Spacer driven morphological twist in Phe-Phe dipeptide conjugates

Article abstract of DOI:10.1016/j.tet.2012.12.063

Chemists and material scientists are interested in complex biological systems and morphologies for fabrication of new materials. Bioinspired design of short peptide conjugates produces various self-assembled structures, depending on their structural diver

Full text of DOI:10.1016/j.tet.2012.12.063

Tetrahedron 69 (2013) 2004e2009
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Spacer driven morphological twist in Phe-Phe dipeptide conjugates
,
,
a,
Surajit Ghosh a y, Lihi Adler-Abramovich b, Ehud Gazit b , Sandeep Verma
*
*
a Department of Chemistry and DST Thematic Unit of Excellence on Soft Nanofabrication, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
b Department of Molecular Biology and Biotechnology, Tel-Aviv University, Tel-Aviv 69978, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemists and material scientists are interested in complex biological systems and morphologies for
fabrication of new materials. Bioinspired design of short peptide conjugates produces various self-
assembled structures, depending on their structural diversity. Phe-Phe aromatic dipeptide is an in-
teresting candidate for designing variety of molecular scaffolds. This report describes the synthesis of
two symmetrical peptide conjugates by a coupling with C2 and C3 symmetric functional linker’s, which
spontaneously generate different self-assembled ultra-structures. C2 Symmetric, ethylene diamine
linker, conjugated two Phe-Phe dipeptides and produced Phe-Phe-EDA-Phe-Phe (5), which undergoes
spontaneous self-assembly to form fibrillar morphology, while C3 symmetric tris(2-aminoethylamine)
conjugated three Phe-Phe dipeptides, produced (Phe-Phe)3TREN (7) resulted in spherical morphology
structures interconnected by fibers. A rational design of two peptide conjugates, by introducing two
different linker’s, direct different self-assemble pathway, which result in distinct morphologies.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 20 September 2012
Received in revised form 17 December 2012
Accepted 26 December 2012
Available online 3 January 2013
1. Introduction
superhydrophobic coatings for microfluidic devices and electro-
static ultracapacitors, which significantly extended the electrode
The organization of molecules into highly ordered assemblies in
chemistry and biology, is a phenomenon of great interest to re-
searchers of both disciplines. The process ‘autonomous organiza-
tion’ also known by the term ‘molecular self-assembly’1 is mediated
through weak inter-molecular forces such as van der Waals, elec-
capacitance.7 In addition, various methodologies were used for the
vertical and horizontal alignment of peptide nanotubes.8 Moreover,
extensive work was done to study the role of p-stacking interaction
in the self-assembly process.9 Another interesting aspect in de-
signing the peptide based nanostructures is the fusion of multiple
short peptides by an intelligent selection of spacers, which allows
for high flexibility and conformational freedom in the construct.
Interesting reports by our group and others showed that the use of
symmetric and asymmetric bifunctional linkers as well as a tri-
functional linkers between peptides affect the self-assembly pro-
cess.10 Here, we report the fascinating role of various spacers fused
in between the short homo dipeptide, Phe-Phe, on the production
of different self-assembled nanostructures.
trostatic interactions, hydrogen bonds and
pep stacking in-
teractions. The combined effect of these weak non-covalent
interactions plays an intriguing role in the fabrication of well-
ordered supramolecular architectures. In recent years, extensive
studies on the fabrication of new materials using natural building
blocks, such as, nucleic acids, lipids, and amino acids have been
performed.2 Several studies have demonstrated that short peptides,
which possess specific molecular recognition modules may form
well organized assemblies such as tubes, tapes, and spheres.3 These
assemblies could be utilized for various applications, including
molecular electronics, tissue engineering, and material science.4
Recent reports showed that short aromatic dipeptides derived
from natural fibril forming peptides form nanometric tubular
structures, which have been used as a template for the fabrication
of metallic nanowires and coaxial metal nano-cables.5 The large
surface area of these nanostructures was utilized for the formation
of a sensitive sensor, which detects phenol,6 as well as for
2. Results and discussion
2.1. Synthesis of Phe-Phe peptide conjugates
Phe-Phe-EDA-Phe-Phe (Compound 5) and (Phe-Phe)3TREN
conjugate (Compound 7) were synthesized by the solution phase
peptide synthesis method. Scheme 1 shows the stepwise synthesis
of compounds 5 and 7. Compound 1 was hydrolyzed with the 1 N
NaOH in methanol to form compound 2, which was converted to its
active NHS ester resulting in compound 3. Compound 3 was treated
with the ethylene diamine to form compound 4. Compound 4 was
treated with the 1 N HCl in EtOAc resulting in compound 5. For the
* Corresponding authors. E-mail addresses: ehudg@post.tau.ac.il (E. Gazit),
sverma@iitk.ac.in (S. Verma).
