[System] polypeptide self-assembly literature information extraction

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Self-assembly of peptides and amyloid fibrils offers an appealing approach for
creating chiral nanostructures, which has promising applications in the fields
of biology and materials science. Although numerous self-assembled chiral
materials have been designed, the precise control of their twisting tendency and
their handedness is still a challenge. Herein, we report the self-assembly of
chiral nanostructures with precisely tailored architectures by changing the
amino acid sequences of the peptides. We designed a series of self-assembling
tripeptides bearing different l-amino acid sequences. The peptide with
l-Phe-l-Phe sequence preferred to self-assemble into left-handed nanohelices,
while with l-Phe-l-Trp right-handed nanohelices would be formed. Moreover, the
diameter of the self-assembled nanohelices could be tailored by changing the
terminal amino acids (His, Arg, Ser, Glu, and Asp). Circular dichroism (CD) and
molecular dynamics simulations (MDSs) revealed that both of the right- and
left-handed nanohelices formed by the tripeptides showed negative Cotton effects
in the peptide adsorption region but exhibited nearly opposite CD Cotton effects
in the aromatic regions. These results indicated that the handedness of the
self-assembled helical nanofibers was not only determined by the chirality of
the peptide backbone but also closely related to the aromatic stacking, hydrogen
bonding and steric interactions induced by the side chains. The findings deepen
our understanding on the chiral self-assembly of peptide and offer opportunities
for the creation of highly functional chiral nanomaterials.

Preparation of Fmoc-tripeptide Chiral Nanostructures. In a
typical experiment, the lyophilized Fmoc-FWH powder (2 mmol, 1.42
mg) was dissolved in an aqueous solution (950 μL) under stirring at
room temperature through the addition of an appropriate amount of
0.5 M NaOH (50 μL). Then, 0.1 M HCl was added to the mixture
solutions and adjusted to pH 6. Afterward, an appropriate amount of
ddH2O was added to the solutions to a ﬁnal concentration of 2 mM.
The samples were then incubated at 25 °C for 24 h without any
disturbance. For the other Fmoc-tripeptide, the peptide assemblies
were obtained following the similar preparation procedure of Fmoc-
FWH as described above. However, because the Fmoc-FWR and
Fmoc-FFR did not dissolve in H2O by adding HCl and NaOH, we
investigated the self-assembly of Fmoc-FWR and Fmoc-FFR in a 5:95
(v/v) methanol/H2O mixture. The peptides were ﬁrst dissolved in
methanol at a concentration of 100 mM, which was then diluted in
950 μL phosphate buﬀer at pH 6.
Scanning Electron Microscopy. The morphologies and
structures of the Fmoc-tripeptide assemblies were investigated using
scanning electron microscopy (SEM, Hitachi S-4800, Japan) at an
acceleration voltage of 3 or 5 kV. To prepare the samples, the Fmoc-
tripeptide assemblies were spun on glass plates to dry and sputter-
coated with platinum using an E1045 Pt-coater (Hitachi High-
technologies Co., Japan).
Transmission Electron Microscopy. The samples for the TEM
analysis were prepared as follows: the assemblies were dispersed into
ddH2O and then 10 μL aliquots of the dispersion were spread onto a
300-mesh carbon-coated copper grid. After air-drying, the samples
were negatively stained with 1 wt % phosphotungstic acid solution for
Fourier Transform Infrared Spectroscopy. Fourier transform
infrared (FTIR) spectroscopy was recorded on a Nicolet-560 FTIR
spectrometer (Nicolet Co., U.S.A.) across the range of 400−4000
cm−1 at room temperature. The peptides were dissolved in D2O to
eliminate the interference of H2O. The samples were adjusted to pD
value to 6 with diluted NaOH and HCl in D2O.
pH Titration. Fmoc-FWH and Fmoc-FFH peptides were dissolved
in ddH2O by adding 0.5 M NaOH at a concentration of 0.5, 2, 5, or
10 mmol L−1 at pH ≈ 12. The titration curves were measured by
monitoring the pH values during the stepwise addition of 0.1 M HCl.
The tripeptides samples were stirred during the titration process.
Zeta Potential Measurement. Zeta potential measurements of
these Fmoc-tripeptide assemblies were investigated using a Zetasizer
Nano-ZS (Malvern Instruments Ltd., U.K.). The samples of the
assemblies were synthesized as mentioned before at pH 6.
Molecular Dynamics Simulation. Molecular dynamics simu-
lations (MDSs) were performed using the Gromacs program67,68 and
the Amber99SB-ILDN force ﬁeld.69 Initial molecular structures of
Fmoc-FFH and Fmoc-FWH were constructed using Accelrys
Materials Studio 2.5 software. The tLeaP tool was used to add the
hydrogen atom of the Fmoc-tripeptide residue. Models was centered
in an orthorhombic periodic boundary condition (PBC) box with a
margin of 1 nm and ﬁlled with SPCE water molecules.70 Na+ and Cl−
ions were added to make the system electrically neutral. The system
was performed with 5000 steepest descent steps and 5000 conjugate
gradient minimization steps to achieve energy minimization and then
simulated for 40 ns at a time step of 1 fs in the NPT ensemble at a
temperature of 300 K. Nonbonded and long-range electrostatic
interactions were calculated using the Particle Mesh Ewald (PME)
method71 with a cutoﬀ value of 1.0 nm. The MD traces were further
analyzed using Gromacs analysis tools and VMD.72

[Assistant] [{"peptide_sequence": "FWH", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.745", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FWR", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 Methanol/H2O", "peptide_concentration(mg/ml)": "0.765", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FWS", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.695", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FWE", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.74", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FWD", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.745", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FFH", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.71", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FFR", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 Methanol/H2O", "peptide_concentration(mg/ml)": "0.745", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FFS", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.76", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FFE", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.7", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FFD", "self-assembly_phase": "fiber", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.685", "PH": "6.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
© 2011 American Chemical Society

