Concentration dependent transformation of oligopeptide based nanovesicles to nanotubes and an application of nanovesicles

Article abstract of DOI:10.1002/asia.200900274

The concentration dependent transformation of an oligopeptide nanostructure from nanovesicles to nanotubes at neutral pH is presented. The oligopeptide Acp-Tyr-Glu (Acp: 6-aminohexanoic acid) forms nanovesicles at a concentration of 6.9 mgmL^-1. At a concentration of 2.3 mgmL^-1 these vesicular structures completely disappear and nanotubular structures are observed. We have also successfully optimized an intermediate concentration (3.4 mgmL^-1) where an ordered array of fused vesicular structures are formed, which actually leads to the transition from nanovesicles to nanotubes. These vesicular structures are very much sensitive toward metal ions and pH. Biocompatible calcium ions and high pH (10.7) can trigger the rupturing of these nanovesicles. One important property of these nanovesicular structures is the encapsulation of a potent anticancer drug doxorubicin, which can also be released in the presence of calcium ions promising a future use of these nanovesicles as vehicles for carrying biologically important molecules.

Full text of DOI:10.1002/asia.200900274

DOI: 10.1002/asia.200900274
Concentration Dependent Transformation of Oligopeptide based
Nanovesicles to Nanotubes and an Application of Nanovesicles
Jishu Naskar and Arindam Banerjee*[a]
Abstract: The concentration dependent
tion (3.4 mgmLꢀ1) where an ordered
array of fused vesicular structures are
formed, which actually leads to the
transition from nanovesicles to nano-
tubes. These vesicular structures are
very much sensitive toward metal ions
and pH. Biocompatible calcium ions
and high pH (10.7) can trigger the rup-
transformation of an oligopeptide
nanostructure from nanovesicles to
nanotubes at neutral pH is presented.
The oligopeptide Acp-Tyr-Glu (Acp: 6-
aminohexanoic acid) forms nanovesi-
cles at a concentration of 6.9 mgmLꢀ1.
turing of these nanovesicles. One im-
portant property of these nanovesicular
structures is the encapsulation of a
potent anticancer drug doxorubicin,
which can also be released in the pres-
ence of calcium ions promising a future
use of these nanovesicles as vehicles
for carrying biologically important mol-
ecules.
At
a
concentration of 2.3 mgmLꢀ1
these vesicular structures completely
disappear and nanotubular structures
are observed. We have also successfully
optimized an intermediate concentra-
Keywords: fluorescence
·
nano-
tubes · oligopeptide · self-assembly ·
vesicles
ꢀ
Introduction
a very short peptide, diphenylalanine (Phe Phe), the aro-
matic core of b-amyloid polypeptide, can be self-assembled
to form a novel class of peptide nanotubes. These nanotubes
have a long length and unique mechanical properties with a
Youngꢁs modulus of about 19 GPa.[5] These peptide nano-
tubes not only can serve as a degradable mold for the fabri-
cation of silver nanowires[3c] but also enable the formation
of platinum nanoparticle composites.[3a] Recent study has
demonstrated that a peptide nanotube can be used as a part
of an electrochemical biosensor platform.[6] By varying the
self-assembling building block, nanostructural transforma-
tion can be performed from spherical to tubular or cylindri-
cal.[7] There are a few reports of chirality induced unprece-
dented transition from vesicles to helical tubules.[8] Gazit
et al. have reported the dipeptide based nanotube to hollow
nanocage structural transition observed by simply attaching
a thiol group at the terminal position of the dipeptide.[9]
Other examples of peptide based nanostructural transitions
include pH-sensitive[10] and concentration dependent[11]
transformation of nanotubes to nanovesicles using the con-
trolled self-association of a singular peptide building block.
