Solvent-mediated morphological transformations in peptide-based soft structures

Article abstract of DOI:10.1016/j.tet.2008.05.003

Self-assembly is routinely used for the design and fabrication of new and advanced materials. Biological building blocks such as short peptides undergo self-assembly to reveal a variety of hierarchical structures such as nanotubes, fibers, and spherical structures. Peptide-based soft spherical structures are potentially useful for a variety of applications including vehicles for drug delivery and biological molecules. This report describes a tripalmitoylated triskelion ditryptophan peptide conjugate (5) that self-assembles to form hollow spheres along with pores on the surface when higher concentration of the dichloromethane was used, but forms fiber structures when heated in toluene solvent.

Full text of DOI:10.1016/j.tet.2008.05.003

Tetrahedron 64 (2008) 6202–6208
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Solvent-mediated morphological transformations in peptide-based soft structures
*
Surajit Ghosh, Sandeep Verma
Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Self-assembly is routinely used for the design and fabrication of new and advanced materials. Biological
building blocks such as short peptides undergo self-assembly to reveal a variety of hierarchical structures
such as nanotubes, fibers, and spherical structures. Peptide-based soft spherical structures are potentially
useful for a variety of applications including vehicles for drug delivery and biological molecules. This
report describes a tripalmitoylated triskelion ditryptophan peptide conjugate (5) that self-assembles to
form hollow spheres along with pores on the surface when higher concentration of the dichloromethane
was used, but forms fiber structures when heated in toluene solvent.
Received 29 January 2008
Received in revised form 30 April 2008
Accepted 1 May 2008
Available online 3 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
expanded the physico-chemical properties of these molecules by
introducing three palmitoyl chains at the N-terminal arms of the
Molecular self-assembly is a tunable methodology for the design
and fabrication of functional materials with applications ranging
from electro-optics to nanomedicine.1 The latter application re-
quires biocompatibility, which can be achieved by self-association
of biomolecular building blocks. ‘Bottom-up’ approaches using
natural building blocks have played a key role in the discovery of
new materials and scaffolds,2–11 and importantly, self-assembling
peptide-based hollow cylinders, spheres and gelatinous scaffolds
are particularly useful as substrates for electronics, optics, energy
storage, tissue-cell attachment, neurite outgrowth, and formation
of active nerve connections, to name a few.11 These novel archi-
tectures also inspire us toward an interest in fundamental phe-
nomenon specific to confined nanospaces.12
triskelionconjugate. Herein, wereportsynthesisofatripalmitoylated
peptide 5 and its ultrastructures in the different solvent regimes.
2. Results and discussion
2.1. Synthesis of compound 5
Compound
5 was synthesized by solution phase peptide
synthesis method as described in Scheme 1.
Palmitic acid was coupled with tryptophan ethyl ester hydro-
chloride in the presence of DCC and HOBt to produce compound 1,
followed by hydrolysis of the C-terminal ester group to produce
compound 2. Compound 2 was coupled with tryptophan ethyl ester
hydrochloride in the presence of DCC and HOBt to yield compound
3, which on ester group hydrolysis produced 4. Compound 4 was
converted to its active N-hydroxysuccinimide ester and its conju-
gation with tris (2-aminoethyl) amine gave the target compound 5.
Detailed experimental procedures are given in Section 4.
The important interactions, which are responsible for well
formed molecular scaffolds are p–p stacking, Van der Waals forces,
hydrogen bonding, etc.13 These interactions facilitate self-assembly
and allow stable growth of supramolecular architectures.
We have recently reported a dipeptide triskelion conjugate that
formed caged structures in methanol–water and exhibited ability to
entrap guest molecules in solution.14,15 As one long-term goal to
develop synthetic virus like particles, we wished to lipidate the core
with long alkyl chains. In the meantime we also became aware of
several reports that reveal the lipid-like peptide self-assembled
structures, depending on the composition of a mixture of solvent.16
The presence of primary amino groups in synthetic triskelion offered
significant scope for modification as an entry into other interesting
material properties of this conjugate and with this premise, we
2.2. Formation of hollow spheres
With three palmitoyl group and six tryptophan moieties, com-
pound 5 shows high degree of hydrophobicity and is able to adapt
lipid-like conformation.17,18 Freshly prepared solution of 5 (0.5 mM)
in 60% dichloromethane–methanol displayed formation of hollow
spheres with an average diameter of w500 nm. Scanning electron
micrographs revealed that these hollow spheres morphed during
drying process and changed their shape to bowl-like structures
with a depression on the spherical surface (Fig. 1a–c). Atomic force
microscopic image also revealed similar morphology for compound
* Corresponding author. Tel.: þ91 512 259 7643; fax: þ91 512 259 7436.