Materials. All Fmoc(cid:1)dipeptides were purchased from Ba-
chem (Bubendorf, Switzerland) and used without further pur-
ification. The purity of the compounds was verified by HPLC
(>98%) and mass spectrometry. HPLC grade water was pur-
chased from Merck and deuterated water (99.9 atom % D) from
Sigma-Aldrich.
Potentiometry. pH measurements were performed using a
Hanna Instruments pH210 pH-meter equipped with a Hamilton
Spintrode pH-probe (reference system, Ag/AgCl; electrolyte,
3 M KCl; diaphragm, ceramic; sensitivity, 58 mV/pH unit at
25 (cid:1)C). The pH-meter was calibrated before each experiment to
check the response of the electrode with two buffer solutions
purchased from Fisher Scientific: phthalate pH 4.01 and phos-
phate pH 7.01 buffer solutions.
“Titration” Experiments. For 5, 10, and 20 mmol L
samples, the required amount of Fmoc(cid:1)dipeptide was directly
suspended into 2 mL of HPLC grade water. Sodium hydroxide
(0.5 M) was added to the aqueous suspensions of Fmoc(cid:1)dipep-
tide until pH 10.5 was reached (e.g., ∼55 μL for 10 mmol L
sample). The samples were vortexed and sonicated (VWR
ultrasonicator bath, 30 W) for 1 min until fully dissolved. For
(cid:1)1 samples at pH 10.5 were used as
stock solutions and diluted to the desired concentration in a final
volume of 2 mL. The pH of the final solutions was then adjusted
to pH 10.5. The titration experiments were performed by
stepwise addition of small volumes of diluted HCl (0.085 M;
2(cid:1)60 μL depending on pH and system), up to a total added
14439dx.doi.org/10.1021/la202113j|Langmuir2011,27,14438–14449LangmuirARTICLEvolume of 750 μL. After each addition the samples were heated to
75(cid:1)80 (cid:1)C for 1 min, vortexed, sonicated, and subsequently
cooled back to room temperature using a water bath. pH values
were recorded before and after heating of the samples. The
samples were deemed fully mixed when no significant difference
((0.3 pH unit) was observed between two pH measurements.
Due to the strong and constant agitation applied, samples were
liquid at all times during the titration experiments. As a control,
water was also titrated using the same methodology described
above; that is, NaOH was added to the water in order to bring the
pH of the solution to 10.5, and then HCl was added stepwise.
Sample Preparation. Depending on the desired concentra-
tion, the required amount of Fmoc(cid:1)dipeptide was suspended
into 2 mL of HPLC grade water. Sodium hydroxide (0.5 M) was
gradually added to the aqueous suspensions of Fmoc(cid:1)dipeptide
(cid:1)1 sample).
until pH 10.5 was reached (∼55 μL for 10 mmol L
The samples were vortexed and sonicated for 1 min to fully
dissolve the short peptide amphiphiles. Depending on the
concentration and on the target pH, a required volume of dilute
hydrochloric acid (0.085 M) was then added dropwise while the
solution was vortexed and sonicated until the target pH was
obtained. Next, the samples were heated to 75(cid:1)80 (cid:1)C until fully
dissolved (2.5, 1.0, and 2.0 min for Fmoc(cid:1)FG, Fmoc(cid:1)GG, and
Fmoc(cid:1)GF, respectively) and homogenized. The samples were
subsequently cooled and maintained at 4 (cid:1)C for ∼12 h (overnight)
to promote or maintain gelation. To allow comparison between
the studied samples, all were investigated after this same aging
time. Reported pH values were those measured after storage. They
were found to be identical to the pH values measured before
heating within (0.3 units.
Fluorescence Spectroscopy. Fluorimetry experiments were
undertaken using a Perkin-Elmer LS55 luminescence spectro-
meter equipped with a Julabo F25 temperature control device.
After their preparation, the samples were transferred into PMMA
disposable cuvettes of 1 cm thickness (Fisher Scientific) and kept
at 4 (cid:1)C overnight. Emission and excitation slit widths were set at
3 and 10 nm, respectively. Emission spectra (excitation at
265 nm) were acquired at 25 (cid:1)C in the 300(cid:1) 600 nm range
(cid:1)1 using FL WinLab software.
with a scan speed of 300 nm min
Changes in the pH conditions and therefore in the self-assembled
structures formed are likely to modify the refractive index of the
samples and the quenching effect resulting from self-assembly.
These effects would have a direct effect on the intensity of the
transmitted light and hence on the emission peaks’ intensity. For
this reason, fluorescence spectra were all normalized as a function
of their respective maximum emission peak. The unnormalized
spectra are presented in the Supporting Information.
Fourier Transform Infrared Spectroscopy (FTIR). Multiple
bounce attenuated total reflectance (ATR) FTIR experiments
were undertaken using samples prepared in deuterated water.
Spectra were recorded on a Thermo Nicolet 5700 spectrometer
equipped with a trough plate comprising of a zinc selenide
crystal, which permitted 12 reflections with a 45(cid:1) angle of
incidence. The samples were spread directly on the surface of
the trough plate. Spectra were acquired in the 4000(cid:1)400 cm
(cid:1)1 over 128 scans. The deuterated
range with a resolution of 4 cm
water spectrum was used as background and subtracted from all
spectra (software used: Omnic version 7.2, Thermo Electron
Corporation).
using a parallel-plate geometry (40 mm diameter). To ensure the
measurements were made in the linear regime, amplitude sweeps
were performed and showed no variation in G0 and G00 up to a
strain of 1%. The dynamic moduli of the hydrogel were measured
(cid:1)1 with a
as a function of frequency in the range 0.01(cid:1)100 rad s
strain of 1%. To keep the sample hydrated, a solvent trap was used
and the atmosphere within the sample chamber was saturated with
water. Unless otherwise stated the experiments were performed at
25 (cid:1)C and repeated at least three times (each time on new samples)
to ensure reproducibility. The standard deviation of multiple
experiments is represented by error bars in the dynamic frequency
Transmission Electron Microscopy (TEM) and Image Anal-
ysis. A carbon coated copper grid (400 mesh, Agar Scientific)
was glow discharged for 30 s and then placed on 10 μL of
sample for 15 s. After blotting on Whatman 50 filter paper, the
loaded grid was washed in double distilled water for 30 s and
blotted. The sample was then stained with 10 μL of 2% (w/v)
uranyl acetate (centrifugated for 5 min beforehand) for 1 min
and blotted for 10 s. Data were collected at high vacuum on a
JEOL 1220 transmission electron microscope connected to a
high resolution (up to 3 Å/pixel resolution) Gatan Orius CCD
scattering experiments were conducted using 10 mmol L
samples. Wet samples were spread onto glass slides as thin films
and allowed to air-dry for 48 h prior to data collection. Experi-
ments were performed on a Philips X’Pert diffractometer
equipped with a copper source (wavelength of 1.54 Å), applying
scans from Bragg angles of 1(cid:1)(cid:1)33(cid:1). The glass slide spectrum was
used as background and subtracted from all spectra.

