[System] polypeptide self-assembly literature information extraction

[User]
An N-terminally Boc (tert-butyloxycarbonyl) group-protected synthetic tripeptide
(Boc-Phe-Phe-Ala-OH) has been found to form a translucent hydrogel in basic
aqueous medium. This hydrogel material has been characterized using field
emission scanning electron microscopy (FE-SEM), transmission electron microscopy
(TEM), Fourier transformed infrared spectroscopy, differential scanning
calorimetric, X-ray diffraction (XRD), and rheological studies. FE-SEM and TEM
studies have revealed the formation of a nanofibrillar network structure upon
gelation. Thiol (-SH) containing ligands (amino acid/peptide) have been used to
stabilize small silver nanoparticles (AgNPs), and these thiol-capped silver
nanoparticles have been incorporated into this hydrogel to prepare hybrid
hydrogels. Morphological study of silver nanoparticles containing a hybrid
hydrogel (using TEM experiments) has indicated the nice fabrication of AgNPs
along the gel nanofibers. Fabrication of nanoparticles upon the gel nanofibers
is due to noncovalent interactions between the capping ligands of the
nanoparticles and the peptide-based hydrogel nanofibers. Rheological
investigations of these hybrid hydrogels have shown the weakening of the
mechanical strength of the hydrogel after incorporation of AgNPs within the
native hydrogel system. Our studies have vividly shown the dependence of the
elastic modulus (G') and yield stress (σ(y)) on three factors: (a) the nature of
the stabilizing ligands used for AgNPs, (b) the size of the AgNPs, and (c) the
amount of AgNPs used for the preparation of hybrid hydrogel systems. Modulation
of the mechanical strength of the hybrid hydrogel can be successfully achieved
by varying these above-mentioned factors. This modulation of the mechanical
properties keeps a future promise to make tunable soft materials with
interesting properties.

