Aromatic-aromatic interactions induce the self-assembly of pentapeptidic derivatives in water to form nanofibers and supramolecular hydrogels

Article abstract of DOI:10.1021/ja9088764

In this paper we report the conjugation of an aromatic moiety (pyrene (P), fluorene (F), or naphthalene (N)) to pentapeptides GAGAS (1), GVPVP (2), VPGVG (3), VTEEI (4), VYGGG (5), and YGFGG (6) to afford peptidic derivatives for exploring pentapeptide-based hydrogels as potential biomaterials. Most of these compounds (1F, 1P, 2F, 2P, 4F, 4P, 4N, 5F, 5N, 6F, 6P, and 6N) behave as molecular hydrogelators and can form hydrogels at minimum concentrations of gelation from 0.5 to 2.8 wt %. The fluorescence spectra of the hydrogels exhibit a significant red shift, indicating the interactions between the aromatic moieties in those hydrogels. The circular dichroism spectra indicate that the self-assembly of the hydrogelators affords helical β-sheet-like structures. Transmission and scanning electron microscopic examination reveals the nanofiber networks of these hydrogelators. In addition, rheological measurements show fair to excellent viscoelastic properties of these hydrogels. The balance of intermolecular aromatic-aromatic interactions and hydrogen bonds of these hydrogelators leads to their self-assembly in water and the formation of nanofibers as the matrixes of hydrogels. A total of 6 of these 18 pentapeptide derivatives-1N, 2N, 3F, 3P, 3N, and 5P-fail to form hydrogels under the conditions tested, likely due to unbalanced intermolecular interactions. This work suggests that aromatic-aromatic interactions are useful and favorable forces for creating molecular nanofibers and supramoleeular hydrogels.

Full text of DOI:10.1021/ja9088764

Published on Web 02/04/2010
Aromatic-Aromatic Interactions Induce the Self-Assembly of
Pentapeptidic Derivatives in Water To Form Nanofibers and
Supramolecular Hydrogels
Manlung Ma,†,‡ Yi Kuang,† Yuan Gao,† Yan Zhang,‡ Ping Gao,§ and Bing Xu*,†,‡
Department of Chemistry, Brandeis UniVersity, Waltham, Massachusetts 02454, and
Departments of Chemistry and of Chemical and Biomolecular Engineering, The Hong Kong
UniVersity of Science and Technology, Clear Water Bay, Hong Kong, China
Received October 17, 2009; E-mail: bxu@brandeis.edu
Abstract: In this paper we report the conjugation of an aromatic moiety (pyrene (P), fluorene (F), or
naphthalene (N)) to pentapeptides GAGAS (1), GVPVP (2), VPGVG (3), VTEEI (4), VYGGG (5), and
YGFGG (6) to afford peptidic derivatives for exploring pentapeptide-based hydrogels as potential
biomaterials. Most of these compounds (1F, 1P, 2F, 2P, 4F, 4P, 4N, 5F, 5N, 6F, 6P, and 6N) behave as
molecular hydrogelators and can form hydrogels at minimum concentrations of gelation from 0.5 to 2.8 wt
%. The fluorescence spectra of the hydrogels exhibit a significant red shift, indicating the interactions between
the aromatic moieties in those hydrogels. The circular dichroism spectra indicate that the self-assembly of
the hydrogelators affords helical or ꢀ-sheet-like structures. Transmission and scanning electron microscopic
examination reveals the nanofiber networks of these hydrogelators. In addition, rheological measurements
show fair to excellent viscoelastic properties of these hydrogels. The balance of intermolecular
aromatic-aromatic interactions and hydrogen bonds of these hydrogelators leads to their self-assembly in
water and the formation of nanofibers as the matrixes of hydrogels. A total of 6 of these 18 pentapeptide
derivativess1N, 2N, 3F, 3P, 3N, and 5Psfail to form hydrogels under the conditions tested, likely due to
unbalanced intermolecular interactions. This work suggests that aromatic-aromatic interactions are useful
and favorable forces for creating molecular nanofibers and supramolecular hydrogels.
as a change in temperature,6 ligand-receptor interaction,7
Introduction
variation of the pH value,8 enzymatic conversion,9 and irradia-
tion of light.10 Gels have found applications in many areas
including biocatalysis,11 bioanalysis,12 chemical sensing,13 food
processing,14 cosmetics,15 lubrication,16 drug delivery,17 tissue
engineering,18,19 wound healing,20 and controlling the fate of
In this paper we report the evaluation of a series of
pentapeptidic derivatives to elucidate the minimum structural
prerequisites and driving forces of a molecule containing a
pentapeptidic motif to act as a hydrogelator.1-4 Composed of
three-dimensional, elastic networks and a liquid in their
interstitial spaces, gels possess many useful properties, such as
forming hierarchical structures, flowing in response to a shear
force,5 and exhibiting phase transitions upon other stimuli such
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† Brandeis University.
