Self-assembling tripeptide as organogelator: The role of aromatic π-stacking interactions in gel formation

Article abstract of DOI:10.1080/10610270903254142

While the terminally protected tripeptide Boc-Phe-Gly-m-ABA-OMe I (m-ABA, meta-amino benzoic acid) is an excellent gelator of aromatic organic solvents, another similar tripeptide Boc-Leu-Gly-m-ABA-OMe II, where the Phe residue of peptide I is replaced by Leu, cannot form gels with the same solvents. The morphology of the gels of peptide I, characterised by the field-emission scanning electron microscopy and high-resolution transmission electron microscopy, reveals the formation of nanofibrous networks which are known to encapsulate solvent molecules to form gels. The wide-angle X-ray scattering studies of the gels suggest the β-sheet-mediated self-assembly of peptide I in the formation of a nanofibrous network, where π-stacking interactions of Phe play an important role in the self-assembly and gel formation. The dried gel of peptide I observed between crossed polarisers after binding with a physiological dye, Congo red, shows a bluish-green birefringence, a characteristic of amyloid fibrils.

Full text of DOI:10.1080/10610270903254142

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Supramolecular Chemistry
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Self-assembling tripeptide as organogelator: the role of
aromatic π-stacking interactions in gel formation
Arpita Dutta a , Dipankar Chattopadhyay b & Animesh Pramanik a
a Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata, 700009, India
b Department of Polymer Science and Technology, University of Calcutta, 92, A.P.C. Road,
Kolkata, 700009, India
Version of record first published: 18 Jan 2010.
To cite this article: Arpita Dutta , Dipankar Chattopadhyay & Animesh Pramanik (2010): Self-assembling tripeptide as
organogelator: the role of aromatic π-stacking interactions in gel formation, Supramolecular Chemistry, 22:2, 95-102
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Supramolecular Chemistry
Vol. 22, No. 2, February 2010, 95–102
Self-assembling tripeptide as organogelator: the role of aromatic p-stacking
interactions in gel formation
Arpita Duttaa, Dipankar Chattopadhyayb and Animesh Pramanika*
b
aDepartment of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India; Department of Polymer Science and
Technology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India
(Received 30 April 2009; final version received 4 August 2009)
While the terminally protected tripeptide Boc-Phe-Gly-m-ABA-OMe I (m-ABA, meta-amino benzoic acid) is an excellent
gelator of aromatic organic solvents, another similar tripeptide Boc-Leu-Gly-m-ABA-OMe II, where the Phe residue of
peptide I is replaced by Leu, cannot form gels with the same solvents. The morphology of the gels of peptide I, characterised
by the field-emission scanning electron microscopy and high-resolution transmission electron microscopy, reveals the
formation of nanofibrous networks which are known to encapsulate solvent molecules to form gels. The wide-angle X-ray
scattering studies of the gels suggest the b-sheet-mediated self-assembly of peptide I in the formation of a nanofibrous
network, where p-stacking interactions of Phe play an important role in the self-assembly and gel formation. The dried gel
of peptide I observed between crossed polarisers after binding with a physiological dye, Congo red, shows a bluish-green
birefringence, a characteristic of amyloid fibrils.
Keywords: peptide; gelator; p-stacking interactions; self-assembly; nanofibrous network
Introduction
observed that dipeptides containing m-ABA, a substituted
g-amino butyric acid with an all trans-extended configur-
ation, can generate amyloid-like fibrils through b-sheet-
mediated self-assembly (40). Therefore, tripeptides I and
II have been designed to incorporate m-ABA for creating
nanofibrous networks necessary for trapping solvent
molecules in gel formation. Since aromatic p–p
interactions provide favourable energetic contributions as
well as order and directionality in the self-assembly of
amyloid structures (41), peptide I has been designed to
incorporate Phe. In peptide II, the Phe residue of peptide I
has been replaced by Leu to examine the role of p-stacking
interactions in the gelation process. The peptides were
synthesisedusingconventionalsolution-phasemethodology.
