution, indicative of DA oxidation, in addition to blue
PDA. The relatively less hydrophobic EAA sequence of 5
promotes the formation of smaller nanostructures that expose
more DA units to the aqueous media.22 There is a clear balance
needed between structures large enough to bury the DA units
from water while being small enough to prevent aggregates
from precipitating out of solution. Sterically demanding
sequences can also foster polymerization as observed with
biotin-containing DA peptides (SI). In all cases, the long axes of
the PDA chains can be envisioned to run coincident with the
length of the 1D amyloid-like nanostructures.
Figure 4. AFM images of PDA nanostructures derived from peptide 4
on SiO2 substrates.
The hydrogen-bonding networks within the PDA nanostruc-
tures were disrupted by raising the pH and inducing charge
repulsion among the resulting carboxylates that forced the PDA
chains to adopt less planar conformations (Figure S19, SI).
This disruption led to a blue shift in the low-energy PDA UV−
vis absorptions and a visual color change from blue (602, 558
nm) to reddish purple (592, 543 nm) (Figure 3a). Likewise, the
circular dichroism (CD) spectra of the PDA nanostructures
showed intense signals (345, 368 nm) with a weak excitonic
Cotton effect at lower energy (Figure 3b). These CD signals
were dramatically diminished upon raising the pH (pH ∼ 8).
Reacidification of the solution then recovered the initial UV−
vis and CD spectral signatures, indicating that the local
planarity and chirality of the polymer chromophores enforced
by β-sheet hydrogen bonding were re-established. These
switching responses establish that the peptide−PDA nanoma-
terial conjugation is responsive to environmental stimuli, and
tetrapeptide sequence in 4 promotes hierarchical aggregation
beyond that of single β-sheet tapes as to be expected for an
amyloid-like assembly process. The less hydrophobic EAA
tripeptide sequence in 5 limited this process to individual and
better-dispersed nanostructures rather than the larger diameter
objects (Figure S13, SI). These AFM images depict bundled
PDA chains as well as what we believe to be the first examples
of single PDA chains embedded within discrete nanostructures
(Figure 4, left panel).
Electrostatic force microscopy (EFM) was used to probe the
electronic behavior of the PDA core inside the peptide
nanostructures. This technique uses an AFM to first collect
the height profile in tapping mode. The measured height profile
is then used in the second scan in order to maintain the tip at a
constant height above the sample, while applying a constant
voltage between the tip and the sample, analogous to gating a
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field effect transistor. Electrostatic force between the tip and the
sample shifts the phase of the oscillation of the tip, allowing the
measurement of the electrostatic force gradient as a function of
the tip position. This technique has been used to differentiate
between conductive carbon nanotubes (CNTs) and insulating
λ-DNA nanostructures on SiO2 surfaces.24 The height profiles
are comparable to those found in standard AFM (3−15 nm,
Figure S14, SI). EFM images of dispersed peptide−PDA
nanostructures were obtained for a range of tip voltages from
−5 to +5 V to probe the response of the charge carriers to the
electric field over the length of the nanostructures (Figure 5). It
experimentally simple technique. We thus reasoned that we
could effect alignment of conjugated polymers for intra-
molecular delocalization provided that DA-based peptide
noodles could also undergo topochemical polymerization.
Scanning electron microscopy (SEM) shows the random
network formation usually observed for self-assembled peptide
hydrogels (Figure 6a). SEM of a peptide hydrogel formed using
Figure 6. SEM images of polymerized gel of peptide 4 as an unaligned
hydrogel (a) and as an aligned peptide noodle (b).
the extrusion technique revealed globally aligned peptide−DA
nanostructures within macroscopic noodles (Figure 6b).
