Fabrication of nanoporous material from a hydrophobic peptide

Article abstract of DOI:10.1039/c0ce00701c

A pentapeptide containing hydrophobic residues self-assembles to form nonporous materials in solid state. The X-ray crystallography reveals that there is no pore in the crystal and the peptide exhibits supramolecular helical architecture prompted by the formation of intra- and intermolecular hydrogen bonds. But the phase-selective gelation of the peptide from xylene-water leads to the formation of nanoporous material with different internal diameter. The field emission scanning electron micrographs (FE-SEM) show that the average pore size is in the range of 20 to 50 nm. Moreover, the nanoporous xerogel can efficiently adsorb I₂ and remove organic dyes from wastewater.

Full text of DOI:10.1039/c0ce00701c

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PAPER
Fabrication of nanoporous material from a hydrophobic peptide†
*
Sibaprasad Maity, Poulami Jana and Debasish Haldar*
Received 7th October 2010, Accepted 8th February 2011
DOI: 10.1039/c0ce00701c
A pentapeptide containing hydrophobic residues self-assembles to form nonporous materials in solid
state. The X-ray crystallography reveals that there is no pore in the crystal and the peptide exhibits
supramolecular helical architecture prompted by the formation of intra- and intermolecular hydrogen
bonds. But the phase-selective gelation of the peptide from xylene–water leads to the formation of
nanoporous material with different internal diameter. The field emission scanning electron micrographs
(FE-SEM) show that the average pore size is in the range of 20 to 50 nm. Moreover, the nanoporous
xerogel can efficiently adsorb I2 and remove organic dyes from wastewater.
synthesized hexagonal gold nanoparticles.8 Herein, we present
Introduction
the conversion of a nonporous peptide into a nanoporous
material in the presence of water. From X-ray crystallography,
there is no pore in the crystal and the peptide 1 exhibits supra-
molecular helical architecture prompted by the formation of
intra- and intermolecular hydrogen bonds. Water mediated
gelation of the peptide from xylene leads to the formation of
nanoporous material with average pore size in the range of 20 to
50 nm. Moreover, the nanoporous xerogel can efficiently adsorb
I2 and remove organic dyes from wastewater.
Fabrication of nanoporous materials is a highly interesting area
of recent research due to their potential application in gas
storage, sensors, ion exchange, purifications, catalysis and
separation.1 In particular, the nanoporous materials have
a strong propensity to interact with atoms, ions, molecules and
adsorb on their nanometre sized pore space and in the large
interior surfaces.2 Up to now, several effective strategies, such as
emulsion processes, surface-protected etching, supramolecular
self-assembly, as well as the replication method, have been
developed to prepare organic microporous (below 2 nm),
mesoporous (2–50 nm) and macroporous (>50 nm) materials
containing predictable porous topologies.3 But it has proved
difficult to form organic polymer networks with perfectly
controlled pore dimensions.4
Results and discussion
Synthesis and solid state analysis
The pentapeptide Boc-Leu(1)-Val(2)-Aib(3)-Phe(4)-Aib(5)-OMe
(1) consisting of hydrophobic amino acid residues, has been
synthesized by conventional solution-phase methodology and
purified, characterized and studied. There are two conforma-
tionally constrained Aib residues to increase the helical nature of
the peptide backbone and enhance the crystallinity.9 Solid-state
FT-IR spectroscopy was performed to study the secondary
structure of the peptide 1. The region 1800–1500 cmꢀ1 is
important for the stretching band of amide I, bending peak of
amide II and hydrogen bonded urethane groups.10 Another
informative frequency range is 3500–3200 cmꢀ1, corresponding
to the N–H stretching vibrations of the peptide.10 An intense
band at 3319 cmꢀ1 indicates the presence of strongly hydrogen-
bonded NH groups. No band has been observed at around
3400 cmꢀ1, indicating that all NH groups are involved in intra-
and intermolecular hydrogen bonding.11 The amide I band at
1654 cmꢀ1 and amide II band at 1528 cmꢀ1 suggest that the
peptide adopts helical structure in solid state (Fig. 1).12 The
helical structure of the peptide 1 was further studied by single
crystal X-ray diffraction. The colorless orthorhombic crystal of
the peptide 1 was obtained from methanol solution by slow
evaporation.13 The ORTEP diagram of peptide 1 with the atomic
Many industries including textile, leather, paper, plastic,
printing, foods and cosmetics, use large quantities of dyes and
produce toxic wastewater with residual dyes. This toxic color
interferes with the penetration of sunlight into water, gas solu-
bility in water and also pollutes the aquatic ecological system. So,
the removal of toxic dyes from wastewater is highly important.