Shelnutt and co-workers have reported the synthesis of di-
peptide nanotubes (DPNTs) using nanopure water instead
of fluoropropanol and they observed that these nanotubes
turned into vesicles upon dilution using water.[3a] Zhang and
co-workers reported several surfactant-like oligopeptides
(each with ~2 nm in length) undergo self-assembly to form
Molecular self-assembly has emerged as a powerful tool for
making various nanostructures and this leads to the develop-
ment of advanced materials.[1] Manipulation of nanoscale
structures can be performed with precision by the proper se-
lection of molecular building blocks and the control of their
self-assembly. Molecules, such as lipids,[2a] amino acid deriva-
tives,[2b] peptides,[2c,d,e,f] and proteins play an increasingly im-
portant role for this purpose. Peptide based scaffolds are
very interesting candidates for constructing nanostructures
owing to their well-defined structure,[2c,d,e,f,h] biocompatibil-
ity,[3] and molecular recognition property.[2b,4] There are nu-
merous examples of short acyclic peptide based nanotube
formation. The first example of simple dipeptide based
nanotube formation in crystal was reported by Gçrbitz and
these nanotubes have chiral hydrophilic channels with van
der Waals diameter up to 10 ꢀ.[2a] Gazit et al. reported that
[a] J. Naskar, Dr. A. Banerjee
Department of Biological Chemistry
Indian Association for the Cultivation of Science
Jadavpur, Kolkata-700032, West Bengal (India)
Fax : (+91)33-24732805
E-mail: bcab@iacs.res.in
arindambolpur@yahoo.co.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200900274.
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FULL PAPERS
nanotubes and nanovesicles having an average diameter of
30–50 nm.[3b] Li et al. reported that a positively charged di-
peptide nanotube (H-Phe-Phe-NH2·HCl) undergoes a spon-
taneous conversion into vesicles upon dilution at physiologi-
cal condition. These positively charged nanotubes with fluo-
rescently labeled ssDNA could enter cells readily after their
conversion into vesicles with attached ssDNA.[11] This transi-
tion is reversible in nature.[12] A recent study also demon-
strated the structural regulation of tubes into vesicles by
molecular coassembly manipulated by a charge-transfer in-
teraction.[13] In our laboratory, we are involved in studying
the self-assembly of short synthetic peptides into various
nanoscopic species including nanotubes,[14a] nanorods,[14b]
nanovesicles,[10] and the transition of one nanostructure into
the other.[10] However, the self-assembly of short synthetic
peptides into well defined nanostructures and the breaking
of these nanostructures by interaction with biocompatible
metal ions like Ca2+, Mg2+, K+, and so forth, is very inter-
esting. To the best of our knowledge this type of study is
very rare in the literature.[15] This report describes the self-
assembly of short water-soluble tripeptides containing 6-
aminohexanoic acid (Acp) as the N-terminal residue, Acp-
Tyr-Glu (Figure 1), into nanovesicles and nanotubes at dif-
ferent concentrations at neutral pH. The formation of these
Figure 2. DLS study at peptide concentration (a) 6.9 mgmLꢀ1
2.3 mgmLꢀ1
, (b) at
.
structures by various electron microscopic techniques, such
as SEM and TEM.
To investigate the morphological features of these self-as-
sembled water-soluble tripeptide, a field emission scanning
electron microscopic (FE-SEM) study was performed. Pep-
tide (6.9 mg) was dissolved in 1 mL water (pH 6.96) and the
solution was allowed to age for 6 h. The FE-SEM study elu-
cidated the formation of nanospheres of various sizes
(Figure 3).
Figure 1. Chemical structure of the compound.
nanovesicles is very much sensitive toward metal ions and
pH. The presence of biocompatible Ca2+ ions and high pH,
trigger the rupturing of these nanovesicles. These nanovesi-
cles can encapsulate a potent anticancer drug (doxorubicin)
and the drug can be released in the presence of Ca2+ ions
by rupturing these vesicles indicating a probable use as a
nanovehicle for carrying drugs and other biologically impor-
tant molecules.
Figure 3. FE-SEM microscopic images of nanospheres at concentration
6.9 mgmLꢀ1
.
Results and Discussion
To access the morphological insight of these self-assem-
bled nanospheres, transmission electron microscopic (TEM)
studies were performed. From the TEM study it was re-
vealed that the spheres were hollow, and they form nanove-
sicles with diameters ranging from 30 to 80 nm (Figure 4).