ABSTRACT: An N-terminally Boc (tert-butyloxycarbonyl)
group-protected synthetic tripeptide (Boc-Phe-Phe-Ala−OH)
has been found to form a translucent hydrogel
aqueous medium. This hydrogel material has been charac-
terized using ﬁeld emission scanning electron microscopy (FE-
transmission electron microscopy (TEM), Fourier
transformed infrared spectroscopy, diﬀerential
calorimetric, X-ray diﬀraction (XRD), and rheological studies.
FE-SEM and TEM studies have revealed the formation of a
nanoﬁbrillar network structure upon gelation. Thiol (-SH)
containing ligands (amino acid/peptide) have been used to
stabilize small silver nanoparticles (AgNPs), and these thiol-capped silver nanoparticles have been incorporated into this hydrogel
to prepare hybrid hydrogels. Morphological study of silver nanoparticles containing a hybrid hydrogel (using TEM experiments)
has indicated the nice fabrication of AgNPs along the gel nanoﬁbers. Fabrication of nanoparticles upon the gel nanoﬁbers is due
to noncovalent interactions between the capping ligands of the nanoparticles and the peptide-based hydrogel nanoﬁbers.
Rheological investigations of these hybrid hydrogels have shown the weakening of the mechanical strength of the hydrogel after
incorporation of AgNPs within the native hydrogel system. Our studies have vividly shown the dependence of the elastic
modulus (G′) and yield stress (σ
y) on three factors: (a) the nature of the stabilizing ligands used for AgNPs, (b) the size of the
AgNPs, and (c) the amount of AgNPs used for the preparation of hybrid hydrogel systems. Modulation of the mechanical
strength of the hybrid hydrogel can be successfully achieved by varying these above-mentioned factors. This modulation of the
mechanical properties keeps a future promise to make tunable soft materials with interesting properties.
Formation of supramolecular gels1−18 from small organic
molecules has attracted signiﬁcant research interest over the
past several years. Low molecular weight gelators are self-
assembled using diﬀerent kinds of noncovalent interactions
including π−π stacking, hydrogen bonding, hydrophobic, ionic,
van der Waals, and others to form gels.1−18 Hydrogel formation
has often been triggered by diﬀerent kinds of external stimuli,
such as enzymes,19,20 pH,21,22 solvents,23−25 metal ions,26,27 salt
concentrations,28,29 and others. Adams and co-workers have
recently developed a new technique to trigger the hydro-
gelation of several short peptides utilizing the hydrolysis of
glucono-δ-lactone by controlling the pH of
dium.30,31Hydrogel materials have been extensively studied
owing to their potent applications in vast areas,
including
sustained release of drugs and bioactive molecules,24,32−34
scaﬀolds for tissue growth,35,36 repair and wound healing,37
catalysis of organic reactions,38 pollutant
wastewater,39−41 and others. Hamley42−44and co-workers have
studied the self-assembly and hydrogelation of several model
short peptide sequences of the amyloidal β-peptide.
Construction of a nanohybrid system using gel nanoﬁbers
and inorganic nanomaterials has become an active area of
current research.45−59 Our research group has been involved in
the discovery and development of peptide-based supra-
molecular gels11,24,39,40,47,48,59,63,64 and preparation of
gel-based diﬀerent nanohybrid systems.11,47,48 Incorporation of
diﬀerent nanomaterials into the supramolecular gel matrix can
aﬀect and modulate the properties of the native gel.45−58 The
hybrid gel can exhibit physicochemical properties that are
interesting and diﬀerent from that of the native gel. There are
several reports on the modulation of the mechanical property of
gel phase materials by incorporating diﬀerent nanomaterials,
including nanoparticles, carbon nanotubes, graphene, metal
ions, and others.11,26−29,50−58
An increase in the inorganic salt concentration has also
triggered the tuning of the mechanical strength of gel phase
materials.26−29 Inclusion of metal ions (coming from inorganic
salts) generally screens the negative charge of carboxylate
groups of the corresponding gelator in gelation, and this assists
in the formation of a more rigid gel system.27 Previously,
Bhattacharya and co-workers examined the mechanical strength
of hybrid gels by incorporating diﬀerent ligand-capped gold
nanoparticles into the native organogel matrix.52,53 They
reported that incorporation of gold nanoparticles into the gel
matrix initially enhanced the rigidity of the hybrid gel. Then,
rigidity of the hybrid gel was diminished with a further increase
in the concentration of nanoparticles. According to them, the
organization of
the gel materials was perturbed in the
accommodation of an increased number of nanoparticles, and
as a consequence of that, gel rigidity was decreased.
Guerzo and co-workers51 have also demonstrated that the
stiﬀness of anthracene-based organogel has been gradually
decreased upon the incorporation of long-chain thiol-capped
gold nanoparticles. Han and co-workers showed that
incorporation of a graphene sheet into the copolymer-based
hydrogel remarkably diminished the elastic modulus (G′);
viscous modulus (G″); and ﬁnally, the strength of the gel.57
In the course of our investigation on self-assembly and
gelation of peptide based molecules,11,24,39,40,47,48,59,63 an N-
terminally protected tripeptide (Boc-Phe-Phe-Ala−OH) has
been discovered to form a translucent, self-supporting gel in a
basic aqueous medium. Moreover, silver nanoparticles contain-
ing hybrid hydrogels have been prepared by incorporating
diﬀerent cysteine-based-ligand-capped AgNPs into the native
hydrogel. Although the gel strength can be easily tunable by
varying the gelator concentration,65 in this study, we are curious
to investigate an alternative way to modulate the gel strength
without changing the concentration of the gelator. In this
regard, AgNP-containing hybrid gels have been prepared to
address the point, whether gel strength can be modulated by
the incorporation of AgNPs into the hybrid gel, and if yes, to
ﬁnd out the factors that are responsible for governing the
modulation of the gel strength using rheological experiments.
Rheological investigations reveal that the mechanical strength
of the hybrid hydrogel depends on the nature of the capping
ligands of the silver nanoparticles, the size of the nanoparticles,
and the amount of nanoparticles added into the native gel.
Materials. L-Phenylalanine, L-alanine, L-cysteine, L-leucine,
formic acid (HCOOH), Boc anhydride, N,N′-dicyclohexylcar-
bodiimide (DCC), 1-hydroxybenzotriazole (HOBt), sodium
borohydride (NaBH4), silver nitrate (AgNO3), and triphenyl
methanol were purchased from Sigma Chemicals. All other
common solvents were purchased from SRL, India.
Gelator Peptide (Boc-Phe-Phe-Ala−OH). Boc-Phe-Phe-
Ala−OMe (6.39 g, 15.0 mmol) was dissolved in 20 mL of
MeOH, and then 10 mL of 1 M NaOH was added. The
reaction mixture was
then stirred, and the progress of
saponiﬁcation was monitored by thin layer chromatography.
After 10 h, the methanol was removed under vacuum, the
residue was placed in 50 mL of water and washed with diethyl
ether (2 × 50 mL). After that, the pH of the aqueous layer was
adjusted to pH 2 using 1 M HCl, and it was extracted with ethyl
acetate (3 × 50 mL). The ethyl acetate extract was pooled,
dried over anhydrous sodium sulfate, and evaporated in vacuum
to yield a white solid sample. Yield: 5.34 g (12.9 mmol, 86.0%).
1H NMR (300 MHz, DMSO-d6, 25 °C, δ): 12.38 (1H, br.),
8.19 (1H, d, J = 7.5), 7.82 (1H, d, J = 7.9), 7.17−6.98 (10H,
m), 6.77 (1H, d, J = 8.3), 4.49−4.47 (1H, m), 4.14−4.10 (1H,
m), 4.02−3.99 (1H, m), 2.96−2.93 (1H, m), 2.74−2.51 (2H,
m), 2.47−2.31 (1H, m), 1.16 (9H, s), 1.06−1.00 (3H, m). 13C
NMR (75 MHz, DMSO-d6, 25 °C, δ): 174.5, 172.0, 171.9,
171.5, 171.3, 155.8, 155.6, 138.6, 138.4, 138.1, 130.0, 129.7,
128.5, 128.4, 126.8, 126.7, 78.7, 78.5, 53.8, 48.0, 38.3, 38.1,
37.9, 28.6, 17.7. Anal. calcd. for C26H33N3O6 (483.2369): C,
64.58; H, 6.88; N, 8.69. Found C, 64.55; H, 6.89; N, 8.67.
HRMS (m/z): 506.3529 [M + Na]+, 507.3451 [M + Na + H]+.
Synthesis of Amino Acid/Dipeptide-Based Silver
Mercaptides. Dipeptides were synthesized by conventional
solution phase methodology by using the racemization-free
fragment condensation strategy (Scheme S1, Supporting
Information). The N-terminus was protected by the Boc
group, the C-terminus was protected as a methyl ester, and the
thiol (−SH) group was protected by the trityl group. Couplings
were mediated by DCC/HOBt. Deprotection of the methyl
ester was performed using the saponiﬁcation method,
deprotection of the Boc was performed by HCOOH, and
deprotection of the trityl group was performed by treatment
with AgNO3 in the presence of pyridine. All compounds were
characterized by mass spectrometry (HRMS) and 1H NMR
spectroscopy (300 MHz).
Hydrogel Preparation. A requisite amount of gelator
peptide was taken in a screw-capped glass vial. Aqueous NaOH
solution was then added into that glass vial. This resultant
mixture was heated until the gelator molecules were dissolved
completely, and this upon cooling at room temperature for 2
min gave a self-supported translucent hydrogel. This hydrogel is
stable over 15 days at room temperature without any visual
Field Emission Scanning Electron Microscopy (FE-
SEM). FE-SEM experiments were performed by placing a small
portion of gel samples on a microscope cover glass. These
samples were then dried ﬁrst in air and then in vacuum and
coated with platinum for 90 s at 10 kV voltages and 10 μA
current. The thickness of the coating layer of platinum was 3−4
nm. After that, micrographs were taken by using a SEM
apparatus, Jeol scanning microscope JSM-6700F.
Transmission Electron Microscopy (TEM). Morpholo-
gies of native and hybrid hydrogels were studied using TEM
experiments. A drop of dilute solution of the gel-phase material
of the corresponding gel was placed on carbon coated copper
grids (300 mesh) and dried by slow evaporation. Each grid was
then allowed to dry in a vacuum at 30 °C for two days. Images
were taken with an FEI (Tecnai Spirit) instrument.
Congo Red Binding Study. A 2.0% (w/v) hydrogel was
prepared, and Congo red solution (1 mmol) was added into the
gel vial. Congo red-stained hydrogel was then prepared by a
heating−cooling cycle. This hydrogel was allowed to age for 1
day, and a small piece of the gel was taken out in a spatula, and
this gel was placed on a glass side upon shearing. Excess dye
was removed by washing using distilled water, then the sample
was dried in vacuum at room temperature for 24 h. The dried
X-ray Diﬀraction Study. Hydrogel was prepared at a
concentration of 3.0% (w/v), and it was then subjected to
lyophilization to obtain the xerogel sample. X-ray diﬀraction
study of this xerogel was carried out by placing the sample on a
glass plate. The experiment was carried out by using an X-ray
diﬀractometer (Bruker AXS, model no. D8 Advance). The
instrument was operated at a 40 kV voltage and 40 mA current
using Ni-ﬁltered Cu Kα radiation, and the instrument was
calibrated with a standard Al2O3 (corundum) sample before
use. For 5−30° scans, the LynxEye super speed detector was
used with scan speed 0.3 s and step size 0.02°.
Diﬀerential Scanning Calorimetry (DSC). Thermal
behavior of the gel was studied using a Perkin-Elmer diﬀerential
scanning calorimeter (Diamond DSC-7) working under N2
atmosphere. The gel sample was taken in a large volume
capsule (LVC) tightly sealed with an O-ring. It was then heated
from 20 to 90 °C at a heating rate of 5 °C/min. A cooling run
was also taken after waiting 10 min at 90 °C, and then the
sample was cooled to 20 °C at a rate of 2 °C/min. The
instrument was calibrated with indium and cyclohexane.
FT-IR Study. FT-IR spectra were recorded using a Nicolate
380 FT-IR spectrophotometer (Thermo Scientiﬁc). The FT-IR
spectrum of the gelator in solid and xerogel state (obtained
from 3.0% (w/v) hydrogel) were recorded using the KBr disk
technique. Xerogel was dissolved in D2O to get the hydrogel
(3.0%, w/v), and this wet gel was used for the FT-IR study
using a CaF2 cell.
Rheology. Rheological experiments were performed with an
AR 2000 advanced rheometer (TA Instruments) using cone
plate geometry in a Peltier plate. The cone diameter was 40
mm, the cone angle was 1° 59′ 50″, and truncation was 56 μm.
NMR Experiment. All NMR studies were carried out on a
Brüker DPX 300 MHz spectrometer at 300 K using a cryo
probe in CDCl3 and DMSO-d6 maintaining a concentration of