The gels were characterised using field-emission scanning
electron microscopy (FE-SEM), high-resolution trans-
mission electron microscopy (HRTEM), wide-angle X-ray
scattering (WAXS) studies and Congo red binding assay.
Low-molecular-weight organogelators are important due
to their potential applications in the field of biomaterials
(1, 2), nanotechnology (3, 4), pollution control (5), oil-spill
recovery (6), food or cosmetic additives (7), preparing
vehicles for drug delivery system (8), light-harvesting
materials (9, 10), dental composite carriers (11), in
preparing dye-sensitive solar cells (12) and others (13–16).
Generally, 3D fibrous networks derived from the self-
assembly of low-molecular-mass organic compounds
encapsulate bulk solvents under appropriate conditions
to form gels. A suitable molecular building block
self-assembles through various non-covalent interactions
such as hydrogen bonds (17–19), p–p-stacking (20–22),
metal coordination (23, 24) and van der Waals interactions
(25–27) to form supramolecular gels. Although there are
several examples of the formation of organogels through
the self-assembly of small organic molecules (28–33),
examples of low-molecular-weight acyclic peptide-based
organogelators are relatively less (34–39).
Results and discussion
It has been observed that p–p interactions play an
important role in gel formation (20–22, 34). Therefore,
studies are necessary with designed molecules to gain
more insights into the influence of p-stacking interactions
in gel formation. In this context, we chose two tripeptides
Boc-Phe-Gly-m-ABA-OMe (I) and Boc-Leu-Gly-m-
ABA-OMe (II) (m-ABA, meta-amino benzoic acid) to
study the gel formation (Figure 1). Previously, it has been
Gelation property of peptides
In the course of the investigation, it has been observed
that peptide I can form thermoreversible transparent gels
in various organic solvents such as benzene, toluene,
m-xylene and 1,2-dichlorobenzene (Table 1). The gelating
capabilities of the terminally protected tripeptide I
have been examined in a variety of organic solvents
*Corresponding author. Email: animesh_in2001@yahoo.co.in
ISSN 1061-0278 print/ISSN 1029-0478 online
q 2010 Taylor & Francis
DOI: 10.1080/10610270903254142
http://www.informaworld.com

96
A. Dutta et al.
Figure 1. Schematic diagrams of peptides I and II.
by dissolving a small amount of the compound in 0.5 ml of
the solvent under investigation by heating. Upon cooling
below the temperature of gelation (Tgel), the complete
volume of the solvent is immobilised to form gel.
The gelation has been confirmed by the inverted test-tube
method (Figure 2) (42). Peptide I is found to be a quite
efficient organogelator to gelify many toxic aromatic
solvents at very low concentration (,2.5%, w/v), in
certain solvents (Table 1), which is a characteristic feature
of a potential gelator. The gels included in Table 1 are
stable at room temperature for few weeks and stable
towards shaking. The change in sol–gel phase transition
temperature with respect to gelation concentration of
peptide I in toluene is presented in Figure 3, where a
steady increase of Tgel is observed with the increase in
gelation concentration (Table 2). It is noteworthy that only
peptide I can form gels, but peptide II, where the Phe
residue of peptide I has been replaced by Leu, cannot form
gels. Another important feature is that peptide I generally
forms gels with aromatic solvents. It fails to form gels with
non-aromatic solvents such as chloroform, methanol,
acetonitrile, acetone and ethylacetate (Table 1). Therefore,
it is apparent that the Phe residue of peptide I plays an
important role in self-association and gelation with
aromatic organic solvents.