Recently organic–inorganic hybrid gels,5 activated charcoal,
clay, porous silica, different polymers6 and such types of different
scaffolds are used for removing dyes from wastewater.7
Recently, we have reported the formation of polydisperse
nanopores by the self-assembly of a b-turn forming tripeptide as
an associating sub-unit that also acts as in situ reducing agent and
Department of Chemical Sciences, Indian Institute of Science Education
and Research, Kolkata, Mohanpur, West Bengal, 741252, India. E-mail:
deba_h76@yahoo.com; deba_h76@iiserkol.ac.in; Fax: +91 34 73
279131; Tel: +91 34 73 279130
† Electronic supplementary information (ESI) available: Synthesis and
1
characterization of peptides, H NMR and 13C NMR (Fig. S1–S4 and
S5–S13). CCDC reference number 772430. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c0ce00701c
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handed helical region of the Ramachandran diagram [except f5
(ꢀ56.8ꢁ) and j5 (142.5ꢁ) values of Aib(5). From Fig. 2 there are
two intramolecular hydrogen bonds N1–H1/O5 and N2–H2/
O4 resulting in a helical conformation for individual peptide
molecules in the solid state. The individual helical sub-units of
peptide 1 are themselves regularly inter-linked through an
intermolecular hydrogen bonding interaction N5–H5b/O7 and
thereby form a supramolecular left handed helix along the
crystallographic a direction (Fig. 3).15 Moreover, in higher order
packing, the supramolecular helical columns are interdigitated
by non-covalent interactions and formed a supramolecular
helical bundle according to the crystallographic c axis (Fig. 4).16
The space filling presentation of higher order crystal packing
(ESI, Fig. S1†) shows that there are no pores in the crystal. The
hydrogen bonding parameters of peptide 1 are listed in Table 2.
Fig. 1 FT-IR spectra of pentapeptide 1 in (a) solid and (b) xerogel from
xylene–water.
Phase-selective gelation and thermal behaviour
The phase-selective gelation from a given mixture of solvents is
an interesting task due to its many applications.17 The peptide 1
forms a clear solution at elevated temperatures in aromatic
solvents and microcrystals are precipitated out of the solution on
cooling. In the present study, peptide 1 was found to be suitable
for selective gelation from an organic solvent/water mixture. In
a typical procedure, 1 mL of xylene and 1 mL of water were
shaken in a sample tube and 80 mg of the peptide 1 was added.
The tube was heated to dissolve the compound in the organic
Fig. 2 The ORTEP diagram of pentapeptide 1 showing the atomic
numbering scheme. Ellipsoids are drawn at the 30% probability level.
Intramolecular hydrogen bonds are shown as dotted line.
Table 1 Selected backbone torsion angles (ꢁ) for peptide 1
C12–N5–C11–C10
N5–C11–C10–N4
C10–N4–C9–C8
N4–C9–C8–N3
C6–C7–N3–C8
66.2 f1
33.4 j1
66.5 f2
22.6 j2
53.3 f3
N3–C7–C6–N2
C6–N2–C5–C4
N2–C5–C4–N1
C4–N1–C3–C2
N1–C3–C2–O1
33.2 j3
75.5 f4
15.9 j4
ꢀ56.8 f5
142.5 j5
numbering scheme is shown in Fig. 2. The solid state confor-
mation of peptide 1 shows that there are two consecutive b-turn
structures where Aib(2) occupies the i + 2nd position of the first
turn and i + 1st position for the second turn.14 Most of the
backbone torsion angles (Table 1) of peptide 1 are in the left
Fig. 3 Supramolecular helix from self-assembly of peptide 1 along the
crystallographic a direction. Intermolecular hydrogen bonds are shown
as dotted line.
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Fig. 6 FE-SEM images of peptide 1 microcrystal from xylene solution
(a) and the surface of microcrystal (b) exhibits no pores.
with increasing gelator concentration (ESI, Fig. S2†), a feature
generally observed for low molecular weight (LMW) gels.18
Morphology and characterization
To obtain insight into the morphology of the reported peptide in
gel state, field-emission scanning electron microscopic (FE-SEM)
measurements were carried out.19 For FE-SEM experiments,
peptide 1 solution in xylene was drop cast on a microscopic glass
slide and dried under vacuum and studied. Fig. 6 exhibits the
microcrystals obtained from the xylene solution. Moreover, there
are no pores visible on the surface of the microcrystal.
Fig. 4 Packing diagram of peptide 1 showing supramolecular helical
columns which are further self-assembled to form helical bundle.
For FE-SEM studies, xerogel of peptide 1 from xylene–water
was placed on a microscopic glass slide and then further dried
under vacuum for two days. Fig. 7 depicts the FE-SEM image of
the peptide xerogel. In Fig. 7, the micrograph shows the porous
nanostructure morphology in their self-assembled state and the
pores are polydisperse in nature with different diameters.
Average pore size is 20–50 nm from solid state. The selected area
electron diffraction (SAED) experiment (transmission electron
microscopy) of xerogel (from xylene–water) exhibits the many
ordered scattering spots (Fig. 8).