[Assistant] [{"peptide_sequence": "FFA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Boc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "14.3", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "2.0"}, {"peptide_sequence": "FFA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Boc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "15.6", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "2.0"}, {"peptide_sequence": "FFA", "self-assembly_phase": "none", "N-terminal_modification": "Boc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "11.7", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "2.0"}, {"peptide_sequence": "FFA", "self-assembly_phase": "none", "N-terminal_modification": "Boc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PB", "peptide_concentration(mg/ml)": "10.4", "PH": "7.46", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "2.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
Peptide nanostructures compose a new class of materials that have gained
attention due to their interesting properties. Among them, nanotubes of
diphenylalanine (FF) and its analogues have been one of the most studied
structures in the last few years. Their importance originates from the need to
better understand the formation of β-amyloid fibrils which are associated with
Alzheimer's disease. In this work, the FF self-assembly process was probed using
time-resolved Raman microscopy. The changes in the Raman spectra are followed
over time after injecting water into a FF-film until micro/nanotubes (MNTs) are
formed. Specific features of the Raman spectra clearly suggest that FF-molecules
after water injection form an intermediate species before forming FF-MNTs. The
broad Raman bands observed for the intermediate species suggest the presence of
very heterogeneous structures based on FF. The FF-MNTs appear almost
instantaneously (detected via the rise of the typical Raman bands of FF-MNTs at
761, 1249 and 1426 cm-1) after the intermediate structures are formed. This
delayed formation of FF-MNTs supports a nucleation process. The formation via
nucleation of FF-MNTs is further corroborated by a simulation of the Raman
spectra based on a 2-step kinetic model and the respective vibrational Raman
modes are identified using Density Functional Theory vibrational calculations.
Our results indicate that the driving force for the FF-MNT patterning process is
the electric dipole re-orientation originating from the FF dipeptide unit
connectivity over time.

The lyophilized form of L-diphenylalanine (FF) was purchased
from Bachem and the 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)
solvent from Sigma Aldrich. A FF:HFIP solution was prepared
(100 mg ml(cid:2)1 concentration). Then, an aliquot of 2 ml of this
solution was placed onto a glass slide. Due to the solvent
evaporation under ambient conditions (typically T = 20 1C
and relative humidity of about 20%) a transparent amorphous
film was obtained in a few seconds. A 0.3 ml aliquot of deionized
water was deposited over the film in order to start the MNT
formation. The Raman triggering of the process started (t = 0 s
in all measurements) at this point. To ensure the reproducibility of
the experiments a micro-syringe (62RNR, Hamilton) was installed
on a xyz-stage to control the position of the water drop relative to
the laser spot position at the sample (see ESI,† Fig. S1). In order to
investigate the formation of MNTs, Raman measurements were
performed using a confocal micro-Raman spectrometer (XploRA
PLUS, Horiba) with an excitation wavelength of 532 nm and a
40(cid:3) air microscope objective (N.A. 0.60, LUCPlanFLN, Olympus).
The spectrometer was equipped with a diﬀraction grating of
2400 gr mm(cid:2)1 which provides a spectral resolution of 1 cm(cid:2)1
per pixel. The spectral acquisition was started before dropping
water and finished after the complete formation of the tubes,
totaling typically 20–100 spectra for each measurement set. The
integration time was set to 1 s per spectrum and the laser power at
the sample was about 5 mW. The repeatability of the process was
checked and the precision was about 0.5 s.
Computer simulations (vibrational modes, molecular electrostatic
potential and electric dipole) on FF peptides [1FF, 2FF,. . ., 6FF],
hydrated FF monomers [FF:1H2O; FF:2H2O; FF:3H2O] and water
cluster [(H2O)n, n = 1–6] structures were performed. In the ESI,†
detailed pieces of information concerning these calculations are
reported.

[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": "100 HFP", "peptide_concentration(mg/ml)": "100.0", "PH": "7.0", "temperature(celsius)": "20.0", "heating_cooling": "No", "incubation_period(min)": "0.1"}


[System] polypeptide self-assembly literature information extraction

[User]
Peptide-based vesicular structures have been the focus of research in the past
decade for their potential application as drug delivery agents. We here report
the self-assembly of amphiphilic dipeptides containing conformation-constraining
alpha,beta-dehydrophenylalanine into nanovesicles. The vesicles can encapsulate
small drug molecules such as riboflavin and vitamin B(12), bioactive peptides,
and small protein molecules. The nanovesicles are resistant to treatment of a
nonspecific protease, proteinase K, and are stable at low concentrations of
monovalent and divalent cations. The vesicles are effectively taken up by
actively growing cells in culture and show no observable cytopathic effects.
These peptide-based nanostructures can be considered as models for further
development as delivery agents for different biomolecules.