[Assistant] [{"peptide_sequence": "FG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.48", "PH": "10.5", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.4", "PH": "10.5", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.8", "PH": "10.5", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "9.6", "PH": "10.5", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.8", "PH": "9.0", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.8", "PH": "7.6", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FG", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.8", "PH": "5.1", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "GG", "self-assembly_phase": "ribbon", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "3.91", "PH": "7.2", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "GG", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "3.91", "PH": "4.6", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "GG", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "3.91", "PH": "7.3", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "GG", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "3.91", "PH": "9.0", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "GF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.8", "PH": "4.4", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "GF", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.8", "PH": "6.9", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "GF", "self-assembly_phase": "none", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.8", "PH": "8.8", "temperature(celsius)": "4.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
Addition of divalent cations to a solution of a naphthalene-diphenylalanine that
forms worm-like micelles at high pH results in the formation of a rigid,
self-supporting hydrogel.

Addition of divalent cations to a solution of a naphthalene-
diphenylalanine that forms worm-like micelles at high pH results
in the formation of a rigid, self-supporting hydrogel.
Low molecular weight hydrogels are formed via the assembly
of suitable molecules into nanoﬁbres, which then entangle and
form the matrix of the gel.1 Many low molecular weight
hydrogelators are known, with current signiﬁcant interest in
peptide-based gelators.2 This interest stems from the ease of
inducing the necessary one-dimensional interactions via hydrogen-
bonding between peptides as well as the potential biocompatibility
of such gelators. To induce gelation, a trigger is normally applied
to induce a sol-to-gel transition. Commonly, temperature,3 pH4 or
solvent composition5 are used to induce gelation.
We have been examining suitably functionalised dipeptides as
potential hydrogelators. These include dipeptides functionalised
at the N-terminus with aromatic groups such as naphthalene4b,6
or ﬂuorenylmethoxycarbonyl (Fmoc).4a,7 We and others have
shown that such molecules can form gels when a DMSO
solution of the gelator is diluted with water2,4a,5,8 or when
the pH of a dipeptide solution is decreased from high pH,
through the apparent pKa of the C-terminus.6 During this
work, we routinely utilise dipeptide concentrations of 0.5 wt%.
We recently demonstrated that this concentration is above the
critical micelle concentration (cmc) of some of the dipeptides
examined at high pH.6b At this high pH, solutions of many of
the dipeptides examined are free-ﬂowing liquids with the
apparent viscosity of water at the same pH. However, other
dipeptide solutions were signiﬁcantly viscous at high pH. We
hypothesised that this was the result of aggregation of the
dipeptides at high pH into worm-like micelles, presumably
with a hydrophobic core consisting of the naphthalene and
hydrophobic amino acids, stabilised by the carboxylates
(Fig. 1a). Assuming such a morphology, we rationalised that
cross-linking with divalent cations should lead to gelation for
this sub-class of functionalised dipeptide (Fig. 1b), similar to
that observed for biopolymers such as pectin and alginate, for
which an egg-box based model has been proposed.9 Here, we
demonstrate that this is the case and it is therefore possible to
prepare gels over a wide range of pH using a small number of
gelators. This greatly expands the utility of these gelators.
Here, we discuss a subset of naphthalene-functionalised
dipeptides (1–5, Scheme 1). We have previously demonstrated
that 1–5 are all eﬀective gelators when the pH of a solution is
Fig. 1 (a) Schematically, the carboxylate of 1 assembles into worm-like
micelles above the cmc. (b) Schematic of worm-like micelles cross-linked
on addition of a divalent ion (blue). (c)–(e) 2D SAXS for dipeptides 1, 2,
and 3 on horizontal shear showing horizontal orientation of the long axis
of the micelles. (f) TEM of 1 at high pH. (g) TEM of 1 on addition of
CaCl2. The scale bar is 200 nm for (f) and (g).
decreased from 11 to approximately 4.6b pH 4 is below the
apparent pKa of the terminal carboxylic acid; we have showed
in detail for 5 that this pKa is where assembly of the peptide
segments into b-sheets occurs, driving ﬁbre formation and
gelation.6a
At pH 10 or above and a concentration of 0.5 wt%, 4 and 5
are free-ﬂowing liquids, with a viscosity similar to water (Fig. S1,
ESIw). Cryo-TEM of solutions of 4 at high pH demonstrated
that this dipeptide forms spherical aggregates under these
conditions (Fig. S2, ESI;w conventional TEM with stain shows
no aggregates at high pH6a). However, at the same pH and
concentration, solutions of the naphthalene-diphenylalanines
1–3 are noticeably viscous, with viscosities of 33, 122 and
56 mPa s respectively at 1 s(cid:2)1 (Fig. S1, ESIw). All solutions
exhibit signiﬁcant shear thinning,
indicative of alignment of
aggregates under the ﬂow when the deformation is faster than
the time required to re-form the equilibrium network structure.
Similarly, small-angle X-ray scattering experiments demonstrate
strong orientation under shear, showing the presence of highly
anisotropic species (Fig. 1c–e). The presence of long, worm-like
micelles was further conﬁrmed by TEM (Fig. 1f).
At an initial pH of 11.7, addition of a number of salts as
solids to a solution of 1 generated self-supporting hydrogels.
More homogeneous gels could be prepared via the addition of
small amounts of concentrated solutions of salts to a solution
of 1 at high pH (Table 1). Gel formation presumably occurs
via the formation of salt bridges between carboxylates on
diﬀerent worm-like micelles, forming cross-links and hence a
three-dimensional network. TEM shows similar structures to
those seen before salt addition (Fig. 1g). Visually, there
appears to be similar dispersity in ﬁlament widths and lateral
associations between pH-triggered10 and salt-triggered assembly.