E-mail address: sverma@iitk.ac.in (S. Verma).
5
(Fig. 1d). After observing soft hollow spheres in 60%
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.05.003

S. Ghosh, S. Verma / Tetrahedron 64 (2008) 6202–6208
6203
However, room temperature solubility of 5 in toluene proved to
be rather poor. Therefore, we decided to heat up 5 in toluene until it
R
R
O
NH
dissolved completely at 85 ꢀC (0.85 mg in 500
mL toluene), and the
NH
NH
i
ii
NH
O
R
O
OH
microscopic analysis of toluene solution indicates appearance of
dense fibrous morphology (Fig. 4). AFM image and TEM image
suggested that fibers were composed of small particles fused
together to reveal ribbon-like fibers (Fig. 4d) with an inclination
toward formation of gel-like structure (data not shown). In-
terestingly, control experiments with non-palmitoylated triskelion
revealed lack of fibril formation in toluene. Table 1 represents
different morphologies obtained in various solvents.
14
OH
2
1
O
R= Palmitoyl group
R
NH
NH
NH
NH
O
O
iv
iii
2
It appears that when compound 5 is heated in toluene, a mor-
phological conversion occurs from small spherical structures to
fibers. Eventual coalescence during the cooling phase leads to the
fusion of spherical structures to fibers with a tendency toward
gelation (data not shown). Three palmitoyl chains connected at the
N-terminal of the molecule may facilitate the fusion process by
interdigitating between each other leading to the coalescence of
spherical structures.
HN
HN
O
HO
NH
O
NH
O
4
3
NH
R
NH
HN
2.4. Encapsulation of the guest molecule and rupture of the
hollow spheres
O
NH
O
v, vi
HN
After morphological studies, we planned to evaluate entrap-
ment of guest molecules within the confines of these hollow
spheres. Rhodamine B dye was chosen as a guest molecule. Com-
4
NH
NH
H
N
O
H
N
N
O
R
pound 5 was dissolved in 1 mM rhodamine B solution and incubated
O
N
for 24 h. The solution was loaded on a glass slide and dried,
followed by fluorescence microscope imaging. The fluorescence
micrographs indicated the entrapment of rhodamine B inside the
hollow spheres (Fig. 5a).
Fluorescence micrograph suggested rupture of the hollow
spheres and release of the dye (Fig. 5b) by simple acidification with
methanolic HCl, further lowering down the pH, hollow spheres
ruptured completely (Fig. 5c and d). The reason behind the loss
of structural integrity may be attributed due to the protonation of
tertiary nitrogen on lowering down the pH of the solution. Finally,
electrostatic repulsion between two positive charged molecule
results in the rupturing of hollow spheres.
N
H
H
NH
HN
HN
R
O
N
H
5
Scheme 1. Synthetic route for compound 5. (i) Tryptophan ethylester hydrochloride,
DCC, HOBt, DMF, 0 ꢀC–rt, 18 h; (ii) 1 N NaOH, MeOH, 3 h; (iii) tryptophan ethylester
hydrochloride, DCC, HOBT, DMF, 0 ꢀC–rt, 18 h; (iv) 1 N NaOH, MeOH, 3 h; (v) DCC,
N-hydroxysuccinimide, DME, 0 ꢀC, 2 h; (vi) TREN [tris (2-aminoethyl) amine], DMF, rt,
24 h.
dichloromethane–methanol we decided to vary the methanol
concentration to see its effect on the morphology of 5. In 80%
dichloromethane, we observed hollow spheres with multiple pores
3. Conclusion
with an average diameter 1–10 mm (Fig. 2a–d). SEM image of the
cracked and opened-up hollow spheres further confirmed that the
structures are hollow in nature. These porous structures clearly
indicate that after the formation of hollow spheres, trapped volatile
solvent dichloromethane escapes from the surface during the
drying process to reveal porous channels.19 This observation is
similar to engendering porosity in polymer samples with the help
of porogens. The use of volatile solvents as porogens has been
reported in contest polymeric materials such as aqueous solution of
poly(o-methoxy aniline).20 We believe self-assembly of compound
5 in dichloromethane–methanol traps volatile solvents in the in-
terior core of the hollow spheres, and while drying, volatile solvent
moves toward the surface resulting in shrunken hollow spheres.