Table 2 Hydrogen bonding parameters of peptide 1
ꢁ
ꢀ
ꢀ
D–H/A
H/A/A
D/A/A
D–H/A/
N1–H1/O5
N2–H2/O4
N5–H5a/O7
2.27
2.5
2.24
3.03
3.33
2.90
148
164
133
a
Symmetry equivalent ꢀ1+x, y, z.
Though it is apparent from the FT-IR spectrum (Fig. 1) that
both the solid compound (crystal) and xerogel (from xylene–
water) have almost identical structure. Fig. 9 shows the wide
angle X-ray scattering (WAXS) patterns of solid powder as
synthesized (a), xerogel from xylene–water (b) and powder
pattern from single crystal X-ray (c). From the three spectra it is
clear that there is a significant change in position and intensity of
the major peaks for as-synthesized solid material, xerogel and
crystal. In fact, sharp reflections are observed in the 20–25ꢁ 2q
phase (xylene) and then it was shaken vigorously to ensure
homogeneous dispersion. After cooling at room temperature the
organic phase gelated entrapping the water inside the tube
(Fig. 5). The gelation was confirmed by the inverted test tube
method. The gels are white, opaque and stable for months at
room temperature. The gel is thermoreversible. The sol–gel
transition temperature (Tgel) of the peptide 1 in xylene increases
Fig. 5 Selective organic liquid gelation over water. (a) Clear solution of
peptide 1 in xylene over water after heating. (b) The opaque gel after
cooling. The aqueous phase (top) is trapped by the gel.
Fig. 7 Field emission scanning electron microscopic image of peptide 1
xerogel showing nanoporous morphology in the solid state.
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Table 3 Analysis and comparison of major X-ray peaks obtained from
peptide 1 crystal, as synthesized solid powder and xerogel from xylene–
water
ꢀ
d-spacing/A
Crystal
Solid
5.59, 4.98, 4.93, 4.84, 4.53, 4.23,
4.19, 3.95, 3.80, 3.65, 3.38
5.74, 5.61, 5.53, 5.39, 5.24, 5.10,
4.98, 4.88, 4.80, 4.64, 4.63, 4.56,
4.42, 4.33, 4.27, 4.14, 3.94, 3.85,
3.74, 3.49, 3.37
Xerogel
5.77, 5.68, 5.44, 5.21, 4.99, 4.83,
4.64, 4.55, 4.33, 3.76, 3.63, 3.54
Fig. 8 SAED (TEM) pattern of peptide 1 xerogel from xylene–water.
range for the as-synthesized materials and crystal whereas only
a very broad feature was observed in the xerogels (Table 3). Also
in the 15–20ꢁ 2q range, there are more sharp reflections in
xerogels than in crystal.
Adsorption in nanopores
In order to examine the pores in xerogel, I2 absorption studies
have been performed. The peptide 1 crystals and nanoporous
xerogel were exposed to I2 vapors.20 Exposure of the sample was
carried out in a closed chamber for 24 h. It was observed that the
nanoporous xerogel of peptide 1 turned dark brown (Fig. 10),
whereas the crystals turned yellowish brown. The nature of the
absorption was verified by crushing the xerogel of peptide 1 after
iodine exposure. The dark brown color inside the nanoporous
xerogel indicates uniform absorption of I2.
Fig. 10 Pentapeptide 1 (a) crystals and (b) xerogel from xylene–water
showing colour change after exposure to I2 vapours.
violet solution. For Rhodamine 6G, 3 mL of 0.005 mM dye
solution was treated with 5 mg of xerogel. The UV-visible
spectroscopy shows that the maximum amount of the crystal
violet (ESI, Fig. S3†) and Rhodamine 6G (Fig. 11) was absorbed
by nanoporous xerogel leaving a nearly clear solution after 24 h.
The maximum amount of crystal violet absorbed by xerogel is
Moreover, we have used nanoporous peptide 1 xerogel
obtained from xylene–water as a filter for dye removal.
Absorption of dye molecules from their respective aqueous
solutions was monitored by UV-visible spectroscopy. 5 mg of
nanoporous xerogel were added to 3 mL of 0.001 mM crystal
7.0 mg gꢀ1 and Rhodamine 6G is 4.5 mg gꢀ1
.
Fig. 11 UV-vis spectra for time dependent absorption of Rhodamine 6G
from aqueous solution by the pentapeptide 1 xerogel from xylene–water.
Fig. 9 WAXS spectra of pentapeptide 1 in (a) solid, (b) xerogel from
xylene–water and (c) powder pattern from single crystal X-ray.
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and brine (2 ꢂ 50 mL) and dried over anhydrous sodium sulfate,
and evaporated under vacuum to yield Boc-Leu-Val-OMe as
a white solid. Purification was done by silica gel column (100–
200 mesh size) and ethyl acetate and hexane 1 : ꢁ3 as eluent.
Yield: 3.85 g (11.26 mmol, 70.37%); mp: 116 C.