Figure 5 illustrates the vesicle size distribution plot showing
the population of different sized vesicles formed by this
water-soluble tripeptide. This distribution plot shows that
exclusively vesicles having diameters between 40–60 nm
have been formed.
To access the formation of supramolecular structures
through molecular self-assembly, the dynamic light scatter-
ing (DLS) experiment was performed. It is a rapid screening
method used to define nanostructures. Without the presence
of discrete peak intensity, no nanostructure can be observed.
From the DLS study, it was found that nanostructures of
various sizes (within the range 80–120 nm) were formed
(Figure 2a) at a peptide concentration of 6.9 mgmLꢀ1. How-
ever, at lower concentration (2.3 mgmLꢀ1) nanostructures of
very narrow sizes (100–110 nm) were observed (Figure 2b).
These observations encouraged us to further study these
To access an independent indication about the topography
of nanostructures, atomic force microscopic (AFM) studies
have been performed. The sample was deposited on a mica
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Transformation of Oligopeptide based Nanovesicles to Nanotubes
peptide self-assembled into vesicles at a concentration of
6.9 mgmLꢀ1.
The solution of nanovesicles was diluted in such a way
that the final concentration was 2.3 mgmLꢀ1. After aging
this solution for 6 h, transmission electron microscopic stud-
ies were performed and it was observed that tubular struc-
tures were formed with an average diameter of 70 nm and
the length of these tubes was ~2.5 mm (Figure 7a–d).
Figure 4. TEM images showing the formation of nanovesicles at concen-
tration 6.9 mgmLꢀ1
.
Figure 5. Vesicle size distribution plot showing the population of different
size vesicles at concentration 6.9 mgmLꢀ1
surface and investigated by an atomic force microscope. The
calculated height of the vesicle was about 15.34 nm (Fig-
ure 6c). All these studies confirmed that the reported tri-
Figure 7. TEM images of nanotubular structure formed at peptide con-
centration 2.3 mgmLꢀ1. (a) and (b) showing a single nanotube with a
hollow channel, (c) showing the length of a tube, (d) showing the coexis-
tence of more than one nanotube.
For further clarification of this transition the stock pep-
tide solution (6.9 mgmLꢀ1) was diluted to make a final con-
centration of 3.45 mgmLꢀ1. This diluted solution was aged
for 3 h and then transmission electron microscopic studies
were performed. Interestingly, the formation of an ordered
array (Figure 8) of fused nanovesicles were observed. Here
it can be thought that the vesicle to tube transition passes
through an intermediate structure of a fused orderly aligned
array of nanostructures, as depicted in Figure 8. TEM ex-
periments were also performed using samples with a concen-
tration of 12 mgmLꢀ1 (above 6.9 mgmLꢀ1) as well as at a
concentration below 2.3 mgmLꢀ1 (1.6 mgmLꢀ1) to investi-
gate whether any new nanofeature is observed. However,
vesicular morphology is still retained at higher concentra-
tions (12 mgmLꢀ1) and tubular morphology is also retained
at lower concentration (1.6 mgmLꢀ1) (Figure S6 in the Sup-
porting Information). It is interesting to note that the
change in concentration plays a vital role to illustrate the
Figure 6. Atomic force microscopic image of vesicle (a), topographic
view along z-axis (b), height profile plot (c).
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A. Banerjee and J. Naskar
FULL PAPERS
Figure 10. CD spectra of the peptide at different concentration,
(a) 6.9 mgmLꢀ1, (b) 3.4 mgmLꢀ1, (c) 2.3 mgmLꢀ1
.
this reported tripeptide at different concentrations[17] and
this supports the data obtained from the FTIR study. Only
the intensity of the peaks changed with concentration whilst
the basic features of the peak remained unaltered.
Figure 8. TEM image showing the transition of nanovesicles to nanotube
at intermediate concentration (3.4 mgmLꢀ1). Circle showing an isolated
vesicle.