Figure 2. Images of inverted test tubes containing transparent
stable tripeptide I gels in: (a) benzene; (b) toluene; (c) m-xylene
and (d) 1,2-dichlorobenzene.
pattern of self-assembly in small peptides. In order to
investigate the existence of intramolecular hydrogen
bonding and peptide conformation in aromatic organic
medium, the solvent dependence of the NH chemical shifts
was examined by NMR titrations (43). In this experiment,
a solution of peptide I in C6D6 (3 mM) was gradually
titrated against polar (CD3)2SO. The changes in the
chemical shifts are presented in Figure 4. The solvent
titration shows that by increasing the percentage of
(CD3)2SO in C6D6 from 0 to 6.7% (v/v), the net changes in
the chemical shift (Dd) values for Phe(1)ZNH,
Gly(2)ZNH and m-ABA(3)ZNH are 1.7, 2.3 and
1.4 ppm, respectively (Figure 4). The Dd values demon-
strate that all the NH groups of peptide I are free and
solvent exposed. In a similar experiment with peptide II,
the net changes in the chemical shift (Dd) values for
Leu(1)ZNH, Gly(2)ZNH and m-ABA(3)ZNH are found
to be 1.8, 2.1 and 1.3 ppm, respectively (Figure 5). Here
also, the Dd values imply that all the three NH groups of II
Proposed model of molecular packing in gel
The self-assembly of peptide I is responsible for producing
supramolecular gels. Therefore, it is necessary to know the
conformation of the peptide which generally controls the
Table 1. Gelation properties of tripeptide I in various organic
solvents.
Melting point
of the gel (8C)
Solvent
w/v %
State
Benzene
Toluene
m-Xylene
1,2-Dichlorobenzene
Chloroform
Methanol
Acetonitrile
Acetone
Ethylacetate
3.90
1.12
2.40
2.10
–
–
–
–
–
Gel
Gel
Gel
Gel
Soluble
Soluble
Soluble
Soluble
Soluble
71
66
68
68
–
–
–
–
–
Figure 3. The change in sol–gel phase transition temperature
with respect to the gelation concentration of peptide I in toluene.

Supramolecular Chemistry
Table 2. The sol–gel phase transition temperature (Tgel) of
tripeptide I in toluene at various gelation concentrations.
97
Concentration (w/v %)
Tgel
3.70
3.05
2.24
1.77
1.46
1.24
1.12
71.0
70.2
69.0
68.0
67.0
66.2
66.0
are solvent exposed. The results show that peptides I and II
do not possess any intramolecular hydrogen bond,
indicating an extended conformation in the aromatic
solvent C6D6, which is further supported by far-UV
circular dichroism (CD) measurement in methanol.
The CD pattern of peptides I and II corresponds to
extended structures (Figure 6). Therefore, the results of
solvent-dependent NMR titrations and CD measurement
favour the conclusion that peptides I and II mostly remain
in extended conformations. Generally, the extended
structure of small peptides promotes b-sheet-mediated
self-assembly (40).
Figure 5. NMR solvent titration curve for NH protons in
peptide II (initial concentration of II, 3 mM in C6D6).
presence of the molecular duplex in those states also
(Table 3). The duplexes of peptide I can stack in the c
direction through the intermolecular hydrogen bonds
between the peptides’ backbones and through the p–p
interactions between the phenyl rings of m-ABAs and the
phenyl rings of the Phe residues to form b-sheet layers
(Figure 8(b)). Several such b-sheet layers of peptide I can
stack in the a direction to form supramolecular b-sheet
structures. The formation of this type of molecular duplex
and the packing like ‘cross b-model of amyloid fibrils’
(44–48) of peptide containing m-ABA have also been
reported previously (40). The d-spacings at 5.33, 4.99,
The WAXS pattern of peptide I was measured to
obtain the information on the molecular packing in the gel
state. The gel of peptide I prepared from toluene (4.0%
w/v) exhibits a well-resolved X-ray diffraction pattern
(Figure 7). A sharp diffraction signal corresponding to a
˚
d-spacing of 41.66 A in the wet gel (Table 3) is
approximately close to the length of the molecular duplex
˚
4.07 and 3.26 A at wide angles (2u , 168) in the WAXS
˚
of peptide I, 39.5 A, calculated by the INSIGHT II graphic
pattern are characteristic of the p–p stacking distances
between Phe/Phe, Phe/solvent, m-ABA/m-ABA and
m-ABA/solvent, etc. observed in the bulk solid, dried
gel and wet gel state of peptide I (Figure 7; Table 3).
