[System] polypeptide self-assembly literature information extraction

[User]
We demonstrate that a tripeptide hydrogelator, Nap-FFG, can selectively
self-assemble at the surface of platelets, thus inhibiting ADP-, collagen-,
thrombin- and arachidonic acid (AA)-induced human platelet aggregations with the
IC(50) values of 0.035 (41), 0.14 (162), 0.062 (68), and 0.13 mg/mL (148 μM),
respectively. Other tripeptide hydrogelators with chemical structures of Nap-FFX
(X = A, K, S, or E) could not or possessed less potencies to inhibit platelet
aggregations. We observed higher amounts of Nap-FFG at the platelet surface by
the techniques of LC-MS and confocal microscopy. We also observed self-assembled
nanofibers around the platelet incubated with the Nap-FFG by cryo-TEM. The ζ
potential of Nap-FFG treated platelets was a little bit more negative than that
of untreated ones. The amount of Nap-FFG at the surface of NIH 3T3 cells was
much less than that of platelets. These observations suggested that Nap-FFG
could selectively self-assemble through unknown ligand-receptor interactions and
form thin layers of hydrogels at the surface of platelets, thus preventing the
aggregation of them. This study not only broadened the application and opened up
a new door for biomedical applications of molecular hydrogels but also might
provide a novel strategy to counteract infection diseases through selective
surface-induced hydrogelations at pathogens, such as bacteria and virus.

Chemicals and Materials. Fmoc amino acids were obtained from
GL Biochem (Shanghai). All the other starting materials were obtained
from Alfa. Commercially available reagents and solvents were used
without further puriﬁcation, unless noted otherwise. The synthesized
compounds were characterized by 1H NMR (Bruker ARX-300) using
DMSO-d6 as the solvent. HPLC was conducted at LUMTECH HPLC
(Germany) system using a C18 RP column with MeOH (0.05% of
TFA) and water (0.05% of TFA) as the eluents. LC-MS was
conducted at
the LCMS-20AD (Shimadzu) system, HR-MS was
performed at the Agilent 6520 Q-TOF LC/MS using ESI-L low-
concentration tuning mix (lot no. LB60116 from the Agilent Tech.).
Peptide Synthesis. The peptide derivative was prepared by solid-
phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and
the corresponding N-Fmoc-protected amino acids with side chains
properly protected by a tert-butyl group or Pbf group. During
deprotection of Fmoc group, 20% piperidine in anhydrous N,N′-
dimethylformamide (DMF) was used. Then the next Fmoc-protected
amino acid was coupled to the free amino group using O-
(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexaﬂuorophos-
phate (HBTU) as the coupling reagent. The growth of the peptide
chain was according to the established Fmoc SPPS protocol. After the
last coupling step, excessive reagents were removed by a single DMF
wash for 5 min (5 mL per gram of resin), followed by ﬁve steps of
washing using DCM for 2 min (5 mL per gram of resin). The peptide
derivative was cleaved using 95% of triﬂuoroacetic acid with 2.5% of
trimethylsilane (TMS) and 2.5% of H2O for 30 min. Twenty mL per
gram of resin of ice-cold diethylether was then added to cleavage
reagent. The resulting precipitate was centrifuged for 10 min at 4 °C at
10 000 rpm. Afterward the supernatant was decanted, and the resulting
solid was dissolved in DMSO for HPLC separation using MeOH and
H2O containing 0.1% of TFA as eluents.
Nap-FFG. 1H NMR (300 MHz, DMSO-d6): δ 8.50−8.05 (m, 3H,
N−H), 7.99−7.65 (m, 3H, Ar−H), 7.57 (s, 1H, Ar−H), 7.51−7.34
(m, 2H, Ar−H), 7.31−7.03 (m, 11H, Ar−H), 4.53 (m, 2H), 3.78 (d, J
= 5.8 Hz, 2H), 3.52 (dd, J = 28.0, 14.0 Hz, 2H), 3.10−2.92 (m, 2H,
−CH2Ph), 2.76 (ddd, J = 28.9, 13.7, 9.8 Hz, 2H, −CH2Ph). HR-MS:
calcd M+ = 537.2336, obsvd (M + H)+ = 538.2332.
Nap-FFA. 1H NMR (300 MHz, DMSO-d6): δ 8.52−8.06 (m, 3H,
N−H), 7.93−7.68 (m, 3H, Ar−H), 7.57 (s, 1H, Ar−H), 7.50−7.35
(m, 2H, Ar−H), 7.32−6.95 (m, 11H, Ar−H), 4.55 (m, 2H), 4.26−
4.14 (m), 3.52 (dd, J = 28.4, 14.0 Hz, 2H), 3.01 (dd, J = 26.1, 10.5 Hz,
2H, −CH2Ph), 2.85−2.65 (m, 2H, −CH2Ph), 1.29 (d, J = 7.2 Hz, 3H,
−). HR-MS: calcd M+ = 551.2493, obsvd (M + H)+ = 552.2491.
Nap-FFS. 1H NMR (300 MHz, DMSO-d6): δ 8.38−8.08 (m, 3H,
N−H), 7.91−7.66 (m, 3H, Ar−H), 7.56 (s, 1H, Ar−H), 7.51−7.37
(m, 2H, Ar−H), 7.27−7.13 (m, 11H, Ar−H), 4.64 (dd, J = 10.5, 7.0
Hz, 1H), 4.50 (m, 1H), 4.30 (dd, J = 8.0, 4.4 Hz, 1H), 3.69 (ddd, J =
14.8, 10.8, 4.5 Hz, 2H, CH2OH), 3.60−3.46 (m, 2H), 3.02 (ddd, J =
17.1, 14.0, 3.8 Hz, 2H, −CH2Ph), 2.75 (ddd, J = 32.4, 13.7, 10.1 Hz,
2H, −CH2Ph). HR-MS: calcd M+ = 567.2442, obsvd (M + H)+ =
568.2439.
Nap-FFK. 1H NMR (300 MHz, DMSO-d6): δ 8.30 (d, J = 6.0 Hz,
2H, N−H), 8.15 (d, J = 9.0 Hz, 1H, N−H), 7.85−7.71 (m, 3H, Ar−
H), 7.56 (s, 1H, Ar−H), 7.51−7.40 (m, 2H, Ar−H), 7.34−7.06 (m,
11H, Ar−H), 4.54 (m, 2H), 4.20 (d, J = 4.6 Hz), 3.52 (dd, J = 30.4,
14.0 Hz, 2H), 3.09−2.92 (m, 2H, −CH2Ph), 2.76 (ddd, J = 31.0, 18.9,
11.4 Hz, 4H), 1.62 (ddd, J = 25.6, 22.1, 7.3 Hz, 4H, −CH2CH2
−). HR-MS: calcd M+ = 608.3071, obsvd (M +
1.36 (m, 2H, −CH2
H)+ = 609.3064.
Nap-FFE. 1H NMR (300 MHz, DMSO-d6): δ 8.25 (m, 3H, N−H),
7.88−7.64 (m, 3H, Ar−H), 7.56 (s, 1H, Ar−H), 7.45 (m, 2H, Ar−H),
7.33−6.97 (m, 11H, Ar−H), 4.53 (m, 2H), 4.26 (m, 1H), 3.51 (dd, J =
29.9, 11.2 Hz, 2H), 3.09−2.92 (m, 2H,-CH2Ph), 2.85−2.65 (m, 2H,
−CH2Ph), 2.29 (d, J = 7.4 Hz, 2H, −CH2COOH), 2.00 (m, 1H), 1.82
(m, 1H). HR-MS: calcd M+ = 609.2548, obsvd (M + H)+ = 610.2542.
Hydrogel Formation. 1.0 mg of each peptide and equal molar of
sodium carbonate to the peptide were ﬁrst suspended in PBS buﬀer
(pH = 7.4); the sodium carbonate was used to neutralize the terminal
carboxylic acid of peptides. The suspensions were then heated to 80
°C to form clear solutions. The gels would form after cooling back to
room temperature within 3 min.
CMC. CMC values of peptides in PBS buﬀer solutions were
determined by dynamic light scattering (DLS). Solutions containing
diﬀerent concentration of peptides were tested, and the light scattering
intensity was recorded for each concentration analyzed. DLS was
performed on a laser
light scattering spectrometer (BI-200SM)
equipped with a digital correlator (BI-9000AT) at 532 nm under
room temperature (22−25 °C).
for Platelet
Aggregation Inhibition Study. Brieﬂy, fresh blood obtained from
healthy donors was centrifuged at 175g for 10 min at 22 °C, and PRP
was obtained. Fresh platelet preparations could be used for aggregation
studies for as long as 6−8 h. The concentration of fresh PRP was 107/
Platelet Aggregation Inhibition Studies. Aggregation was
measured at 37 °C by a turbidimetric method in a Chrono-Log
platelet aggregometer (Chrono-Log Corporation, Havertown, PA). A
450 μL aliquot of PRP was stirred at 1100 rpm and activated by
addition of diﬀerent agonists (10 μM of ADP, 2 μg/mL of collagen,
0.5 mM of arachidonic acid, or 1 U/mL of thrombin), in the presence
or absence of peptides, in a ﬁnal volume of 500 μL with diﬀerent
incubation times (1, 5, and 30 min). The extent of aggregation was
estimated quantitatively by measuring the maximum curve height
above baseline level. The sample of the suspending medium without
any platelets (PPP) was present to serve as a 100% aggregation
standard. Concentrations of peptides ranged from 0.01 to 0.2 mg/mL.
PRP sample with PBS was used as control
to calculate relative
magnitude. When thrombin was used, to inhibit ﬁbrin polymerization,
5 μL of 2.0 mmol/L (ﬁnal concentration) of the peptide glycyl-L-
prolyl-L-arginyl-L-prolin (GPRP) was added.
Commercially Available Platelet Concentrate (PRP) from
Tianjin Blood Bank. The platelet concentrate was used within its
validity period (24 h). The concentration of platelets in platelet
concentrate was 108/mL. This PRP was used in the experiments that
Determination of Peptide Concentration in Centrifuged Pellet of
Platelets by LC-MS. Solutions containing diﬀerent concentrations of
peptides were prepared in PBS with equal molar of sodium carbonate.
To 450 μL PRP, 50 μL of peptide solution was added and then
incubated at 37 °C for 5 min (ﬁnal peptide concentrations were 0.035,
0.1, and 0.2 mg/mL). After being centrifuged at 1570g for 10 min, the
precipitate was washed 3 times by PBS and then dissolved into 200 μL
of DMSO by sonication. The amount of peptides in the platelet
precipitate was characterized by LC-MS.
Laser Scanning Confocal Microscopy. The solutions containing
100 mg/mL of Nap-FFG and other peptides were prepared in DMSO
and diluted to 2 mg/mL by PBS with equal molar of sodium
carbonate. Thioﬂavin T was added into diﬀerent peptide solution with
100 μM of the ﬁnal concentration. The platelets without peptides and
peptide solutions without platelets were also stained with equal
Thioﬂavin T as negative controls. 450 μL PRP with or without 50 μL
peptide solutions was then incubated at 37 °C for 5 min. After
centrifuged at 1570g for 10 min, the solution was washed 3 times by
PBS, and resulting platelets were collected into 500 μL PBS. The
distribution of diﬀerent peptides with platelets was observed under
laser scanning confocal microscopy (CLSM, Leica TCS SP5).
Cryo-TEM. Cryo-TEM samples (PRP with 0.2 mg/mL Nap-FFG
and NapFFA) were prepared at 22 °C and stored at 4 °C. A
micropipet was used to load 5 mL sample solution onto a lacey
support TEM grid, which was held by tweezers. The excess solution
was blotted with a piece of ﬁlter paper, resulting in the formation of
thin ﬁlms suspended the mesh holes. After waiting for about 10 s to
ITC measurements were performed using a VP-ITC
microcalorimeter (MicroCal) in PBS (pH 7.4) at 20 °C. Nap-FFG
and four coagulants were dissolved in PBS and adjusted to pH 7.4,
then used directly in experiments. To measure the binding constants
of thrombin, ADP, AA, and collagen with Nap-FFG, 20 injections of
peptide (0.5 mM) into the calorimeter cell, which was completely
ﬁlled with coagulants solution (1468.5 μL; 0.05 mM), were collected
at 180 s intervals while being stirred at 307 rpm. The titration data and
binding plot were analyzed using MicroCal Origin software.
ζ Potential. The ζ potential of peptide solutions in PBS or in PRP
at 0.2 mg/mL concentration was measured using a BI-90 plus
Zetasizer. The measurements were performed automatically using an
aqueous dip cell. To prepare the samples of peptide in PRP, the
DMSO solution containing 100 mg/mL of peptide was ﬁrst diluted to
2 mg/mL by PBS with equal molar of sodium carbonate. The solution
was then diluted with PRP to make the ﬁnal peptide concentration to
be 0.2 mg/mL.
Determination of Peptide Concentration in Centrifuged
Pellet of 3T3 Cells by LC-MS. Solutions containing diﬀerent
concentrations of peptides were prepared in PBS with equal molar of
sodium carbonate. To 450 μL Dulbecco’s modiﬁed Eagle’s medium
(DMEM) with 10% of FBS containing 3T3 cell (106 cells/mL), 50 μL
of peptide solution was added and then incubated at 37 °C for 5 min
(ﬁnal peptide concentrations were 0.035, 0.1, and 0.2 mg/mL). After
being centrifuged at 1570g for 10 min, the precipitate was washed 3
times by PBS and then dissolved into 200 μL of DMSO by sonication.
The amount of peptides in the 3T3 cells precipitate was characterized
by LC-MS.