‡ Department of Chemistry, The Hong Kong University of Science and
Technology.
§ Department of Chemical and Biomolecular Engineering, The Hong
Kong University of Science and Technology.
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A R T I C L E S
Ma et al.
cells.21 In the area of bioengineering and drug delivery, most
of hydrogels result from cross-linked natural or synthetic
polymers,22 which serve as the networks for encapsulating water
and therapeutic agents. In addition, the elastin-like polypeptides
have attracted numerous interest as smart biomaterials for drug
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Although they received less attention previously, small or
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molecular weight hydrogelators.28,33
hydrogelators consist of aromatic moieties and rely on
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Compared to alkyl chains, aromatic-aromatic interactions
possess several distinct merits: (i) Aromatic-aromatic interac-
tion normally is stronger than the van der Waals interaction
(London dispersion force) between alkyl chains, and it has been
well-established as a stabilizing force for proteins.36 Aromatic-
aromatic interaction should allow the formation of more stable
supramolecular polymers, thus resulting in mechanically strong
or stable hydrogels. (ii) Aromatic rings have relatively compact
volumes, which reduce the steric repulsion usually associated
with the bulky and long alkyl chains. (iii) The overlap between
aromatic rings normally adopts a plane-to-plane or an edge-to-
plane orientation,36 which leads to more predicable and efficient
self-assembly of the molecules in either the organic or aqueous
phase. To obtain the qualitative correlations, we chose to study
the gelation of pentapeptidic derivatives because pentapeptides
are a common motif of the epitope(s) that dictates the biological
function of proteins.37 Understanding and controlling the self-
assembly of pentapetides may lead to a new type of biomaterials
(in the form of hydrogels, nanofibers, or micelles) that mimic
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Encouraged by the promising prospects of oligopeptide-based
hydrogels demonstrated in recent works,19,30,39–42 we chose to
investigate the supramolecular hydrogels based on six pen-
tapeptidic sequences (Scheme 1). Some sequences of these
pentapeptides act as the repetitive epitopes in antigens or other
biomacromolecules. For example, pentapeptide VTEEI is the
repeat sequence from the Plasmodium falciparum blood stage
antigen Pf332 recognized by certain parasite-neutralizing anti-
bodies,43 sequences GVGVP and VPGVG are short, elastin-
like peptides and the epitopes found frequently in elastin,44 and
VYGGG exhibits an inhibitory effect in the binding monoclonal
antibody 10D11.45 These pentapeptides are selected in a random
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Scheme 1. Chemical Structures and Synthesis of Different Pentapeptide Epitopes To Attach on a Fluorenyl, Pyrenyl, or Naphthyl Group
way to evaluate the generality of the aromatic-aromatic
interaction for promoting self-assembly in water and hydroge-
lation.
water, which afford the networks of nanofibers and lead to
hydrogelation. A total of 12 of these 18 peptidic derivatives
behave as small-molecule hydrogelators and afford hydrogels
at a minimum gelation concentration of less than 2.8 wt %. A
total of 4 of these 18 pentapeptidess3F, 3P, 3N, and 5Psonly
form precipitates upon cooling their solution from around 70
°C to room temperature. 2N melts and forms an oily layer
separating from water, and 1N only results in a clear solution
instead of a hydrogel.
We chose three kinds of aromatic moieties, pyrene (P),
fluorene (F), and naphthalene (N), to covalently attach to
pentapeptides GAGAS (1), GVPVP (2), VPGVG (3), VTEEI
(4), VYGGG (5), and YGFGG (6). In their native forms, without
the attachment of any groups at either the N-termini or the
C-termini of the six different types of pentapeptides, none of
these pentapeptides act as hydrogelators.46 The proper balance
of intermolecular aromatic-aromatic interactions and hydrogen
bonds of these molecules result in molecular self-assembly in
The fluorescence spectra of the hydrogels and their corre-
sponding solutions indicate that aromatic-aromatic interactions
among the fluorenyl, pyrenyl, and naphthyl rings play a
significant role for hydrogelation, the circular dichroism spectra
show helical structure and/or ꢀ-sheet-like features in the
hydrogels formed by the self-assembly of these pentapeptidic
derivatives, and the transmission electron microscopy (TEM)
and scanning electron microscopy (SEM) images indicate the
presence of networks of the nanofiber of these hydrogelators in
the hydrogels. Some of the hydrogels also exhibit excellent
mechanical properties as demonstrated by the rheological study
of these hydrogels. Our results suggest that the well-balanced
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Table 1. Optical Images of the Hydrogels of the Pentapeptide
Derivatives
hydrophobicity and hydrophilicity of the small-molecule hy-
drogelatorsinwater,thestronginteractions(i.e.,aromatic-aromatic
interactions or other noncovalent interaction) between the
pentapeptidic chains of the hydrogelators, and the evenly
distributed nanofibers contribute to the observed excellent
mechanical property. As proved by the rheology test, 1F, 1P,
4F, 4P, and 4N exhibit an excellent mechanical property
compared with other pentapeptidic hydrogelators in this work.