It is well known that various interactions, such as hydro-
gen bonding, p–p interaction, and hydrophobic interactions
play a significant role in the self-assembly of peptides in
water and this ultimately leads to the formation of various
peptide based nanostructures including nanotubes, nanorods,
nanovesicles, and others.[2d,e,f,3c,10,14a,b] It is evident from CD
and FTIR experiments that the reported oligopeptide Acp-
Tyr-Glu has a turn structure in solution, which does not
change upon dilution. Figure 11 provides a schematic repre-
type of nanostructure formation from the self-assembling
tripeptide (Figure 9). This is a unique example of a nanove-
sicle to nanotube transition that is triggered by dilution.
Figure 9. Schematic illustration of the concentration dependent nano-
structural transformation from nanovesicles to nanotubes with the ap-
pearance of an intermediate nanostructure of orderly array of fused
nanovesicles: (a) nanovesicles, (b) intermediate, orderly array of fused
nanovesicles, (c) a nanotube.
Figure 11. Schematic presentation showing a tentative model for the for-
mation of a nanotube and a nanovesicle: (a) turn shaped molecular con-
formation, (b) molecular assembly having a curvature, (c) two dimension-
al layer closure of this curved structure generates a nanovesicle, (d) roll-
ing of this curved structure in one direction leads to the formation a
nanotube.
An FTIR study (Figure S5 in the Supporting Information)
has been performed in order to access the secondary struc-
ture of this water-soluble tripeptide at different concentra-
tions. From the FTIR spectra, it was found that an intense
sentation illustrating the formation of nanovesicles and
nanotubes from a curved shaped assembly. This turn struc-
ture can be self-assembled in one direction to form a
curved, assembled structure (Figure 11b). Two-dimensional
layer closure of this type of curved shaped supramolecular
structure leads to the formation of a vesicular structure (Fig-
ure 11c). Furthermore, rolling up of this curved structure re-
peak (owing to amide I stretching) appeared at 1673 cmꢀ1
ꢀ1
ꢀ
and an N H stretching frequency appeared at 3400 cm in-
dicating a hydrogen bonded turn (type II b turn)[16] confor-
mation of the reported peptide at different concentrations.
Circular dichroic (CD) spectra (Figure 10) of this water-
soluble tripeptide at different concentrations showed two
positive bands. A band arising from an n–p* transition ap-
peared at 225 nm (for various concentrations) and the other
peaks (for p–p*) appeared at 205 and 207 nm, clearly indi-
cating the presence of a turn-like secondary structure for
sults in the formation of
ure 11d).[3b,9,18]
a tubular structure (Fig-
Formation of soft vesicular structures and their disruption
is very important in drug delivery. For that purpose, we
became interested in optimizing the condition for the rup-
turing of these soft vesicular structures. From the TEM
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Transformation of Oligopeptide based Nanovesicles to Nanotubes
study, it was observed that the presence of biocompatible
Ca2+ ions (at a peptide/metal ion concentration ratio 2:1)
could trigger the rupturing of these nanovesicles (Fig-
ure 12a) and energy dispersive X-ray (EDX) analysis
proved the presence of calcium within the ruptured vesicles
(Figure S4 in the Supporting Information). We have also
tested the pH sensitivity of these nanovesicles. This vesicular
morphology was completely lost at pH 10.7 (Figure 12b).
Figure 13. Confocal fluorescence microscopic image of doxorubicin
loaded vesicles (a), doxorubicin released from vesicles in the presence of
calcium ions (b).
Conclusions
In summary, we have described the self-assembly of a water-
soluble tripeptide that leads to the formation of two nano-
scopic spieces, nanovesicles and nanotubes, at neutral pH.
At a peptide concentration of 6.9 mgmLꢀ1, exclusive forma-
tion of nanovesicles was observed and at 2.3 mgmLꢀ1, the
peptide is self-assembled into nanotubes. This indicates that
this vesicle to tube transition is concentration dependent.
These peptide nanovesicles were able to entrap small drug
molecules, doxorubicin. Formation of nanovesicles was very
sensitive toward metal ions and pH. These vesicular struc-
tures can be ruptured in the presence of biocompatable
Ca2+ ions and also by an increase of the pH of the solution.