1H NMR (500 MHz, CDCl3, d ppm): 6.61–6.60 [d, 1H, J ¼ 7.5
Hz, Val(2) NH], 4.91–4.89 [d, 1H, J ¼ 8 Hz, Leu(1) NH], 4.53–
4.51 [m, 1H, Leu(1) CaH], 4.11–4.10 [m, 1H, Val(2) CaH], 3.72
[s, 3H, OCH3], 2.18–2.13 [m, 1H, Val(2) CbH], 1.69–1.63 [m, 2H,
Leu(1) CbH], 1.48–1.47 [m, 1H, Leu(1) CgH], 1.45 [s, 9H, Boc
CH3], 0.94–0.88 [m, 12H, Leu(1) Cd and Val(2) Cg]. 13C NMR
(125 MHz, CDCl3, d ppm) 172.09, 171.56, 155.74, 79.87, 59.36,
57.00, 52.10, 36.90, 31.20, 28.26, 24.77, 18.89, 17.69, 15.48, 11.31.
FT-IR (KBr): 3330, 3082, 2966, 2874, 2873, 1750, 1687, 1661,
1557, 1458, 1438, 1393, 1367, 1290, 1242, 1208, 1180, 1048, 1025,
Experimental
General methods and materials
All L-amino acids were purchased from Sigma chemicals. HOBt
(1-hydroxybenzotriazole) and DCC (dicyclohexylcarbodiimide)
were purchased from SRL.
Peptide synthesis
The peptides were synthesized by conventional solution-phase
methodology using racemization free fragment condensation
strategy.21 The Boc group was used for N-terminal protection
and the C-terminus was protected as a methyl ester. Couplings
were mediated by dicyclohexylcarbodiimide/1-hydroxybenzo-
triazole (DCC/HOBt). Methyl ester deprotection was performed
by the saponification method. All the intermediates were char-
925, 877, 785, 767, 647 cmꢀ1
.
acterized by 500 MHz H and 13C NMR and FT-IR spectros-
Anal calcd for C17H32N2O5 (344.45): C, 59.28; H, 9.36; N,
8.13. Found: C, 59.25; H, 9.40; N, 8.22%.
1
copy. The final compound was fully characterized by 500 MHz
1H NMR spectroscopy, 13C NMR spectroscopy, FT-IR spec-
troscopy and mass spectrometry. The peptide 1 was character-
ized by X-ray crystallography.
(c) Boc-Leu-Val-OH (4). To 3.82 g (11.17 mmol) of Boc-Leu-
Val-OMe, 25 mL MeOH and 2 M 15 mL NaOH were added 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; the residue was
dissolved in 50 mL of water, and washed with diethyl ether (2 ꢂ
50 mL). Then the pH of the aqueous layer was adjusted to 2 using
1 M HCl and it was extracted with ethyl acetate (3 ꢂ 50 mL). The
extracts were pooled, dried over anhydrous sodium sulfate, and
evaporated under vacuum to obtain the compound as a waxy
solid.
(a) Boc-Leu-OH (2). A solution of L-Leucine (2.62 g, 20 mmol)
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-
butylpyrocarbonate (4.8 g, 22 mmol) was added and stirring was
continued at room temperature for 6 h. Then the solution was
concentrated in vacuum to about 20–30 mL, cooled in an ice-
water bath, covered with a layer of ethyl acetate (about 50 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 and dried over anhydrous Na2SO4
and evaporated under vacuum. The pure material was obtained
as a waxy solid.
Yield: 3.56 g (10.77 mmol, 96.41%).
1H NMR (DMSO-d6, 500 MHz, d ppm): 12.67 [br, 1H,
COOH], 7.71–7.69 [d, 1H, J ¼ 8 Hz, Val(2) NH], 7.00–6.99
[d, 1H, J ¼ 8.5 Hz, Leu(1) NH], 4.17–4.14 [m, 1H, Leu(1) CaH],
4.03–3.99 [m, 1H, Val(2) CaH], 2.06–2.02 [m, 1H, Val(2) CbH],
1.63–1.57 [m, 2H, Leu(1) CbH], 1.45–1.40 [m, 1H, Leu(1)
CgH], 1.38 [s, 9H, Boc CH3], 0.88–0.87 [m, 12H, Leu(1) CdH
and Val(2) CgH]. 13C NMR (DMSO-d6, 125 MHz, d ppm):
172.82, 172.61, 155.25, 78.01, 56.72, 52.84, 40.56, 30.14, 28.14,
24.21, 22.96, 21.53, 18.97, 17.80. FT-IR (KBr): 3321, 2969, 2935,
2880, 1716, 1684, 1656, 1538, 1466, 1419, 1392, 1367, 1292, 1245,
Yield: 3.81 g (16.5 mmol, 82.5%).