y
Present address: Chemistry Division, Indian Institute of Chemical Biology, 4,
Raja S.C. Mullick Road, Kolkata 700032, India.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.12.063

S. Ghosh et al. / Tetrahedron 69 (2013) 2004e2009
2005
O
O
O
O
H
H
H
O
N
O
i
O
N
O
O
N
OH
ii
N
N
H
N
N
H
H
O
O
O
O
O
O
O
1
3
2
O
O
H
H
N
O
O
O
N
H2N
H
H
H
N
N
N
NH2
O
N
N
iv
H
H
N
N
H
N
H
O
O
O
iii
H
O
O
O
3
5
4
H2N
O
O
O
NH
O
O
HN
HN
O
NH
NH2
NH
NH
O
O
HN
vi
v
O
O
HN
N
O
N
O
NH
3
NH
HN
HN
O
O
NH
NH
O
O
7
HN
O
H2N
O
6
Scheme 1. Synthesis of compound 5 and 7, Phe-Phe-EDA-Phe-Phe and (Phe-Phe)3TREN, respectively.
synthesizing of the triskelion derivative of Phe-Phe conjugate,
compound 3 was again treated with tris(2-aminoethyl) amine to
form compound 6, which was further treated with 1 N HCl in EtOAc
to form compound 7. Detailed experimental procedures are given in
the Experimental section.
2.3. Morphological structure characterization of the tri-
skelion: (Phe-Phe)3TREN conjugate
Interestingly, we have also investigated the morphology of the
self-assembled triskelion structures, which composed of three
diphenylalanine peptide modules. The triskelion dissolving in 60%
methanol in water resulted in the formation of fibers emanating
from the spherical structures. Scanning electron microscopy image
reveals the fibers radiating from the spherical surface (Fig. 2a).
Similar observation was obtained using atomic force microscopy,
which exhibited spherical structures with a diameter ranging from
2.2. Morphological structures characterization of Phe-Phe-
EDA-Phe-Phe conjugate
As mentioned above, the Phe-Phe dipeptide forms discrete
nanotube structures.5 Here, we observe that two Phe-Phe conju-
gated with ethylene diamine, Phe-Phe-EDA-Phe-Phe (5) shows an
extensive fiber formation in 60% methanol in water. Optical mi-
croscopy [OM] images show the extensive fiber formation in 60%
methanol in water (Fig. 1a), and a magnified view of the OM image
shows rod-shaped fibers (Fig. 1b). Atomic force microscopy images
exhibit the fibrous bundles, and a higher magnification reveals the
fibers approximated width of 60 nm (Fig. 1c, d). Transmission
electron microscopy micrograph also shows the fibrous bundle
with an averaged width of 10e20 nm (Fig. 1e). We have further
investigated this fiber morphology in environmental scanning
microscopy, which also confirmed the rod shaped fiber formation
(Fig.1f). Therefore, Phe-Phe-EDA-Phe-Phe in 60% methanol in water
exhibited extensive nanofiber formation with a varied diameter of
10e500 nm.
600 nm to 1 mm, and fibers radiating from the vesicular surface
with an average diameter of 60e100 nm (Fig. 2b). In addition, it was
also apparent that the fibers interconnect the spherical structures.
