Supramolecular parallel β-sheet and amyloid-like fibril forming peptides using δ-aminovaleric acid residue

Article abstract of DOI:10.1016/j.tet.2005.03.100

Four terminally blocked tripeptides containing δ-aminovaleric acid residue self-assemble to form supramolecular β-sheet structures as are revealed from their FT-IR data. Single crystal X-ray diffraction studies of two representative peptides also show that they form parallel β-sheet structures. Self-aggregation of these β-sheet forming peptides leads to the formation of fibrillar structures, as is evident from scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images. These peptide fibrils bind to a physiological dye, Congo red and exhibit a typical green-gold birefringence under polarized light, showing close resemblance to neurodegenerative disease causing amyloid fibrils.

Full text of DOI:10.1016/j.tet.2005.03.100

Tetrahedron 61 (2005) 5906–5914
Supramolecular parallel b-sheet and amyloid-like fibril forming
peptides using d-aminovaleric acid residue
Arijit Banerjee,a Apurba Kumar Das,a Michael G. B. Drewb and Arindam Banerjeea,
*
aDepartment of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
bSchool of Chemistry, The University of Reading, Whiteknights, Reading RG6 6AD, UK
Received 24 December 2004; revised 10 March 2005; accepted 23 March 2005
Abstract—Four terminally blocked tripeptides containing d-aminovaleric acid residue self-assemble to form supramolecular b-sheet
structures as are revealed from their FT-IR data. Single crystal X-ray diffraction studies of two representative peptides also show that they
form parallel b-sheet structures. Self-aggregation of these b-sheet forming peptides leads to the formation of fibrillar structures, as is evident
from scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images. These peptide fibrils bind to a
physiological dye, Congo red and exhibit a typical green-gold birefringence under polarized light, showing close resemblance to
neurodegenerative disease causing amyloid fibrils.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
agents against amyloidoses. Due to non-crystallinity and
extremely poor solubility of real amyloidogenic sequences,
there are no significant studies that clearly demonstrate the
mechanism of b-sheet formation and its aggregation at
atomic resolution using single crystal X-ray diffraction
studies. So, it is worthwhile to design supramolecular
b-sheet forming model peptides that can form amyloid-like
fibrils and are capable of forming single crystals.
Supramolecular b-sheet forming peptides are important for
their numerous potential uses in material1 and biological2
sciences. Self-aggregating b-sheet forming peptides some-
times provide a fibrous network to form gels.3 Under
suitable conditions, they can also provide suitable molecular
scaffolds for growing neurons2a and cartilage.2b
Many fatal neurodegenerative diseases like Alzheimer’s
disease,4 Parkinson’s disease,5 and prion related diseases6
are believed to occur due to protein misfolding and
subsequent protein/protein fragment aggregation to form
b-sheet rich amyloid fibrils. This fact has motivated
extensive research on the general mechanism of b-sheet
formation and the subsequent aggregation to form fibrillar
quaternary structures. Recent evidence suggests that not
only disease-related proteins, but also other non-disease
related proteins can be induced to form aggregated b-sheet
rich amyloid fibrils under appropriate conditions.7 More-
over, some very recent results have established that not the
matured fibrils but the intermediates, that is the protofibrils,
are potent neurotoxic agents for Alzheimer’s disease8 and
even for prion related diseases.9 Thorough knowledge of
b-sheet aggregation is thus, important to understand the
mechanism of fibrillogenesis in order to design therapeutic
It is still a controversial issue whether amyloid fibril
formation proceeds preferentially through the self-assembly
of parallel or antiparallel b-sheets. Recent studies have
suggested that Ab10-35 give rise to parallel10a b-strand
whereas Ab16-22 forms antiparallel b-strands.10b In our
previous study, we have demonstrated that short peptides
composed of non-coded amino acids can self-assemble to
form amyloid-like fibril forming supramolecular parallel
b-sheet structure in crystals.11
d-Ava (d-aminovaleric acid) has been used for confor-
mational interest to design new foldamers. Previous reports
described that an oligopeptide with a centrally located
d-Ava residue forms a helical conformation in solution12a
and an octapeptide with a d-Ava residue at the central
position can form a b-hairpin structure in solution.12b Very
recently, Baldauf et al. have demonstrated all possible helix
types in oligomers of d-amino acids (d-peptides) and their
stabilities employing various methods of ab initio MO
theory.13 However, in this paper, we report the use of the
d-aminovaleric acid residue (d-Ava) in model terminally
Keywords: d-Aminovaleric acid; Supramolecular parallel b-sheet;
Amyloid-like fibril.
*
Corresponding author. Tel.: C91 33 2473 4971; fax: C91 33 2473 2805;
e-mail: bcab@mahendra.iacs.res.in
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.03.100