The observed broad peak in the diffraction pattern of the
molecular modelling program. In the duplex, two
antiparallel molecules of peptide I are locked through
the intermolecular hydrogen bonds between two m-ABA
˚
moieties (Figure 8(a)). Similar d-spacing values, 41.11 A
˚
in the bulk solid and 41.62 A in the dried gel, indicate the
Figure 4. NMR solvent titration curve for NH protons in
peptide I (initial concentration of I, 3 mM in C6D6).
Figure 6. CD curves of peptides I and II.

98
A. Dutta et al.
Figure 7. Comparative WAXS patterns of the bulk solid of
peptide I, dried gel obtained from peptide I–toluene (4.0% w/v)
and wet gel of peptide I–toluene (4.0% w/v).
Figure 8. A schematic representation of a tentative model for
the molecular packing of the gel phase material of peptide I. (a)
Showing the formation of a molecular duplex and (b) showing the
stacking of the duplexes in the c direction to form a b-sheet layer.
wet gel is due to heavy scattering from solvent molecules
(Figure 7).
The FT-IR spectroscopy is a useful tool to get
information about the peptide conformation and inter-
molecular hydrogen bonding. In the solid state (using the
KBr matrix), intense bands have been observed for peptide
I (dried gel) at 3340 cm21 and for peptide II (bulk solid) at
3342 cm21, which indicates the presence of strongly
hydrogen-bonded NH groups (Figure 9). No band has been
observed around 3400 cm21 for any of these two peptides
in the solid state. The absence of this band suggests that all
the NHs for peptides I and II in the solid state are involved
in intermolecular hydrogen bonding. The CO stretching
band around 1654–1695 cm21 (amide I) and the NH
bending and the CZN stretching peak near 1527 cm21
(amide II) suggest the presence of an intermolecularly
hydrogen-bonded b-sheet conformation for peptides I and
II (49).
The regular, parallel alignment of the phenyl rings of
Phe in the b-sheet structure of peptide I (Figure 8(b)) helps
in constructing a nanofibrous network which interacts with
the aromatic solvents through p–p interactions to form
gels. On the other hand, peptide II, where the Phe residue
of peptide I has been replaced by Leu, cannot form gels
due to the lack of p–p interactions.
Morphological studies of gels
The morphology of the assembled peptides I and II in
organic solvents was studied using FE-SEM and HRTEM.
The FE-SEM image of the dried gel of peptide I obtained
from toluene shows the formation of a nanofibrous
network (Figure 10(a)). These fibres are found to be
100–120 nm in width and several hundreds of micro-
metres long (Figure 10(b)). It indicates that peptide I
possesses the directional forces required for the uni-
directional intermolecular interactions between the gelator
molecules to form the fibrous nanoscale architecture (50).
The HRTEM images of the dried gel of peptide I also
exhibit a nanofibrous network that can entrap suitable
Table 3. Major peaks in the WAXS pattern for peptide I.
˚
d-Spacing in A (position in 82u)
Bulk solid
41.11 (2.15), 35.18 (2.51), 5.33 (16.63),
4.99 (17.75), 4.07 (21.85), 3.26 (27.32),
2.15 (41.99)
Wet gel
41.66 (2.12), 35.19 (2.51), 5.42 (16.33),
4.99 (17.75), 2.08 (43.54)
Dried gel
41.62 (2.12), 35.12 (2.51), 5.11 (17.34),
2.15 (42.00), 2.08 (43.56)

Supramolecular Chemistry
99
network which is necessary to form gels. When the stained
dried gels of peptide I are placed under cross polarisers,
the Congo red-bound nanofibrils exhibit a typical bluish-
green birefringence, a characteristic of amyloid fibrils
(Figure 13) (51). The result supports the proposed model of
b-sheet-mediated self-assembly of peptide I (Figure 8(b))
in creating a nanofibrous network in the gel state.