Escape of solvent from the spheres leads to pore formation.
In conclusion, we have showed the N-terminal modification of
the triskelion ditryptophan peptide conjugate by introducing three
palmitoyl chains, which is known to be a cell penetrating group,
leads to intriguing morphological changes governed by solvent
interactions. It is anticipated that such fine-tuning of the self-
assembly may lead to the construction of a wide array of nano-
structures thereby fostering innovations in the development of
functional peptide scaffolds.
4. Experimental
4.1. General
2.3. Morphological studies in other solvents
Dichloromethane, N,N-dimethylformamide, methanol, triethyl-
amine, and 1,2-dimethoxy ethane were distilled following standard
procedures prior to use. N,N0-Dicyclohexylcarbodiimide (DCC),
N-hydroxybenzotriazole (HOBT), tert-butyloxycarbonyl carbonate,
Self-assembly of compound 5 was evaluated in other pure and
mixed solvents (Table 1) to investigate the morphological changes
induced by solvent change. Formation of hollow spheres also
dominated in other solvents such as in chloroform–methanol
mixture (data not shown) and in DMF (Fig. 3b). Uniformly distrib-
uted hollow spheres were observed in case of dichloroethane–
methanol (80:20) (Fig. 3a) and dichloromethane–trifluoroethanol
(80:20) solvent mixture (data not shown).
and 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.

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S. Ghosh, S. Verma / Tetrahedron 64 (2008) 6202–6208
Figure 1. (a,b) SEM micrograph of 5 formed in dichloromethane–methanol mixture. Red arrow indicates the bowl shaped hollow spheres with a surface depression; (c) magnified
image of a bowl; (d) AFM image of the hollow spheres. Scale bar corresponds to 500 nm.
4.2. Synthesis of the compound 1
29.57, 31.83, 33.67, 36.47, 52.93, 61.34, 109.66, 111.28, 118.37, 119.33,
121.91, 122.77, 127.63, 136.07, 172.04, 172.97; FTIR (KBr, cmꢂ1): 1647
(amide II), 1741 (amide I), 3307 (–NH str); HRMS calcd for
Palmitic acid (1 g, 3.89 mM) and N-hydroxy benzotriazole
(0.525 g, 3.89 mM) were dissolved in dry DMF and reaction mixture
was cooled to 0 ꢀC under nitrogen atmosphere. Solution of DCC
(0.96 g, 4.67 mM) in dichloromethane was added into the reaction
mixture at 0 ꢀC under nitrogen atmosphere. The reaction mixture
was stirred at 0 ꢀC for 1 h. After 1 h tryptophan ethyl ester hydro-
chloride (1.04 g, 3.89 mM) was added into the reaction mixture
followed by triethylamine (0.65 mL, 4.67 mL) and reaction mixture
was stirred for 24 h at room temperature. Reaction mixture was
filtered to remove N,N0-dicyclohexylurea (DCU) and filtrate was
concentrated in reduced pressure. The residue was dissolved in
dichloromethane and organic layer was washed with 1 N HCl
(3ꢁ30 mL), 10% NaHCO3 (3ꢁ30 mL), and brine (30 mL). Organic
layer was dried over anhydrous sodium sulfate and concentrated
under reduced pressure. The crude compound was purified
through silica gel column chromatography by using dichloro-
methane and methanol (97:3) to give pure compound 1 (1.5 g,
C
29H46N2O3 (MþH): 471.3508. Found (MþH)þ: 471.3586.
4.3. Synthesis of the compound 2
Compound 1 (0.81 g, 1.7 mM) was dissolved in methanol
(10 mL) and 1 N NaOH (1.6 mL) was added into the solution at room
temperature. The reaction mixture was stirred for 3 h at room
temperature. Reaction mixture was concentrated to completely
remove methanol under reduced pressure. The residue was acidi-
fied with 1 N HCl (5 mL) and extracted with dichloromethane
(3ꢁ20 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 get compound
2 (0.72 g, 1.62 mL), which was directly used for the synthesis of 3.