A. Banerjee et al. / Tetrahedron 61 (2005) 5906–5914
5907
Figure 1. Schematic representation of peptides 1, 2, 3 and 4.
blocked tripeptides to design and construct supramolecular
b-sheet structures in the solid state and subsequent self-
assembly of these b-sheets leads to the formation of
amyloid-like fibrils. The schematic presentation of all
terminally protected tripeptides are shown in Figure 1. To
the best of our knowledge this is the first, crystallographic
study of oligopeptides containing a d-Ava residue.
Figure 2. FT-IR spectra at the region 3000–4000 cmK1 (a) and 1000–
2000 cmK1 (b) of peptides 1, 2, 3 and 4 in solid state.
conformational heterogeneity and to increase the crystal-
linity of the corresponding peptides. The third residues for
peptides 1 and 2 are leucine (Leu) and valine (Val),
respectively, having hydrophobic side chains that might
help these peptides to aggregate also using van der Waals
interactions. Similarly, for peptides 3 and 4 we have used
Phe and Ile, respectively. Out of the four reported
peptides, we obtained suitable single crystals for peptides
1 and 2 and obtained their structures. All peptides were
studied using FT-IR, NMR, scanning electron microscopy,
transmission electron microscopy and optical microscopy.
2. Results and discussion
In our previous results we have demonstrated that the
combination of conformationally flexible amino acid Acp
(3-aminocaproic acid) and conformationally constrained
Aib (a-aminoisobutyric acid) provides overall extended
structures14 whereas by contrast a combination of confor-
mationally flexible g-Abu (g-aminobutyric acid) and Aib
gives a turn structure.15 In this paper, a series of terminally
blocked tripeptides containing conformationally conflicting
amino acid residues (d-aminovaleric acid and a-amino-
isobutyric acid) have been designed and synthesized. The
d-aminovaleric acid residue has been used to exploit its CO
and NH groups as hydrogen bonding functionalities and the
centrally located tetramethylene unit provides sufficient
flexibility to the peptide backbone that might help these
peptides to adopt an extended backbone conformation. The
conformationally restricted a-aminoisobutyric acid (Aib)
residue has been used in each of the peptides to decrease
2.1. Solid-state FT-IR study
Preliminary information on the conformational preferences
of all peptides were obtained from solid-state FT-IR studies
(Fig. 2). In solid state (KBr matrix), intense bands at 3275–
3375 cmK1 have been observed for all reported peptides
indicating the presence of strongly hydrogen bonded NH
groups. Important IR data of all these reported peptides are
listed in Table 1. The bands corresponding to NH stretching
appear over 3430 cmK1 suggesting the occurrence of free
NH groups for peptide 2 and peptide 4.16 The absence of a
band attributable to free NH (over 3430 cmK1) indicates
Table 1. Infrared (IR) absorption frequencies (cmK1) for all reported peptides in solid state (on KBr pellet)
Peptide
CO stretch (cmK1
)
NH bend (cmK1
)
NH stretch (cmK1
)
Boc-d-Ava-Aib-Leu-OMe 1
Boc-d-Ava-Aib-Val-OMe 2
Boc-d-Ava-Aib-Phe-OMe 3
Boc-d-Ava-Aib-Ile-OMe 4
1656 (s)
1666 (s)
1652 (s)
1659 (s)
1536 (m)
1536 (m)
1541 (m)
1539 (m)
3399 (m), 3382 (m), 3292 (s)
3443 (m), 3403 (m), 3269 (s)
3401 (w), 3290 (s)
3431 (w), 3332 (m), 3287 (s)
sZstrong, wZweak, mZmedium.