Nanovesicles Based on Self-Assembly of Conformationally Constrained
Aromatic Residue Containing Amphiphilic Dipeptides
Peptide-based vesicular structures have been the focus of research in the past decade for their potential application
as drug delivery agents. We here report the self-assembly of amphiphilic dipeptides containing conformation-constraining
R,(cid:226)-dehydrophenylalanine into nanovesicles. The vesicles can encapsulate small drug molecules such as riboflavin
and vitamin B12, bioactive peptides, and small protein molecules. The nanovesicles are resistant to treatment of a
nonspecific protease, proteinase K, and are stable at low concentrations of monovalent and divalent cations. The
vesicles are effectively taken up by actively growing cells in culture and show no observable cytopathic effects. These
peptide-based nanostructures can be considered as models for further development as delivery agents for different
biomolecules.
Introduction
Self-assembly of soft materials into nanostructures offers
exciting applications. “Peptosomes”,1-3 vesicles formed by the
self-assembly of peptides, offer superior stability in addition to
tailorability of physical, chemical, and biological properties along
with unmatched biocompatibility.4 Encapsulation, controlled
release, and biological stability of bioactive compounds are some
of the potential application areas of vesicular structures.5 Specific
biomedical applications involving actuator functions for tem-
perature, pH, and salt-triggered release as well as incorporation
of adhesion and recognition sites in the assembled structures can
be better met with potent designs of peptosomes.5-7 Recent studies
have demonstrated that spontaneous assemblies of peptide
molecules into vesicles not only form at an appropriate
concentration of the amphiphilic molecule, but can also be induced
by incorporation of conformational rigidity, resulting in restricted
conformational entropy.5,8-13 Medium-sized and short peptide
sequences have been shown to assemble into vesicles in aqueous
medium; however, the recent and elegant demonstration of a
dipeptide assembling into stable nanovesicular structures that
are stable in acidic and alkaline conditions is noteworthy.2
H-Lys-¢Phe-OH (hereafter denoted as Glu-¢Phe and Lys-¢Phe,
respectively) which self-assembled into anionic and cationic
vesicular structures, respectively. Our design was based on
previous observations that (i) peptides with a hydrophobic tail
and a charged amino acid at one end self-assembled into vesicles
and tubes,4 (ii) aromatic residue containing peptides as small as
dipeptides could also self-assemble2,10 into nanotubes and
nanovesicles, and (iii) incorporation of the ¢Phe residue, the
unsaturated analogue of phenylalanine, with a double bond
between the CR and C(cid:226) atoms provides structural14-16 and
proteolytic stability to the self-assembled structures.17 We found
that the vesicles formed by the amphiphilic dipeptides Glu-¢Phe
and Lys-¢Phe exhibited stability to nonspecific protease treat-
ment, were able to encapsulate bioactive molecules, were not
cytotoxic, and were easily taken up by cells in culture. The novel
self-assembled nanostructures could thus have potential ap-
plications as vehicles for delivery of biologically relevant
molecules.
H-Glu-¢Phe-OH. Boc-Glu(OtBu)-OH (Novabiochem) (1.52 g,
5 mM) was dissolved in dry tetrahydrofuran (Sigma-Aldrich) and
the resulting solution stirred in an ice-salt bath at -15 (cid:176) C.
N-Methylmorpholine (Sigma) (0.65 mL, 5 mM) was added to the
solution followed by isobutyl chloroformate (Sigma) (0.7 mL, 5
mM). After 10 min, a precooled aqueous solution of dl-threo-(cid:226)-
phenylserine (Sigma-Aldrich) (1 g, 5.5 mM) and sodium hydroxide
(0.22 g, 5.5 mM) was added and the resulting mixture stirred overnight
at room temperature. The reaction mixture was concentrated in vacuo,
acidified with citric acid to pH 3.0, and extracted with ethyl acetate
(Spectrochem) (3 (cid:2) 20 mL). The ethyl acetate layer was washed
with water (2 (cid:2) 15 mL) and with saturated sodium chloride (1 (cid:2)
20 mL), dried over anhydrous sodium sulfate, and evaporated to
yield Boc-Glu(OtBu)-DL-threo-(cid:226)-phenylserine as an oily compound
(2.3 g, (cid:24)100%). The compound, Boc-Glu(OtBu)-DL-threo-(cid:226)-
phenylserine, was then mixed with anhydrous sodium acetate (0.53
g, 6.5 mM) in freshly distilled acetic anhydride (50 mL) and the
resulting mixture stirred for 36 h at room temperature. The thick
slurry obtained was poured over crushed ice and stirred till the oily
suspension gave rise to a yellow-colored solid. The precipitate was
filtered, washed with 5% NaHCO3 and cold water, and dried under
vacuum. The resulting azalactone, Boc-Glu(OtBu)-¢Phe-Azl, was
dissolved in methanol and treated with 1.5 equiv of a 1 N NaOH
solution and the resulting mixture stirred at room temperature for
3-4 h. The mixture was then partially evaporated to remove methanol,
acidified with citric acid to pH 3.0, and extracted with ethyl acetate
(3 (cid:2) 30 mL). The combined ethyl acetate extract was washed with
water (2 (cid:2) 20 mL), dried over anhydrous sodium sulfate, and
evaporated to yield Boc-Glu(OtBu)-¢Phe-OH as a white solid.
Deprotection at the R-amino group and side chain protection were
achieved by treatment with 98% formic acid (30 mL) for 3 h at room
temperature. The reaction mixture was evaporated to dryness, and
the residue was precipitated with anhydrous diethyl ether. The
resulting precipitate was filtered, washed several times with dry
ether, and subsequently lyophilized from 10% acetic acid-water
(20 mL) to yield the final compound H-Glu-¢Phe-OH as a white
powder, overall yield 0.6 g (48%), Rf ) 0.15 (CHCl3-MeOH, 9:1).
The peptide was purified on a preparative reversed-phase C18
column (Deltapak, C18, 15 (cid:237)m, i.d. 300 (cid:2) 19 mm) using an
acetonitrile-water linear gradient of 5-45% acetonitrile (0.1% TFA)/
water (0.1% TFA) at a flow rate of 4 mL/min over 25 min. The
purified peptide was reinjected into an analytical reversed-phase C18
column (Phenomenex, C18, 5 (cid:237)m, i.d. 250 (cid:2) 4.6 mm) using a an
acetonitrile-water linear gradient of 5-45% acetonitrile (0.1% TFA)/
water (0.1% TFA) at a flow rate of 1 mL/min over 25 min and was
found to be 98% pure with a retention time of 13 min. The purified
peptide was analyzed by mass spectrometry (Applied Biosystems
QStar (Q-TOF)), observed mass 293 Da, expected mass 292.3 Da.
NH2-Lys-¢Phe-COOH. The peptide was synthesized as described
above starting with Boc-Lys(Boc)-OH, overall yield 0.57 g (39%),
Rf ) 0.1 (CHCl3-MeOH, 9:1). The peptide was purified as described
above and analyzed by mass spectrometry, retention time 15 min,
observed mass 292 Da, expected mass 291.38 Da.
Circular Dichroism (CD) Spectroscopy. All spectra were
recorded on a JASCO-810 polarimeter equipped with a Peltier-type
thermostat and purged continuously with dry N2 gas at 10 L/min
(LPM) during data acquisition. Data were collected in a quartz cuvette
with a path length of 0.1 or 1 cm between 190 and 360 nm at a scan
speed of 25 nm/min and a response time of 16 s. The average of
50 scans was used for analysis of the spectrum. The titration studies
were carried out at a peptide concentration between 1 and 10 mM.
The concentration of the peptide was estimated by UV spectroscopy
with (cid:15)205 ) 5167 per peptide bond.18 CD studies were also carried
out at different temperatures and in the presence of monovalent and
divalent cations.
Fourier Transform Infrared (FTIR) Spectroscopy. Spectra
were collected on a Perkin-Elmer Spectrum BX FTIR spectrometer.
The assembled peptide samples were spotted on a CaF2 window and
air-dried at 99% relative humidity. The samples were then rehydrated
with D2O, and hydrogen-deuterium exchange was monitored every
minute for 10 min in the spectral range of 1400-1900 cm-1. The
FTIR spectra were smoothed with a smoothing length of 20 units.
Two-dimensional correlation spectroscopy (2D COS) maps were
generated from the stack of spectra using the freely available software
package 2D-Shige. All spectral assignments were done according
to published reports.19-22
Electron Microscopy. For transmission electron microscopy
(TEM), the peptide samples were prepared at a concentration of
1-10 mg/mL in doubly distilled water and incubated for 24 h to
make sure that the process of assembly was complete. The sample
was adsorbed on a 400 mesh copper grid with a carbon-coated
Formvar support, stained with 1% uranyl acetate, and viewed under
the 120 kV mode of a transmission electron microscope (Tecnai 12
BioTWIN, FEI Netherlands). Photomicrographs were digitally
recorded using a Megaview II (SIS, Germany) digital camera. Image
analysis was carried using Analysis II (Megaview, SIS, Germany)
and ImageJ (http://rsb.info.nih.gov/ij/) software packages.
Proteolytic Stability Assays. The proteolytic stability of the
peptide vesicles was assayed using reversed-phase HPLC. The peptide
vesicles were incubated with a nonspecific protease, proteinase K,
for 24 h. The samples were then injected into a reversed-phase C18
column (Phenomenex, C18, 5 (cid:237)m, i.d. 250 (cid:2) 4.6 mm) using an
acetonitrile-water linear gradient of 5-45% acetonitrile (0.1% TFA)/
water (0.1% TFA) at a flow rate of 1 mL/min over 25 min. A decrease
in the peak area of the native peptide was used as an indicator of
proteolytic degradation.
Encapsulation Studies. The relevant bioactive molecules were
mixed with preformed vesicles in equal weight ratios and subjected
to sonication (Branson 200, 19 W) for 2-5 min or aspirated through
a pipet. The sample was then ultracentrifuged at 120 000 rpm for
5 h at 10 (cid:176) C in a Beckman Coulter OptiMax MLA-130 rotor. A
small volume was removed from the supernatant, and the concentra-
tion of the free test molecules was estimated spectrophotometrically.
The percentage encapsulation was calculated using the following
formula: encapsulation (%) ) (absorbance of bound molecules/
absorbance of total molecules) (cid:2) 100. To visualize protein
encapsulation in the nanovesicles, the proteins were tagged to 20
nm gold particles (Ted Pella) by adsorption. After overnight
incubation of 1 mg/mL protein with 50 (cid:237)L of nanogold, the gold
nanoparticles with adsorbed proteins were washed three times with
buffer/water with repeated centrifugation. The conjugates were then
mixed with the nanovesicles in a manner similar to that for
encapsulation of small molecules (described above). The samples
were then imaged under a Phillips technai 120k.