These results indicate that the combination of aromatic-aromatic
interaction and hydrogen bonding provides a general and useful
methodology to understand and explore molecular self-assembly
in water for designing supramolecular nanomaterials.
Results and Discussion
Synthesis. As shown in Scheme 1, all the pentapeptides were
prepared by solid-phase peptide synthesis (SPPS) using 2-chlo-
rotrityl chloride resin and the corresponding NR-Fmoc-protected
amino acids with side chains properly protected by a tert-butyl
group. Followed by the removal of the Fmoc group, the first
amino acid was loaded onto the resin at the C-terminal. After
the resins were treated with 20% piperidine to remove the
protecting group, the next Fmoc-protected amino acid was
coupled to the free amino group using O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate/1-hydroxy-
benzotriazole (HBTU/HOBt)46 as the coupling reagent. The
growth of the peptide chain followed the established Fmoc SPPS
protocol.47 Each process was monitored by a Kaiser test46 or
chloranil test46 during the cleavage of the protecting group or
the loading of an amino acid. At the final step, the N-termini
of the pentapeptides were either protected by Fmoc or coupled
with 1-pyrenebutyric acid or 2-naphthaleneacetic acid to attach
the aromatic group (fluorenyl, pyrenyl, or naphthyl group) to
the pentapeptides.
Gelation Properties. Because the pentapeptidic derivatives
prepared in this work possess the carboxylic acid, a change of
pH (deprotonation or protonation) is able to trigger hydroge-
lation. Table 1 shows the optical images of the hydrogels of
these pentapeptidic derivatives. Most of the compounds (shown
in Scheme 1) are able to form hydrogels at a certain pH and
temperature (Table S-1, Supporting Information46). When the
pH values are higher than the listed values in Table S-1, the
hydrogels transform into clear solutions (i.e., gel-sol transition).
The general procedure for making the hydrogels in this work
is to dissolve the pentapeptide derivatives at a high pH solution
(deprotonating the carboxylic acid) and then to lower the pH
of the solution to trigger hydrogelation. Therefore, the pen-
tapeptidic derivatives can only self-assemble and gel with water
at a certain range of pH and ionic strength, similar to the
mechanism of the hydrogelation of other amphiphilic oligo-
peptides.30,40
pentapeptide, shows poor solubility in water.48,49 Carefully
adjusting the pH of the solution to 4.8 results in the hydrogel
of 2F. Compound 2F melts to become an oil above 70 °C and
remains separated from the aqueous phase even after the sample
is cooled back to room temperature, indicating that the proline
residue at the end of the pentapeptide likely hinders efficient
molecular packing. Compound 2N either dissolves in water at
pH higher than 4.0 or becomes a suspension at pH lower than
<2.5. Upon heating, it also melts and becomes an oil that
separates from water. This result indicates that proline is a poor
candidate to serve as a residue for the backbone of this type of
hydrogelators. Such an observation is supported by the fact that
none of the three compounds containing VPGVG (as the
pentapeptide segment) form a hydrogel at all the conditions we
tested. The proline residue, at the middle part of the pentapeptide
(VPGVG), apparently further disfavors the supramolecular order
that is necessary for the formation of the self-assembled
nanofibers and the hydrogelation. In addition, according to
molecular dynamic studies,50 VPGVG likely undergoes a
conformational transition from an open to a more compact
closed state upon an increase in temperature, which results in
precipitation of the derivatives of VPGVG (3F, 3P, and 3N).
Compounds 3F, 3P, and 3N all show a dramatic solubility
change with pH and exhibit low melting points, agreeing with
the fact that the bulky, nonplanar proline residue prevents the
efficient molecular packing.51
GAGAS, the epitope consisting of the least amount of bulk
side chains, appears to be quite hydrophilic, as indicated by
the fact that 1N is soluble in water even under low pH (<3.0)
and at high concentration (6.0 wt %). Bearing more hydrophobic
and larger aromatic rings than the naphthyl group, 1F and 1P
are hydrogelators that form hydrogels in an acidic condition
(pH < 3) at minimum concentrations of 1 wt % and 0.5 wt %,
respectively.
VTEEI, in which the pentapeptide bears two short alkyl side
chains and three carboxylic acids, shows the ability to form a
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A R T I C L E S
hydrogel over quite a wide range of pH when it attaches to a
fluorenyl, pyrenyl, or naphthyl group, suggesting that multiple
carboxylic acids, which can serve as both donors and acceptors
of hydrogen bonds, facilitate the self-assembly and hydrogelation
when there are sufficient hydrophobic interactions originating
from the pentapeptides themselves and the aromatic moieties.