[Assistant] [{"peptide_sequence": "FFG", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "3.3", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFG", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "3.6", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFG", "self-assembly_phase": "none", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "2.7", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFG", "self-assembly_phase": "none", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "2.4", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "5.5", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "6.0", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFA", "self-assembly_phase": "none", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "4.5", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFA", "self-assembly_phase": "none", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "4.0", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFS", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "0.88", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFS", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", 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"Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "1.8", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}, {"peptide_sequence": "FFK", "self-assembly_phase": "none", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "1.6", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "3.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
Self-assembled synthetic materials are typically disordered, and controlling the
alignment of such materials at the nanometer scale may be important for a
variety of biological applications. In this study, we have applied directional
freeze-drying, for the first time, to develop well aligned three dimensional
(3D) nanofibrous materials using amino acid like L-phenylalanine (Phe). 3D
free-standing Phe nanofibrous monoliths have been successfully prepared using
directional freeze-drying, and have presented a unique hierarchical structure
with well-aligned nanofibers at the nanometer scale and an ordered compartmental
architecture at the micrometer scale. We have found that the physical properties
(e.g. nanofiber density and alignment) of the nanofibrous materials could be
tuned by controlling the concentration and pH of the Phe solution and the
freezing temperature. Moreover, the same strategy (i.e. directional
freeze-drying) has been successfully applied to assemble peptide nanofibrous
materials using a dipeptide (i.e. diphenylalanine), and to assemble Phe-based
nanofibrous composites using polyethylenimine and poly(vinyl alcohol). The
tunability of the nanofibrous structures together with the biocompatibility of
Phe may make these 3D nanofibrous materials suitable for a variety of
applications, including biosensor templates, tissue scaffolds, filtration
membranes, and absorbents. The strategy reported here is likely applicable to
create aligned nanofibrous structures using other amino acids, peptides, and
polymers.

Phe, Phe-Phe, Rhodamine B (RhoB), poly(vinyl alcohol) (PVA, Mw = 90,000), and
branched polyethylenimine (PEI, average Mw = ∼25,000) were purchased from Sigma (St.
Louis, MO). Distilled water was used as a solvent. RhoB labeled PEI (RhoB-PEI) was
obtained by mixing PEI and RhoB in 50 mM phosphate buffered solution at a molar ratio of
1:100 of PEI to RhoB, stirring for 2 h at room temperature, and dialyzing in deionized water
using a cellulose dialysis sack with a cutoff of 10 kDa for 2 days. Fresh stock solutions of
Phe were prepared by dissolving Phe in distilled water at concentrations of 10, 25, 50, 100,
and 150 mM. A Phe-Phe solution was prepared by dissolving Phe-Phe in distilled water with
vigorous stirring at a concentration of 2 mM. Phe-PVA and Phe-PEI solutions were obtained
by adding PVA or RhoB-PEI, respectively, into 100 mM Phe solution at a Phe/PVA or Phe/
RhoB-PEI weight ratio of 5/1.
1.5 mL of Phe, Phe-Phe, Phe-PEI, or Phe-PVA solution was pipetted into a 2 mL centrifuge
tube. The tube was placed into liquid nitrogen or a −20 or −80 °C freezer. Frozen samples
were then freeze-dried using a Virtis automatic freeze-dryer.
Field emission scanning electron microscopy or FE-SEM (JEOL, JSM-7600F) was
performed at 10 kV to characterize the morphology of nanofibrous materials. Before
imaging, samples were coated (Hummer IV Sputtering System) with a thin layer of gold to
promote conductivity. The diameters of nanofibers were measured using an image analyzer
(Adobe Photoshop CS2 9.0). A nanoscope multimode scanning probe microscope (Bruker
Instruments) was used for the atomic force microscopy (AFM) measurements. RhoB stained
samples were examined under a confocal laser scanning microscopy or CLSM (LSM 510,
Carl Zeiss, Jena, Germany). Circular dichroism (CD) spectra were recorded using a Jasco
J-810 spectropolarimeter (Japan) with instrument settings of 100 mdeg sensitivity, 1 nm
bandwidth, 1 s response time, 1 nm data pitch, and 100 nm/min scan rate. A total of 50-80
scans were accumulated and averaged in each experiment. Materials were prepared at a
concentration of 100 μM and analyzed in a 10 mm quartz cell at 25°C. The CO 2 capture
performance of Phe nanofibrous monoliths was determined using thermal gravimetric
analysis (TGA) as we previously reported.37-39 In brief, samples were placed into the middle
of the TGA microbalance quartz-tube reactor and heated to 105°C in a N2 atmosphere at a
flow of 200 mL/min for about 60 min. The temperature was then adjusted to 40°C and pure
dry CO2 was introduced at a gas flow rate of 200 mL/min until no obvious weight gain was
observed. The weight change in mg of the sorbents was recorded and the weight change in
percentage was defined as the ratio of the amount of the gas adsorbed over the total amount
of CO2 adsorbed. Adsorption capacity in mmol/(g sorbent) was calculated from the weight
change of the samples during adsorption.