[Assistant] [{"peptide_sequence": "EF", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "EF", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "2.8", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "EF", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "4.6", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "EF", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "6.4", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "EF", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "8.2", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "EF", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "10.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "2.8", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "4.6", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "6.4", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "8.2", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KF", "self-assembly_phase": "vesicle", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "Delta-F", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "5/95 HFP/H2O", "peptide_concentration(mg/ml)": "10.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
Further understanding of the interactions between nanoparticles (NPs) and
biological molecules offers new possibilities in the applications of
nanomedicine and nanodiagnostics. The properties of NPs, including size, shape,
and surface functionality, play a decisive role in these interactions. Herein,
we evaluated the influences of gold NPs (AuNPs) with different sizes (5-60 nm)
and shapes (i.e., spherical, rod, and cage) on the self-assembly of
diphenylalanine (Phe-Phe) dipeptides. We found that the size of AuNPs smaller
than 10 nm did not affect the self-assembly process of Phe-Phe, while bigger
AuNPs (>10 nm) caused the formation of starlike peptide morphologies connected
to one center. In the case of shape differences, nanorod and nanocage
morphologies acted differently than spherical ones and caused the formation of
densely packed, networklike dipeptide morphologies. In addition to these
experiments, by combining photothermal properties of AuNPs with a Phe-Phe-based
organogel having a thermo-responsive property, we demonstrated that the
degelation process of AuNPs embedded organogels may be controlled by laser
illumination. Complete degelation was achieved in about 10 min. We believe that
such control may open the door to new opportunities for a number of
applications, such as controlled release of drugs and tissue engineering.