Fluorene and naphthalene are appropriate hydrophobic groups
to assist the hydrogelation of VYGGG derivatives, but pyrene
appears to be too hydrophobic to allow 5P to dissolve in water
even under basic conditions. When the hydrophobic interactions
decrease, the pentapeptidic derivatives 5F and 5N form hydrogels.
In the case of YGFGG, when phenylalanine replaces one
glycine residue in 6P, proper aromatic-aromatic interaction with
the pyrene ring may be allowed, favoring the hydrogelation of
6P at pH around 5. Compound 6N shows a gelation ability
similar to that of 6P. Compound 6F forms a hydrogel at a higher
pH (7.8), suggesting efficient aromatic-aromatic interaction in
6F. To further prove that aromatic-aromatic interaction plays
a necessary role for the molecular self-assembly of the pen-
tapeptides in water, we replaced the naphthyl group with a
cyclohexyl group to connect to YGFGG or VTEEI at the
N-terminal of the pentapeptide. Compounds 4C and 6C only
afford precipitates (Figure S-1, Supporting Information46) and
fail to form a hydrogel under the same condition used for 4N
and 6N, respectively. This result, together with the fact that
neither 1N nor 2N is a hydrogelator, indicates that adequate
aromatic-aromatic interaction is crucial for molecular hydroge-
lation of these pentapeptide derivatives.
The comparison of the hydrogelators and the properties of
the pentapeptide derivative (Table S-1, Supporting Information)
suggest several notable trends of the properties of these
hydrogelators: (i) The increase of the volume of the alkyl side
chains leads to gelation at a relatively high pH. Because all the
pentapeptidic derivatives have a free carboxylic acid terminus,
a higher pH leads to more deprotonation and results in higher
hydrophilicity of the pentapeptidic derivatives. Therefore, the
pentapeptides with larger alkyl side chains need a higher pH to
achieve sufficient hydrophilicity to balance the increased
hydrophobicity caused by the side chains. This trend is also
consistent with the calculated log P values (Table S-2, Sup-
porting Information). (ii) For the same pentapeptide motif, the
derivatives bearing the naphthyl group always form gel under
more acidic conditions than those carrying either a fluorenyl or
a pyrenyl group, agreeing with the fact that naphthalene provides
weaker aromatic-aromatic interaction than the fluorenyl or
pyrenyl group does. (iii) The presence of a proline residue in
the peptide chain apparently disfavors the molecular self-
assembly, likely due to the twisted conformation of proline that
restricts this kind of pentapeptide chains to prevent efficient
packing between the molecules and hydrogelation. This steric
hindrance is amplified when the proline residue closes to the
aromatic ring, for example, in compounds 3F, 3P, and 3N, to
further destabilize the aromatic-aromatic interactions.
rings, suggesting that the aromatic-aromatic interactions are
pronounced in the corresponding hydrogels. The fluorescence
spectra of the hydrogels of 1F and 6F exhibit broad peaks at
348 and 365 nm, respectively. The fluorenyl groups in the
hydrogel of 1F, having less sterically hindered side chains than
those of other pentapeptides, could stack in a head-to-head
manner and also have multiple mosde of aromatic-aromatic
interactions, which likely contributes to the broadness of its
emission peak in the gel phase. The two emission peaks at 344
and 383 nm in the gel phase of 2F suggest two dominate modes
of aromatic-aromatic interactions of fluorenyl groups (likely
to be head-to-head and head-to-tail52). The hydrogels of 4F and
5F exhibit relatively narrow emission peaks at 343 and 346 nm,
respectively, suggesting fewer modes of aromatic-aromatic
overlap between the fluorenyl groups.
The emission peaks of 1P at around 453 nm indicate
dimerizationofpyrenylgroupsandsuggestefficientaromatic-aromatic
interaction between the pyrenyl groups in the hydrogel of 1P.
Other pentapeptide hydrogelators that have the pyrenyl groups
also shows peaks around 450 nm in the gel phase and the
absence of the same peaks in the corresponding solution phase,
indicating the dimerization of pyrene groups in the gel phase.
This observation is also consistent with the dimerization of
pyrene in other supramolecular hydrogels.41,53 Generally, there
are smaller red shifts in the hydrogels containing fluorenyl or
naphthyl groups compared to those containing pyrenyl ones.
As shown in Table S-4 (Supporting Information), the red shifts
for the hydrogelators containing pyrenyl groups are about 65
nm, and about 55 and around 35 nm for the hydrogelators
containing naphthyl groups and fluorenyl groups, respectively.