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A. Banerjee et al. / Tetrahedron 61 (2005) 5906–5914
2.2. Single crystal X-ray diffraction study
These preliminary conformational data were further,
supported by single crystal X-ray diffraction studies. Single
crystals of peptide1 and peptide 2 were grown from
methanol–water solution. The molecular conformations of
the peptides 1 and 2 in the crystals are illustrated in Figure 3.
This reveals that the reported peptides are unable to form
any kind of intramolecularly hydrogen bonded (N–H/O or
N–H/N) turn structures despite the fact that there is a
centrally positioned helicogenic Aib17 residue for both
peptides and the f and j values of the majority of the
constituent amino acid residues fall within the helical region
of the Ramachandran map (Table 2 for peptide 1 and Table
3 for peptide 2). Self-assembly of each individual monomer
leads to the formation of parallel b-sheet columns along the
crystallographic a axis (Fig. 4). For peptide 1 the parallel b-
sheet column is stabilized by three intermolecular hydrogen
bonds (N3–H3/O8, N9–H9/O13 and N14–H14/O22)
(Table 2) exploiting all its hydrogen bonding functionalities.
However, only two intermolecular hydrogen bonds (N3–
H3/O8 and N9–H9/O11) are present in peptide 2
connecting individual peptide molecules to form the
supramolecular parallel b-sheet column (Table 3). In
peptide 2, one N–H and one carbonyl group do not form
any type of hydrogen bond. These data confirm our initial
insight from the solid-state FT-IR data, that both peptides 1
and 2 possess an intermolecularly hydrogen-bonded b-sheet
structure, although peptide 2 has one free NH while in
peptide 1 all NHs are engaged in hydrogen-bonding.
Parallel b-sheet columns self-assemble along the crystal-
lographic b axis for peptide 1 (Fig. 5a) and along the
Figure 3. The ORTEP diagram of (a) peptide 1 and (b) peptide 2 with the
atomic numbering scheme. Ellipsoids at 20% probability.
that all NHs are involved in intermolecular hydrogen
bonding for peptides 1 and 3.16 The CO stretching band at
around 1650–1660 cmK1 (amide I) and the NH bending
peak near 1535 cmK1 (amide II) suggest the presence of
intermolecularly hydrogen bonded supramolecular b-sheet-
like conformations16 for all peptides in the solid state. So,
from solid-state FT-IR data it can be concluded that all the
peptides share a common structural feature, the intermole-
cularly hydrogen-bonded sheet.
Table 2. Characteristics of peptide 1 (Boc-d-Ava-Aib-Leu-OMe)
(a) Selected torsional anglesa (8) of peptide 1
Residue
f
j
u
q1
q2
q3
d-Ava(1)
Aib(2)
Leu(3)
K67.14
K49.12
62.74
135.37
K44.92
36.43
169.55
179.26
176.95
K55.06
—
—
K69.7
—
—
K60.79
—
—
(b) Intermolecular hydrogen bonding parameters of peptide 1
˚
˚
D–H/A
H/A (A)
D/A (A)
D–H/A (8)
N3–H3/O8b
2.25
2.04
2.53
3.023(10)
2.904(8)
3.358(9)
150
179
161
N9–H9/O13b
N14–H14/O22b
a The torsion angles for rotation about the bonds of peptide backbone: f, j, u. Torsions in the main chain in the N-terminal d-Ava residue about Ca-Cb, Cb–Cg
and Cg–Cd q3 to q1, respectively.
b Symmetry element K1Cx, y, z.
Table 3. Characteristics of peptide 2 (Boc-d-Ava-Aib-Val-OMe)
(a) Selected torsional anglesa (8) of peptide 2
Residue
f
j
u
q1
q2
q3
d-Ava(1)
Aib(2)
Val(3)
80.09
56.73
K73.76
K129.31
40.79
150.88
K175.43
170.98
173.46
59.27
—
—
76.26
—
—
57.75
—
—
(b) Intermolecular hydrogen bonding parameters of peptide 2
˚
˚
D–H/A
H/A (A)
D/A (A)
D–H.A (8)
N3–H3/O8b
N9–H9/O11b
2.31
2.12
3.028(8)
2.915(6)
142
153
a The torsion angles for rotation about the bonds of peptide backbone: f, j, u. Torsions in the main chain in the N-terminal d-Ava residue about Ca–Cb, Cb–Cg
and Cg–Cd q3 to q1, respectively.
b Symmetry element K1Cx, y, z.