Synthesis of Gold Nanoparticles. The 5 nm AuNPs were
prepared through the well-established reduction reaction.44 Brieﬂy, a
10 mL aqueous solution containing 2.5 × 10−4 M HAuCl4 and 2.5 ×
10−4 M trisodium citrate was ﬁrst prepared. While stirring, ice-cooled
0.3 mL of 0.1 M sodium borohydride was added to this solution. The
color change in the solution was an indication of
the particle
formation. The nanoparticles were used within 2−3 h.
The 10−60 nm gold nanoparticles were also synthesized as follows:
to 49 mL of deionized water was added 1 mL of 12.7 mM aqueous
HAuCl4 solution. The mixture was heated to boiling while stirring.
Afterward, diﬀerent amounts of 38.8 M trisodium citrate solution
(200−600 μL) were added to the boiling mixture. After cooling to
room temperature, the samples were centrifuged and washed with DI
water several times and dispersed in ethanol.
Preparation of Gold Nanorods. The Au nanorods (AuNRs)
were synthesized by a three-step seeding method described by Murphy
and co-workers.45 In the ﬁrst step, 0.5 mL of 0.01 M HAuCl4 was
added to 20 mL of 0.1 M cetyltrimethylammonium bromide (CTAB).
Next, 1.2 mL of ice-cold 0.1 M sodium borohydride was added to the
mixture all at once. The solution was vigorously stirred at 25 °C for 5
min. The brownish color indicates the seed formation. The solution
was then used for the synthesis of gold nanorods within 20 min of its
synthesis. In the second step, a 54 mL growth solution containing 10−4
M HAuCl4 with 0.1 M CTAB was prepared. Afterward, three ﬂasks
each containing 18 mL growth solution was labeled as I, II, or III. To
these ﬂasks were added 0.1 mL of 0.1 M ascorbic acid and stirred
gently. A 2.0 mL of seed solution was then mixed with ﬂask I. Next, 2.0
mL of solution I was added to ﬂask II. Finally, 2 mL of solution II was
mixed with solution III. The synthesized nanorods were centrifuged
and washed with water several times to remove excess CTAB.
Synthesis of Gold Nanocages. The Au nanocages (AuNCs)
were synthesized by the galvanic replacement reaction between silver
(Ag) nanocubes and chloroauric acid (HAuCl4). In the ﬁrst step, Ag
nanocubes were synthesized by a previously described protocol.46
Afterward, a ﬁxed amount of Ag nanocubes was dispersed in water
containing poly(vinylpyrrolidone) (PVP) in a ﬂask under magnetic
stirring and then heated to a boil for 10 min. A 3 mL 0.2 mM HAuCl4
aqueous solution was added to the ﬂask through a syringe pump under
magnetic stirring. The solution was heated for another 10 min until the
the system was stable. Once cooled down to room
temperature, the sample was centrifuged and washed with saturated
NaCl solution to remove AgCl and with water several times to remove
PVP and NaCl before characterization by TEM and UV−vis.
Self-Assembly of Dipeptides. 2.0 mg of lyophilized diphenyla-
lanine dipeptide (Phe−Phe; Bachem AG, Switzerland) was ﬁrst
dissolved in 0.9 mL of ethanol at 70 °C for 10 min and then cooled to
room temperature. To the peptide solution was then added 100 μL
aliquot amounts of AuNPs (i.e., spherical, rod, or cage) dispersed in
ethanol. After 3−4 h, each peptide−AuNP mixture (100 μL) was
placed onto a glass slide or silicon wafer surfaces and dried at ambient
temperature, until the solvent evaporated. The resultant dipeptide
structures were characterized by SEM.
Preparation of Organogels. For organogel fabrication, we used
Phe−Phe dipeptides, which have no protecting group or any end
functionalization, as a building block. Typical Phe−Phe organogels
with or without AuNPs were fabricated as follows: a 0.40 M stock
Phe−Phe solution was ﬁrst prepared in 1,1,1,3,3,3-hexaﬂuoro-2-
propanol (HFIP) and then diluted by AuNPs containing chloroform
(3.0 × 1010 to 5.6 × 1011 particles/mL) to give a ﬁnal concentration of
8.0 mM. We chose HFIP/chloroform solvent medium for
fabrication of organogels because our Phe−Phe dipeptides do not
have any side chain or end group to promote gelation in a water
medium. In general, HFIP is often used to dissolve Phe−Phe and then
initiate the molecular self-assembly in the organic phase (i.e., toluene
and chloroform). The AuNPs (∼20 nm) were synthesized, as
mentioned above. Subsequently, an organogel network having
AuNPs was rapidly formed. Plain organogels were also obtained in
the same manner without using AuNPs. Laser-triggered degelation of
AuNPs containing organogels was investigated by using a green laser
at a power density of 50 mW/cm2 with a wavelength of 532 nm.
Characterizations. All peptide morphologies and fabricated
organogels were characterized by a JEOL JSM-6060 scanning electron
microscope (SEM) with an acceleration voltage of 10 kV after the
samples were coated with a thin layer of gold. A Fourier transform
infrared (FTIR) spectrometer (Thermo Nicolet IR) operated under an
attenuated total reﬂection mode was also used to collect spectra of the
peptide samples. The IR spectra were recorded with a 4 cm−1
resolution. Thermogravimetric measurements of organogels with and
without AuNPs were also carried out using a SII 7300 Exstar thermal
analyzer. Samples were heated from room temperature to 350 °C at a
constant rate of 2 °C/min under N2

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[System] polypeptide self-assembly literature information extraction

[User]
Dipeptide derivative molecules can self-assemble into space-filling nanofiber
networks at low volume fractions (<1%), allowing the formation of molecular gels
with tunable mechanical properties. The self-assembly of dipeptide-based
molecules is reminiscent of pathological amyloid fibril formation by naturally
occurring polypeptides. Fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) is
the most widely studied such molecule, but the thermodynamic and kinetic
phenomena giving rise to Fmoc-FF gel formation remain poorly understood. We have
previously presented evidence that the gelation process is a first order phase
transition characterized by low energy barriers to nucleation, short induction
times, and rapid quasi-one-dimensional crystal growth, stemming from
solvent-solute interactions and highly specific molecular packing. Here, we
discuss the phase behavior of Fmoc-FF in different solvents. We find that
Fmoc-FF gel formation can be induced in apolar solvents, in addition to
previously established pathways in aqueous systems. We further show that in
certain solvent systems anisotropic crystals (nanofibers) are an initial
metastable state, after which macroscopic crystal aggregates with no preferred
axis of growth are formed. The molecular conformation is sensitive to solvent
composition during assembly, indicating that Fmoc-FF may be a simple model
system to study complex thermodynamic and kinetic phenomena involved in peptide
self-assembly.