The use of divalent cations resulted in gels with higher moduli
than monovalent cations (Fig. S4, ESIw) and for divalent ions,
G0 was almost one order of magnitude greater than G0 0.
Table 1 Rheological properties for gels formed on addition of salt
solutions (ﬁnal concentration of 0.05 M) to a solution of 1
The eﬀect of salts on gelators has been documented for a
number of cases, but is extremely complex.11 Kuang et al. have
recently reported an Fmoc-pentapeptide that is sensitive to
potassium over sodium ions.12 In their case, the peptide
sequence was derived from a K+-binding epitope. Here,
similar G0 were obtained with sodium and potassium salts.
From the perspective of applications such as cell culturing, the
use of buﬀers is common and hence the eﬀect of salts on
gelators such as 1 is an important observation. In some cases, we
note that the ﬁnal pH was aﬀected by the addition of the salt.
Within the chloride salts, G0 correlated with the Hofmeister
series, with Ca2+ leading to the gel with the highest G0, followed
by Mg2+, Na+, Li+, K+ and NH4
+. For the Ca2+ salts, G0
decreased in the order of nitrate, chloride to sulfate as would
also be expected from the Hofmeister series. Focusing on
calcium nitrate, addition at pH 11.7 to solutions of 1 or 2
led to the formation of invertable hydrogels over a period of
approximately 90 min. For solutions of 3, a gel-like phase
co-existed in the presence of some liquid. In contrast, for 4 a
homogeneous clear liquid resulted and for 5, a precipitate
formed. This clearly demonstrates the importance of the
micellar structure at high pH. For 1, G0 was controlled by
the concentration of calcium nitrate used (Fig. 2). G0 reached a
maximum at a ratio of calcium: carboxylic acid of approxi-
mately 2 : 1. For 1, G0 exceeded G0 0 across a wide range of
calcium nitrate concentrations, with tan d being between 0.10
and 0.19 from 1.0 mg mL(cid:2)1 to 60 mg mL(cid:2)1 (Fig. S5, ESIw),
similar to values obtained for pH-triggered or solvent-triggered
hydrogels.7,8 The addition of calcium salts has also previously
been used to induce gelation using a number of peptide-
amphiphiles.4c,13 However, in these cases, the peptides carry
multiple carboxylic acids rather than the single C-terminus
present in the current case.
Gels were formed on addition of calcium nitrate from a
pH of 12 to a pH of 7. The apparent pKa of 1 is 6.0 (Fig. S6,
ESI) and, as described elsewhere,6b for pH-driven gelation this
is the pH around which gelation starts to occur. In the presence
of calcium nitrate, there was a gradual decrease in G0 with
decreasing pH until the apparent pKa (Fig. S7, ESIw). This is to
be expected. The cross-links between worm-like micelles require
the presence of both salts and deprotonated carboxylates. As
the pH decreases, the proportion of carboxylates will decrease
and hence the number of cross-links will be lower. The presence
12072Chem.Commun.,2011,47,12071–12073ThisjournaliscTheRoyalSocietyofChemistry2011Published on 17 October 2011. Downloaded by MIT Library on 3/21/2024 2:19:20 PM. View Article Onlinegel with a signiﬁcant pH gradient within the system. Over 24 h,
equilibration occurred such that the entire system was at a
uniform pH of 3.8, very close to the 3.7 expected.
Finally, we note that whilst gels are only formed at
high pH on salt addition for dipeptide solutions that exhibit
shear-thinning, the examples are not restricted to 1–3. Fmoc-
diphenylalanine (which form gels on pH decrease from high pH4a)
also exhibits shear-thinning behavior at high pH and indeed forms
a gel on addition of calcium nitrate (G0 = 10.5 kPa).
In conclusion, we have shown that a subset of dipeptide-
based hydrogelators can form stiﬀ, self-supporting hydrogels
at high pH on addition of salts. There are analogies here with
pectin-based gels, where gelation can be induced by Ca2+ or
by acid depending on the molecular weight of the pectin, as
well as with the peptide-amphiphiles developed by Stupp.4c,13
Gels can now be prepared from dipeptide-gelators across the
whole pH range either by acid or salt addition. This expands
the range of dipeptide-based gelators that can be used at
physiological pH.
2 (a) D. J. Adams, Macromol. Biosci., 2011, 11, 160; (b) D. J. Adams
and P. D. Topham, Soft Matter, 2010, 6, 3707; (c) S. Bhattacharya,
U. Maitra, S. Mukopadhyay and A. Srivastava, in Molecular Gels,
ed. R. G. Weiss and P. Terech, Spring, Dordrecht, Netherlands,
2006, p. 613.
(a) Rheological time sweep showing gel formation on addition
of NaNO3 to a solution of 1 at pH 11.7. At 60 min, GdL was added.
G0, black data; G0 0, red data. Insert: pH trace for identically-treated
solution. (b) Ca-induced gel at pH 11.7 formed on top of pH-induced
gel of 1 at pH 3.4. Gels contain Universal Indicator. Over time, the
pH equilibrates.
of calcium nitrate below the apparent pKa resulted in a slightly
increased G0 if included prior to a decrease in pH; adding
calcium nitrate after pH adjustment resulted in G0 being very
similar to that in the absence of added salt (Fig. S8, ESIw).
A transition between the salt-induced and pH-induced gels
was possible. To exemplify this, glucono-d-lactone (GdL) was
added as a solid to the top of a gel formed via the addition of
sodium nitrate to a solution of 1 (a monovalent salt was
chosen for experimental ease as the initial gel is therefore
weak and the eﬀect of acid easy to observe). We have
previously used GdL to induce a slow, uniform pH drop as
dissolution of GdL is signiﬁcantly quicker than hydrolysis to
gluconic acid.6,7a,b After addition of sodium nitrate, a gel with
a G0 of 340 Pa was formed after 40 min (Fig. 3). Suﬃcient
GdL was then added to the top of the gel to result in a ﬁnal
pH of 3.4. This caused the pH to initially drop quickly (see Fig. 3
insert). G0 dropped quickly to 90 Pa and then started to rise
dramatically. This correlated with the gel strength dropping as
the pH dropped to the apparent pKa and then, on reaching
the pKa, starting to increase as pH induced gelation started.
Gel-to-gel transitions are very rare.14 Based on the rheological
data, it currently seems more likely that this is a gel-to-sol-to-
gel transition, but absolute determination is diﬃcult from the
current data and is the subject of continuing work.
This approach can be used to prepare interesting single-
gelator systems with dramatic pH gradients. To exemplify this,
a gel was prepared from 1 at a ﬁnal pH of 3.4 using GdL. After
gel formation, a solution of 1 at pH 11.7 was placed on top of
the ﬁrst gel and gelation induced by addition of a solution of
calcium nitrate. Gelation of the upper layer was complete
within 90 min, allowing the formation of a two-layer invertable