2.4. Secondary structures analysis of the self-assembled
nanostructures
In order to study the secondary structures of the two com-
pounds, we have performed Fourier-transform infrared (FTIR)
spectroscopy of the conjugates in 60% methanol in water. The
amide I spectral peak obtained for Phe-Phe-EDA-Phe-Phe conjugate
at around 1652 cmꢀ1, indicates the typical
a-helix structure, while
(Phe-Phe)3TREN conjugate peak appeared at 1643 cmꢀ1 indicates
the typical 310 helix structure (Fig. 3).11

2006
S. Ghosh et al. / Tetrahedron 69 (2013) 2004e2009
Fig. 3. FTIR spectroscopic studies of the Phe-Phe conjugate in 60% methanol in water:
(a) absorption maxima of Phe-Phe-EDA-Phe-Phe at 1652 cmꢀ1 indicates the typical
-helix structures; (b) absorption maximum of (Phe-Phe)3TREN at 1643 cmꢀ1 indicates
a
the typical 310 helix structures.
fluorescence fibers (Fig. 4a) while (Phe-Phe)3TREN conjugate
shows stained spherical structures (Fig. 4b).
The experimental results presented in the current article pro-
vide an interesting evidence for the morphological and structural
features of the Phe-Phe conjugates. A key point in these simple
systems is the ability to form well-ordered assemblies by the
facilitation of the geometrically-restricted
pep
stacking.5,7,13 Fol-
lowing is a potential model for the mode of interaction of the
peptide conjugate building-block (Fig. 5).
Fig. 1. Microscopy images of the Phe-Phe-EDA-Phe-Phe conjugates in 60% methanol in
water: (a) optical microscopy image shows the extensive fiber formation; (b) high
magnification image of the fibers (c) atomic force microscopy [AFM] image of fibrous
aggregates; (d) high magnification AFM image of the fibers; (e) TEM image of the fibers
aggregates; (f) ESEM image of the fibers.
Fig. 4. Fluorescence microscopy image of Phe-Phe conjugates with Thioflavin T (ThT)
in 60% methanol water: (a) Phe-Phe-EDA-Phe-Phe shows fiber morphology and (b)
(Phe-Phe)3TREN shows spherical structures.
2.5. Nanostructures stained with thioflavin T
Thioflavin T is a benzothiazole dye, which is well known for
exhibiting enhanced fluorescence upon binding to amyloid fibrils
and is commonly used to identify and quantify amyloid fibrils,
both ex vivo and in vitro.12 For investigating the ordered nature of
the nanostructures, we have stained the fibers with thioflavin T.
Phe-Phe-EDA-Phe-Phe stained with Thioflavin T shows green
Fig. 2. Microscopy image of the (Phe-Phe)3TREN conjugate in 60% methanol in water:
(a) SEM image shows the fibers radiating from the vesicular surface; (b) atomic force
microscopy image also shows the fibers radiating from the vesicular surface and
interconnects to each other.
Fig. 5. Model for the molecular organization of the Phe-Phe conjugates. (a) Schematic
illustration of Phe-Phe-EDA-Phe-Phe and (Phe-Phe)3TREN. (b) Possible intermolecular
interaction through
pep
stacking in Phe-Phe-EDA-Phe-Phe. (c) Possible in-
stacking in (Phe-Phe)3TREN.
termolecular interaction through
pep

S. Ghosh et al. / Tetrahedron 69 (2013) 2004e2009
2007
3. Conclusion
mp¼82e84 ꢁC, Rf [5% methanol in dichloromethane]¼0.3, ¼ꢀ00.02
[c 0.12, methanol]. 1H NMR (400 MHz, CD3OD, TMS,
d ppm): 1.23 (s,
In conclusion we have reported and demonstrated novel design,
synthesis and microscopic evaluation of self-assembled structures
of two different peptide conjugates. Direction of self-assembly was
shown to be solely dependent on the symmetry of linkers, and as
a consequence, a morphological switch was observed. Bioinspired
selection of linkers offers the design of novel peptide conjugates,
which could open up new avenues of smart materials.
9H); 2.5e2.6 (m, 1H); 2.70e2.95 (m, 2H); 3.0e3.2 (m, 1H);
4.15e4.18 (m, 1H); 4.53e4.57 (m, 1H); 7.09e7.15 (m, 10H); FTIR
(KBr, cmꢀ1): 1520 (amide II); 1660 (amide I); FABMS (Mþ1): 413;
Anal. Calcd for C23H28N2O5, C, 66.97; H, 6.84; N, 6.79; found, C,
67.11; H, 6.66; N, 6.12.