Calcium ion triggered rupturing of drug loaded nanovesicles
holds future promise to use these nanovesicles as nanovehi-
cles of carrying drugs to the target site.
Figure 12. TEM images of ruptured nanovesicles (a) in the presence of
calcium ions (b) at high pH 10.7 (c) stability of vesicles at pH 5.2.
Biomolecule based nanostructures are very promising
candidates as excellent vehicles for carrying various bioac-
tive molecules into living systems owing to their biocompati-
bility and tailorability for targeted delivery.[3c,9,19] There are
various reports of peptide based self-assembled nanostruc-
tures. However, the encapsulation of bioactive molecules
into peptide vesicles is yet to be explored. Here, a simple
enclosed environment of peptide nanovesicles has been uti-
lized for encapsulation of a potent anticancer drug, doxoru-
bicin. In this study the peptide solution was incubated with
doxorubicin for seven days to permit fluorescence detection
of drug-loaded nanovesicles. Fluorescence microscopic stud-
ies clearly affirmed the drug entrapment from the appear-
ance of fluorescent circular structures (Figure 13a) viewed
under a fluorescence microscope. It is interesting to note
that the entrapped drug can be released easily by adding
Ca2+ ions into the drug loaded vesicle solution (Figure 13b).
The presence of Ca2+ ions brings about the complete rap-
ture of these drug-filled vesicles and allows release of the
drug into the surroundings. Thus, these nanovesicles may
serve as delivery vehicles for drugs and other biologically
important molecules.
Experimental Section
The reported peptide was synthesized by conventional solution phase
methods by using recimization free fragment condensation strategy. The
Boc group was used for N-terminal protection and the C-terminus was
protected with a methyl ester. Deprotections of the ester group were per-
formed using the saponification method, and removal of the Boc group
with trifluoro acetic acid (TFA). Couplings were mediated by dicyclohex-
ylcarbodiimide and 1-hydroxybenzotriazole (DCC/HOBT). The final
compound was fully characterized by NMR, mass, and IR spectroscopy.
Synthesis
Boc-Acp-OH: A solution of 6-aminocaproic acid (2.62 g, 20 mm) in a
mixture of dioxane (40 mL), water (20 mL), and 1 m NaOH (20 mL) was
stirred and cooled in an ice water bath. Di-tert-butyl pyrocarbonate
(4.8 g, 22 mm) was added, and stirring was continued at room tempera-
ture for 6 h. The solution was then concentrated in vacuum to about 20–
25 mL, cooled in an ice-water bath, covered with a layer of ethyl acetate
(about 20 mL), and acidified with a dilute solution of KHSO4 to pH 2–3
(congo red). The aqueous phase was extracted with ethyl acetate, and
this operation was done repeatedly. The ethyl acetate extracts were
pooled, washed with water, dried over anhydrous Na2SO4, and evaporat-
ed in vacuum. The pure material was obtained as a waxy solid. Yield:
3.93 g (17 mm, 85%); elemental analysis: calcd (%) for C11H21NO4 (231):
C 57.79, H 9.76, N 6.06; found: C 56.77, H 9.78, N 5.99.
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A. Banerjee and J. Naskar
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Boc-Acp-Tyr-OMe (1): A total of 3.69 g (16 mm) of Boc-Acp-OH was
dissolved in a mixture of 25 mL of dichloromethane (DCM) in an ice
water bath. H-Tyr-OMe was isolated from 7.36 g (32 mm) of the corre-
sponding methyl ester hydrochloride by neutralization, subsequent ex-
traction with ethyl acetate, and concentration (15 mL). This was added to
the reaction mixture, followed immediately by 3.3 g (16 mm) of DCC.
The reaction mixture was allowed to come to RT and was stirred for
48 h. DCM was evaporated, the residue was taken in ethyl acetate
(60 mL), and dicyclohexylurea (DCU) was filtered off. The organic layer
was washed with 2 m HCl (3ꢃ50 mL), brine (2ꢃ50 mL), and 1 n sodium
carbonate (3ꢃ50 mL) and brine (2ꢃ50 mL), dried over anhydrous
sodium sulfate, and evaporated under vacuum to yield as a white solid.