[Assistant] [{"peptide_sequence": "FF", "self-assembly_phase": "fiber", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.625", "PH": "7.0", "temperature(celsius)": "-20.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "fiber", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "0.625", "PH": "7.0", "temperature(celsius)": "-80.0", "heating_cooling": "No", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
1.50 ppm (s, OC(CH3)3, 9H). 13C NMR (CDCl3): 168.2,
156.5, 133.2, 130.8, 130.1 (d, J = 7.2 Hz), 129.2, 128.9, 120.1,
117.9, 107.5, 83.0, 66.2, 28.5, 28.3 ppm. MS (CI, NH3) 354 ([M þ
NH4]þ). Accurate mass calculated: 354.070 48. Found: 354.069 75.
Analysis calculated for C16H17O3Br: C, 56.99%; H, 5.08%.
Found: C, 56.90%; H, 5.06%.
The tert-butyl protecting group was removed by dissolving tert-
butyl 2-(6-bromonaphthalen-2-yloxy)acetate (8.0 g, 23.7 mmol)
in chloroform (ca. 30 mL). Trifluoroacetic acid (11.08 mL, 6.1
equiv, 145 mmol) was added, and the solution was stirred for 24 h.
Hexane (200 mL) was added, and the resulting white precipitate
was collected. After washing with hexane, the resulting solid was
dried in vacuo to give 2-(6-bromonaphthalen-2-yloxy)acetic acid
in a 92% yield. 1H NMR (d6-DMSO): 8.13 (d, ArH, 1H, 3JHH =
1.5 Hz), 7.85 (d, ArH, 1H, 3JHH = 9.0 Hz), 7.78 (d, ArH, 1H,
3JHH = 8.8 Hz), 7.57 (dd, ArH, 1H, 3JHH = 2.2 Hz), 7.32 (d, ArH,
1H, 3JHH = 2.6 Hz), 7.26 (dd, ArH, 1H, 3JHH = 2.6 Hz), 4.81 (s,
OCH2, 2H), 3.39 ppm (s, OH). 13C (d6-DMSO): 170.6, 150.7,
134.9, 132.2, 128.7, 127.1, 126.2, 126.1, 124.3, 123.0, 122.5, 72.3,
59.9 ppm. MS (CI, NH3) 298 ([M þ NH4]þ), Accurate mass
calculated: 298.007 88. Found: 298.007 99. Analysis calculated for
C12H9O3Br: C, 51.27%; H, 3.23%. Found: C, 50.91%; H, 3.18%.
The C-ethyl-protected alanine was coupled to 2-(6-bromo-
naphthalen-2-yloxy)acetic acid using standard coupling meth-
odologies.28 To a stirred solution of 2-(6-bromonaphthalen-2-
yloxy)acetic acid (1.00 g, 3.56 mmol) in chloroform (20 mL) was
added N-methylmorpholine (0.40 mL, 3.60 mmol), and isobutyl
chloroformate (0.49 mL, 3.60 mmol) at 0 (cid:1)C. The solution was
stirred at 0 (cid:1)C for 5 min. A solution of alanine ethyl ester (0.35 g,
2.5 mmol) and N-methylmorpholine (0.40 mL, 3.60 mmol) in
chloroform (20 mL) was added. The solution was allowed to
warm to room temperature and stirring continued overnight. The
solution was washed with distilled water (2 (cid:2) 100 mL), hydro-
chloric acid (2 (cid:2) 100 mL, 0.1 M), aqueous potassium carbonate
solution (100 mL, 0.1 M), and distilled water again (4 (cid:2) 100 mL)
and dried over magnesium sulfate, and the solvent was removed in
vacuo. The solid was then washed with petroleum ether (10 mL)
and dried under vacuum for 48 h to give ethyl 2-(2(6-bromo-
naphthalen-2-yloxy)acetamido)propanoate in a 73% yield. 1H
NMR (CDCl3): 7.94 (s, ArH, 1H), 7.70 (d, ArH, 1H, 3JHH = 8.8
Hz), 7.62 (d, ArH, 1H, 3JHH = 8.3 Hz), 7.53 (d, ArH, 1H, 3JHH =
8.3 Hz), 7.26 (m, ArH, 2H, 3JHH = 8.3 Hz), 7.12 (s, ArH, 1H),
4.71 (m, CH3CHCdO, 1H), 4.62 (s, OCH2CdO, 2H), 4.23 (m,
OCH2CH3, 2H, 3JHH = 7.0 Hz), 1.49 (d, NHCH(CH3), 3H,
3JHH = 6.6 Hz), 1.29 ppm (t, OCH2CH3, 3H, 3JHH = 7.0 Hz). 13C
NMR (CDCl3): 172.9, 167.8, 155.7, 133.1, 130.9, 130.4, 130.1,
129.0, 119.7, 118.3, 108.0, 67.7, 62.1, 48.2, 18.9, 14.5 ppm. MS
(ESþ, CH3OH) 402 ([M þ Na]þ), Accurate mass calculated:
402.0317. Found: 402.0317. Analysis calculated for C17H18NO4-
Br: C, 53.70%; H, 4.77%; N, 3.68%. Found: C, 54.04%; H,
4.78%; N, 3.66%.
To deprotect the C-terminus, lithium hydroxide (0.2 g) was
added to a solution of ethyl 2-(2(6-bromonaphthalen-2-ylox-
y)acetamido)propanoate (0.5 g) in a THF:water solution (3:1
mixture, 25 mL), and the solution was stirred for 18 h. After this
time, distilled water (ca. 100 mL) was added, and then hydro-
chloric acid (1.0 M) was added dropwise until the pH was lowered
to pH 3. The resulting white precipitate was collected by filtration
and washed well with water and petroleum ether before being
dried in vacuo to give 2-(2-(6-bromonaphthalen-2-yloxy)acetam-
ido)propanoic acid as a white powder in 89% yield. 1H NMR (d6-
DMSO): 8.44 (d, ArH, 1H, 3JHH =7.5 Hz) 8.13 (d, ArH, 1H,
3JHH = 1.6 Hz), 7.86 (d, ArH, 1H, 3JHH = 8.8 Hz), 7.75 (d, ArH,
1H, 3JHH = 8.8 Hz), 7.58 (dd, ArH, 1H, 3JHH = 1.9 Hz), 7.34 (m,
NH, 1H), 7.31 (dd, ArH, 1H, 3JHH = 2.4 Hz), 4.65 (m, OCH2-
CdO, 2H), 4.30 (m, (CH3)CHCdO, 1H), 3.42 (s, OH), 1.33 (d,
(CH3)CHC=O, 3H, 3JHH = 7.2 Hz) ppm. 13C (d6-DMSO):
174.3, 167.5, 156.4, 133.0, 130.3, 129.7 (d, J = 5.7 Hz), 129.3,
129.0, 120.2, 116.9, 107.8, 67.1, 47.8, 17.6 ppm. MS (ESþ,
CH3OH) 374 ([M þ Na]þ). Accurate mass calculated: 374.0005.
Found: 374.0004. Analysis calculated for C15H14NO4Br: C,
51.16%; H, 4.01%; N, 3.96%. Found C, 51.16%; H, 4.02%; N,
Ethyl 2-(2-(2-(6-bromonaphthalen-2-yloxy)acetamido)propa-
namido)-3-methylbutanoate was prepared as a white solid in a
91% yield. 1H NMR (CDCl3): 7.92 (d, ArH, 1H, 3JHH = 1.6 Hz),
7.68 (d, ArH, 1H, 3JHH = 9.8 Hz), 7.59 (d, ArH, 1H, 3JHH =
8.7 Hz), 7.51 (dd, ArH, 1H, 3JHH = 8.7 Hz, 3JHH = 2.0 Hz), 7.22
2-(2-(2-(6-Bromonaphthalen-2-yloxy)acetamido)propanamido)-
3-methylbutanoic acid was collected as a white solid in a 88%