[Assistant] {"peptide_sequence": "FA", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "11.7", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "90.0"}


[System] polypeptide self-assembly literature information extraction

[User]
Peptide nanostructures are expected to serve as a major tool in future
nanotechnological applications owing to their excellent self-assembly
properties, biological and chemical flexibility and structural simplicity. Yet
one of the limiting factors for the integration of peptide assemblies into
functional electro-organic hybrid devices is the controlled patterning of their
assemblies. Here we report the use of inkjet technology for the application of
peptide nanostructures on nonbiological surfaces. The aromatic dipeptides
nanotubes (ADNT) which readily self-assemble in solution were used as an 'ink'
and patterned on transparency foil and ITO plastic surfaces using a commercial
inkjet printer. While inkjet technology was used in the past for the patterning
of carbon nanotubes, it was not used for the deposition of biomolecular
nanostructures. Furthermore, during the development of the application we were
able to produce two types of nanostructures, i.e. nanotubes and nanospheres by
the self-assembly of the same aromatic dipeptide, tertbutoxycarbonyl-Phe-Phe-OH
(Boc-Phe-Phe-OH), under different conditions. Both spherical and tubular
structures could be efficiently patterned on surfaces into predesigned patterns.
The applications of such technology are discussed.

The studied peptides, diphenylalanine (H-Phe-Phe-OH) and
two N -terminal modiﬁed analogues: Boc-Phe-Phe-OH and
Fmoc-Phe-Phe-OH, were purchased from Bachem (Buben-
dorf, Switzerland). Fresh stock solutions were prepared by
dissolving lyophilized form of the peptides in 1,1,1,3,3,3-HFP
(Sigma–Aldrich) at a concentration of 50 or 100 mg/ml. To
avoid any preaggregation, fresh stock solutions were prepared
for each experiment.
For the preparation of nanospheres, a peptide stock solution
was diluted in ethanol to a concentration of 10 mg/ml then
immediately diluted in ddH2O to a ﬁnal concentration of
5 mg/ml. For the preparation of nanotubes the 100 mg/ml
peptide stock solutions in HFP was diluted into a ﬁnal
concentration of 2 mg/ml in ddH2O. Immediately after dilution
in ddH2O, the peptide solution was allowed to dry at room
temperature over glass cover slip for SEM analysis.
Diphenylalanine peptide, Fmoc-Phe-Phe-OH and Boc-Phe-
Phe-OH stock solutions were diluted in ethanol
concentration of 10 mg/ml and then immediately diluted in
ddH2O to a ﬁnal concentration of 5 mg/ml.
HP Business Inkjet 2300 printer was used for ‘printing’ (i.e.
patterned deposition) of the nanotube patterns on commercial
transparency foil or on the conductive side of ITO plastic.
An empty and cleaned cartridge was ﬁlled with one of the
nanotubes or nanospheres solution, then ‘printed’ single or
multiple times. Furthermore, different cartridges were ﬁlled
with different solutions allowing the dispensation of different
nanostructure on the same surface. All the surfaces were
recorded using digital photography.

[Assistant] [{"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "15/85 HFP/H2O", "peptide_concentration(mg/ml)": "10.0", "PH": "4.3", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Boc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "4/96 HFP/H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "4.9", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
This journal is © The Royal Society of Chemistry 2012