1H NMR (500 MHz, DMSO-d6, d ppm): 12.36 [br, 1H,
–COOH], 7.03–7.02 [d, 1H, J ¼ 8 Hz, Leu(1) NH], 3.91–3.87
[m, 1H, Leu(1) CaH], 1.64–1.61 [m, 2H, Leu(1) CbH], 1.51–1.47
[m, 1H, Leu(1) CgH], 1.36 [s, 9H, Boc CH3], 0.87–0.83 [m, 6H,
Leu(1) CdH]. 13C NMR (125 MHz, DMSO-d6, d ppm): 174.73,
155.64, 77.76, 66.41, 51.81, 28.24, 24.38, 22.92, 21.24. FT-IR
(KBr): 3462, 3355, 3311, 2983, 2968, 2934, 2877, 2525, 1727,
1699, 1675, 1533, 1456, 1419, 1385, 1368, 1296, 1197, 1172, 1087,
1172, 1045, 1020, 929, 868, 782, 650 cmꢀ1
.
Anal calcd for C16H30N2O5 (330.42): C, 58.16; H, 9.15; N,
8.48. Found: C, 58.14; H, 9.18; N, 8.50%.
1048, 1018 cmꢀ1
.
(d) Boc-Leu-Val-Aib-OMe (5). 3.56 g (10.774 mmol) of
Boc-Leu-Val-OH was dissolved in 15 mL dry DCM in an ice-
water bath. H-Aib-OMe 3.38 g (22 mmol) was isolated from
corresponding methyl ester hydrochloride by neutralization,
subsequent extraction with ethyl acetate and concentrated to
7 mL. Then it was added to the reaction mixture, followed
immediately by 2.22 g (10.77 mmol) dicyclohexylcarbodiimide
(DCC) and 1.64 g (10.77 mmol) HOBt. The reaction mixture was
allowed to come to room temperature and stirred for 72 h. The
residue was taken in 30 mL ethyl acetate and dicyclohexylurea
(DCU) was filtered off. The organic layer was washed with 2 M
HCl (3 ꢂ 50 mL), brine (2 ꢂ 50 mL), then 1 M sodium carbonate
(3 ꢂ 30 mL) and brine (2 ꢂ 30 mL) and dried over anhydrous
sodium sulfate and evaporated under vacuum to yield the
(b) Boc-Leu-Val-OMe (3). 3.698 g (16 mmol) of Boc-Leu-OH
was dissolved in 25 mL dry DCM in an ice-water bath. H-Val-
OMe was isolated from 5.365 g (32 mmol) of the corresponding
methyl ester hydrochloride by neutralization, subsequent
extraction with ethyl acetate and the ethyl acetate extract was
concentrated to 10 mL. It was then added to the reaction
mixture, followed immediately by 3.3 g (16 mmol) of dicyclo-
hexylcarbodiimide (DCC) and 2.45 g (16 mmol) of HOBt. The
reaction mixture was allowed to come to room temperature and
stirred for 48 h. DCM was evaporated and the residue was dis-
solved in ethyl acetate (60 mL) and dicyclohexylurea (DCU) was
filtered off. The organic layer was washed with 2 M HCl (3 ꢂ
50 mL), brine (2 ꢂ 50 mL), 1 M sodium carbonate (3 ꢂ 50 mL)
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tripeptide as a white solid. Purification was done by silica gel
column (100–200 mesh size) and ethyl acetate and hexane (1 : 2)
as eluent.
and evaporated under vacuum to yield compound 7 as a white
solid. Purification was done by silica gel column (100–200 mesh
size) and ethyl acetate and hexane (1 : 3) as eluent.
ꢁ
Yield: 3.04 g (7.077 mmol, 65.68%); mp: 114 C.
Yield: 1.51 g (2.61 mmol, 46.19%).