A. Banerjee et al. / Tetrahedron 61 (2005) 5906–5914
5909
Figure 4. (a) Packing diagram of peptide 1 along a projection and (b) Packing diagram of peptide 2 along a projection illustrating intermolecular hydrogen
bonding in solid state and the formation continuous b-sheet columns. Hydrogen bonds are shown as dotted lines.
Figure 5. (a) Packing of peptide 1 along crystallographic b direction and (b) packing of peptide 2 along crystallographic c direction forming b-sheet layer
structures. Hydrogen bonds are shown as dotted lines.
crystallographic c axis for peptide 2 (Fig. 5b) to form two-
dimensional b-sheet layers. Each b-sheet layer is then
regularly stacked along crystallographic c axis for peptide 1
(Fig. 6) and along crystallographic b axis for peptide 2
(Fig. 7) using van der Waals’ interactions forming complex
quaternary b-sheet structures. Crystal data for peptides 1
and 2 are listed in Table 4.
conformations of peptides 1 and 2 are flat without any
noticeable bend or turn.
2.3. Scanning electron microscopic study
The morphological studies of all peptides were carried out
using a scanning electron microscope (SEM). The scanning
electron microscopic (SEM) images of peptides 1, 2, 3 and 4
(Figs. 8–11, respectively) of the dried fibrous material
grown from methanol–water clearly demonstrate that the
aggregates in the solid state are bunches of long small
filaments, resembling amyloid fibrils.18
Except the j1 for peptide 1 and j1 and j3 for peptide 2, all
f, j values of these peptides 1 and 2 fall within the helical
region of the Ramachandran plot (Tables 2 and 3).
However, neither of these peptide backbones adopts turn
or turn-like structure. No intramolecular hydrogen bond has
been observed. Instead, these peptides self-aggregate to
form intermolecularly hydrogen-bonded supramolecular
b-sheet structures. It is also noteworthy that C–C bonds
along the polymethylene units of the d-Ava residue adopt
gauche conformations. However, the overall molecular
2.4. Transmission electron microscopic study
Transmission electron microscopy of all the peptides at high
magnification provides information concerning their
detailed structure although only a two dimensional
Figure 6. Higher order packing of peptide 1 along crystallographic c axis
and forming quaternary b-sheet structures. Hydrogen bonds are shown as
dotted lines.
Figure 7. Higher order packing of peptide 2 along crystallographic b axis
and forming quaternary b-sheet structures. Hydrogen bonds are shown as
dotted lines.

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A. Banerjee et al. / Tetrahedron 61 (2005) 5906–5914
Table 4. Crystallographic data for peptide 1 and 2
Peptide 1
Peptide 2
Formula
C21H39N3O6
429.55
Methanol–water
Orthorhombic
293
P212121
6.100 (8)
14.325 (16)
29.94 (3)
2616 (5)
4
C20H37N3O6
415.53
Methanol–water
Orthorhombic
293
P212121
6.143 (8)
13.156 (15)
29.50 (3)
2384 (5)
4
Formula weight
Crystallizing solvent
Crystal system
Temperature (K)
Space group
˚
a (A)
˚
b (A)
˚
c (A)
3
U (A )
˚
Z
Dcalcd (g cmK3
)
1.091
1.158
˚
l (A)
R1 (IO2s(I))
wR2 (IO2s(I))
0.71073
0.1050
0.2643
0.71073
0.0958
0.2041
projection of the specimen could be imaged. All the
peptides exhibit fibrillar morphology under the TEM. The
representative TEM image of peptide 3 (Fig. 12) reveals that
the peptide exists as a bunch of long unbranched filaments
having diameter w20–40 nm.
2.5. Congo red binding study
Air-dried drops of the solution of all these peptides were
stained with a physiological dye Congo red and under cross
polarizers these peptide fibrils exhibit green birefringence,
characteristic feature of amyloid fibrils when investigated
Figure 8. SEM image of dried material of peptide 1 obtained from
methanol–water solution by slow evaporation.
Figure 11. Typical SEM image of dried material of peptide 4 showing
amyloid-like fibrillar morphology obtained from methanol–water solution
by slow evaporation.
Figure 9. Typical SEM image of dried fibrous material of peptide 2
obtained from methanol–water solution by slow evaporation.
Figure 12. Transmission electron micrograph of peptide 3 showing
amyloid-like morphology. The sample was prepared on a carbon coated
copper grid by slow evaporation of methanol–water solution of the
peptide 3.
Figure 10. Typical SEM image of dried material of peptide 3 showing
amyloid-like fibrillar morphology obtained from methanol–water solution
by slow evaporation.