4.4. Synthesis of compound 3
4. Experimental
4.1. General
(BOC)FFeNHS (3): Compound 2 (0.5 g, 1.2 mmol) and N-Hy-
droxy succinimide (0.153 g, 1.3 mmol) were dissolved in 1,2-
dimethoxy ethane (10 ml) and reaction mixture was cooled to
0
ꢁC under nitrogen atmosphere. A solution of DCC (0.274 g,
Dichloromethane, N,N-dimethylformamide, methanol, triethyl-
amine and 1,2-dimethoxy ethane were distilled following standard
procedures prior to use. N, N’-dicyclohexylcarbodiimide (DCC),
N-hydroxybenzotriazole (HOBT), t-butyloxycarbonyl carbonate,
1.3 mmol) in 1,2-dimethoxy ethane (5 ml) was added into the re-
action mixture dropwise and reaction mixture was stirred for 2 h at
0
night. The reaction mixture was filtered and filtrate was concen-
trated under reduced pressure. Solid material was washed with
diethyl ether and dried under high vaccum. Crude compound 3
(0.6 g, 1.1 mmol) was directly used for synthesis of compounds 4
and 6.
ꢁC. After which the reaction mixture was kept in a freezer over-
L-amino acids were purchased from Spectrochem, Mumbai, India,
and used without further purification. Tris (2-aminoethyl) amine
(TREN) was purchased from Sigma. 1H and 13C NMR spectra were
recorded on JEOL-JNM LAMBDA 400 model operating at 400 and
100 MHz, respectively. HRMS spectra were recorded on Waters Q-
TOF Premier Micromass MS Technology.
4.5. Synthesis of compound 4
4.2. Synthesis of compound 1
[(BOC)FF]2EDA (4): Compound 3 (2.5 g, 4.9 mmol) was dissolved
in dry DMF (30 ml) at room temperature under nitrogen atmo-
sphere. Solution of ethylene diamine (0.144 g, 2.4 mmol) in dry
DMF (1.0 ml) was added into the reaction mixture dropwise and
stirred under nitrogen atmosphere at room temperature for 24 h.
The reaction mixture was concentrated under reduced pressure
and water was added. Solid material was washed with 1N HCl
(3ꢂ15 ml), 10% NaHCO3 (3ꢂ10 ml), water 20 ml and diethyl
ether 20 ml and dried by vaccum to form pure compound 4
(2.0 g, 2.3 mmol). mp¼202e204 ꢁC, Rf [5% methanol in
dichloromethane]¼0.5, ¼ꢀ00.02 [c 0.48, methanol]. 1H NMR
(BOC)FFeOMe (1): N-(BOC)-L-Phenylalanine (5 g, 18.8 mmol),
and N-Hydroxybenzotriazole (2.53 g, 18.8 mmol) were dissolved in
dry DMF (25 ml) and reaction mixture was cooled to 0 ꢁC under
nitrogen atmosphere. Solution of DCC (4.26 g, 20.6 mmol) in
dichloromethane was then added into the reaction mixture. The
reaction mixture was stirred at 0 ꢁC for 1 h. After which,
L-Phe-
nylalanine methyl ester hydrochloride (3.3 g,18.8 mmol) was added
into the reaction mixture followed by triethylamine (3.13 ml,
22.5 mmol) and the reaction mixture was stirred for 24 h at room
temperature. Reaction mixture was filtered to remove DCU and
filtrate was concentrated in reduced pressure. The residue was
dissolved in dichloromethane and the organic layer was washed
with 1 N HCl (3ꢂ30 ml), 10% NaHCO3 (3ꢂ30 ml) and brine (30 ml).