Circular Dichroism. As a helpful tool to determine the
secondary structures of proteins, circular dichroism (CD) is
useful for assessing the superstructures of these self-assembled
pentapeptide derivatives in the gel phase. The differences in
the circular dichroism spectra of the gels indicate that even two
hydrogels with the same sequence of amino acids can self-
assemble in different ways when the hydrophobic groups
differ.46 As shown in Figure 2, 1F and 1P share the common
feature of ꢀ-sheet structure according to the CD spectra. Both
of them exhibit a peak near 197 nm (ππ* transition) and a trough
near 216 nm (nπ* transition), suggesting the backbones of the
pentapeptides adopt similar ꢀ-sheet-like configurations.54 The
measured CD spectrum of 2F shows a distinct trough at 205
nm (ππ* transition) (characteristic peak of a random coil) and
a less pronounced trough at 240 nm (nπ* transition), indicating
that it is also contains a ꢀ-turn-like arrangement. This observa-
tion also resembles the reported results of other short, elastin-
like peptides.49,55 Compound 2P exhibits weak CD signals,
which agrees with the proline residue’s poor tendency for self-
assembly. Compound 4F possesses a ꢀ-turn structure, character-
ized by a peak near 218 nm and a trough near 240 nm in its
CD spectrum.56 With a different hydrophobic group attached
(52) Schweitzer, D.; Hausser, K. H.; Haenel, M. W. Chem. Phys. 1978,
29, 181–185. Channon, K. J.; Devlin, G. L.; Magennis, S. W.;
Finlayson, C. E.; Tickler, A. K.; Silva, C.; MacPhee, C. E. J. Am.
Chem. Soc. 2008, 130, 5487–5491.
Fluorescence Emission Spectra. Although it remains a chal-
lenge to determine the precise molecular arrangements of these
hydrogelators in the gel phase, the emission spectra of the
hydrogelators in the gel and solution phase offer useful and
relevant information regarding the interaction of the aromatic
rings. Table S-4 (Supporting Information) summarizes the
emission peaks in the solution and gel phases of all pentapeptide
derivatives that form hydrogels. Emission spectra of the
hydrogels (Figure 1) clearly indicate the existence of the
excimers of the naphthyl rings, the fluorenyl rings, or the pyrenyl
(53) Tovar, J. D.; Claussen, R. C.; Stupp, S. I. J. Am. Chem. Soc. 2005,
127, 7337–7345.
(54) Behanna, H. A.; Donners, J.; Gordon, A. C.; Stupp, S. I. J. Am. Chem.
Soc. 2005, 127, 1193–1200.
(55) Reiersen, H.; Clarke, A. R.; Rees, A. R. J. Mol. Biol. 1998, 283, 255–
264.
(56) Ford, S. J.; Wen, Z. Q.; Hecht, L.; Barron, L. D. Biopolymers 1994,
34, 303–313. Hecht, L.; Barron, L. D. Farady Discuss. 1994, 99, 35–
47.
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A R T I C L E S
Ma et al.
Figure 1. Fluorescence spectra of the hydrogels of the pentapeptidic derivatives (λex ) 265, 330, and 272 nm for fluorenyl, pyrenyl, and naphthyl groups,
respectively).
to the epitope VTEEI, the CD signals of 4P suggest more
R-helix rather than ꢀ-turn structure. The hydrogel of 4N only
exhibits weak CD signals, suggesting few ordered structures.
Compound 5F shares the common feature of a ꢀ-sheet of a
polypeptide in the CD signal (below 240 nm), and 5N exhibits
a very weak CD signal. The peak of 5F arising from 220 nm is
due to a peptide nπ* transition, and the trough at 205 nm
indicates a less ordered conformation and a characteristic peak
of a random coil. As shown in Figure 2E, 6F, 6P, and 6N share
similar ꢀ-sheet structure.57 They all have a trough in 217 nm
in the CD spectra due to the peptide nπ* transition. The peak
near 200 nm with comparable magnitude is due to exciton
splitting of the lowest peptide ππ* transition. The prominent
ꢀ-sheet characteristic was observed in the hydrogel of 6N.