yield. 1H NMR (d6-DMSO): 8.20 (bd, NH, 1H, 3JHH = 7.6 Hz),
8.12 (d, ArH, 1H, 3JHH = 1.6 Hz), 8.06 (bd, NH, 1H, 3JHH =
8.5 Hz), 7.86 (d, ArH, 1H, 3JHH = 8.9 Hz), 7.76 (d, ArH, 1H,
3JHH = 8.8 Hz), 7.58 (dd, ArH, 1H, 3JHH = 8.8 Hz, 3JHH = 2.1
Hz), 7.34 (d, ArH, 1H, 3JHH = 1.6 Hz), 7.30 (dd, ArH, 1H,
3JHH = 8.9 Hz, 3JHH = 2.1 Hz), 4.66 (d, OCH2, 2H, 3JHH =
2.2 Hz), 4.53 (m, CHNH, 1H), 4.15 (dd, CHNH, 1H, 3JHH =
8.6 Hz,3JHH = 5.9 Hz), 2.04 (m, CH(CH3)2, 1H), 1.28 (d,
CHCH3, 3H, 3JHH = 7.1 Hz), 0.85 ppm (d, CH(CH3)2, 6H,
3JHH = 6.6 Hz). 13C NMR (d6-DMSO): 172.8, 172.2, 166.9,
155.9, 132.6, 129.9, 129.3, 129.2, 128.9, 128.6 119.7, 116.5, 107.5,
66.8, 57.1, 47.6, 29.8, 19.0, 18.4, 17.9 ppm. MS (CI) 468/470 ([M þ
NH4]þ). Analysis calculated for C20H23N2O5Br: C, 53.23%; H,
5.14%; N, 6.21%. Found C, 52.93%; H, 5.11%; N, 6.18%.
Gelation Studies. The dipeptide derivative (25.0 mg) was
suspended in deionized water (5.00 mL). An equimolar quantity
of NaOH (0.1 M, aq) was added, and the solution was gently
stirred for 30 min until a clear solution was formed. The pH of this
solution was measured to be 10.7. To prepare hydrogels, solutions
were added to measured quantities of glucono-δ-lactone (GdL)
according to the requirements of final pH, and the samples were
left to stand for 24 h.
pH Measurements and pKa Determination. A FC200 pH
probe (HANNA instruments) with a (6 mm (cid:2) 10 mm) conical tip
was employed for all the pH measurements. The stated accuracy
of the pH measurements is (0.1. The pH changes during the
gelation process were recorded every 1 min for 24 h. All measure-
ments were conducted at room temperature. The pKa values of
dipeptide derivative (0.5 wt %) solutions were determined by
titration via the addition of aliquots of a 0.1 M HCl solution. pH
values were also recorded every 2 s during the titration process. To
prevent the formation of gel, the solutions were gently stirred, thus
keeping the solution liquid during the whole “titration” process.
Circular Dichroism and Linear Dichroism. Measurements
were performed at room temperature (21 (cid:1)C) on a Jasco J-815
spectropolarimeter modified for linear dichroism (Jasco UK,
Great Dunmow, UK). Spectra were measured between 350 and
180 nm with a data pitch of 0.2 nm. The bandwidth was set to 2 nm
with a scanning speed of 100 nm min-1 and a response time of 1 s.
Spectra were collected at 2 min intervals. For clarity, the circular
dichroism (CD) and linear dichroism (LD) figures show spectra
only every 10 min. The path length was 0.1 mm in a demountable
Spectrosil quartz cuvette (Starna Optiglass, Hainault, UK). Note
that the sample was placed directly adjacent to the photomulti-
plier tube in order to minimize loss of light due to scattering. All
measurements were performed in duplicate.
Rheology. Initial tests for gelation were carried out by simple
vial inversion.29 Typically, 2 mL of solution (with added GdL)
was placed in a sample tube (22 mm diameter, 60 mm high). After
standing for 24 h, the sample tube was inverted, with gelation
being shown by a lack of flow. Dynamic rheological experiments
were performed on an Anton Paar Physica MCR101 rheometer.
In the oscillatory shear measurements, a sandblasted parallel top
plate with a 50 mm diameter and 1.0 mm gap distance was used.
Gels for rheological experiments were prepared on the bottom
plate of the rheometer by loading a 2.0 mL solution of the gelator
immediately after GdL addition. At this point, the sample is still a
free-flowing liquid. Hence, sample uniformity and reproducibility
are high. Evaporation of water from the hydrogel was minimized
by covering the sides of the plate with low-viscosity mineral oil.
The measurements of the shear modulus (storage modulus G0 and
loss modulus G00) with gelation were made as a function of time at
a frequency of 1.59 Hz (10 rad/s) and at a constant strain of 0.5%
for a period of 2 h, followed by an amplitude sweep from 0.01% to
100% under a frequency of 10 rad/s. The strain amplitude
measurements were performed within the linear viscoelastic re-
gion, where the storage modulus (G0) and loss modulus (G00) are
independent of the strain amplitude. All the experiments were
conducted at 25 (cid:1)C.
Fluorescence Spectroscopy. Fluorescence spectra were ob-
tained on a PerkinElmer luminescence spectrometer LS55. Thio-
flavin T (ThT) (0.1 mL of a 0.5 μM solution in water) was added to
a solution of the dipeptide derivative (2 mL of a 0.5 wt % solution
at pH 10.7). This was added to the required amount of GdL and
immediately loaded into a 1.0 cm path-length cuvette. Emission
spectra were collected every 5 min in the first 3 h and then every
30 min for 21 h. The emission spectra were recorded between 460
and 600 nm by excitation at 455 nm. The maxima emission peaks
were at 485 nm. The slit width for emission and excitation was
2.5 nm, and the scan rate was 100 nm/min.
FTIR. IR spectra were collected on Bruker FTIR at 2 cm-1
resolution by averaging over 64 scans. All hydrogels were pre-
pared in D2O using NaOD (0.1 M) to change the pD. The
hydrogels were loaded onto a CaF2 windows, and another CaF2
window was placed on the top