Although Figure 6b shows an area of ca. 44 μm2, comparable
alignment can be envisioned throughout the macroscopic
structure (mm−cm scales). These noodle macrostructures were
subjected to UV irradiation leading to the formation of blue
hydrogel noodles (Figure 7a), a color that was observed above
Figure 5. EFM images showing the (a) topography and phase shift
images of peptide−PDA nanostructures from peptide 4 (b) without an
applied tip voltage and with (c) −5 and (d) + 5 V applied to the tip.
is clear that in the absence of tip voltage (Figure 5b), there is
no tip/fiber interaction indicating no electric field in the
nanostructure. The observed phase shift images and their
dependence on the tip voltage (Figure 5c,d) indicate that
charge carriers can be induced within these structures that
move freely over their lengths despite the presence of the
peptides.
The phase shift images for both negative and positive tip
voltage show distinct ‘negative−positive−negative’ contrast
across the short axis of each individual structure. This contrast
has been observed in other organic conducting nanofibers and
is thought to arise from the difference in the capacitance of the
system as the tip scans over the nanofiber: a horizontal
attractive force is felt by the tip as it approaches the side of the
nanofiber, followed by a repulsive force when the tip travels
directly above it.25
The present peptide−PDA assemblies are comprised of
randomly oriented 1D nanostructures. There are many known
techniques to align π-conjugated polymers or self-assembled
nanostructures,26 including those that use physical rubbing,27
Langmuir−Blodgett films,28 extrusion from mesoporous
inorganic crystals,29 the addition of small molecules such as
liquid crystals,30 “aligner”31 and “twimer”32 compounds, and
the application of strong electric30 or magnetic fields.12 More
recently, we employed a solution dispensing route first reported
by Stupp33 to achieve macroscopic (cm scale) domains of
aligned π-conjugated peptide nanostructures within physically
manipulatable “noodle-like” hydrogels.34 This method is
attractive when compared to the above techniques because it
is performed completely in aqueous media using an
Figure 7. Peptide 4 hydrogel noodles before (colorless) and after
(blue) DA polymerization as seen under a light microscope (a)
without and (b) with crossed polarizers. Spatially controlled
polymerization of a peptide−DA noodle performed by shielding
with a TEM grad shadow mask (c, d). Arrows indicate regions shielded
from UV light.
during the polymerization of randomly aligned peptide−DA
nanostructures. The present system thus offers an exper-
imentally facile method to align peptide nanostructures poised
for topochemical polymerization to yield highly aligned arrays
of conjugated polymers within nanostructured 1D fibrils, all
available from one simple solution manipulation.
Polarized optical microscopy (POM) was used investigate
the extent of global alignment of the peptide−PDA noodles as
well as differentiate between the birefringence before and after
polymerization (Figure 7b). Although both samples are
birefringent, there is a clear color contrast in the polymerized
material leading to a reddish appearance. This color is not
associated with a resonant process but reflects how the
nanostructures within the noodles interact with the polarized
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ASSOCIATED CONTENT
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■
S
Experimental, characterization, and instrumental details, and
additional UV−vis and AFM images. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
tovar@jhu.edu
■
Present Address
⊥Macromolecular Science and Engineering, Case Western
Reserve University, 2100 Adelbert Road, Cleveland, Ohio
44106, United States.
ACKNOWLEDGMENTS
■
We thank Dr. J. Michael McCaffery (JHU IIC) and Ms. Julie
Bitter for help acquiring and interpreting POM images. S.R.D.
was an NSF-IGERT Fellow through JHU’s INBT. We thank
Johns Hopkins University, JHU’s Institute for NanoBioTech-
nology, the National Science Foundation (DMR-1106167,
N.M.), and the Department of Energy Office of Basic Energy
Sciences (DE-SC0004857, J.D.T. peptide nanomaterials) for
generous support.
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2010, 9, 594.
(34) Wall, B. D.; Diegelmann, S. R.; Zhang, S.; Dawidczyk, T. J.;
Wilson, W. L.; Katz, H. E.; Mao, H. Q.; Tovar, J. D. Adv. Mater. 2011,
23, 5009.
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