4.4. Synthesis of the compound 3
3.1 mM). Mp: 56–58 ꢀC, Rf [5% methanol in dichloromethane]¼0.6,
25
[
a
]
þ0.11 (c 6.1, dichloromethane). 1H NMR (400 MHz, CDCl3,
D
Compound 2 (0.7 g, 1.58 mM) and N-hydroxy benzotriazole
(0.256 g, 1.89 mM) were dissolved in dry DMF and reaction mixture
was cooled to 0 ꢀC under nitrogen atmosphere. Solution of DCC
(0.389 g, 1.89 mM) in dichloromethane was added. The reaction
mixture was stirred at 0 ꢀC for 1 h. After 1 h tryptophan ethyl ester
hydrochloride (0.425 g, 1.58 mM) was added into the reaction
TMS,
d ppm): 0.84–0.87 (m, 3H), 1.13–1.23 (m, 26H), 2.09–2.13 (m,
2H), 3.28–3.34 (m, 2H), 4.06–4.17 (m, 2H), 4.90–4.95 (m, 1H), 5.96–
5.98 (m, 1H), 6.95 (s, 1H), 7.06–7.10 (m, 1H), 7.14–7.18 (m, 1H), 7.31–
7.33 (d, 1H, 8 Hz), 7.51–7.53 (d, 1H, 8 Hz), 8.31 (br s, 1H); 13C NMR
(100 MHz; CDCl3,
d ppm): 13.98, 22.61, 24.83, 25.41, 27.54, 29.27,

S. Ghosh, S. Verma / Tetrahedron 64 (2008) 6202–6208
6205
Figure 2. (a) SEM micrograph of hollow spheres of compound 5 formed in dichloromethane–methanol mixture (80:20); (b,c) magnified image showing multiple pores; (d) cracked
and opened-up spheres.
mixture followed by triethylamine (0.26 mL, 1.89 mM) and reaction
mixture was stirred for 24 h at room temperature. Reaction mixture
was filtered to remove DCU and filtrate was concentrated under
reduced pressure. The residue was dissolved in dichloromethane
and organic layer was washed with 1 N HCl (3ꢁ30 mL),10% NaHCO3
(3ꢁ30 mL), and brine (30 mL). Organic layer was dried over anhy-
drous sodium sulfate and concentrated under reduced pressure.
The crude compound was purified through silica gel column
chromatography by using dichloromethane and methanol
(s, 1H), 6.92 (s, 1H), 7.02–7.12 (m, 2H), 7.15–7.19 (m, 1H), 7.23–7.25
(m, 2H), 7.27–7.31 (d, 1H, 8 Hz), 7.61–7.68 (d, 1H, 8 Hz), 8.31–8.39
(d, 2H); 13C NMR (100 MHz; CDCl3,
d ppm): 13.97, 14.08, 22.64,
24.82, 25.44, 27.27, 28.1, 29.12, 29.26, 29.31, 29.43, 29.61, 29.66,
31.86, 33.75, 36.45, 52.88, 53.5, 61.43, 109.13, 110.21, 111.28, 118.22,
118.81, 119.27, 119.64, 121.96, 122.06, 123.12, 123.59, 127.28, 127.5,
135.98, 136.14, 170.01, 171.26, 173.2; FTIR (KBr, cmꢂ1): 1493 (amide
II), 1629 (amide I), 3326 (–NH str); HRMS calcd for C40H56N4O4
(MþH): 657.4302. Found (MþH)þ: 657.4389.
(98.5:1.5) to give pure compound 3 (0.88 g, 1.3 mM). Mp: 68–78 ꢀC,
25
Rf [5% methanol in dichloromethane]¼0.8, [
a
]
ꢂ0.03 (c 9.9,
D
4.5. Synthesis of the compound 4
dichloromethane). 1H NMR (400 MHz, CDCl3, TMS,
d ppm): 0.83–
0.87 (m, 3H), 1.14–1.17 (t, 3H), 1.23–1.31 (m, 26H), 1.88–1.96 (m, 2H),
3.01–3.14 (m, 3H), 3.29–3.34 (m,1H), 3.47 (m,1H), 4.0–4.08 (m, 2H),
4.69–4.76 (m, 1H), 6.15–6.20 (dd, 2H), 6.60–6.61 (d, 1H, 4 Hz), 6.75
Compound 3 (0.74 g, 1.1 mM) was dissolved in methanol (10 mL)
and 1 N NaOH (1.5 mL) was added into the solution at room tem-
perature. The reaction mixture was stirred for 3 h at room tem-
perature. Reaction mixture was concentrated to remove methanol
under reduced pressure. The residue was acidified with 1 N HCl
(5 mL) and extracted in dichloromethane (3ꢁ20 mL). Combined
organic layer was washed with water followed by brine (20 mL),
dried over anhydrous sodium sulfate, and concentrated under re-
duced pressure to get compound 4 (0.68 g, 1.08 mM), which was
directly used for the synthesis of 5.