Microstructures. According to the SEM and TEM images
of the hydrogels, the pentapeptidic hydrogelators self-assemble
into nanofibers that physically cross-link to form a fibrous
network as the matrix of the hydrogels. For example, the fibers
in 1P (Figure 3A,B) are short but entangled to form a dense
network, which could contribute to its relatively high storage
modulus value. The SEM image of 2F shows flat ribbons (Figure
3C), which could result from the aggregation of the nanofibers
of 2F (Figure 3D). In the SEM image of 2F, those aggregates
of the fibers appear quite random and exhibit a disordered
microstructure (Figure 3C), agreeing with the fact that the
hydrogel at 2F lacks thermoreversibility. On the other hand,
the cryo-dried hydrogel of 2P exhibits a quite ordered nano-
structure (Figure S-3, Supporting Information46) likely due to
the dimerization of the pyrenyl group. As shown in Figure 3E,F,
the fibrils in the hydrogel of 4P consist of bundles of the
supramolecular chains. Unlike 4F and 4P, the fibrils in the
hydrogel of 4N form discrete, short, and coil bundles (Figure
S-4F, Supporting Information46) after cryo-drying, which could
be responsible for its poor mechanical strength and little
difference between G′ and G′′ in the frequency sweep. As
revealed by SEM and TEM of 5F and 5N, the fibers of 5F
(57) Woody, R. W. Biopolymers 1969, 8, 669. Madison, V.; Schellma, J.
Biopolymers 1972, 11, 1041.
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Self-Assembly of Pentapeptidic Derivatives
A R T I C L E S
Figure 2. CD spectra of the hydrogels formed by the pentapeptidic hydrogelators.
spread out, although some of them randomly aggregate into
bundles. The randomness also contributes to the quite scattered
fibrous network (Figure 3G). We observed the 5F gel with a
3D network structure under SEM (Figure 3G), but the TEM
image shows an inefficiently cross-linked network (Figure 3H)
at a smaller scale. The rheology property also coincides with
the TEM image to suggest that 5F is a weak hydrogelator. In
the case of the derivatives of pentapeptide YGFGG, TEM
reveals that the three different hydrophobic groups afforded three
different kinds of microstructures in their hydrogel matrixes.
For example, 6P seems to self-assemble into short-stick-like
microcrystals (Figure 3J) that aggregate randomly to afford a
quite irregular packing, as shown in the SEM image (Figure
3I); 6F, forming the gel at a concentration as low as 1.8 wt %,
self-assembles into long and well-organized fibers (Figure S-6,
Supporting Information46).
the strain sweep and frequency sweep provide sufficient data
for studying the deformation and flow of materials, especially
in gel states. During strain sweep, the rheological properties of
hydrogels are independent of the strain up to a critical strain
level. In a frequency sweep, measurements are made at different
oscillation frequencies at a constant oscillation amplitude and
temperature. As shown in the experimental data of this work,
12 out of the 18 pentapeptide derivatives self-assemble to form
the supramolecular hydrogels. To understand their elastic
properties, we measured the storage modulus (G′) and the loss
modulus (G′′) of these supramolecular hydrogels. All 12
hydrogels studied here show linear viscoelastic responses at
strain amplitudes up to 1%. There is a clear domination of G′
over G′′ in all 12 hydrogels tested, and in addition, all show
negligible frequency dependence. These results should suggest
effective and viscoelastic hydrogels. As shown in Table 2, 1P
and 4P show rather large storage moduli though the concentra-
tions of the peptides in the hydrogels are only 1 and 1.5 wt %,
respectively. The storage moduli of 1P and 4P are much higher
Rheology. One of the most characteristic properties of gels
is their viscoelasticity, which reflects the mechanical properties
of the hydrogels. According to the rheological measurement,
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J. AM. CHEM. SOC. VOL. 132, NO. 8, 2010 2725

A R T I C L E S
Ma et al.
viscoelasticity. Compound 4F (2.35 wt %) is mechanically
stronger than 4N (1.34 wt %) in terms of viscoelastic behavior,
as shown in frequency sweep and strain sweep. Although it
forms a clear hydrogel upon heating or changing the pH, 5F
gives a relatively weak elasticity response during the test. In
the hydrogel of 6P, the phenyl ring on the tyrosine or
phenylalaninemayreducetheprobabilityofthearomatic-aromatic
overlap of the pyrenyl group, resulting in a weak viscoelastic
property of the gel.
According to the dynamic frequency sweep data, the G′ values
of each type of pentapeptide increase in the order of F < N <
P, which follows the extent of the red shifts (nearly constant
for F (35 nm), N (55 nm), and P (65 nm)) of the emission
spectra of the substituted pentapeptides. This agreement supports
the fact that aromatic-aromatic interaction-caused dimerization
is an important factor for the mechanical strength of the
hydrogels.
Cytotoxicity Assay. To verify the biocompatibility of the
pentapeptidic derivatives, we use the MTT assay to examine
the cell viability after incubating HeLa cells with these
pentapeptidic derivatives (Table 3). All the pentapeptides show
little cyctotoxicity before connecting to the aromatic groups,
as indicated by their IC50 values. Among all pyrenyl pentapep-
tidic derivatives, 1P and 5P exhibit lower cytotoxicity than the
others. After 3 days of incubation of HeLa cells with GAGAS,
1F, 1P, or 1N, the IC50 of 1P is 300 µM while the IC50 of 1F
and 1N is 190 and 193 µM, respectively, indicating that 1P is
more biocompatible than the derivatives carrying a fluorenyl
or naphthyl group. These results suggest that the dimerization
of 1P decreases the cytotoxicity associated with the pyrene
motif.