Conclusions
This study shows that tripeptide I, where the backbone is
modified by the extended structure of m-ABA, forms
molecular duplexes, which on stacking through intermole-
cular hydrogen bonds and p–p interactions create layers of
b-sheets. The layers upon further aggregation generate a
nanofibrous network which encapsulates a variety of
aromatic organic solvents at very low concentration to
form thermoreversible transparent organogels. On the other
hand, another similar tripeptide II, where the Phe residue of
peptide I is replaced by Leu, cannot entrap organic solvents.
Although peptide II generates filamentous fibrils through
b-sheet-mediated self-assembly, the non-aromatic Leu
residue cannot provide favourable energy, order and
directionality in the self-assembly to form a nanofibrous
network which is necessary to entrap solvent molecules.
From this observation, it is evident that the Phe residue is
crucial for the self-assembly and gelation. The present study
also shows that even small peptides containing only one Phe
can form gels if the backbone of the peptide is suitably
modified for b-sheet formation. This observation holds
future promise to design and construct low-molecular-
weight gelators.
Figure 9. FT-IR spectra at the regions 3200–3500 and
1500–1750 cm21 of peptides I (dried gel) and II (bulk solid).
solvent molecules effectively (Figure 11(a,b)).
The FE-SEM image of peptide II obtained from toluene
also shows the formation of filamentous fibrillar
morphology. These fibres are found to be 400–500 nm
in width and several hundreds of micrometres long
(Figure 12), therefore they fail to generate a nanofibrous
Experimental
Synthesis of peptides
Peptides I and II were synthesised by conventional
solution-phase methods (52). The Boc group was used for
Figure 10. (a) FE-SEM image of the dried gel derived from peptide I in toluene showing a nanofibrous network; (b) the enlarged image
of the fibrils of the dried gel of peptide I with an average diameter of 100–120 nm.

100
A. Dutta et al.
Figure 11. HRTEM images (a) and (b) of the dried gel derived from peptide I in toluene showing the formation of a nanofibrous
network.
N-terminal protection and the C-terminus was protected as
a methyl ester. N-terminal deprotection was performed
using CF3CO2H. Couplings were mediated by dicyclohex-
ylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt).
All intermediates were characterised by thin layer
chromatography on silica gel and used without further
purification. The final peptides were purified by column
chromatography using silica gel (100–200 mesh) as the
stationary phase and ethyl acetate–petroleum ether
mixture as the eluent. Peptides I and II were characterised
by NMR and IR.
in water and washed with diethyl ether. The pH of the
aqueous solution was adjusted to 8 with sodium
bicarbonate and extracted with ethyl acetate. The extracts
were pooled, washed with saturated brine, dried over
sodium sulphate and concentrated to a highly viscous
liquid that gave a positive ninhydrin test. This dipeptide
free base was added to a well ice-cooled solution of Boc-
Phe-OH (1.29 g, 4.87 mmol) in DMF (6 ml) followed by
DCC (1.50 g, 7.30 mmol) and HOBt (0.66 g, 4.87 mmol).
The reaction mixture was stirred at room temperature for
3 days. The residue was taken up in ethyl acetate and DCU
was filtered off, and, to the filtrate, 20 ml of ethyl acetate
was added. The organic layer was washed with 2 M HCl
(3 £ 50 ml), 1 M Na2CO3 solution (3 £ 50 ml) and brine,
dried over anhydrous Na2SO4 and evaporated in vacuo, to
yield a white solid. Purification was done using silica gel
as the stationary phase and ethyl acetate–petroleum
ether mixture as the eluent. Yield: 1.8 g (81.1%).