COMMUNICATION
Silvia Marchesan et al.
Tripeptide self-assembled hydrogels: unexpected twists of chirality
Change of chirality of the ﬁrst N-terminal amino acid of
tripeptides VFF and FFV from L to D results in self-assembled
hydrogels at physiological pH from non-assembling L-analogues.
Interestingly, changing the chirality of F yields very diﬀerent
nanostructures; nanotapes are observed for DVFF, twisted ﬁbers
for DFFV.
The self-assembly behaviour of amyloid peptides is the topic of
intense research due to both pathological relevance and
potential application in the production of nanostructured
materials.1 In particular, the core sequence KLVFF has been
identiﬁed as the shortest key fragment responsible for the
formation of b-sheet ﬁbrils, which can yield hydrogels.2 At
present, there are no reported examples of KLVFF fragments
capable of self-assembly and hydrogelation in the absence of
organic solvents: uncapped di- and tripeptides are either too
hydrophilic and readily dissolve in water, or too hydrophobic, and
thus precipitate. In the case of FF alone, an elegant study revealed
discrete nanotubes of persistent length, when a concentrated
solution of the dipeptide in hexaﬂuoroisopropanol is diluted
in water.3 Later, a few examples were reported of capped
F derivatives, including di- and tripeptides, capable of self-assembly
when ﬁrst dissolved at high concentration in an organic solvent.4
This is not surprising as it is known that the self-assembly process is
strongly dependent on solvent.5 Diﬀerent is the case for Fmoc-FF
and naphthalene-FF, that do not require the presence of organic
solvents to hydrogelate, but where self-assembly is driven by the
synthetic aromatic moiety.6
Here, we report that while the short tripeptides VFF and FFV
indeed do not self-assemble into ﬁbrils in phosphate buﬀer, it is
suﬃcient to change the conﬁguration from L to D of the N-terminal
amino acid in both sequences to obtain self-supporting hydrogels.
To the best of our knowledge, these are the ﬁrst examples of
uncapped FF-containing tripeptides capable of self-assembly into
hydrogels at physiological pH without the aid of organic solvents.
Interestingly, Cryo-TEM and AFM data show that the change of
Fig. 1 Dynamic time sweep rheological data of freshly made hydrogels
(left). The hydrogels formed are self-supporting (right).
chirality in the case of DFFV results in highly twisted nano-
structures, and CD, FT-IR and Thioﬂavin T ﬂuorescence
conﬁrm they consist of b-sheets.
In this study, we used a pH switch to promote self-assembly:
peptides were ﬁrst dissolved in a 0.1 M sodium phosphate
solution at pH 12, while subsequent dilution to ﬁnal physiological
pH at B7.4 triggered the formation of macroscopic hydrogels
only for DVFF and DFFV, but not for their L-analogues, as
conﬁrmed by visual observation and rheology (Fig. 1). While
both gels were initially very soft (G0 of 10 kPa was reached only
after 1 hour for both peptides), it is worth noting that gelation of
DFFV could be markedly accelerated by sonication (see ESIw).
CryoTEM (Fig. 2) of the tripeptides containing a D-amino acid
showed nanoscale ﬁbrous morphologies, whilst no structures
could be identiﬁed for their L-analogues. Interestingly, the resulting
nanostructures of the two non-natural peptides were markedly
diﬀerent from each other. DVFF yielded long nanotapes, often
running in parallel (Fig. 2A, white arrowheads) and displaying
lateral stacking into wider structures (Fig. 2A, black arrowheads).
DFFV self-assembled into thicker ﬁlaments, usually running in
twisted pairs with a recurrent helix period of B150–200 nm
(Fig. 2B and C). These nanostructures were conﬁrmed by
AFM on dried gel samples (Fig. 3A–D). In particular, a helix
period of B150–200 nm is compatible with the AFM linescan
of DFFV (line drawn in Fig. 3B and analysis in Fig. 3C) and is
shown in the image in Fig. 3D. Occasionally, multiple DFFV
ﬁlaments could also twist into coiled ﬁbrils (see ESIw). In further
experiments, the precursor solutions were dried onto a silicon wafer
before gelation without sonication, in an attempt to gain insights
into the initial phases of self-assembly. Interestingly, DVFF yielded
similar structures to those observed after sonication (compare
Fig. 3A and E), while DFFV formed short nanorods which
suggest partial, short order alignment (Fig. 3F). It is possible
the natural peptides (see ESIw), in agreement with Cryo-TEM.
It is worth noting how the diﬀerent behaviour (self-assembling
or not) and morphology (i.e. tapes versus twisted ﬁbrils) results
from very subtle
to tripeptide
composition.
(A) CryoTEM of DVFF hydrogel. Black arrowheads: wide
nanotapes. White arrowheads: parallel double ﬁlaments. (B) CryoTEM
of DFFV hydrogel. Black arrowheads: helix period, more clearly visible
in the higher magniﬁcation image C. Scale bar = 200 nm.
Thioﬂavin T ﬂuorescence is a well-known method to assess
b-sheet structures, although it is only recently that a binding
mode has been proposed suggesting that the dye binds to an
aromatic–hydrophobic groove spanning across at least four
consecutive b-strands.7 Confocal
imaging (Fig. 4) of our
samples treated with Thioﬂavin T revealed ﬁbers of several
mms in length for both DVFF and DFFV, with the latter
yielding a denser network, as expected from our data. In
contrast, the lack of ﬂuorescence for the two natural analogues
suggests they are devoid of extended b-sheets (not shown).
CD was used to better assess the nature of the secondary
structure of the peptide self-assembled materials (Fig. 5).
Surprisingly, the CD spectra of all four peptides showed an
evident maximum centered at 225–230 nm, indicative of p–p
stacking of the aromatic phenylalanine residues. It is plausible
that this is due to the formation of aggregates for the two
L-peptides, as their limited solubility led to slow precipitation.
It is worth noting that in the case of the self-assembling
peptide DFFV, the intensity of the maximum was signiﬁcantly
reduced, due to the presence of the D-aromatic amino acid.
More importantly, as expected only the two self-assembling
structures containing non-natural amino acids displayed the
b-sheet minimum in the region 210–220 nm (Fig. 5, bottom).
The DFFV self-assembled peptide was the only one showing a
broad positive signal in the near-UV region (250–270 nm),
which can be attributed to phenylalanine residues with tertiary
structure. The b-sheet nature of the self-assembling peptides
Fig. 3 AFM images of dried gels of DVFF (A) and DFFV (B) obtained
after sonication; an AFM linescan (C) and a detailed view (D) of a helix
period of B150–200 nm for DFFV. Precursor solutions which were not
sonicated and were dried before gelation display similar structures for
DVFF (E) but only short nanorods for DFFV (F).
Fig. 5 CD spectra of the four tripeptides: each sample displays p–p
stacking (maximum at 225–230 nm); only the two self-assembling hydrogels
(bottom) display the b-sheet minimum in the region 210–220 nm.
2196Chem.Commun.,2012,48,2195–2197ThisjournaliscTheRoyalSocietyofChemistry2012Published on 24 November 2011. Downloaded by MIT Library on 3/21/2024 2:24:04 PM. View Article Online(Cryo-TEM, AFM and confocal) and spectroscopic (CD, FT-IR)
techniques. In addition, these gels are promising candidates
for the development of biomaterials as they form at physiolo-
gical pH without the aid of organic solvents, or of synthetic
capping end-groups, and the display of a D-amino acid at their
N-terminus may be a convenient feature to extend their
resistance to protease degradation.3 Investigation of the struc-
tural properties of a series of analogues is currently underway in
our laboratories to further elucidate how the chiral features
described in this study inﬂuence intermolecular interactions
and translate into the observed dramatic eﬀects on the supra-
molecular structures. It can be envisaged that chirality will be a
key tool to control self-assembly behavior and also to induce
designed morphologies in nanostructured materials.
was also conﬁrmed by FT-IR spectral analysis of the amide
I–II regions (Fig. 6). The natural peptide VFF revealed a
mixture of secondary structures,
including a predominant
maximum at 1610–1620 cm(cid:2)1 that can be attributed to antiparallel
aggregates, and which is absent or barely visible in all the other
samples. In comparison, the spectrum of FFV is very diﬀerent,
with a broad maximum centered at 1640 cm(cid:2)1 ascribed mainly
to random coils, and a dominant TFA peak in the region
1670–1680 cm(cid:2)1 that makes it diﬃcult to assess the presence of
b-structures. Nevertheless, both VFF and FFV are likely devoid
of extended b-sheets with supramolecular order, as suggested by
CD analysis and lack of binding and ﬂuorescence of Thioﬂavin T.
TFA presence was apparent also in the spectra of the two
non-natural self-assembling peptides, however, in this case
the antiparallel b-sheet signature was evident at least for
DFFV (1637 and 1680–1690 cm(cid:2)1).
Moreover, DVFF and DFFV both displayed a broad signal
in the N–H amide II region, shifted to lower frequencies due to
extended hydrogen bonding, indicative once again of b-sheets.
More speciﬁcally, the signal at 1570 cm(cid:2)1 suggests COO(cid:2)/
+ head-to-tail interactions in the self-assembling tripeptides,
as previously observed for the gel-forming Ile-Phe but not for
the non-self-assembling Val-Phe.4d It is interesting to note that
a maximum centered at 1523 cm(cid:2)1 was present in the spectrum
of VFF, but not FFV. It appears that the overall lack of
supramolecular order for the two natural peptides can be
ascribed to the co-existence of a number of secondary structures
for VFF, and their absence for FFV, which appears to be
predominantly random coil.
this study reports two examples where
chirality acts as a key tool for the self-assembly of short
peptides. This eﬀect is not limited to isolated nanostructures,
but extends to the formation of macroscopic self-supporting
hydrogels, as conﬁrmed by rheology and a variety of microscopic