The cytotoxicity of other compounds also supports the above
observation. For example, the pyrenyl derivatives of GVGVP
and VPGVG show higher cytotoxicity than the other pyrene
derivatives (i.e., 4P, 6P, and 5P). According to the result of
the MTT assay, 3P exhibits the highest cytotoxicity, which is
consistent with the fact that the proline residue in the middle
of the peptide sequence disrupts the dimerization of the pyrenyl
group more significantly than in the other cases. These results
also agree with the fact that 2P exists mainly as a monomer at
a concentration of 140 µM (Figure 4).
In the case of 4P, the presence of three carboxylic acid groups
and one hydroxyl group apparently affects the dimerization of
the pyrenyl groups, which makes 4F or 4N more biocompatible
than the pyrenyl one.
In the case of the hydrogelators derived from VYGGG, the
hydrogelators consist of the three smallest amino acids and a
tyrosine residue, with the hydrophobic part (VY) at one end
and the hydrophilic part (GGG) at the other end. The dimer-
ization of the pyrenyl group and the solubility of the peptide
sequence reach an optimal point; therefore, all the derivatives
of VYGGG appear to be biocompatible.
In the case of YGFGG, the presence of two hydrophobic and
aromatic amino acids (Y, tyrosine; F, phenylalanine) in the
epitope YGFGG affects the aromatic-aromatic interaction that
is critical for dimerization of the pyrenyl groups. With the
presence of valine in the case of 5P, the glycine residues possess
less impact on the aromatic-aromatic interaction of the pyrenyl
group than the phenylalanine does in 6P. The two hydrophobic
aromatic parts are separated from Gly; thus, the toxicity of 6P
originates from the dominant monomeric form of 6P rather than
a dimer at 231 µM (when compared with 5P, the most
biocompatible one (Figure 4)). Similar to other pyrene-contain-
Figure 3. Scanning electron micrographs (A, C, E, G, I) and transmission
electron micrographs (B, D, F, H, J) of the self-assembled nanofibers in
the cryo-dried hydrogels of 1P (A, B), 2F (C, D), 4P (E, F), 5F (G, H),
and 6P (I, J).
than those of polymeric hydrogels at similar concentration (e.g.,
the polymeric hydrogels formed by self-assembly of copolypep-
tides58 and the cross-linking of peptides59). Because of the least
steric bulk of the side chains on pentapeptide epitope GAGAS
in 1P, multiple carboxylic acid groups (VTEEI) in 4P, and the
efficient packing via aromatic-aromatic interaction through
pyrenyl groups, compounds 1P and 4P exhibit excellent
(58) Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.;
Pochan, D.; Deming, T. J. Nature 2002, 417, 424–428.
(59) Hu, B. H.; Messersmith, P. B. J. Am. Chem. Soc. 2003, 125, 14298–
14299.
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Self-Assembly of Pentapeptidic Derivatives
A R T I C L E S
Table 2. Summary of the Rheological Properties of the Hydrogels
dynamic strain sweep (Pa)
dynamic frequency sweep (Pa)
compd
G′
G′′
G′
G′′
1F
1P
2F
2P
4F
4P
4N
5F
5N
6F
6P
6N
224.87 ( 29.74
1600.23 ( 31.02
196.58 ( 3.10
130.77 (8.19
653.79 ( 26.54
20190.24 ( 1172.84
865.16( 102.63
93.18 ( 2.98
22.77 ( 1.77
205.83( 16.29
21.48 ( 1.43
15.47 ( 2.76
113.52( 15.15
3174.13 ( 389.64
133.22 ( 5.48
24.31 ( 2.06
3.24 ( 0.26
158.22 ( 15.14
1171.56 ( 19.69
79.80 ( 5.25
12.93 ( 2.93
124.09( 34.41
12.93 ( 2.93
4.33 ( 1.69
20.12 ( 3.16
450.42 ( 62.57
17945.33 ( 580.61
611.84( 89.52
49.32 ( 4.67
66.71( 8.93
13382.41 ( 239.35
108.37 ( 24.78
31.06 ( 2.45
3.41 ( 0.49
10.65 ( 1.31
8.65 ( 1.31
12.73 (0.43
3.45 ( 0.22
11.02 ( 1.64
4.06 ( 0.31
14.31 ( 0.52
3.29 (0.17
5.58 ( 0.08
13.63 ( 2.84
3.69 ( 0.41
13.18( 0.11
8.22 (1.87
2.65 ( 0.36
Table 3. Summary of the IC50 Values of All the Compounds
hydrogels should be instructive for designing peptide hydrogels
as promising nanomaterials for biomedical applications. To
further investigate the correlation between dimerization of
pyrenyl derivatives and their cytotoxicity, we obtained the
emission spectra (Figure 4) of the pyrene derivatives at 140
µM (the lowest concentration for 3P to exhibit toxicity). Figure
4 indicates that the intensities of the emissions of the monomeric
forms follow a decreasing order: 3P > 4P > 2P ≈ 6P > 1P >
5P. This trend matches well with the decrease of the cytotoxicity
of those compounds. Compound 5P shows no toxicity at high
concentration (1.5 mM), which has the lowest intensity of
emission originating from the monomeric pyrene. The dimer-
ization of pyrene can enhance the biocompatibility of the
compounds and minimize the toxicity caused by the pyrene
monomer. This result may provide a useful guide to minimize
the unintended cytotoxicity of pyrene.