Mp ¼ 141–1438C; Elemental analysis calcd for
C24H29N3O6 (455.51): C, 63.28; H, 6.42; N, 9.22%;
Found: C, 63.18; H, 6.33; N, 9.15%. IR (KBr): 3340, 2928,
Boc-Gly-m-ABA-OMe (I). Peptide I was prepared follow-
ing the reported procedure (53).
Boc-Phe-Gly-m-ABA-OMe (I). To compound 1 (1.50 g,
4.87 mmol), trifluoroacetic acid (3 ml) was added at 08C
and stirred at room temperature. The removal of the Boc
group was monitored by TLC. After 3 h, trifluoroacetic
acid was removed under reduced pressure to afford the
crude trifluoroacetate salt. The residue was taken up
Figure 13. A dried gel of peptide I–toluene observed between
crossed polarisers after binding with Congo red shows a bluish-
green birefringence.
Figure 12. FE-SEM image of the fibrils of peptide II generated
from toluene with an average diameter of 400–500 nm.

Supramolecular Chemistry
1H NMR 300 MHz (CDCl3,
NMR experiments
101
1695, 1654, 1527 cm21
.
1
All H and 13C NMR spectra of peptides I and II were
recorded on a Bruker Avance 300 model spectrometer
operating at 300 and 75 MHz, respectively. The peptide
d ppm): 9.11 (m-ABAZNH, 1H, s), 8.21 [Ha (m-ABA),
1H, s], 7.96 [Hd (m-ABA), 1H, d, J ¼ 7.8 Hz], 7.74 [Hb
(m-ABA), 1H, d, J ¼ 7.8 Hz], 7.41 [Hc (m-ABA), 1H, t,
J ¼ 8.1 Hz], 7.21–7.30 (phenyl ring protons), 7.01
(GlyZNH, 1H, t), 5.25 (PheZNH, 1H, d, J ¼ 7.6 Hz),
4.45 (CaHs of Phe, 1H, m), 4.25–4.02 (CaHs of Gly, 2H,
m), 3.90 (ZOCH3, 3H, s), 3.25–3.01 (CbHs of Phe, 2H,
m), 1.44 (CH3s Boc, 9H, s); 13C NMR 75 MHz (CDCl3,
d ppm): 171.84, 167.26, 166.12, 155.35, 138.00, 136.50,
130.00, 128.72, 128.27, 127.91, 126.19, 124.37, 123.91,
120.34, 79.34, 55.77, 51.52, 43.08, 37.26, 27.72; HR-MS
(MþNaþ) ¼ 478.19, Mcalcd ¼ 455.51.
1
concentrations were 10 mM in CDCl3 for H NMR and
40 mM in CDCl3 for 13C NMR.
CD spectroscopy
Solutions of peptides I and II in methanol (1.5 mM as the
final concentration) were used for obtaining the spectra.
Far-UV CD measurements were recorded at 258C with a
0.5 s averaging time and a scan speed of 50 nm/min, using
a JASCO spectropolarimeter (J 720 model) equipped with
a 0.1 cm path length cuvette. The measurements were
taken at 0.2 nm wavelength intervals and 2.0 nm spectral
bandwidth, and five sequential scans were recorded for
each sample.
Boc-Leu-Gly-m-ABA-OMe (II). To compound 1 (1.50 g,
4.87 mmol), trifluoroacetic acid (3 ml) was added at 08C
and stirred at room temperature. The removal of the Boc
group was monitored by TLC. After 3 h, trifluoroacetic
acid was removed under reduced pressure to afford the
crude trifluoroacetate salt. The residue was taken up in
water and washed with diethyl ether. The pH of the
aqueous solution was adjusted to 8 with sodium
bicarbonate and extracted with ethyl acetate. The extracts
were pooled, washed with saturated brine, dried over
sodium sulphate and concentrated to a highly viscous
liquid that gave a positive ninhydrin test. This dipeptide
free base was added to a well ice-cooled solution of Boc-
Leu-OH (1.12 g, 4.87 mmol) in DMF (6 ml) followed by
DCC (1.50 g, 7.30 mmol) and HOBt (0.66 g, 4.87 mmol).