1H NMR (CDCl3, 500 MHz, d ppm): 6.66 [s, 1H, Aib(3) NH],
6.60–6.58 [d, 1H, J ¼ 8.5 Hz, Val(2) NH], 4.87 [b, 1H, Leu(1)
NH], 4.19–4.16 [m, 1H, Leu(1) CaH], 4.07–4.06 [m, 1H, Val(2)
CaH], 3.71 [s, 3H, –OCH3], 2.27–2.21 [m, 1H, Val(2) CbH], 1.65–
1.65 [m, 3H, Leu(1) CbH & CgH], 1.52 [s, 6H, Aib(3) CH3], 1.44
[s, 9H, Boc CH3], 0.953–0.919 [m, 12H, Leu(1) CdH and Val(2)
CgH]. 13C NMR (CDCl3, 125 MHz, d ppm): 174.61, 172.55,
170.04, 155.74, 79.87, 58.27, 56.31, 52.41, 40.76, 31.20, 28.26,
24.77, 19.10, 17.47, 15.48. FT-IR (KBr): 3328, 2959, 2932, 2870,
1749, 1690, 1648, 1542, 1388, 1367, 1290, 1243, 1172, 1045,
1H NMR (500 MHz, CDCl3, d ppm): 7.27–7.20 [m, 5H,
aromatic ring protons], 7.16–7.14 [d, 1H, J ¼ 7 Hz, Phe(4) NH],
6.74 [s, 1H, Aib(3) NH], 6.59–6.57 [d, 1H, J ¼ 7.5 Hz, Val(2)
NH], 4.88–4.87 [d, 1H, J ¼ 8 Hz, Leu(1) NH], 4.80–4.76 [m, 1H,
Phe(4) CaH], 4.10–4.07 [m, 1H, Leu(1) CaH], 4.05–4.03 [m, 1H,
Val(2) CaH], 3.69 [s, 3H, –OCH3], 3.18–3.05 [m, 2H, Phe(4)
CbH], 2.31–2.21 [m, 1H, Val(2) CbH], 1.71–1.65 [m, 2H, Leu(1)
CbH], 1.63 [s, 3H, Aib(3) CH3], 1.48 [s, 3H, Aib(3) CH3], 1.44 [s,
9H, Boc CH3], 1.41[m, 1H, Leu(1) CgH], 0.95–0.88 [m, 12H,
Leu(1) CdH Val(2) CgH]. 13C NMR (125 MHz, CDCl3, d ppm):
173.88, 172.95, 171.92, 170.34, 155.86, 136.75, 129.33, 128.37,
126.86, 80.67, 59.14, 57.30, 55.83, 53.57, 52.20, 40.41, 37.82,
29.77, 28.23, 25.51, 24.80, 24.70, 22.93, 21.77, 19.32, 17.41. FT-
IR (KBr): 3332, 2961, 2927, 2872, 2346, 1747, 1651, 1524, 1455,
1024 cmꢀ1
.
Anal calcd for C21H39N3O6 (429.55): C, 58.72; H, 9.16; N,
9.78. Found: C, 58.75; H, 9.18; N, 9.76%.
(e) Boc-Leu-Val-Aib-OH (6). To 3.0
g
(7.0 mmol) of
1391, 1366, 1245, 1172, 1118, 1046, 1024 cmꢀ1
.
compound 4, 25 mL MeOH and 2 M 15 mL NaOH were added
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; the residue was
dissolved in 50 mL of water, and washed with diethyl ether (2 ꢂ
50 mL). Then the pH of the aqueous layer was adjusted to 2 using
1 M HCl and it was extracted with ethyl acetate (3 ꢂ 50 mL). The
extracts were pooled, dried over anhydrous sodium sulfate, and
evaporated under vacuum to obtain the compound as a waxy
solid.
Anal calcd for C30H48N4O7 (576.72): C, 62.48; H, 8.39; N,
9.71. Found: C, 62.52; H, 8.45; N, 9.76%.
(g) Boc-Leu-Val-Aib-Phe-OH (8). To 1.5 g (2.61 mmol) of
compound 7, 25 mL MeOH and 2 M 15 mL NaOH were added
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; the residue was
dissolved in 50 mL of water, and washed with diethyl ether (2 ꢂ
50 mL). Then the pH of the aqueous layer was adjusted to 2 using
1 M HCl and it was extracted with ethyl acetate (3 ꢂ 50 mL). The
extracts were pooled, dried over anhydrous sodium sulfate, and
evaporated under vacuum to obtain the compound as a waxy
solid.
Yield: 2.35 g (5.65 mmol, 79.83%).
1H NMR (DMSO-d6, 500 MHz, d ppm): 12.19 [s, 1H,
–COOH], 8.18 [s, 1H, Aib(3) NH], 7.47–7.45 [d, 1H, J ¼ 9 Hz,
Val(2) NH], 7.13–7.12 [d, 1H, J ¼ 8.5 Hz, Leu(1) NH], 4.19–4.16
[m, 1H, CaH Leu(1)], 3.95–3.92 [m, 1H, Val(2) CaH], 1.94–1.90
[m, 1H, Val(2) CbH], 1.59–1.56 [m, 2H, Leu(1) CbH], 1.43–1.40
[m, 1H, Leu(1) CgH], 1.40 [s, 6H, Aib(3) CH3], 1.31 [s, 9H, Boc
CH3], 0.87–0.83 [m, 12H, Leu(1) CdH and Val(2) CgH]. 13C
NMR (DMSO-d6, 125 MHz, d ppm) 175.25, 169.88, 155.84,
78.12, 56.66, 54.67, 53.15, 31.33, 28.13, 24.95, 24.54, 24.26, 22.89,
21.58, 18.96, 17.73. FT-IR (KBr): 3298, 2963, 2936, 2870, 1718,
1654, 1535, 1387, 1367, 1281, 1248, 1170, 1046, 1021, 937, 875,
Yield: 1.46 g (2.59 mmol, 99.2%).