Yield: 4.89 g (12 mm, 75%); 1H NMR (300 MHz, [D]CHCl3): d=6.95 (d,
J=9, 2H; ArH), 6.81 (d, J=9, 2H; ArH), 5.82 (d, J=6, 1H; Tyr
CONH), 4.90–4.83 (m, 1H; Tyr CaH), 4.64 (b, 1H; Acp CONH), 3.77 (s,
3H; COOCH3), 3.24–3.18 (m, 2H; Acp CeH2), 3.02–2.98 (m, 2H; Tyr
CbH2), 2.87–2.79 (m, 2H; Acp CaH2), 2.16–2.05 (m, 2H; Acp CbH2), 1.46
(s, 9H; (CH3)3C), 1.40–1.28 (m, 2H; Acp CdH2), 1.06–1.04 ppm (m, 2H;
Acp CgH2); ESI-MS: 430.92 [M+Na]+, 431.92 [M+H+Na]+; elemental
analysis: calcd (%) for C21H32N2O6 (408): C 61.75, H 7.90, N 6.86; found:
C 50.55, H 7.96, N 6.42.
50 mL). The extracts were pooled, dried over anhydrous sodium sulfate,
and evaporated under vacuum to yield 4. Yield: 2.09 g (4 mm, 66.6%);
1H NMR (300 MHz, [D6]DMSO): d=12.41 (b, 2H; COOH), 8.18 (d, J=
6, 1H; Glu CONH), 7.89 (d, J=9, 1H; Tyr CONH), 7.03 (d, J=9, 2H;
Ar H), 6.71 (b, 1H; Acp CONH), 6.62 (d, J=6, 2H; Ar H), 4.45–4.43
(m, 1H; Glu CaH), 4.22–4.20 (m, 1H; Tyr CaH), 4.06–3.99 (m, 2H; Acp
CeH2), 2.90–2.83 (m, 2H; Tyr CbH2), 2.63–2.55 (m, 2H; Acp CaH2), 2.28–
2.20 (m, 2H; Acp CbH2), 2.00–1.98 (m, 2H; Glu CgH2), 1.85–1.75 (m,
2H; Glu CbH2), 1.36 (s, 9H; (CH3)3C), 1.19–1.15 (m, 2H; Acp CdH2),
1.10–1.08 ppm (m, 2H; Acp CgH2); ESIMS: 545.90 [M+Na]+, 561.86
[M+K]+; elemental analysis: calcd (%) for C25H37N3O9 (523): C 57.35,
H 7.12, N 8.30; found: C 57.34, H 7.01, N 8.28.
H2N-Acp-Tyr-Glu-OH (5): To 2.09 g (4 mm) of Boc-Acp-Tyr-Glu-OH
was added 5 mL of TFA, and the removal of the Boc group was moni-
tored by TLC. After 2 h, TFA was removed under vacuum. The residue
was taken in water (20 mL) and washed with diethyl ether (2ꢃ30 mL).
The pH of the aqueous solution was then adjusted to 8 with liquid NH3.
The aqueous portion was evaporated under vacuum to yield peptide 1 as
a white solid. Yield: 1.26 g (3 mm, 75%); ½aꢁ2D2 =6.82 (c=0.58 in water);
1H NMR (300 MHz, [D6]DMSO): d=8.25 (d, J=6, 1H; Glu CONH),
7.93 (d, J=6, 1H; Tyr CONH), 7.04 (d, J=9, 2H; ArH), 6.63 (d, J=9,
2H; ArH), 4.49–4.44 (m, 1H; Glu CaH), 4.25–4.18 (m, 1H; Tyr CaH),
2.93–2.88 (m, 2H; Acp CeH2), 2.82–2.72 (m, 2H; Tyr Cbh2), 2.64–2.55 (m,
2H; Acp CaH2), 2.31–2.27 (m, 2H; Acp CbH2), 2.05–2.01 (m, 2H; Glu
CgH2), 1.86–1.76 (m, 2H; Glu CbH2), 1.49–1.33 (m, 2H; Acp CdH2), 1.08–
1.04 ppm (m, 2H; Acp CgH2); 13C NMR (75 MHz, [D6]DMSO): d=
173.78 (COOH), 173.12 (COOH), 171.97 (C=O), 171.87 (C=O), 155.72,
130.96, 128.06, 114.81 (aromatic carbon), 53.95 (Glu Ca), 51.20 (Tyr Ca),
36.73 (Acp Ce), 34.93 (Tyr Cb), 30.01 (Acp Ca), 26.76 (Acp Cd), 26.35
(Glu Cg), 25.28 (Glu Cb), 24.63 ppm (Acp Cg); ESIMS: 446.08 [M+Na]+,
468.04 [M+K]+, 424.04 [M+H]+; elemental analysis: calcd (%) for
C20H29N3O7 (423): C 56.73, H 6.90, N 9.92; found: C 56.34, H 6.33, N 9.28.