[Assistant] [{"peptide_sequence": "AV", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Conjugate", "conjugate_partner": "Napthalene", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "3.1", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "AV", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Conjugate", "conjugate_partner": "Napthalene", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "3.6", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "AV", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Conjugate", "conjugate_partner": "Napthalene", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "4.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "AV", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Napthalene", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Conjugate", "conjugate_partner": "Napthalene", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "5.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
We report the first histidine-containing self-assembling tripeptide devoid of
capping groups that forms a thermoreversible hydrogel under physiological
conditions and catalyses hydrolysis of an ester, providing a minimalist building
block for functional soft materials.

COMMUNICATION
S. Marchesan et al.
A biocatalytic and thermoreversible hydrogel from a histidine-containing
tripeptide
Short peptides have emerged as very attractive minimalist
building blocks for supramolecular (bio)materials, in light of
their simplicity of preparation and benign fate in biological
settings and in the environment.1 The vast majority of these
systems exploit synthetic N-capping groups that are rigid
and aromatic (e.g., Fmoc, Nap, etc.), thus eﬀectively templating
self-assembly.2 Recently, useful tools have been developed to
predict their gelation.3 In the case of unprotected (tri)peptides,
a priori self-assembly design in water is notoriously diﬃcult.
Despite intense research eﬀorts have enabled the development
of valid tools to predict their aggregation propensity,4–6 very few
hydrogel examples have been reported thus far. The first
unprotected tripeptides self-assembling into hydrogels were
described in 2012 and consisted of hydrophobic sequences
that exploited appropriate positioning of D- and L-amino acids
in heterochiral sets of three.7,8 The key role played by the amino
acid stereoconfiguration was confirmed in a complete stereo-
isomer series whereby homochiral analogues did not assemble.9,10
In 2015, the first four L-tripeptides able to gel were reported,
consisting of amphiphilic sequences bearing a hydrophilic lysine
and two aromatic amino acids (i.e, Phe, Tyr, and/or Trp).5 Another
four examples soon followed bearing amino acids Glu, Thr, Lys or
Cys besides aromatic Phe residues; however, they all required
small amounts (i.e., 8%) of an organic solvent to assist the
dissolution, and the final pH of the hydrogel was below 5, thus
limiting biological use.11 Considering that hydrogels formed by
Here we report the first example of an unprotected tripeptide
bearing His forming a supramolecular hydrogel under physio-
logical conditions. The hydrogel withstands temperatures used in
cell culture for potential use as a biomaterial, is thermoreversible,
and catalyses ester hydrolysis, thus showing potential towards
functional soft materials.
LHis–DPhe–DPhe formed a thermoreversible hydrogel at 2 wt%
in phosphate buffer following a pH trigger to neutral at room
temperature (Fig. 1). The gel-to-sol transition had an onset
temperature of 45 1C, as observed visually and confirmed
by differential scanning calorimetry (DSC). A homogeneous
solution was obtained at 65 1C and the gel reformed after ca.
10 minutes upon cooling to room temperature (Fig. 1A). DSC
data confirmed a wide endotherm for the gel-to-sol transition,
however, with a remarkably high Tm = 88–90 1C (see the ESI†).
This temperature is compatible with that of the amyloid nano-
structure disassembly, as recently reviewed.12 While gel melting
implies the loss of the three-dimensional network forming the
gel matrix, it does not necessarily correspond to a complete loss
of the amyloid nanostructure, which typically occurs at higher
temperatures.
The viscoelastic properties of the system were assessed by
rheometry that revealed good resistance to applied stress for
the peptide gel, since the gel-to-sol transition was observed at
50 Pa (Fig. 1B). The elastic modulus was also considerably high
for an unprotected tripeptide, and reached 50 kPa and beyond
as the self-assembly proceeded over time (Fig. 1C).
FT-IR, Thioflavin T fluorescence assay, and CD were employed
to assess the peptide conformation (Fig. 2 and the ESI†). The
amide I band region in the FT-IR spectra is very informative of the
secondary structure, with beta-sheets most frequently reported for
self-assembled short peptides.13,14 In the case of His–Phe–Phe
hydrogels, the spectra displayed a signal compatible with the beta-
sheets (1637 cm(cid:2)1) as typically occurs for similar amyloid
peptides,10 although it was not possible to discern the presence
of the characteristic shoulder for the anti-parallel arrangement,
due to the intense signal of the TFA counterion (1677 cm(cid:2)1).
In addition, the presence of other maxima at 1653 cm(cid:2)1 and
1600 cm(cid:2)1 suggested a certain level of heterogeneity in the
peptide secondary structure (Fig. 2A), which is compatible with
the wide gel-to-sol endotherm observed by DSC. The amide II
band region was dominated by a broad signal centred at 1545 cm(cid:2)1
and a shoulder at 1514 cm(cid:2)1. The amyloid structure based on
beta-sheets was also confirmed by the Thioflavin T fluorescence
assay (see the ESI†). The dye is known to bind laterally to
amyloid fibrils composed of at least four consecutive beta-
strands.15 This interaction limits the rotation of a single bond
that connects the two aromatic rings that compose the dye,
resulting in fluorescence.16
CD was also used to monitor the peptide conformation, as the
region of 190–220 nm is indicative of the secondary structure.
However, the concentration required for self-assembly led to signal
saturation below 220 nm even when 0.1 mm cuvettes were used.
Nevertheless, the acquisition of CD spectra in the 220–300 nm
region over time proved to be useful to monitor the self-assembly
(Fig. 2B). Ellipticity evolved during the first 20 minutes until it
reached a plateau. A negative minimum at 225 nm suggested the
D-supramolecular chirality as previously observed for heterochiral
unprotected tripeptides.10 The spectrum was also characterised by
a shoulder at 230–240 nm. In the near-UV region of 255–270 nm,
the weak but distinctive signal of vibronic states for the forbidden
p - p* transition of the aromatic ring indicated that Phe side
chains were located in a chiral environment.17
Atomic force microscopy (AFM) and transmission electron
microscopy (TEM) were used to visualise nanomorphological
features of peptide assemblies (Fig. 3). The hydrogel was formed
by a dense network of bundles of fibres reaching a thickness of
over 1 micron, and as long as tens of microns, thus explaining
the high elastic modulus registered by rheometry.
Next, the catalytic function of the hydrogel was assessed.
In recent years, there has been an increasing interest in the
development of minimalist peptide motifs for biocatalysis.
In particular, esterase-mimicking motifs have been the most
studied ones, and typically employ sequences containing either
the Ser–His–Asp catalytic triad or the His–Zn2+ coordination
motif. While His alone does not exhibit catalytic activity, it is
the most important amino acid playing the role of a proton
donor or acceptor in ester hydrolysis.18 The supramolecular
organisation of His-bearing peptides is a strategy that can
successfully achieve biocatalysis through the creation of multi-
valency, typically featuring ordered arrays of His residues. While
initial reports displayed peptides as long as 42 amino acids,19,20
research efforts have ever since been devoted towards the develop-
ment of conveniently shorter sequences. To date, this approach has
been proven to be successful for nanofibers formed by decapeptide
amphiphiles,21 capped 14-mer22 and 7-mer23,24 peptides, and
even N-acetylated pentamers assembled on the surface of gold
nanoparticles.25 This minimalist approach culminated with
Fmoc–tripeptide nanotubes.26 However, all these examples
feature peptides derivatised at either one or both termini, likely
due to the intrinsic difficulties of the self-assembly design
for unprotected short peptides. Besides, it should be noted
that catalysis by means of isolated nanostructures in solution
is markedly different from the catalysis occurring in bulk
functional materials.
Fig. 3 (A) AFM image of peptide hydrogel. (B) TEM image at high (left) and
low (right) magnifications of the peptide hydrogel.
the yellow-coloured p-nitrophenol (pNP) that is easily quanti-
fied by measuring the absorbance at 405 nm.27 The product
formation was monitored over one hour (Fig. 4 and the ESI†), and
it was negligible for control samples with 1 mM peptide solution
or buﬀer (kobs = 5.0 (cid:3) 10(cid:2)5 s(cid:2)1 in both cases). By contrast, the
presence of the supramolecular hydrogel resulted in marked
biocatalysis (kobs = 9.5 (cid:3) 10(cid:2)4 s(cid:2)1 increased to 1.7 (cid:3) 10(cid:2)3 s(cid:2)1
with 1 mM pNPA), and that reached a plateau corresponding to
complete conversion of the substrate into the product, and visibly
yellow gels. Intermediate peptide concentrations that were
below mgc displayed moderate catalysis (Fig. 4C) and absence
of a linear dependence of kobs on the peptide concentration.
TEM analysis (see the ESI†) revealed the presence of thin
nanofibrils (10 mM sample) and also oligomers and protofibrils
(25 mM sample), which are typical morphologies of early-stage
amyloid assemblies.28,29 Overall, these results confirmed that
the esterase-like activity is associated with the formation of
supramolecular structures.
In conclusion, we reported herein the first unprotected
tripeptide bearing His and forming supramolecular hydrogels
under physiological conditions. In the context of unprotected
tripeptide hydrogels, this is the first example displaying a catalytic
function, thus setting a new benchmark in the quest for minimalist
biocatalysts. The material is thermoreversible at temperatures
higher than those used in cell culture, allowing potential use as a
Fig. 4 (A) Multi-well plate colourimetric screen for ester hydrolysis.
(B) Substrate conversion into product over time and at diﬀerent peptide
concentrations. (C) Esterase-like activity observed rate (kobs) for the catalytic
peptide systems. Note: (nano) refers to formation of nanostructures, while
(gel) refers to the hydrogel system.
biomaterial and oﬀering the opportunity to control assembly and
disassembly by heating/cooling, besides changing of pH values.
Further studies in this area will provide simple building blocks for
supramolecular functional biomaterials.30 If the field advances
towards a catalytic function with a higher level of complexity, an
appropriate choice of amino acid chirality may also oﬀer interesting
opportunities towards stereoselective reactions.

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[System] polypeptide self-assembly literature information extraction

[User]
The search for low molecular weight hydrogelators (LMWH) with varying structural
motif is getting intense because of its potential application in the field of
biomedicines as well as the diversified area of nano-biotechnology. In this
paper, we have developed hydrogels of simple cationic dipeptide amphiphiles that
have a wide range of minimum gelation concentration (MGC), 12-0.25% (w/v) in
plain water. The self-aggregation behavior of these thermoreversible
hydrogelators has been investigated through different spectroscopic and
microscopic techniques. A balanced participation of hydrophilicity and
hydrophobicity is the major driving force for gelation, which could be modulated
by a minute change in the architecture of the amphiphile head. The prospective
use of this material in controlled release suggests that this system could also
be applied as the drug delivery vehicle. Moreover, the presence of a
biodegradable amide linkage susceptible to base or enzyme-catalyzed hydrolysis
increases its probable applications as biomaterials.