1H NMR (DMSO-d6, 500 MHz, d ppm): 12.75 [br, 1H,
–COOH], 7.98 [s, 1H, Aib(3) NH], 7.61–7.59 [d, 1H, J ¼ 8 Hz,
Phe(4) NH], 7.39–7.38 [d, 1H, J ¼ 8 Hz, Val(2) NH], 7.26–7.15
[m, 5H aromatic ring protons], 7.05–7.03 [d, 1H, J ¼ 8.5 Hz,
Leu(1) NH], 4.40–4.35 [m, 1H, Phe(4) CaH], 4.10–4.07 [m, 1H,
Leu(1) CaH], 3.96–3.95 [m, 1H, Val(2) CaH], 3.01–2.90
[m, 2H, Phe(4) CbH], 1.95–1.89 [m, 1H, Val(2) CbH], 1.55–1.53
[m, 2H, Leu(1) CbH], 1.39–1.36 [m, 1H, Leu(1) CgH], 1.34 [s,
9H, Boc CH3], 1.32 [s, 6H, Aib(3) CH3], 0.81–0.78 [m, 12H,
Leu(1) CdH, Val(2) CgH]. 13C NMR (DMSO-d6, 125 MHz,
d ppm): 129.18, 128.07, 126.36, 78.04, 56.00, 53.46, 40.09, 36.77,
28.12, 25.00, 24.20, 23.12, 21.79, 19.13, 17.89. FT-IR (KBr):
3422, 2959, 2928, 2855, 1657, 1532, 1385, 1261, 1169, 1169, 1047,
781, 610 cmꢀ1
.
Anal calcd for C20H37N3O6 (415.52): C, 57.81; H, 8.98; N,
10.11. Found: C, 57.91; H, 9.04; N, 10.0%.
(f) Boc-Leu-Val-Aib-Phe-OMe (7). 2.32 g (5.6 mmol) of
compound 5 was dissolved in 25 mL dry DCM in an ice-water
bath. H-Phe-OMe was isolated from 2.41 g (11.2 mmol) of the
corresponding methyl ester hydrochloride by neutralization,
subsequent extraction with ethyl acetate and the ethyl acetate
extract was concentrated to 10 mL and then added to the reac-
tion mixture, followed immediately by 1.155 g (5.6 mmol) dicy-
clohexylcarbodiimide (DCC) and 0.857 g (5.6 mmol) of HOBt.
The reaction mixture was allowed to come to room temperature
and stirred for 72 h. DCM was evaporated and the residue was
dissolved in ethyl acetate (60 mL) and dicyclohexylurea (DCU)
was filtered off. The organic layer was washed with 2 M HCl (3 ꢂ
50 mL), brine (2 ꢂ 50 mL), 1 M sodium carbonate (3 ꢂ 50 mL)
and brine (2 ꢂ 50 mL) and dried over anhydrous sodium sulfate,
1024, 800 cmꢀ1
.
Anal calcd for C29H46N4O7 (562.7): C, 61.90; H, 8.24; N, 9.96.
Found: C, 62.00; H, 8.25; N, 9.86%.
(h) Boc-Leu-Val-Aib-Phe-Aib-OMe (1). 1.15 g (2.57 mmol) of
compound 7 was dissolved in 25 mL DCM in an ice-water bath.
H-Aib-OMe was isolated from 1.38 g (9.0 mmol) of the corre-
sponding methyl ester hydrochloride by neutralization, subse-
quent extraction with ethyl acetate and the ethyl acetate extract
was concentrated to 10 mL. It was then added to the reaction
mixture, followed immediately by 0.530 g (2.57 mmol) dicyclo-
hexylcarbodiimide (DCC) and 0.393 g (2.57 mmol) of HOBt. The
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reaction mixture was allowed to come to room temperature and
stirred for 72 h. DCM was evaporated and the residue was dis-
solved in ethyl acetate (60 mL) and dicyclohexylurea (DCU) was
filtered off. The organic layer was washed with 2 M HCl (3 ꢂ
50 mL), brine (2 ꢂ 50 mL), 1 M sodium carbonate (3 ꢂ 50 mL)
and brine (2 ꢂ 50 mL) and dried over anhydrous sodium sulfate,
and evaporated in a vacuum to yield Boc-Leu-Val-Aib-Phe-Aib-
OMe as a white solid. Purification was done by silica gel column
(100–200 mesh size) and ethyl acetate and hexane (1 : 3) as
eluent.
Field emission scanning electron microscopy
The morphologies of the reported materials were investigated by
field emission scanning electron microscope (FE-SEM). For the
SEM study, a small amount of the peptide 1 solution in xylene
and the gel materials were drop cast on a glass slide and dried and
coated with platinum. Then the micrographs were taken in an
SEM apparatus (JEOL microscope JSM-6700F).
Transmission electron microscopy
ꢁ
Yield: 1.19 g (1.80 mmol, 69.49%); mp: 144–145 C.
The TEM studies were performed using a small amount of the gel
materials on carbon-coated copper grids (300 mesh) by slow
evaporation and allowed to dry under vacuum at 30 ꢁC for
2 days. Images were taken both in the transmission mode and
diffraction mode. TEM was done by a JEOL JEM 2010 electron
microscope.