Table 1
Solvent responsive morphological transformation
Solvent
Hollow spheres
Fiber
DCM–MeOH (60:40)
DCM–MeOH (80:20)
Chloroform–methanol (60:20)
Dichloroethane–methanol (80:20)
þ (Bowl shape)
ꢂ
ꢂ
ꢂ
ꢂ
þ (Hollow/multiple holes)
þ
þ
(Array of hollow spheres)
4.6. Synthesis of the compound 5
Dichloroethane–trifluoroethanol (80:20)
þ
ꢂ
(Array of hollow spheres)
DMF
Toluene
þ
ꢂ
þ
Compound 4 (0.5 g, 0.79 mM) and N-hydroxysuccinimide (0.1 g,
0.87 mM) were dissolved in 1,2-dimethoxy ethane (20 mL) and
ꢂ

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S. Ghosh, S. Verma / Tetrahedron 64 (2008) 6202–6208
Figure 3. (a) SEM micrograph shows the uniformly arranged hollow spheres of 5 in 1,2-dichloroethane–methanol mixture (80:20); (b) spherical morphology in DMF.
reaction mixture was cooled to 0 ꢀC under nitrogen atmosphere.
Solution of DCC (0.18 g, 0.87 mM) in 1,2-dimethoxyethane (2 mM)
was added into the reaction mixture drop-wise and reaction
mixture was stirred for 2 h at 0 ꢀC followed by storing overnight
0 ꢀC. Reaction mixture was filtered and the filtrate was concen-
trated under reduced pressure. Solid was washed with diethyl ether
Figure 4. Compound 5 in toluene (a, b) AFM image of the fiber; (c) magnified image of the fiber and green marked area indicates puncated spherical structures as substructures of
fibrous structure (blue artwork). (d) TEM image shows the dense fibrous network.

S. Ghosh, S. Verma / Tetrahedron 64 (2008) 6202–6208
6207
Figure 5. (a) Fluorescence microscopy image of hollow spheres in presence of the rhodamine B dye (60% dichloromethane–methanol); (b) rupture of fluorescent spheres at pH 6.2;
(c) SEM image shows the opened-up spherical structures at pH 6.2 and (d) complete rupture at pH 4.2.
and dried under high vaccum pump. Crude compound (0.54 g,
0.74 mM) was dissolved in dry DMF (3.5 mL) at room temperature
under nitrogen atmosphere. Solution of ethylenediamine (31 mL,
5. Microscopic sample preparation
5.1. Optical microscopy
0.2 mL) in dry DMF (0.5 mL) was added into the reaction mixture
drop-wise under nitrogen atmosphere at room temperature. The
reaction mixture was stirred at room temperature under nitrogen
atmosphere for 24 h. The reaction mixture was concentrated under
reduced pressure and dissolved in dichloromethane. Organic layer
was washed with 1 N HCl (3ꢁ10 mL), 10% NaHCO3 (3ꢁ10 mL), and
brine solution. Organic layer was dried over anhydrous sodium
sulfate and concentrated. The crude compound 5 was purified
through silica gel column chromatography by using dichloro-
methane–methanol (97:3) solvent system to give pure compound
Gel solution of the compound 5 in toluene was loaded onto the
glass slide and image was captured under cross-polarized light by
Labomed Digi 3 microscope.
5.2. Scanning electron microscopy
Incubated solution (20 mL) was coated atop metal slides. A gold
coating was applied to the top of the sample to make it conductive
for analysis. Samples in different solvents and gel in toluene were
analyzed by SEM. SEM measurements were performed on FEI
QUANTA 200 Microscope equipped with a tungsten filament gun.
Micrograph was recorded at WD 10.6 mm, in varied magnification.