The reaction mixture was stirred at room temperature for
3 days. The residue was taken up in ethyl acetate and DCU
was filtered off, and, to the filtrate, 20 ml of ethyl acetate
was added. The organic layer was washed with 2 M HCl
(3 £ 50 ml), 1 M Na2CO3 solution (3 £ 50 ml) and brine,
dried over anhydrous Na2SO4 and evaporated in vacuo, to
yield a white solid. Purification was done using silica gel
as the stationary phase and ethyl acetate–petroleum ether
mixture as the eluent. Yield: 1.7 g (82.9%). Mp ¼ 118–
1208C; Elemental analysis calcd for C21H31N3O6 (421.49):
C, 59.84; H, 7.41; N, 9.97%; Found: C, 59.76; H, 7.34; N,
9.88%. IR (KBr): 3342, 2960, 1672, 1526 cm21. 1H NMR
300 MHz (CDCl3, d ppm): 9.15 (m-ABAZNH, 1H, s),
8.23 [Ha (m-ABA), 1H, s], 7.86 [Hd (m-ABA), 1H, d,
J ¼ 7.8 Hz], 7.72 [Hb (m-ABA), 1H, d, J ¼ 7.8 Hz], 7.45
[Hc (m-ABA), 1H, t, J ¼ 8.1 Hz], 7.11 (GlyZNH, 1H, t),
5.39 (LeuZNH, 1H, d, J ¼ 7.6 Hz), 4.25–4.02 (CaHs of
Gly and Leu, 3H, m), 3.89 (ZOCH3, 3H, s), 1.62–1.82
(CbHs and CgHs of Leu, 3H, m), 1.43 (CH3s Boc, 9H, s),
0.95–0.85 (CdHs of Leu, 6H, m); 13C NMR 75 MHz
(CDCl3, d ppm): 173.23, 167.52, 166.25, 155.74, 138.05,
130.11, 128.35, 124.51, 124.06, 120.47, 79.44, 53.41,
51.60, 43.15, 40.79, 27.84, 24.27, 22.52, 21.41; HR-MS
(MþNaþ) ¼ 444.00, Mcalcd ¼ 421.49.
FE-SEM study
The morphologies of the dry gel of peptide I and the
fibrous materials of peptide II generated from toluene
were investigated using FE-SEM. For the FE-SEM study,
the fibrous materials of the peptides were platinum coated.
The micrographs were taken in a FE-SEM apparatus
(JEOL JSM-6700F).
HRTEM
The morphology of the dried gel of peptide I generated
from toluene was investigated using HRTEM. A solution
of peptide I in toluene (1.12% w/v) was incubated
overnight at room temperature. Transmission electron
microscopy (TEM) studies of the peptide were done using
a small amount of the solution of the peptide on carbon-
coated copper grid (300 mesh) by slow evaporation and
allowed to dry in vacuum at room temperature for 1 day.
Images were taken by JEOL JEM-2100.
Congo red binding assay
The dried gel of peptide I generated from toluene was
stained by the addition of alkaline Congo red solution
(80% methanol/20% glass-distilled water containing 10 ml
of 1% NaOH) for 2 min and then excess stain was removed
by rinsing the stained gel material with glass-distilled
water for several times. The stained gel was dried under
vacuum at room temperature for 24 h, then visualised at
40 £ magnification and a birefringence was observed
between crossed polarisers.
WAXS
The WAXS pattern was made on the tripeptide gel of 4.0%
(w/v) peptide I in toluene. The experiment was carried out

102
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Acknowledgements
A.D. would like to thank CSIR, New Delhi, India, for a senior
research fellowship (SRF). The financial assistance of UGC,
New Delhi is acknowledged [Major Research Project, No. 32-
190/2006(SR)]. We acknowledge the financial assistance of
Centre for Research in Nanoscience and Nanotechnology,
University of Calcutta.
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