Dipeptide derivative molecules can self-assemble into space-ﬁlling nanoﬁber networks at low volume
fractions (<1%), allowing the formation of molecular gels with tunable mechanical properties. The self-
assembly of dipeptide-based molecules is reminiscent of pathological amyloid ﬁbril formation by naturally
occurring polypeptides. Fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) is the most widely studied
such molecule, but the thermodynamic and kinetic phenomena giving rise to Fmoc-FF gel formation
remain poorly understood. We have previously presented evidence that the gelation process is a ﬁrst
order phase transition characterized by low energy barriers to nucleation, short induction times, and rapid
quasi-one-dimensional crystal growth, stemming from solvent–solute interactions and highly speciﬁc
molecular packing. Here, we discuss the phase behavior of Fmoc-FF in diﬀerent solvents. We ﬁnd that
Fmoc-FF gel formation can be induced in apolar solvents, in addition to previously established pathways
in aqueous systems. We further show that in certain solvent systems anisotropic crystals (nanoﬁbers) are
an initial metastable state, after which macroscopic crystal aggregates with no preferred axis of growth
are formed. The molecular conformation is sensitive to solvent composition during assembly, indicating
The phase behavior of mixtures containing self-organizing
molecules is a sustained challenge in molecular purifi-
cations and in the study of molecular structure. A number
of studies have shown that generalized phase diagrams can
be used to understand the general features of a binary
mixture of solute and solvent.1–3 In some systems, however,
it is not clear whether phase separation is determined pri-
marily by thermodynamics or kinetics.4–7 In the thermo-
dynamic limit, if solute molecules are assumed to experi-
ence a net attraction of ε, a phase diagram could be devel-
oped where kT/ε (experimentally characterized by measur-
ing solute secondary virial coeﬃcients and solute self-
diﬀusivity)8–12 is plotted as a function of solute volume
fraction (Fig. 1). These generalized phase diagrams have
been shown to be useful predictors of solubility of small
molecules, proteins and colloids, and, surprisingly, capture
solubilities even when the molecules have very anisotropic
interactions.10–12 The general nature of the proposed phase
diagram suggests that at the same value of ε/kT, diﬀerent
solutes will have the same solubility. Solvent–solute inter-
actions play a key role in establishing the magnitude of
ε/kT. These attractions are often broken down into polar,
dispersion and hydrogen bonding interactions. The relative
Self-assembling gelators based on short peptide derivative
molecules form ordered fibrous structures, commonly via π–π
stacking and hydrogen bonding interactions.13–22 The result-
ing bulk properties of the produced materials are characteristic
of soft solids. The most widely studied such gelator molecule
is fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF).13–21
Low-concentration solutions of Fmoc-FF form gels of substan-
tial elastic moduli when mixed with water. Because of their
flexibility for altering chemical composition, Fmoc-FF and
similar short peptide derivative gelators show promise for a
host of uses including tissue engineering, nanofabrication,
biosensing.23–27
However, the design of new gelator molecules with desired
self-assembly properties is limited by the lack of understand-
ing of the gelation mechanism. Hence, these low molecular
weight gelators are less often designed than they are
discovered.
We have previously shown that gelation of Fmoc-FF in
DMSO solutions triggered by addition of water results from a
first order phase transition giving rise to elongated crystals of
high aspect ratios.21 Detailed studies indicate that increasing
the concentration of water increases the strength of attraction
ε/kT, leading to fast nucleation rates and quasi-one-dimen-
sional crystal growth.21 If this is done with suﬃcient rapidity,
the mixture will lie below the spinodal and large amorphous
droplets will form, which over time re-dissolve, nucleate and
grow crystals in coexistence with a dilute solution.
However, the type of solvent can impact the molecular
packing of crystals formed when the solidus is crossed. As we
show here, solvent–solute interactions determine the self-
assembly pathway and structure stability, and therefore play a
crucial role in shaping the energy landscape and phase behav-
ior of dipeptide gelators. Understanding these interactions is
necessary not only for developing guidelines for adequate
solvent environments for existing gelators, but moreover for
generalizing the design rules for novel self-assembling mole-
cules. In addition, widely studied peptides such as Aβ-42, the
Alzheimer’s peptide and initial inspiration for study of Fmoc-
FF, yield highly polymorphic assemblies. Polymorphism in
these systems is assumed to be the result of kinetic trapping,
rather than relaxation to thermodynamic equilibrium. Hence,
Fmoc-FF may present us with an opportunity to not only shed
light upon a highly versatile biomaterial, but to also a near-
equilibrium, self-assembled structure of an amino acid motif
thought to be crucial to the self-assembly of Aβ-42.
With the exception of a few notable studies, the role of sol-
vents in dipeptide gelation has been largely overlooked in the
literature. Raeburn et al. investigated gels of Fmoc-FF in four
diﬀerent solvents mixed with water, reporting their obser-
vations on the resulting mechanical properties.28 Wang et al.
investigated the role of solvent-bridged hydrogen bonding in
fiber formation of diphenylalanine (FF).29 Ramos Sasselli et al.
have developed a simple packing model based on solvophobi-
city of amphiphilic molecules to determine the thermo-
dynamic favorability of fiber stability and applied it to pH-trig-
gered gelation of Fmoc-FF. Previous studies highlighting fiber
versus crystal formation have focused on the structure of
diﬀerent gelator molecules in a fixed system31,32 ( pH-triggered
gelation in aqueous solution); here, we instead explore the
phase behavior of one molecule in a variety of solvent systems
with the aim of understanding what drives the formation of
fibrous gels. While many studies have focused only on biocom-
patible hydrogels, we employ a multitude of solvents with a
range of diﬀerent interaction types, mainly in order to recog-
nize how these interactions lead to specific phase behavior
and stability, as well as to develop guidelines for applications
that do not require cell-friendly solvents.
The larger the value of R, the less soluble A will be in
B. Estimated solubility parameters can be calculated based on
group contributions of the molecule.33,34 Experimentally, they
are established by finding a number of solvents in which the
substance is soluble or insoluble, plotting their coordinates in
Hansen space, and determining a sphere that encompasses as
many solvents as possible in which the solute dissolves. The
coordinates of the center of that solubility sphere are then con-
sidered to be the HSPs of the solute.33,35 Outside of the solubi-
lity sphere, other domains containing information about
phase behavior can be established.36–41 We employ this
approach to study the phase behavior of Fmoc-FF and interpret
the solvent–solute interactions present in the system. Three
solvent systems (DMSO/H2O, methanol/H2O, and toluene) are
selected to illustrate distinct phase behavior and highlight
structural diﬀerences. Our findings indicate that:
(2) Fmoc-FF exhibits distinct polymorphism in certain
solvent systems, where it can transition from a metastable an-
isotropic crystalline structure (fibers) to large crystal aggre-
gates with no preferred axis of growth.
All non-NMR samples were prepared at a fixed Fmoc-FF con-
−1 (minimum concentration to ensure
centration of 15 mg mL
a suﬃcient signal in the CPMAS NMR measurements). The
appropriate mass of the solid peptide was added to 1 mL of
solvent and vortex-mixed. In some cases, the suspension was
heated (to no more than 75 °C) to facilitate dissolution. If
Fmoc-FF did not form a clear solution at this temperature, the
behavior in the given solvent was labeled as “insoluble”. The
heated solutions were allowed to cool back to room tempera-
ture and left to stand overnight, after which the phase behavior
was noted. For ternary systems, the desired mass of Fmoc-FF
was first fully dissolved in the solvent, followed by addition of
the appropriate amount water, and left to stand overnight.
NMR samples were allowed to self-assemble in the following
solvent systems: DMSO/H2O (xH2O = 0.95), methanol/H2O
(xH2O = 0.95 and xH2O = 0.20), pure methanol (no H2O), and
pure toluene. Each sample was prepared at a concentration of
−1, with the exception of the sample in pure metha-
nol, which was prepared at approximately 300 mg mL
A small amount (40 ppm) of Nile Blue fluorescent dye was
added to the solution. Immediately upon mixing the solution
with water, 50 µL of the mixture was transferred into a glass
bottom dish (MatTek Corp.) and covered with a lid. The
sample was loaded onto a Zeiss LSM 700 confocal microscope
and images were taken using a 63× magnification oil immer-
sion objective at 639 nm excitation wavelength. In the case of
solvents in which the dye was insoluble (apolar solvents such
as benzene and toluene), the peptide was pre-dyed. This pro-
cedure involved dissolving the Fmoc-FF and dye in a highly
volatile solvent
(acetone) and heating the solution under
vacuum until all the solvent had evaporated. The dyed solid
powder was then dissolved in the desired apolar solvent.
A 15 μL droplet of the solution was transferred onto a glass
surface, before the onset of gel transition. A 400 mesh carbon
coated copper grid (SPI Supplies) was placed on the sample
droplet for 30 s and the excess liquid was removed. The grid
was then washed and placed on a 15 μL droplet of ammonium
molybdate solution for 60 s. The excess stain was washed and
the sample grid was stored covered overnight. Images were col-
lected using JEOL 2100 Cryo and JEOL 2010 LaB6 transmission
electron microscopes operating at 200 kV.
All NMR experiments were performed on a 500 MHz (11.75 T)
Bruker NMR spectrometer. All samples were prepared as
described previously and lyophilized. We used 1H–13C cross
polarization magic angle spinning (CPMAS) solid-state NMR to
probe the natural abundance 13C populations of Fmoc-FF
assemblies.42 The shapes and linewidths of CPMAS NMR
signals can be used to assess the overall level of molecular
order. The peaks from the Fmoc group were identified by
running CPMAS on two samples assembled in DMSO/H2O
(xH2O = 0.95), one of which was uniformly labeled with 13C iso-
topes on the two phenylalanine residues (see Fig. S1, ESI†).
Each sample was packed into a 3.2 mm Bruker NMR rotor. The
magic angle spinning speed for each experiment was 20 kHz.
A 2 ms 1H–13C CP spinlock was applied concurrently with a 50
kHz radio frequency field on the 13C channel and linear ramp
pulse between 60 and 120 kHz on the 1H channel. During
signal acquisition, a 100 kHz 1H decoupling pulse was applied
using two pulse phase modulation (TPPM).43 Each spectrum
was collected over the course of 12–15 hours of signal aver-
aging time with a recycle delay of 5 s. The NMR chemical
shifts were referenced to adamantane. All measurements were
repeated to ensure reproducibility.