Materials. Silica gel of 60-120 mesh, L-tryptophan, L-
phenylalanine, l-proline, n-hexadecylamine, N,N-dicyclohexy-
lcarbodiimide (DCC), 4-N,N-(dimethylamino)pyridine (DMAP),
N-hydroxybenztriazole (HOBT), iodomethane, solvents, and all
other reagents were procured from SRL, India. Water used
throughout the study was Milli-Q water. Thin-layer chroma-
tography was performed on Merck precoated silica gel 60-F254
plates. All the deuterated solvents for NMR experiments and
8-anilino-1-naphthalenesulphonic acid (ANS) were obtained
from Aldrich Chemical Co. Amberlyst A-26 chloride ion-
exchange resin was obtained from BDH, U.K. Mass spectro-
metric (MS) data were acquired by electron spray ionization
(ESI) technique on a Q-tof-Micro Quadruple mass spectropho-
tometer, Micromass.
Preparation of Hydrogel. The aqueous dispersions of the
required amount of compounds 1-6 were slowly heated to
dissolve the compounds in water and then allowed to cool slowly
(undisturbed) to room temperature in a vial with i.d. ) 10 mm.
After 20-30 min the solid aggregate mass was stable to
inversion of the glass vial when the glass vial was turned upside
down; the compound was recognized to form a hydrogel.
Determination of Gel-Sol Transition Temperature (Tgel).
The gel to sol transition temperature (Tgel) was determined by
placing a hydrogel containing an inverted screw-capped glass
vial with i.d. ) 10 mm in a thermostatted oil bath, and then the
temperature was raised at a rate of 2 (cid:176) C/min. Here, the Tgel was
defined as the temperature ((0.5 (cid:176) C) at which the hydrogel
melts and starts to flow out of the gel.
Microscopic Studies. Scanning electron microscopy (SEM)
and transmission electron microscopy (TEM) measurements
were performed on JEOL-6700F and JEOL JEM 2010 micro-
scopes, respectively. A piece of gel was mounted on a glass
slide and a 300-mesh carbon-coated copper grid for SEM and
TEM sampling, respectively, and dried for few hours under
vacuum before imaging. For bright-field and fluorescence
microscopic images of the gels at MGC, the following method
was used: a small quantity of gel was placed on a microscope
slide, which was sealed with a glass coverslip, and images were
taken. Fluorescence microscopy was conducted using a light
microscope (BX61, Olympus) equipped with a filter set consist-
ing of a BP330-385 nm for an exciter and a band absorbance
filter covering wavelengths below 420 nm.
Circular Dichroism. The circular dichroism (CD) spectra
of 6 with concentration variation (from 0.005 to 0.05%, w/v)
at room temperature and with varying temperatures (from 40
to 90 (cid:176) C) at 0.05%, w/v aqueous solution, were recorded by
using a quartz cuvette of 1 mm path length with a Jasco J-815
spectropolarimeter.
Fluorescence Spectroscopy. The emission spectra of ANS
were recorded on a Varian Cary Eclipse fluorescence spectro-
photometer by adding the probe molecules in aqueous solutions
of transparent hydrogels, i.e., 2 and 4-6 gels with varying
concentrations at room temperature. ANS was initially dissolved
in MeOH, and from this super stock the required amount of
ANS solution was added to the experimental solutions [5 (cid:237)L
of super stock (0.01 M) was added to a 5 mL aqueous solution
of the gelators to reach the probe concentration of 1 (cid:2) 10-5
M]. The ANS solutions were excited at (cid:236)ex ) 365 nm.
FTIR Measurements. FTIR measurements of the gelators
in CHCl3 solution and dried gel from D2O were carried out in
a Shimadzu FTIR-8100 spectrophotometer using a 1 mm KBr
cell and silicon wafer, respectively, at the MGC values.
Controlled Release of Entrapped Vitamin B6 andB12 from
Hydrogel. The controlled release of entrapped biomolecules was
carried out as follows: 0.2 mL of 2 mM vitamin solutions
(vitamin B6 and B12) in pH 7.0 phosphate buffer (1 mM) was
added to 0.5 and 3.0% (w/v) gelators in different vials, heated
to their Tgel, and held for a few hours undisturbed to attain the
Figure 1. Variation of the gel to sol transition temperature (Tgel) with
the gelator concentrations.
gel state at room temperature. Then, a 0.2 mL, pH 7.0 phosphate
buffer (1 mM) solution was added on top of each gel. The 0.2
mL supernatant buffer solution was removed each time, and
the concentrations of the released vitamins were analyzed by
time-dependent UV-vis spectra using a Shimadzu UV-1700
analyzer. The release rate of vitamins was investigated in
triplicate at room temperature.

[Assistant] [{"peptide_sequence": "FP", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "132.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "FP", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "144.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "FP", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "108.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "FP", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "96.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "PY", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "110.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "PY", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "120.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "PY", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "90.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "PY", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "80.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "WP", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "77.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "WP", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "84.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "WP", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "63.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "WP", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "56.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "PW", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "33.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "PW", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "36.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "PW", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "27.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "PW", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "24.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "WF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.4", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "WF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "4.8", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "WF", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "3.6", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "WF", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "3.2", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "FW", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.75", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "FW", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "3.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "FW", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.25", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}, {"peptide_sequence": "FW", "self-assembly_phase": "none", "N-terminal_modification": "Chlorination", "C-terminal_modification": "N-hexadecylamine", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "25.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

gradient of 0.1% TFA in H2O to 0.1% TFA in acetonitrile : isopropanol
(iPrOH) (7 : 3) in 55 min. The molecular masses were conﬁrmed
on an electrospray ionization mass spectrometer with a linear ion
trap mass analyzer (LTQ-IT, Thermo Fischer, Waltham, MA, USA).
The purity of all the HPLC-puriﬁed peptides was ≥98%. HPLC-
puriﬁed peptides were used for all studies.
Methanol solutions of both Boc-protected and Boc-deprotected
peptide esters (10 mg/ml, w/v) were prepared and centrifuged
in an Eppendorf centrifuge at 20879 g for 30 min at room tempera-
ture. No residue was observed visually. The 10-mg/ml solutions were
diluted to 5 and 2 mg/ml. The peptide solutions were immediately
used for morphological characterization.
The peptide solutions were adsorbed for approximately 30 s
at ambient conditions onto formvar/carbon-coated 200-mesh
copper grids and then stained with 2% (w/v) uranyl acetate for
30 s before blotting out excess solvent. The MeOH samples
dried in 5–10 min. The samples were examined with a JEM-2100
(M/S JEOL Ltd, Tachikawa, Tokyo, Japan) transmission electron
microscopy (TEM) at 100 kV accelerating voltage.
The average hydrodynamic radii were measured by recording the
scatter at 90(cid:3), within a few minutes of dissolving the peptides,
using Flexible Correlator Photocor-FC (JDS Uniphase) Dynamic
Light Scattering (DLS) instrument (Photocor Instruments, Beltsville
MD, USA). The hydrodynamic radii were calculated using the distri-
bution analysis method based on multipass algorithm to process
the time-dependent correlation function data (light scattering
intensity) using DYNALS software.
repetitive scans at a rate of 100 nm/min, band width of 1 nm,
response time of 0.5 s and a sample interval of 0.2 nm in 1-mm
light path demountable cells. The instrument was calibrated with
(+)-10-camphorsulfonic acid (1 mg/ml), and the ellipticity was
expressed as the mean residue ellipticity, [θ]MRE (deg cm2/dmol).
The electrical conduction of peptides dried from MeOH stocks
was measured using two-terminal transport experiments on
interdigitated electrodes using Keithley 236 source I/V measure
unit (Keithley Instruments, Inc. Cleveland, OH, USA). The devices
for these measurements consisted of interdigitated electrodes
with 0.5-mm gaps. Peptides were deposited on the electrodes
using cast deposition of a 10-ml drop of 10 mg/ml. Measurements
were carried out after solvent evaporation. The voltage was
increased linearly from 0.1 to 2 V in 0.1-V steps, and the resultant
current was measured. Control experiments on empty devices
(i.e. without peptides) and also with Boc-protected amino acids
were carried out.

[Assistant] [{"peptide_sequence": "LII", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "5.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "LII", "self-assembly_phase": "nanosphere", "N-terminal_modification": "Boc", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "10.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "II", "self-assembly_phase": "fiber", "N-terminal_modification": "Free", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "5.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}, {"peptide_sequence": "II", "self-assembly_phase": "fiber", "N-terminal_modification": "Free", "C-terminal_modification": "Methoxy", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 Methanol", "peptide_concentration(mg/ml)": "10.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "30.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
Energy transfer between a naphthalene-dipeptide and either a hosted dansyl
acceptor or a co-assembling anthracene-dipeptide acceptor occurs on formation of
a hydrogel by decreasing the pH.