Boc-Acp-Tyr-OH (2): To 4.48 g (11 mm) of Boc-Acp-Tyr-OMe was added
25 mL of MeOH and 15 mL of 2m NaOH, and the progress of saponifica-
tion was monitored by thin layer chromatography (TLC). The reaction
mixture was stirred. After 10 h, methanol was removed under vacuum
and the residue was taken in 50 mL of water and washed with diethyl
ether (2ꢃ50 mL). Then the pH of the aqueous layer was adjusted to 2
using 1 m HCl, and it was extracted with ethyl acetate (3ꢃ50 mL). The
extracts were pooled, dried over anhydrous sodium sulfate, and evaporat-
ed under vacuum to yield 3.92 g of 2. Yield: 3.92 g (10 mm, 90.6%);
1H NMR (300 MHz, [D6]DMSO): d=12.55 (b, 1H; COOH), 8.01 (d, J=
6, 1H; Tyr CONH), 6.99 (d, J=6, 2H; ArH), 6.75–6.73 (t, J=6, 1H; Acp
CONH), 6.63 (d, J=9, 2H; ArH), 4.34–4.26 (m, 1H; Tyr CaH), 4.06–3.99
(m, 2H; Acp CeH2), 2.87–2.81 (m, 2H; Tyr CbH2), 2.74–2.66 (m, 2H; Acp
CaH2), 2.05–1.99 (m, 2H; Acp CbH2), 1.36 (s, 9H; (CH3)3C), 1.31–1.28
(m, 2H; Acp CdH2), 1.19–1.11 ppm (m, 2H; Acp CgH2); ESI-MS: 417.57
[M+Na]+, 433.55 [M+K]+; elemental analysis: calcd (%) for C20H30N2O6
(394): C 60.90, H 7.61, N 7.10; found: C 61.05, H 7.66, N 7.00.
Experiments
NMR experiments: NMR studies were carried out on a Brꢄker DPX
300 MHz spectrometer at 300 K. Compounds concentrations were in the
range 1–10 mmol in CDCl3 and [D6]DMSO
Mass Spectroscopy: Mass spectra were recorded on a HEWLETT PACK-
ARD series 1100MSD and Micromass Qtof micro YA263 mass spectrom-
eter by positive mode electron spray ionisation.