The organic layer was dried over anhydrous sodium sulfate and
concentrated in reduced pressure. The crude compound was puri-
fied through silica gel column chromatography by using dichloro-
methane and methanol (94:6) solvent system to form the pure
compound 1 (6.2 g, 14.5 mmol). mp¼102e105 ꢁC, Rf [5% methanol
in dichloromethane]¼0.5, ¼ꢀ00.04 [c 0.46, methanol]. 1H NMR
(400 MHz, DMSO-d6, TMS, d ppm): 1.27 (s, 18H); 2.82e2.85 (m, 4H);
2.85e2.98 (m, 4H); 3.32 (4H, overlapped signal for linker’s H and
DMSO-d6 peak); 4.03e4.10 (m, 2H); 4.44e4.49 (m, 2H); 6.88e6.90
(m, 2H); 7.10e7.24 (m, 20H); 7.95 (broad signal, 4H); 13C NMR
(100 MHz; DMSO-d6,
d ppm): 28.09, 33.35, 37.43, 38.01, 47.5, 53.8,
55.8, 78.1, 126.3, 127.9, 129.29, 137.4, 138.0, 155.12, 170.7, 171.2; FTIR
(KBr, cmꢀ1): 1526 (amide II); 1648 (amide I); 3302 (eNH str);
FABMS (Mþ1): 850; Anal. Calcd for C48H60N6O8, C, 67.90; H, 7.12; N,
9.90; found, C, 67.55; H, 6.89; N, 9.63.
(400 MHz, CDCl3, TMS,
(s, 3H); 4.2 (m, 1H); 4.70e4.71 (m, 1H); 6.89e6.91 (m, 2H);
7.08e7.23 (m, 10H, overlapped aromatic signal and CDCl3 peak); 13
NMR (100 MHz; CD3OD, ppm): 28.6, 38.4, 39.2, 52.6, 55.0, 57.1,
d
ppm): 1.32 (s, 9H); 2.93e2.99 (m, 4H); 3.6
4.6. Synthesis of compound 6
C
[(BOC)FF]3TREN (6): Compound 3 (0.8 g, 1.5 mmol) was dis-
solved in dry DMF (4.5 ml) at room temperature under nitrogen
atmosphere. Solution of Tris (2-aminoethyl) amine (0.0642 g,
0.43 mmol) in dry DMF (0.5 ml) was added into the reaction mix-
ture dropwise and stirred under nitrogen atmosphere at room
temperature for 24 h. The reaction mixture was concentrated under
reduced pressure and water was added. Solid material was washed
with 1 N HCl (3ꢂ15 ml), 10% NaHCO3 (3ꢂ10 ml), water 20 ml and
diethyl ether 20 ml and dried by vaccum to form pure compound 6
(0.55 g, 0.41 mmol). mp¼178e180 ꢁC, Rf [10% methanol in
dichloromethane]¼0.6, ¼ꢀ00.02 [c 0.26, methanol]. 1H NMR
d
80.5, 127.6, 127.8, 129.3, 129.5, 130.3, 137.8, 138.5, 157.4, 173.0, 174.1;
FTIR (KBr, cmꢀ1): 1523 (amide II); 1655 (amide I); 3329 (eNH str);
FABMS (Mþ1): 427; Anal. Calcd for C24H30N2O5 C, 67.59; H, 7.09; N,
6.57; found, C, 67.19; H, 6.71; N, 6.23.
4.3. Synthesis of compound 2
(BOC)FFeOH (2): Compound 1(3.0 g, 7.0 mmol) was dissolved in
methanol (30 ml) and 1(N) NaOH (10 ml) was added into the so-
lution. The reaction mixture was stirred for 3 h at room tempera-
ture. Reaction mixture was concentrated to completely remove the
methanol under reduced pressure. The residue was acidified with
1 N HCl (15 ml) and extracted in dichloromethane (3ꢂ25 ml). The
combined organic layer was washed with water followed by brine
(20 ml), dried over anhydrous sodium sulfate and concentrated
under reduced pressure to form compound 2 (2.3 g, 5.5 mmol).