Energy transfer between a naphthalene-dipeptide and either a
hosted dansyl acceptor or a co-assembling anthracene-dipeptide
acceptor occurs on formation of a hydrogel by decreasing
Energy transfer between suitably organised aromatic groups
is receiving current interest with potential applications in
light-harvesting and molecular electronics.1 Much of this work
is inspired by attempts to mimic the unidirectional energy
transfer between chromophores in photosynthetic systems.2
One method of organising the chromophores is to utilise
materials that self-assemble to give organogels.3 The formation
of 1-D ﬁbres that leads to gelation results from the organisation
of the molecules into long arrays with speciﬁc distances
between the molecules. This assembly and organisation arises
from cooperative
molecules including hydrogen bonding, van der Waals interactions
and, of relevance here, p-stacking. When the ﬁbre-forming
molecules include a suitable donor chromophore, these ﬁbres
can be used to transfer energy to a second acceptor
chromophore within the system. A number of reports detail
the use of this approach in organic solvents.4 However, the use
of hydrogelators containing ﬁbril-forming donor chromophores
and a suitable hosted acceptor is less common. Light-harvesting
hydrogels have been prepared from cationic glutamate derivatives
that complex with a mixture of anionic naphthalene- and
anthracene-based ﬂuorophores.5 Energy transfer between a
naphthalene-based hydrogelator and a dansyl probe has also
been demonstrated, but required the initial dissolution of the
gelator in DMSO.6 Very recently, MacPhee et al. reported
energy transfer between donor and acceptor chromophores
conjugated to ﬁbre-forming peptides.7 Again, an organic
solvent was utilised for initial dissolution of the peptides.
Here, we show that multicomponent hydrogels can be
prepared that undergo energy transfer by incorporation of a
suitable acceptor moiety or by mixing dipeptide-conjugates.
This second system requires no organic solvent
assembly.
functionalised at
the N-terminus have been shown to be eﬀective hydrogelators.
To enable gelation, a common strategy is conjugation at
the N-terminus with an aromatic group such as ﬂuorenyl-
methoxycarbonyl
In addition
to being integral to gelation, these aromatic groups are
also convenient ﬂuorophores with which to probe energy
transfer. Hydrogelation is often carried out in these systems
via a pH switch; the dipeptide-conjugates are soluble at high
pH and gel at a lower pH. We have recently reported the use of
the hydrolysis of glucono-d-lactone (GdL) to controllably
reduce the pH in such systems which allows the formation of
highly reproducible hydrogels.10 This method also allows the
gelation to be monitored with time since the hydrolysis of GdL
takes a number of hours to come to completion. Using this
method, we have shown that a naphthalene-dipeptide assembles
into ﬁbres via b-sheet formation between the dipeptide segment
with the concomitant arrangement of the naphthalene groups
into a left-handed helical stack.11 We showed that addition
of thioﬂavin T results in incorporation of the dye within
the b-sheets of the self-assembled ﬁbres, resulting in an
increase in ﬂuorescence on formation of b-sheets between
the dipeptides.11 We rationalised that incorporation of an
acceptor chromophore within the aromatic stacks should lead
to eﬃcient energy transfer.
Naphthalene-diphenylalanine was prepared using standard
synthetic protocols from 2-naphthol (see ESIw for details).
A 2.2 mM aqueous solution of naphthalene-diphenylalanine,
I (Fig. 1a), was prepared via the addition of NaOH to an
aqueous suspension of the dipeptide (the sodium salt of I is
completely soluble). Addition of 3.75 mg mL(cid:2)1 of GdL
resulted in the formation of a transparent, self-supporting
hydrogel after approximately 15 min (demonstrated by stability
to vial inversion, Fig. 1b) with the pH dropping to 3.3 over
800 min (see Fig. S1, ESIw). Following the gelation process by
ﬂuorescence, a slight red-shift was observed with time on
excitation of the system at 280 nm as has been reported
elsewhere for related naphthol-dipeptide systems,9 Fig. 2a.
In addition, an increase in intensity was observed compared
to the solution at high pH. This increase in intensity is
presumably due to the diﬀerence in environment at high and
low pH (micellar structures form ﬁbrous structures as the pH
drops, see Fig. S10, ESIw). A shoulder at 370 nm became more
pronounced on gelation, Fig. 2a. No further change was seen
after 120 min. Emission at 355 nm is suitable for energy
transfer to chromophores with a excitation maximum at this
wavelength. These include anthracene and certain dansyl
derivatives. Addition of a dansyl derivative (II, Fig. 1a) to a
concentration of 0.085 mM using a stock solution in methanol
(4.0 mol% relative to I) had no discernable eﬀect on the
hydrogelation. The change in pH was very similar to the
dipeptide solution alone on addition to GdL (Fig. S1, ESIw).
The rheological data for hydrogels prepared with and without
II were also very similar (Fig. S2, ESIw). After 100 min, there is
no change in the rheological data, implying that structure
evolution is complete by this point, despite the fact that the pH
Published on 11 May 2010. Downloaded by MIT Library on 3/21/2024 2:49:11 PM. View Article Online / Journal Homepage / Table of Contents for this issuehas yet
to reach equilibrium. Such behaviour has been
demonstrated previously for such systems.10,11 We probed
the eﬀect of adding II by ﬂuorescence. On excitation of the
naphthalene rings at 280 nm at high pH (before gelation), a
similar emission at 355 nm was observed along with a weak
emission at 540 nm (arising from the dansyl derivative in a
hydrophilic environment). The intensity of emission at 355 nm
was lower than in the absence of II, indicating that energy
transfer occurs to some degree even at high pH. On addition of
GdL, the intensity of the emission at 355 nm increased again
with time (and decreasing pH due to the slow hydrolysis of
GdL), Fig. 2b, but not to the same degree as in the absence
of II. The emission at 540 nm decreased, being replaced with
an increase in emission at 485 nm. This can be attributed to II
environment6
and therefore is strongly indicative of II being incorporated
within the self-assembled ﬁbres. This incorporation into the
self-assembled aggregates and resulting energy transfer can be
monitored with time, Fig. 2b. The ratio of emission at 355 nm
to 485 nm varied as expected with the concentration of II
added, Fig. 2c (see also Fig. S8, ESIw).
the acceptor molecules are simply
incorporated in the system via addition of a stock solution
and presumably interact via the hydrophobicity of the ﬁbres.
In addition to these systems, where a suitable chromophore is
added to a dipeptide-conjugate, we have also found that
similar results could be obtained using a system where both
Fig. 2 (a) Fluorescence spectra for a solution of I (2.2 mM) before
and after GdL addition. (b) Fluorescence spectra for a solution
of I (2.2 mM) with added II (0.085 mM) after GdL addition.
(c) Fluorescence spectra for solutions of I (2.2 mM) with diﬀerent
quantities (as indicated) of added II after 2 h.
donor and acceptor are conjugated to a dipeptide sequence.
Using a similar synthetic approach, a dialanine conjugated to
an anthracene unit was prepared (III, Fig. 3a, see ESIw for full
synthetic details). III alone does not form a stable hydrogel on
pH decrease; instead precipitation occurs (data not shown).
However, addition of 10 mL of a solution of (III) (12.1 mM) to
a solution of I (2.2 mM) at pH 10 resulted in a clear solution
as for I alone. No organic solvents were required for this.
On addition of GdL, a self-supporting hydrogel formed that
was again optically transparent, Fig. 3b. We again followed
the gelation with ﬂuorescence. A complex emission spectrum
between 390 and 500 nm appeared, replacing the emission
Fig. 1 (a) Structure of naphthalene-diphenylalanine (I) and dansyl
derivative (II). (b) I forms a transparent self-supporting hydrogel at a
concentration of 2.2 mM and a pH of 4 (left). A transparent self-supporting
gel was also formed in the presence of II (0.084 mM). (c) Energy transfer
occurs between I and II (white rectangle) hosted within the ﬁbres that form
via hydrogen-bonding between dipeptides leading to emission at 485 nm in
addition to emission at 355 nm from I alone.
Published on 11 May 2010. Downloaded by MIT Library on 3/21/2024 2:49:11 PM. View Article Onlinetuned by substituent. Similarly, a number of diﬀerent aromatic
moieties have been shown to be suitable for the preparation of
dipeptide hydrogels. Hence, future work will concentrate on
tuning the emission to allow energy transfer between a wider
range of molecules. This strategy could be useful for the design
of the next generation of light harvesting materials.
6 M. Montalti, L. S. Dolci, L. Prodi, N. Zaccheroni, M. C. A. Stuart,
K. J. C. van Bommel and A. Friggeri, Langmuir, 2006, 22,
2299–2303.
Fig. 3 (a) Structure of anthracene-dialanine, III. (b) A transparent
self-supporting gel is formed on addition of GdL (3.75 mg/mL) to a
solution of I (2.2 mM) containing III (0.015 mM). (c) Fluorescence
spectra for a solution of I (2.2 mM) with added III (0.060 mM) before
and after GdL addition.
of the naphthalene ring as the pH dropped, Fig. 3c. Again, the
ratio of emission at 355 nm to that at 440 nm varied with
concentration of added III (see Fig. S8, ESIw). Mixing gelators
has been used recently for cell culturing applications;12
the successful energy transfer demonstrated here shows that
co-assembly into ﬁbres is occurring in the current system as
opposed to phase separation into ﬁbres of one gelator and
ﬁbres of the other (where eﬀective energy transfer would not
be expected). This method may therefore be useful not just
for energy transfer applications, but also for probing the
co-assembly of gelators.
In conclusion, we have demonstrated that energy transfer is
possible from a naphthalene-dipeptide to dansyl or anthracene
chromophores as a hydrogel is formed. The formation of the
hydrogel is accompanied by an increase in p-stacking between
chromophores as long ﬁbrous structures are formed. This is a
highly robust system, with the use of GdL to alter the pH
allowing the kinetics of assembly to be probed in addition to
the initial and ﬁnal states. The simplicity of the gelator systems
makes this an attractive method for generating optically
transparent hydrogels capable of energy transfer. We note
that the emission spectra for the naphthalene rings can be

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[System] polypeptide self-assembly literature information extraction

[User]
In our work toward developing ester-containing self-assembling peptides as soft
biomaterials, we have found that a fluorenylmethoxycarbonyl (Fmoc)-conjugated
alanine-lactic acid (Ala-Lac) sequence self-assembles into nanostructures that
gel in water. This process occurs despite Fmoc-Ala-Lac's inability to interact
with other Fmoc-Ala-Lac molecules via β-sheet-like amide-amide hydrogen bonding,
a condition previously thought to be crucial to the self-assembly of
Fmoc-conjugated peptides. Experimental comparisons of Fmoc-Ala-Lac to its
self-assembling peptide sequence analogue Fmoc-Ala-Ala using a variety of
microscopic, spectroscopic, and bulk characterization techniques demonstrate
distinct features of the two systems and show that while angstrom-scale
self-assembled structures are similar, their nanometer-scale size and
morphological properties diverge and give rise to different bulk mechanical
properties. Molecular dynamics simulations were performed to gain more insight
into the differences between the two systems. An analysis of the
hydrogen-bonding and solvent-surface interface properties of the simulated
fibrils revealed that Fmoc-Ala-Lac fibrils are stronger and less hydrophilic
than Fmoc-Ala-Ala fibrils. We propose that this difference in fibril
amphiphilicity gives rise to differences in the higher-order assembly of fibrils
into nanostructures seen in TEM. Importantly, we confirm experimentally that
β-sheet-type hydrogen bonding is not crucial to the self-assembly of short,
conjugated peptides, and we demonstrate computationally that the amide bond in
such systems may act mainly to mediate the solvation of the self-assembled
single fibrils and therefore regulate a more extensive higher-order aggregation
of fibrils. This work provides a basic understanding for future research in
designing highly degradable self-assembling materials with peptide-like
bioactivity for biomedical applications.