1H NMR (CDCl3, 500 MHz, d ppm): 7.39 [s, 1H, Aib(3) NH],
7.19 [s, 1H, Aib(5) NH], 7.15–7.13 [d, 1H, J ¼ 7 Hz, Phe(4) NH],
7.29–7.19 [m, 5H, aromatic ring protons], 6.55–6.54 [d, 1H, J ¼
5.5 Hz, Val(2) NH], 4.94 [b, 1H, Leu(1) NH], 4.66–4.62 [m, 1H,
Phe(4) CaH], 4.01–3.97 [m, 1H, Leu(1) CaH], 3.92–3.91 [m, 1H,
Val(2) CaH], 3.72 [s, 3H, –OCH3], 3.52–2.88 [m, 2H, Phe(4)
CbH], 2.28–2.24[m, 1H, Val(2) CbH], 1.74–1.71 [m, 2H, Leu(1)
CbH], 1.54 [s, 6H, Aib(5) CH3], 1.50 [s, 3H, Aib(5) CH3], 1.46 [s,
9H, Boc CH3], 1.45 [s, 3H, Aib(5) CH3], 1.27 [m, 1H, Leu(1)
CgH], 1.03–0.94 [m, 12H, Leu(1) CdH and Val(2) CgH]. 13C
NMR (CDCl3, 125 MHz, d ppm): 175.17, 174.02, 173.92, 171.03,
170.87, 156.47, 138.38, 129.29, 128.14, 126.22, 81.49, 60.41,
57.16, 55.91, 54.38, 52.20, 40.20, 36.88, 28.92, 28.24, 28.17, 26.72,
25.32, 24.89, 24.67, 23.50, 22.84, 21.63, 19.18, 17.59. FT-IR
(KBr): 3320, 2962, 2936, 2874, 1734, 1685, 1654, 1528, 1466,
X-ray crystallography
Single crystal X-ray analysis of peptide 1 was recorded on
a Bruker high-resolution X-ray diffractometer instrument.
Wide angle X-ray diffraction study
The WAXS pattern was made on the solid and the xerogel of
peptide 1 from xylene–water. The experiment was carried out in
a Seifert X-ray diffractometer (C 3000) with a parallel beam
optics attachment. The instrument was operated at a 45 kV
voltage and 200 mA current and was calibrated with a standard
silicon sample. The sample was scanned from 15ꢁ to 40ꢁ (2q) at
the step scan mode (step size 0.02ꢁ, preset time 2 s) and the
diffraction pattern was recorded using a scintillation scan
1458, 1387, 1366, 1279, 1222, 1163, 1048, 1023, 758, 702 cmꢀ1
.
Anal calcd for C34H55N4O8 (661.83): C, 61.70; H, 8.38; N,
10.58. Found: C, 61.66; H, 8.43; N, 10.62%.
NMR experiments
ꢀ
detector. The wavelength of the X-ray source is 1.5418 A (Ka
value of Cu).
All NMR studies were carried out on a Bruker AVANCE
500 MHz spectrometer at 278 K. Compound concentrations
were in the range 1–10 mM in CDCl3 and (CD3)2SO.
Conclusions
In conclusion, the conversion of a hydrophobic peptide into
a nanoporous material by the presence of water has been dis-
cussed. The X-ray crystallography reveals that there is no pore in
the crystal and the peptide 1 exhibits supramolecular helical
architecture prompted by the formation of intra- and inter-
molecular hydrogen bonds. Water mediated gelation of the
peptide from xylene leads to the formation of nanoporous
material with average pore size in the range of 20 to 50 nm.
Moreover, the nanoporous xerogel can efficiently absorb I2 and
remove organic dyes from wastewater. These findings presented
here may provide useful guidelines for the design of nano-
materials having applications in the fields of absorption, encap-
sulation of active ingredients, and separation techniques.
FT-IR spectroscopy
All reported solid-state FT-IR spectra were obtained with
a Perkin Elmer Spectrum RX1 spectrophotometer with the KBr
disk technique.
Mass spectrometry
Mass spectra were recorded on a Q-Tof Micro YA263 high-
resolution (Waters Corporation) mass spectrometer by positive-
mode electrospray ionization.
UV-visible spectroscopy
UV-visible absorption spectra were recorded on a UV-visible
spectrophotometer (Perkin Elmer).
Acknowledgements
We acknowledge the DST, New Delhi, India, for financial
assistance Project No. (SR/FT/CS-041/2009). S. Maity and
P. Jana wishes to acknowledge the C.S.I.R, New Delhi, India for
research fellowship. We are thankful to Dr Raju Mandal,
Department of Inorganic Chemistry, I.A.C.S., Jadavpur,
Kolkata-700032, India, for his assistance in X-ray crystallo-
graphy data refinement. We gratefully acknowledge the I.A.C.S.,
Phase-selective gelation
80 mg of the peptide 1 was shaken in a sample tube with 1 mL of
xylene and 1 mL of water. The tube was heated and shaken
vigorously to ensure homogeneous dispersion. The organic phase
gelated on cooling.
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ꢀ
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