[Assistant] [{"peptide_sequence": "vFF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "6.7", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "fFV", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "6.7", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "VFF", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "6.7", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "FFV", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "6.7", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
The self-assembly of molecular building blocks into nano- and micro-scale
supramolecular architectures has opened up new frontiers in polymer science.
Such supramolecular species not only possess a rich set of dynamic features as a
consequence of the non-covalent nature of their core interactions, but also
afford unique structural characteristics. Although much is now known about the
manner in which such structures adopt their morphologies and size distributions
in response to external stimuli, the kinetic and thermodynamic driving forces
that lead to their transformation from soluble monomeric species into ordered
supramolecular entities have remained elusive. Here we focus on
Boc-diphenylalanine, an archetypical example of a peptide with a high propensity
towards supramolecular self-organization, and describe the pathway through which
it forms a range of nano-assemblies with different structural characteristics.
Our results reveal that the nucleation process is multi-step in nature and
proceeds by Ostwald's step rule through which coalescence of soluble monomers
leads to the formation of nanospheres, which then undergo ripening and
structural conversions to form the final supramolecular assemblies. We
characterize the structures and thermodynamics of the different phases involved
in this process and reveal the intricate nature of the transitions that can
occur between discrete structural states of this class of supramolecular
polymers.

Phase behaviour. N-(t-butoxycarbonyl)-L-Phe-L-Phe-COOH (Boc-FF, Bachem)
stock solutions were prepared in absolute ethanol and then diluted in the required
ratios in double distilled water, forming an array of peptide concentrations and
solvent compositions. The structural morphologies of the resulting species were
then determined by bright ﬁeld microscopy and SEM observations. In all following
experiments, Boc-FF stock solution in ethanol was diluted to form 10% (v/v)
ethanol in water solution and phase behaviour was monitored by the following
Isolation of different phases. Suspensions of Boc-FF spheres were prepared by
mixing of 1 part in 10 by volume stock solution of its monomers into ddH2O
followed by gentle mixing. The tubular phase was isolated by the vigorous agitation
of this suspension or by allowing this solution to stand for 1 h. The ﬁbrillar phase
was isolated by centrifugation of suspensions of spheres at 4 (cid:2)C and the separation
of the supernatant. The supernatant was stored at 4 (cid:2)C for 15–20 min and was then
ﬂash frozen with liquid nitrogen and dried under reduced pressure.
ultraviolet–visible spectroscopy. Ten-millilitre aliquots of a suspension of Boc-
FF were prepared as described above and allowed to stand in a syringe. At 2-min
intervals, the suspension was gently agitated and 1.5 ml was pushed through a
0.22 mm syringe ﬁlter (Millex Ultrapore, PVDF membrane). The concentration of
Boc-FF in these aliquots was determined from the difference in speciﬁc absorbance
at 257.6 nm and a baseline at 300 nm using Cary-Varian 400 spectrophotometer
running WinUV software. The spectral bandwidth was 1.0 nm.
Dynamic light scattering. One millilitre suspensions of Boc-FF were prepared as
described above, and 100 ml aliquots were introduced into a low volume DLS cell.
The refractive index was estimated to be 1.450 and the absorbance to be 0.001. The
dispersant viscosity was taken to be 1.538 cP, and the dispersant refractive index as
1.339. Size measurements were made at 20 (cid:2)C using a Malvern Zetasizer Nano ZSP
(Malvern Instruments, Malvern, UK). z-potential measurements were performed in
a Malvern disposable folded capillary cell the same parameters as above and with
the additional speciﬁcation of a dispersant dielectric constant at Er ¼ 69.4. Mea-
surements were averaged over the ﬁrst 5 min after sample preparation.
constant temperature of 10–20 (cid:2)C. The reference cell was ﬁlled with 10% by volume
of an ethanol solution in water, and the sample cell was ﬁlled with a sample of
Boc-FF spheres, prepared immediately prior to its introduction into the instrument
by adding 10% by volume of a 7.5 g l (cid:2) 1 Boc-FF stock solution in ethanol into
water. For experiments conducted below room temperature, the water was pre-
cooled and the DSC instrument was pre-set to a temperature of 4 (cid:2)C below that at
which the experiment was to be performed, to minimize the time between sample
introduction and the beginning of each measurement. Baseline measurements were
subtracted from the reaction measurement, and the area of the exothermic peak
integrated to yield the enthalpy of transition.
Microdroplet formation. Microﬂuidic channels were fabricated in poly-
dimethylsiloxane (Dow Corning, Midland, MI, USA) using SU8 on silicon masters
and standard soft lithography techniques, and then plasma bonded to glass slides to
create sealed devices. Boc-FF stock solutions in ethanol were mixed on chip to form
a solution of 10% (v/v) ethanol in water, and microdroplets were formed in an oil
phase of Fluorinert FC-40 (Sigma-Aldrich, Gillingham, UK) and 2.0% (ww) block-
copolymer surfactant. Syringe pumps (Cetoni GmbH, Korbussen, Germany) were
used to control the ﬂow of ﬂuids, and the microdroplets were immediately inserted
into rectangular glass capillaries (CM Scientiﬁc, Silsden, UK) sealed and then
observed under bright ﬁeld microscopy as a function of time.
High-resolution scanning electron microscopy. Samples of Boc-FF solutions at
1:10 ethanol/water ratios at various time points after the initiation of self-assembly
were placed on glass slides and were left to dry at room temperature. Samples were
then coated with Cr and viewed using a JSM-6700 ﬁeld-emission HR-SEM (Jeol,
Tokyo, Japan), equipped with a cold ﬁeld emission gun, operating at 1 kV.
Nuclear magnetic resonance spectroscopy. NMR data were acquired using a
BRUKER Avance 700 Ultrashield spectrometer equipped with TXI Cryoprobe.
Samples of the Boc-FF peptide were dissolved in 100% EtOD and then diluted to
form solutions of 10% (v/v) ethanol in H2O. One-dimensional 1H spectra were
rapidly acquired at 25 (cid:2)C using 180 water-selective pulses to suppress resonances
from H2O. NMR data were processed and analyzed using TOPSPIN.
X-ray powder diffraction. Samples of the spheres, gels and tubes were prepared as
above and mechanically ground into ﬁne powders. The resulting samples were
placed in a ﬂat glass sample holder and XRPD data was collected on a laboratory
Philips X’Pert Pro diffractometer using Bragg-Brentano geometry. X-rays were
generated using Ni-ﬁltered Cu Ka1 radiation (l ¼ 1.54056 Å) generated at 40 kV
and 40 mA at 298 K and a scan range of 3–60(cid:2) 2y and a step size of 0.033(cid:2) 2y.
Continuous scans were done using the gonio scan axis, and data were analyzed
using X’Pert HighScore Plus. The nano-scale ultrastructure of the spheres, ﬁbrils
and tubes was retained in the ﬁne powders as conﬁrmed by electron microscopy.