Concentration of the samples used was 0.5 mM in 60% dichloro-
methane–methanol and for the gel 0.85 mg in 1 mL toluene.
(0.25 g, 0.12 mM). Mp: compound decomposes at 198 ꢀC, Rf [10%
25
methanol in dichloromethane]¼0.6, [
a
]
D
ꢂ0.02 (c 2.0, N,N0-dime-
thylformamide). 1H NMR (400 MHz, DMSO, TMS,
d ppm): 0.81(m,
6H), 1.1–1.2 (merged peaks, 78H, palmitoyl chain H), 1.9 (m, 6H), 2.2
(m, 6H), 3.0 (m, 12H), 3.3 (merged with DMSO peak, 12H), 4.5 (m,
6H), 6.9 (m, 6H), 7.01 (m, 6H), 7.07 (s, 6H), 7.26–7.28 (d, 6H,
J¼8.0 Hz), 7.5 (m, 6H), 7.6 (br s, 3H), 7.8 (m, 6H), 10.7 (m, 6H); 13C
5.3. Atomic force microscopy
NMR (100 MHz; DMSO-d6, d ppm): 13.97, 22.14, 25.15, 27.39, 27.82,
28.61, 28.77, 28.88, 28.98, 29.12, 31.34, 35.26, 37.04, 52.95, 53.47,
61.0, 109.78, 110.21, 11.23, 118.15, 118.20, 118.40, 120.79, 123.53,
127.37, 136.05, 171.14, 171.50, 172.49; FTIR (KBr, cmꢂ1): 1515 (amide
II), 1649 (amide I), 3301 (–NH str); HRMS calcd for C120H168N16O9
(MþH): 1978.3180. Found (MþH)þ: 1978.3280; purity of the com-
pound was >99% checked by FPLC (C-18 column, eluent A: 0.1% TFA
inwaterand eluent B: 0.1% TFA in acetonitrile, flow rate 0.8 mL/min).
Samples were imaged with an atomic force microscope (Molec-
ular Imaging, USA) operating under Acoustic AC mode (AAC), with
the aid of a cantilever (NSC 12(c) from MikroMasch). The force con-
stant was 0.6 N/m, while the resonant frequency was 150 kHz. The
images were taken in airat room temperature, withthe scan speed of
1.5–2.2 lines/s. The data acquisition was done using PicoScan 5Ò
software, whilethedataanalysiswasdonewith theaidofvisualSPM.

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The mixture of the peptide solution was incubated for 0–7 days in
60%dichloromethane–methanolandmicrographswere recorded for
selected incubation periods. Sample solution (10 mL) was transferred
onto freshly cleaved mica surface and uniformly spread with the aid
of a spin-coater operating at 200–500 rpm (PRS-4000). The sample-
coated mica was dried for 30 min at room temperature, followed by
AFM imaging. In similar way gel solution was spread over freshly
cleaved mica surface and dried for 30 min, followed by AFM imaging.
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5.4. Transmission electron microscopy
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Solutions of compound 5 (3 mL) in 60% dichloromethane–
methanol were loaded on carbon coated copper grid, dried and
stained with uranyl acetate, followed by imaging. Gel solution of
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compound 5 (2 mL) in toluene was loaded onto the carbon coated
copper grid, dried and negatively stained with uranyl acetate fol-
lowed by imaging.
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5.5. Fluorescence microscopy
Compound 5 (1 mM) was dissolved in 10
lution in 60% dichloromethane–methanol and incubated for 2 days
at 37 ꢀC. After 2 days, 10
L incubated solution was loaded onto the
mM rhodamine B so-
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m
glass slide and followed by fluorescence microscopic imaging. So-
lution of compound 5 was acidified with the methanolic HCL so-
lution and pH was measured. These dye entrapped vesicular
structures were examined on a fluorescent microscope (Zeiss Axi-
oskop 2 Plus) provisioned with an illuminator (Zeiss HBO 100) and
a rhodamine filter (absorption 540 nm/emission 625 nm). This fil-
ter optimized visualization of rhodamine-treated (positive resolu-
tion) compared with untreated (negative resolution) spherical
structures that were virtually invisible in this light.
Acknowledgements
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S.G. thanks IIT-Kanpur for a pre-doctoral fellowship. This work is
supported through a Swarnajayanti Fellowship in Chemical Sci-
ences (DST) to S.V.
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