(400 MHz, DMSO-d6, TMS, d ppm): 1.26 (s, 27H); 2.73e2.83 (m, 6H);
2.93e3.07 (m, 6H); 3.3 (s, overlapped signal for linker’s 12H and
DMSO-d6 peak); 4.18e4.20 (m, 3H); 4.45e4.52 (m, 3H); 6.87e6.89
(d, 3H, J¼8 Hz), 7.15e7.21 (m, 30H); 7.86 (broad s, 3H); 7.97e7.99
(m, 3H); 13C NMR (100 MHz; DMSO-d6,
d ppm): 27.9, 33.6, 37.3,
38.2, 47.3, 53.8, 56.1, 78.1, 126.3, 127.8, 129.3, 137.4, 138.02, 155.12,
170.74, 171.29; FTIR (KBr, cmꢀ1): 1527 (amide II); 1648 (amide I);

2008
S. Ghosh et al. / Tetrahedron 69 (2013) 2004e2009
3324 (eNH str); FABMS (Mþ1): 1331; Anal. Calcd for C75H96N10O12
,
5.3. Scanning electron microscopy
20 L of the incubated solution of conjugates 5 and 7 in 60%
C, 67.75; H, 7.28; N, 10.53; found, C, 67.55; H, 7.01; N, 9.99.
m
4.7. Synthesis of compound 5
methanol in water at a concentration of 1 mM were deposited on
slides, and then coated with gold. SEM measurements were per-
formed on either FEI QUANTA 200 Microscope equipped with
a tungsten filament gun or a JSM JEOL 6300 SEM operating at 5 kV.
Environmental scanning electron microscopy images were cap-
tured using Quanta 200 FEG Field Emission Gun ESEM operating at
10 kV.
(FF)2EDA (5): Compound 4 (0.17 g, 0.20 mmol) and 1 N HCl
in EtOAc (5.0 ml) were mixed and stirred for 3 h. After 3 h solid
material was separated from the reaction mixture. Solvent was
removed from the reaction mixture by decantation method and
residue was washed with EtOAc twice, followed by two washes
with diethyl ether. Solid material was dried in high vacuum pump
and desolved in (90%) methanol in water passed through ion ex-
change column. Solvent was removed in reduced pressure and solid
material was dried under high vacuum. Crude solid material was
dissolved in methanol and precipitated out with diethyl ether. Solid
material was dried under high vacuum to form pure compound 5
(0.05 g, 0.07 mmol). mp¼above 210 ꢁC, Rf [10% methanol in
dichloromethane]¼0.5, ¼ꢀ00.02 [c 0.26, methanol]. 1H NMR
5.4. Atomic force microscopy
Solutions of conjugates 5 and 7 were imaged with an atomic
force microscope (Molecular Imaging, USA) operating under
acoustic AC mode (AAC), with the aid of a cantilever (NSC 12(c)
from MikroMasch). The force constant was 0.6 N/m, while the
resonant frequency was 150 kHz. The images were taken in air at
room temperature, with a scan speed of 1.5e2.2 lines/s. The data
acquisition was done using PicoScan 5Ò software, while data was
analyzed with the aid of visual SPM. The solutions of conjugates
were incubated for 0e7 days in 60% methanol in water and mi-
(400 MHz, CD3OD, TMS, d ppm): 2.86e3.13 (m, 8H); 3.20e3.22 (m,
4H, overlapped signal for CD3OD and linker’s H); 4.06e4.09 (m,
2H); 4.48e4.52 (m, 2H); 7.18e7.32 (m, 20H); 1H NMR (400 MHz,
DMSO-d6, TMS,
d ppm): 2.84e3.13 (m, 8H); 3.15 (s, 4H, linker’s H);
3.95e4.05 (broad signal, 2H); 4.42e4.48 (m, 2H); 7.18e7.29 (m,
20H); 8.15e8.18 (m, 6H, overlapped signal for amine’s 4H and
amide’s 2H); 8.90e8.92 (m, 2H, amide); 13C NMR (100 MHz;
crographs were recorded for selected incubation periods. 10 mL of
sample solution was transferred onto freshly cleaved mica surface
and uniformly spread with the aid of a spin-coater operating at
200e500 rpm (PRS-4000). The sample-coated mica was dried for
30 min at room temperature, followed by AFM imaging.
CD3OD,
d ppm): 38.54, 39.08, 39.79, 50.2, 55.49, 56.7, 127.9, 128.8,
129.6, 130.1,130.3, 130.6, 135.49,138.14,169.4, 172.9; FABMS (Mþ1):
650; Anal. Calcd for C38H44N6O4, C, 70.35; H, 6.84; N, 12.95; found,
C, 70.11; H, 6.95; N, 13.11.