Fmoc-L-Ala-L-Lac−OH Synthesis. Fmoc-L-Ala-L-Lac−OH was
synthesized using the general strategy for depsipeptide unit synthesis
introduced by Nguyen et al.6 Synthesis and puriﬁcation details as well
as identiﬁcation and purity analysis though NMR and HPLC-MS can
be found in the Supporting Information.
Gel Preparation. All gels characterized in this study were prepared
using a pH switch method. To a solution of either Fmoc-AA or Fmoc-
ALac, 1 equiv of 0.5 M NaOH was added in order to deprotonate and
dissolve the molecules. This mixture was vortex mixed and sonicated
just until clear and then immediately added to a small volume of 1
equiv of 0.1 M HCl in a separate vial to reduce the gel inhomogeneity.
Gelator solutions at high pH were always prepared fresh and gelled
immediately to prevent Fmoc removal. Although related work would
indicate that ester-containing Fmoc-ALac gels are relatively stable to
hydrolysis over the time course of analysis experiments,9 to minimize
these eﬀects only freshly prepared gels were analyzed. For 5 mg/mL
gels, the ﬁnal pH of Fmoc-AA gels was between 3.1 and 3.5, and the
ﬁnal pH of Fmoc-ALac gels was between 3.2 and 3.4.
Transmission Electron Microscopy (TEM). TEM samples were
prepared using 5 mg/mL gels. Approximately 10 μL of gel was placed
on the shiny side of a non-glow-discharged 300-mesh carbon-coated
copper grid (Electron Microscopy Sciences, Hatﬁeld, PA) and allowed
to adsorb for 2 min. Excess gel and water were wicked away using ﬁlter
paper, and the grid was placed shiny side down on top of a 10 μL drop
of DI water for 30 s to wash away any salts. After wicking the washing
water with ﬁlter paper, grids were placed shiny side down on top of a
10 μL drop of 4% uranyl acetate in water for 1 min. Finally, the uranyl
acetate solution was wicked away using ﬁlter paper, and the grids were
allowed to dry completely before imaging. Samples were imaged on
instrument with an
the nanometer
acceleration voltage of 80 kV, and the AMT Advantage HR 1kX1k
digital camera associated with the instrument was used to produce
digital micrographs.
Wide-Angle X-ray Scattering (WAXS). All gel samples were
prepared at 5 mg/mL as described above, allowed to dry slowly on
single-crystal quartz slides over 48 h in a humid environment, and then
dried completely in vacuo. Dried gel ﬁlms were subjected to X-rays
operating at a wavelength of 1.54059 Å (Cu Kα1) on a Scintag X1
θ−θ powder diﬀractometer. Diﬀraction patterns were analyzed using
Jade v9.1.1 (Materials Data, Inc.) software.
Fourier Transform Infrared Absorption (FTIR) Spectroscopy.
Gel samples for FTIR were prepared at 5 mg/mL and were prepared
by tightly sandwiching gels between two calcium ﬂuoride (CaF2)
windows, which were then seated within a ﬂow cell. Solution-phase
samples were prepared at 20 mg/mL in ethanol to ensure adequate
signal and were loaded into a sample cell composed of two CaF2
windows separated with 50-μm-thick PET spacers. All samples were
measured using a transmission conﬁguration. A Bruker Vertex 70
FTIR instrument equipped with a liquid-nitrogen-cooled MCT
detector collected and averaged 250 scans (4000 to 400 cm−1) of
each sample spectrum at a 2 cm−1 resolution. The ethanol background
spectrum was subtracted from all solution-phase sample spectra, and
an empty ﬂow cell background spectrum was subtracted from all gel-
phase sample data.
Circular Dichroism (CD) Spectroscopy. A Jasco J-815 instru-
ment was used to collect CD spectra. Gels (5 mg/mL) were placed in
a cylindrical demountable quartz cuvette with a path length of 0.1 mm
for analysis. Background scans consisted of water containing the same
concentrations of NaOH and HCl used to induce the gelation of
Fmoc-AA and Fmoc-ALac, and all scans were performed at a scan
speed of 200 nm/min with a data pitch of 0.5 nm and a response time
of 1 s. For each scan, three spectra were taken with background
subtraction and then averaged to give the ﬁnal spectrum. Linear
dichroism artifacts were characterized by rotating the cuvette 90, 180,
and 270° from the original orientation but were found to be small for
both Fmoc-AA and Fmoc-ALac gels.
Rheological Characterization. The storage and loss moduli of
Fmoc-AA and Fmoc-ALac gels were determined using an Anton-Paar
Physica MCR-101 rheometer with a parallel-plate geometry (top plate
diameter of 8 mm). Gels (5 mg/mL) for rheological analysis (n = 4 for
each system) were formed within polystyrene cloning cylinders (∼8
mm inner diameter) adhered to a polystyrene Petri dish with silicone
grease. Each gel had a volume of 200 μL. For all tests, the operating
height of the top plate was 2.5 mm to ensure good contact with the
gel’s top surface, which was often concave due to the meniscus formed
Figure 1. Both Fmoc-Ala-Ala (A, top) and Fmoc-Ala-Lac (A, bottom) self-assemble upon a pH change to form hydrogels (B, left) composed of
ﬁbrillar structures seen in TEM micrographs (B, right). Scale bar = 100 nm. X-ray diﬀraction patterns obtained from dried gel ﬁlms (C) show similar
peak positions and intensities, indicating that characteristic dimensions (such as ﬁbril diameter) are similar between the two systems. The green
arrows point out a unique, non-higher-order reﬂection in each diﬀraction pattern that may correspond to diﬀerent Fmoc−Fmoc or strand−strand
spacing in each system.
within the cloning cylinder during gelation. Because the gel diameter
matched that of the top plate, gels were not trimmed prior to analysis.
The strain amplitude used in the tests was 1%, and the angular
frequency was varied from 0.2 to 5 rad/s over eight points. For each
gel, the storage and loss moduli were calculated as the average of the
eight points.
Molecular Dynamics (MD) Simulations. MD simulations were
performed on Fmoc-AA and Fmoc-ALac ﬁbrils. The simulated ﬁbril
structures were based on previous results derived for the Fmoc-AA
ﬁbril (100 molecules that had already undergone a 160 ns simulation
and here proven to be stable).8 MD simulations were performed using
GROMACS (version 4.5.4). The atomic force ﬁeld parameters were
transformed from OPLS-AA,10 OPLS-AA/L,11 MM3,12 and our
previous work.8 Molecular dynamics simulations were performed on
the Texas Advanced Computing Center’s “Lonestar” supercomputer.
All simulations were applied periodic boxes with explicit TIP3P water
molecules, and all were subjected to steepest-descent energy
minimizations with 5000 maximum steps to remove high-energy
contacts. In order to keep the system stable at 300 K and 1 bar, 100 ps
NVT and 100 ps NPT equilibrations were applied, respectively. The
LINCS algorithm was applied to constrain bonds.13 Further details can
be found in our previous paper.8 Modeling parameters were derived
from previous studies,8,14 and ﬁnal simulated ﬁbril structures in .pdb
format are provided in the Supporting Information.
Melting-Temperature Simulations. For DNA and similar
materials stabilized largely by noncovalent intermolecular interactions,
the measurement of a melting temperature can be used to quantify the
material’s stability and predict sequence-dependent thermodynamic
behavior.15−17 In this work, we simulated a similar melting procedure
and obtained the melting curves to understand the eﬀect of replacing
the amide bond of Fmoc-AA with an ester bond. When the
temperature is gradually increased to some point (the so-called
melting temperature in this article),
the noncovalent interactions
between individual molecules within a ﬁbril structure will be disrupted;
therefore, the ﬁbril structure breaks apart and the fraction of the
molecular surface area exposed to solvent will increase. By measuring
the solvent -accessible surface area (SASA) with increasing temper-
ature from 670 to 870 K, we quantitatively estimated the eﬀect of
changing the terminal
residue (Ala to Lac) on noncovalent
intermolecular interactions between Fmoc-dipeptides within a self-
assembled ﬁbril structure. During the simulations, for each selected
temperature window at least one 50 ns MD simulation was performed
and the last 30 ns SASA results were averaged to plot. The standard
deviation for each point has been indicated as error bars in Figure 5A.
Potential of Mean Force. In our previous study,8 we observed
that the surface of a ﬁbril
is composed of both hydrophobic and
hydrophilic regions, which may lead to higher-order aggregation

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