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[System] polypeptide self-assembly literature information extraction

[User]
Formation of fibril-type nanostructures of the Alzheimer's beta-amyloid
diphenylalanine (l-Phe-l-Phe, FF) at the organic-aqueous interface and the
factors affecting their structures have been investigated. Such nanostructures
are also formed by bovine serum albumin and bovine pancreas insulin. The
concentration of the precursor taken in the aqueous layer plays an important
role in determining the morphology of the nanostructures. The addition of
curcumin to the organic layer changes the structure of the self-assembled
one-dimensional aggregates of diphenylalanine. By coating the diphenylalanine
dipeptide fibrils with appropriate precursors followed by calcination in air, it
has been possible to obtain one-dimensional nanostructures of inorganic
materials.

Diphenylalanine1Dﬁbrilswerepreparedattheorganic-aqueous
interface as follows. A total of 10 mg of diphenylalanine
(phenylalanine-phenylalanine, NH2-L-Phe-L-Phe-COOH; Bachem,
Switzerland) was dissolved in 2 mL of water at 50 °C in a 5 mL
beaker under sonication, and 2 mL of toluene was added to
form an organic layer above the water layer. After 10 min, a
thin white ﬁlm formed at the interface. The ﬁlm was lifted onto
a Si(111) or quartz substrate for characterization. To obtain
longer and more dense 1D nanostructures of diphenylalanine,
aggregation processes were continued for longer durations (30,
50, 80, and 150 min), keeping the other conditions the same.
Films were also prepared without sonication of the water layer
before the addition of toluene. The effect of the concentration
of the precursor dipeptide in the water layer was examined by
decreasing its concentration to 0.5 mg/mL, keeping the other
reaction parameters the same. The effect of curcumin on the
formation of 1D ﬁbrils was examined by adding 2 mL of a 10-6
M solution of curcumin in toluene, keeping the other parameters
the same. Considering the volume of the organic layer (2 mL),
the concentration works out to be 10-6 M, while the dipeptide
concentration is 0.016 M in the water layer (2 mL).
A total of 10 mg of bovine serum albumin (Sigma-Aldrich,
India) was dissolved in 2 mL of water in a 5 mL beaker, and 2
mL of toluene was added on the top of the water layer. After
20 h, a thin ﬁlm formed at the interface. The ﬁlm was lifted onto
Si(111) for characterization. The concentration of bovine albu-
min in the water layer was decreased to 1 mg/mL, keeping the
other parameters the same to examine the effect of dilution on
the structures at the interface. A total of 10 mg of bovine
pancreas insulin (Sigma-Aldrich, India) was dissolved in 2 mL
of an aqueous acidic solution (pH ) 2), and 2 mL of toluene
was added on top of the water layer in a sealed beaker, which
was kept at 40 °C. After 3 days, a thin ﬁlm formed at the
interface.
In order to examine the formation of 1D nanostructures of
gold by using diphenylalanine ﬁbrils as templates, 2 mg of a
diphenylalanine 1D nanostructure as a dried powder was
treated with HAuCl4 (20 µL, 20 mM aqueous solution) and
ascorbic acid (20 µL, 15 mM), followed by heating at 400 °C in
air for 2 h. To prepare a 1D nanostructure of platinum, H2PtCl6
was used in place of HAuCl4, keeping the other conditions the
same. To prepare a 1D nanostructure of SiO2, 2 mg of a
diphenylalanine 1D nanostructure as a dried powder was
treated with 10 µL of tetraethylorthosilicate (TEOS) followed by
heating at 400 °C in air for 2 h.
Characterization of the thin ﬁlms at the interface was carried
out by transferring them onto Si(111). A ﬁeld-emission scanning
electron microscope (FEI Nova-Nano SEM-600, The Nether-
lands) was used to look at their morphology. In the case of
peptide and amyloid nanostructures, ﬁlms were coated with
gold to render them conducting for FESEM studies. Energy-
dispersive X-ray analysis (EDAX) was carried out by using the
EDAX system attached to the FESEM. Transmission electron
microscopy (TEM) studies of the nanostructures of gold, plati-
num, and SiO2 were carried out with a JEOL JEM 3010 instru-
ment operating at an accelerating voltage of 300 kV. Powder
XRD patterns of nanostructures were recorded with a Philips
X’Pert diffractometer employing the Bragg-Brentano conﬁgu-
ration using Cu KR radiation. TGA of peptide nanostructures was
carried out with a Mettler Toledo thermal analyzer under a O2
atmosphere with a scan rate of 2 °C/min. UV-visible spectra
FIGURE 1. FESEM images of diphenylalanine 1D ﬁbrils obtained at
the toluene-water interface after (a) 10 min and (b) 150 min of the
aggregation process.

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