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[System] polypeptide self-assembly literature information extraction

[User]
DOI: 10.1073/pnas.1217742110
PMCID: PMC3545743
PMID: 23267112 [Indexed for MEDLINE]

Materials and Methods
Peptides. Peptides with conﬁrmed amino acid analysis (purity ≥95%) were
purchased from the American Peptide Company. Net peptide content varied
between 70% and 85%. All peptides were acetylated at the N terminus. Pep-
tide handling and hydrogel preparation were done as reported previously (12).
Fig. 6. X-ray ﬁber-diffraction pattern, simulation
snapshots, CD spectra, and SEM of KE7 peptide. (A)
Diffraction pattern of KE7 peptide with meridional
and equatorial directions deﬁned as for a β-type
ﬁber-diffraction pattern. (B) Meridional
intensity
proﬁle ﬁtted by four Gaussian proﬁles and a 0-order
polynomial. (C) Equatorial intensity proﬁle by four
Gaussian proﬁles and a 0-order polynomial (d values
in nanometers). (D) Final snapshots of the structure
formed from the simulations of the 64 KE7 peptides
in water. The peptides form a larger interconnected
cluster instead of single ﬁbers observed in the other
peptides. (E) CD spectra showing the direct confor-
mational transition of the KE7 peptides from ran-
dom coil to β-sheet with increasing concentration,
without going through the α-helical intermediate
(F) High-resolution SEM image of KE7 re-
vealing lamellar morphology.
Rheology. Viscoelasticity of peptide hydrogels was measured using an ARES-
G2 rheometer (TA Instruments). A serrated stainless steel, parallel-plate
geometry of 8-mm diameter was used and the gap distance was maintained
between 0.8 and 2 mm. Oscillatory frequency sweep studies were performed
for a range of 0.1–100 rad/s, using either a 0.1% or 1% strain. Oscillatory
amplitude sweep studies were conducted from 0.1 to 100% strain with an
angular frequency of 1 rad/s. All measurements were done at 25 °C. The
ring-cast method used for hydrogel preparation is described in SI Materials
and Methods.
X-Ray–Diffraction Experiments. Aqueous solutions of 2.5 mg/mL for KE7 and
15 mg/mL for NL6 at pH 7 were prepared for X-ray ﬁber diffraction. Ap-
proximately 4-μL solution drops were deposited by pipette onto a super-
hydrophobic poly(methyl methacrylate) surface and evaporated within
about 1 h under quasi–contact-free conditions into solid residues (21). Fur-
ther details are given in SI Materials and Methods.
Computer Simulations. Simulations of four peptides, namely LD6, ID3, NL6, and
KE7, were performed, using the Optimized Potentials for Liquid Simulations
(OPLS) force ﬁeld. The extended simple point charge model (SPC/E) model
was used for water molecules and GROMACS version 4.0.5 was used for
molecular dynamics simulations, as reported (12). Further details are given in
SI Materials and Methods.
2. Reches M, Porat Y, Gazit E (2002) Amyloid ﬁbril formation by pentapeptide and
tetrapeptide fragments of human calcitonin. J Biol Chem 277(38):35475–35480.
3. Abedini A, Raleigh DP (2009) A role for helical intermediates in amyloid formation by
10. Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: Lessons
from the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol 8(2):101–112.
11. Glabe CG, Kayed R (2006) Common structure and toxic function of amyloid oligomers
implies a common mechanism of pathogenesis. Neurology 66(2 Suppl 1):S74–S78.
12. Hauser CAE, et al. (2011) Natural tri- to hexapeptides self-assemble in water to am-
yloid β-type ﬁber aggregates by unexpected α-helical intermediate structures. Proc
Natl Acad Sci USA 108(4):1361–1366.
15. Görbitz CH (2006) The structure of nanotubes formed by diphenylalanine, the core
recognition motif of Alzheimer’s β-amyloid polypeptide. Chem Commun (Camb) (22):
2332–2334.
25. Blake C, Serpell L (1996) Synchrotron X-ray studies suggest that the core of the
transthyretin amyloid ﬁbril is a continuous β-sheet helix. Structure 4(8):989–998.
26. Inouye H, Gleason KA, Zhang D, Decatur SM, Kirschner DA (2010) Differential effects
of Phe19 and Phe20 on ﬁbril formation by amyloidogenic peptide A beta 16–22 (Ac-
KLVFFAE-NH2). Proteins 78(10):2306–2321.

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