5.5. Transmission electron microscopy
4.8. Synthesis of compound 7
3 mL solutions of conjugates 5 and 7 in 60% methanol water were
loaded on carbon coated copper grids. After 1 min, excess fluid was
removed and the grids were stained with 2% uranyl acetate in
water. Excess staining solution was removed from the grid after
2 min. Samples were viewed using a JEOL 1200EX electron micro-
scope operating at 80 kV. High resolution samples were viewed and
analyzed with a Philips Tecnai F20 Field Emission Gun electron
microscope operating at 200 kV.
(FF)3TREN (7) : Compound 6 (0.2 g, 0.15 mmol) and 1 N HCl in
EtOAc (6.0 ml) were mixed and stirred for 3 h. After 3 h solid ma-
terial was separated out from the reaction mixture. Solvent was
removed from the reaction mixture by decantation method and
residue was washed with the EtOAc twice, followed by the two
washes with diethyl ether. Solid material was dried by high vacuum
and dissolved in methanol water (90%) passed through ion ex-
change column. Solvent was removed in reduced pressure and solid
material was dried under high vacuum. Crude solid material was
dissolved in methanol and precipitated out with diethyl ether. Solid
material was dried under high vacuum to form pure compound 7
(0.07 g, 0.068 mmol). mp¼Hygroscopic Not Performed, Rf [10%
methanol in dichloromethane]¼0.4, ¼ꢀ00.02 [c 0.23, methanol].
5.6. ThT staining and confocal laser microscopy imaging
10 mL ThT solution (2 mM, PBS buffer) was mixed with 10 mL of
each of the two Phe-Phe conjugates solutions: Phe-Phe-EDA-Phe-
Phe (Compound 5) and (Phe-Phe)3TREN conjugate (Compound 7).
An LSM 510 confocal laser scanning microscope (Carl Zeiss Jena,
Germany) was used at excitation and emission wavelengths of 440
and 485 nm, respectively.
1H NMR (400 MHz, CD3OD, TMS,
d ppm): 2.83e2.92 (m, 6H);
2.98e3.03 (m, 6H); 3.19 (s, 12H, overlapped signal for linker’s H and
CD3OD peak); 3.97e4.00 (m, 3H); 4.44e4.48 (m, 3H); 7.10e7.23 (m,
30H); 13C NMR (100 MHz; CD3OD,
d
ppm): 36.5, 38.3, 39.5, 55.2,
5.7. Fourier-transform infrared spectroscopy
56.4, 57.8, 129.05, 129.8, 130.6, 131.10, 131.12, 131.5, 136.4, 138.8,
170.84, 175.0; FABMS (M ꢀ 1): 1028; Anal. Calcd for C60H72N10O6, C,
70.01; H, 7.05; N, 13.61; found, C, 69.85; H, 6.95; N, 13.33.
Infrared spectra were recorded using Nicolet Nexus 470 FTIR
spectrometer with DTGS detector. Solution samples of Phe-Phe-EDA-
Phe-Phe (Compound 5) and (Phe-Phe)3TREN conjugate (Compound
7) were dried on CaF2 plate to form thin film. The peptide deposits
were resuspended with D2O and dried. The resuspension procedure
was repeated twice to ensure maximal hydrogen to deuterium ex-
change. The measurements were taken using a 4 cmꢀ1 resolution
and 2000 scans averaging. The transmittance minima values were
determined by OMNIC analysis program (Nicolet).
5. Nanostructure sample preparation and imaging
5.1. Nanostructure preparation
The lyophilizes powder of Phe-Phe-EDA-Phe-Phe (Compound 5)
and (Phe-Phe)3TREN conjugate (Compound 7) were dissolved in
60% methanol in water at a concentration of 1 mM.
Acknowledgements
5.2. Optical microscopy
We thank members of the Gazit laboratory for helpful discus-
sions and DST Thematic Unit of Excellence on Soft Nanofabrication,
IIT Kanpur, for access to microscopy facilities. S.G. thanks IIT Kanpur
for a predoctoral research fellowship.
Solution of conjugate 5 in 60% methanol in water was loaded
onto a glass slide and images were captured under cross polarized
light by Labomed Digi 3 microscope.

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