Boc-Acp-Tyr-Glu-OMe (3): A total of 3.54 g (9 mm) of 2 in 10 mL of
N,N-dimethylformamide was cooled in an ice water bath. H-Glu-OMe
was isolated from 4.2 g (20 mm) of the corresponding methyl ester hydro-
chloride by neutralization, subsequent extraction with ethyl acetate, and
concentration (15 mL). This was added to the reaction mixture, followed
immediately by 2.08 g (11.11 mm) of DCC and 1.37 g (10.11 mm) of
HOBT. The reaction mixture was stirred for 3 days. The residue was
taken in ethyl acetate (60 mL), and DCU was filtered off. The organic
layer was washed with 2 mL HCl (3ꢃ50 mL), brine (3ꢃ50 mL), 1m
sodium carbonate (3ꢃ50 mL), and brine (2ꢃ50 mL), dried over anhy-
drous sodium sulfate, and evaporated under vacuum to yield the peptide
as a white solid. Purification was done by a silica gel column (100–200
mesh) using chloroform/methanol (98:2) as the eluent. Yield: 3.30 g
(7 mm, 77.77%); 1H NMR (300 MHz, [D]CHCl3): d=7.02 (d, J=9, 2H;
ArH), 6.96 (d, J=9, 1H; Glu CONH), 6.80 (d, J=9, 2H; ArH), 5.97 (b,
1H; Acp CONH), 4.07–4.67 (m, 1H; Glu CaH), 4.60–4.53 (m, 1H; Tyr
CaH), 3.75 (s, 3H; COOCH3), 3.67 (s, 3H; Glu side chain COOCH3),
3.16–3.05 (m, 2H; Acp CeH2), 3.03–2.97 (m, 2H; Tyr CbH2), 2.89–2.81 (m,
2H; Acp CaH2), 2.40–2.34 (m, 2H; Glu CgH2), 2.23–2.05 (m, 2H; Glu
CbH2), 2.03–1.96 (m, 2H; Acp CbH2), 1.46 (s, 9H; (CH3)3C), 1.37–1.25
(m, 2H; Acp CdH2), 1.03–0.96 ppm (m, 2H; Acp CgH2); ESIMS:
[M+Na]+ 573.99; elemental analysis: calcd (%) for C27H41N3O9 (551): C,
58.79; H, 7.49; N, 7.62. Found: C, 58.34; H, 7.33; N, 7.28.
Dynamic light Scattering (DLS) experiment: DLS has been done in
Nano ZS MALVERN Instrument UK using solution of different peptide
concentration.
Circular Dichroic (CD) study: CD study has been carried out on a
JASCO J-815-150S instrument at a temperature of 258C.
Polarimeter: Perkin–Elmer instruments. Model 341 LC polarimeter.
Fourier Transform IR (FT-IR) spectroscopy: The FT-IR spectra were
taken using Shimadzu (Japan) model FT-IR spectrophotometer. Peptide
solutions were suspended on a CaF2 plate and dried by vacuum. The pep-
tide deposits were resuspended with D2O and subsequently dried to form
thin films and spectra were collected at 258C.
Transmission electron microscopy (TEM) and field-emission scanning
electron microscopy (FE-SEM): TEM and FE-SEM were carried out to
investigate the morphology of the nanostructures. In general, solutions of
peptide at different concentration were taken and a drop of the solution
was placed on a carbon-coated copper grid (300 mesh) and evaporated.
Again, a drop of uranil acetate solution (freshly prepared 2% uranil ace-
tate solution) was added and dried under vacuum for 10 h. With these
grids, TEM studies were carried out using a JEOL JEM 2010 electron
microscope. During SEM, a solution of the reported peptide was taken
on glass cover slips and evaporated to dryness for 24 h. A gold coating
was applied on the top of the sample to make it conductive for analysis.
It was studied on a JEOL JSM 6007F instrument at 3.0 KV voltage and
20000ꢃ magnification.
Boc-Acp-Tyr-Glu-OH (4): To 2.82 g (6 mm) of Boc-Acp-Tyr-Glu-OMe
were added 25 mL of MeOH and 15 mL of 2m NaOH, and the progress
of saponification was monitored by thin-layer chromatography (TLC).
The reaction mixture was stirred. After 10 h, methanol was removed
under vacuum and the residue was taken in 50 mL of water and washed
with diethyl ether (2ꢃ50 mL). The pH of the aqueous layer was then ad-
justed to 2 using 1m HCl, and it was extracted with ethyl acetate (3ꢃ
Atomic force microscopic (AFM) study: The peptide solution was dried
by slow evaporation on a microscopic cover glass and then atomic force
1822
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ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2009, 4, 1817 – 1823

Transformation of Oligopeptide based Nanovesicles to Nanotubes
microscopic (AFM) study was performed using an AUTOPROBE CP
BASE UNIT, di CP-II instrument, Model no. AP-0100 instrument.
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Received: July 14, 2009
Published online: November 17, 2009
Chem. Asian J. 2009, 4, 1817 – 1823
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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