against HeLa Cells
epitope
F
P
N
GAGAS
GVGVP
VPGVG
VTEEI
VYGGG
YGFGG
>1.5 mMa
>1.5 mMa
>1.5 mMa
>1.5 mMa
>1.5 mMa
>1.5 mMa
190 µM
300 µM
231 µM
140 µM
160 µM
>1.5 mMa
231 µM
193 µM
>1.5 mMa
>1.5 mMa
400 µM
>1.5 mMa
>1.5 mMa
350 µM
>1.5 mMa
655 µM
>1.5 mMa
445 µM
a No toxicity detected at the highest concentration tested (1.5 mM).
Conclusion
In summary, we have demonstrated that an aromatic group
(fluorenyl, pyrenyl, or napthyl group), as an effective hydro-
phobic group and an origin of aromatic-aromatic interaction,
brings a powerful driving force for the formation of supramo-
lecular hydrogels. The use of aromatic groups can be a useful
alternative to complement the use of alkyl chains, a well-
established approach that provides hydrophobic force in the
hydrogels of peptide amphiphiles developed by Stupp and co-
workers.60 In addition, the aromatic-aromatic interaction should
be useful for developing peptide-based organogels as Banerjee
et al. has shown that at least two phenylalanine residues are
required for pentapeptide derivatives to form organogels.61
These results not only provide a starting point and platform to
design and study a range of pentapeptide derivatives, but also
offer a simple experimental set for potentially developing the
theoretical treatment of supramolecular hydrogelation. Although
proline-based hydrogelators have been reported,62 the presence
of proline (a well-known ꢀ-sheet blocker63) between the
hydrophilic part strongly affects the noncovalent interaction,
which may offer a useful way to refine and control supramo-
lecular interactions within the hydrogels. Although it remains
Figure 4. (A) Emission spectra of all pyrenyl pentapeptidic derivatives at
140 µM with λex ) 330 nm. (B) Intensity of the emission at 393 nm and
the cytotoxicity of the compounds at 140 µM.
(60) (a) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 5133–5138. (b) Palmer, L. C.; Stupp, S. I. Acc. Chem.
Res. 2008, 41, 1674–1684.
ing pentapeptides, the cytotoxicity of 6P originates from the
monomeric 6P.
As a general trend observed in this work, the dimerization
of pyrenyl groups can improve the biocompatibility of the
hydrogelators. The biocompatibility of these pentapeptide-based
(61) Banerjee, A.; Palui, G. Soft Matter 2008, 4, 1430–1437.
(62) Rodriguez-Llansola, F.; Escuder, B.; Miravet, J. F. J. Am. Chem. Soc.
2009, 131, 11478–11484.
(63) (a) Chou, P. Y.; Fasman, G. D. Annu. ReV. Biochem. 1978, 47, 251–
276. (b) Wood, S. J.; Wetzel, R.; Martin, J. D.; Hurle, M. R.
Biochemistry 1995, 34, 724–730.
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the Hong Kong Research Grant Council (RGC), a start-up grant
from Brandeis University, and the NSF (Grant MRSEC 0820492).
to be verified, this work suggests that the aromatic-aromatic
interaction in water may control and assist the formation of
hydrogen bonding that is critical for the self-assembly of small
molecules in water. The biocompatibility of VYGGG and its
derivatives may allow them to serve as hydrogels for tissue
engineering. Also, elastin-like hydrogels can potentially serve
as attractive building blocks for a range of applications in
nanomedicine.
Supporting Information Available: Abbreviations list, de-
tailed experimental section, synthesis description, and charac-
terization (1H and 13C NMR, high-resolution mass spectrometry
(HRMS), and CD analysis) of the pentapeptidic derivatives. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Acknowledgment. We acknowledge financial support from the
Human Frontier Science Program (HFSP; Grant RGP 0056/2008),
JA9088764
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2728 J. AM. CHEM. SOC. VOL. 132, NO. 8, 2010