A. Banerjee et al. / Tetrahedron 61 (2005) 5906–5914
5911
dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/
HOBt). All the intermediates were characterized by 1H
NMR (300 MHz) and thin layer chromatography (TLC) on
silica gel and used without further purification. The final
products were purified by column chromatography using
silica (100–200 mesh size) gel as stationary phase and ethyl
acetate/ethyl acetate–toluene mixture as eluent. The purified
1
final compounds were fully characterized by 300 MHz H
NMR spectroscopy, mass spectrometry and elemental
analysis.
4.1.1. Boc-d-Ava-OH 5. A solution of d-aminovaleric acid
(3.51 g, 30 mmol) in a mixture of dioxane (60 mL), water
(30 mL) and 1 M NaOH (60 mL) was stirred and cooled in
an ice-water bath. Di-tert-butylpyrocarbonate (7.2 g,
33 mmol) was added and stirring was continued at room
temperature for 6 h. Then the solution was concentrated in
vacuo to about 30–40 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 concentrated in vacuo.
The pure material 5 was obtained as a waxy solid. YieldZ
5.6 g (25.8 mmol, 86%); dH (300 MHz, CD3SOCD3) 11.76
(–COOH, 1H, br), 6.54 (d-Ava(1) NH, 1H, t, JZ9 Hz),
2.64–2.70 (d-Ava(1) CdHs, 2H, m), 1.94–1.99 (d-Ava(1)
CaHs, 2H, m), 1.21–1.26 (d-Ava(1) Cb and Cg Hs, 4H, m),
1.14 (Boc-CH3, 9H, s); mass spectral (ESI) data (MC
Na)CZ240.1, McalcdZ217; (found: C, 55.35; H, 8.72; N,
6.42 C10H19N1O4 (217) requires C, 55.30; H, 8.76; N,
6.45%).
Figure 13. Congo red stained peptide 1 fibrils observed through crossed
polarizers showing green-gold birefringence a characteristic feature of
amyloid fibrils.
microscopically.19 Figure 13 is a representative picture of
peptide 1 stained with Congo red, and exhibits distinct
green-gold birefringence under polarized light.
3. Conclusion
Solid-state FT-IR data of all peptides (peptides 1, 2, 3 and 4)
reveal that, all reported peptides self-associate to form
intermolecularly hydrogen bonded supramolecular b-sheet
structure. Crystal structures of two peptides (peptide 1 and
peptide 2) not only support the preliminary informational
feature obtained from the solid-state FT-IR data but also
suggest that these two peptides self-aggregate in a parallel
orientation. Upon further self-assembly, these beta sheet
structures produce peptide fibrils. All reported peptides
show their morphological similarity with amyloidogenic
proteins or protein fragments. They also bind to the
physiological dye Congo red and exhibit a typical green-
gold birefringence under polarized light like amyloid fibrils.
Previously, it has been shown that fibril formation of
Alzheimer’s-associated Ab peptide fragment proceeds via
parallel b-sheet formation.12a,20a Some very recent results
also demonstrate that fragments of Prion proteins20b,c and
even Tau filament fragments20d also self-aggregate via
parallel b-sheet formation. So, this study of the amyloid-like
fibril forming model peptide with parallel b-sheet structure
at atomic resolution may assist the scientific community
studying amyloid diseases in investigating the pathway(s)
and self-assembly mechanism during amyloid fibril for-
mation. Moreover, insertion of d-Ava residues into the
peptide backbone not only helps to form hydrogen-bonded
supramolecular b-sheet structures, but also provides
proteolytic resistance as d-Ava is homomorphous with the
Gly-Gly segment of any peptide.
4.1.2. Boc-d-Ava(1)-Aib(2)-OMe 6. Boc-d-Ava-OH (5.4 g,
25 mmol) was dissolved in dichloromethane (DCM)
(50 mL) in an ice-water bath. H-Aib-OMe was isolated
from the corresponding methyl ester hydrochloride (7.67 g,
50 mmol) by neutralization, subsequent extraction with
ethyl acetate and concentration to 15 mL and this was added
to the reaction mixture, followed immediately by dicyclo-
hexylcarbodiimide (DCC) (5.15 g, 25 mmol). The reaction
mixture was allowed to come to room temperature and
stirred for 48 h. DCM was evaporated, 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!50 mL), brine (2!50 mL), then 1 M sodium
carbonate (3!50 mL) and brine (2!50 mL) and dried over
anhydrous sodium sulfate, and evaporated in vacuo to yield
Boc-d-Ava-Aib-OMe 6 as a white solid. YieldZ6.68 g
(21.14 mmol, 84.5%); mp 104–106 8C; dH (300 MHz,
CDCl3) 6.24 (Aib(2) NH, 1H, s), 4.72 (d-Ava(1) NH, 1H,
t, JZ9.6 Hz), 3.73 (–OCH3, 3H, s), 3.14–3.10 (d-Ava(1)
CdHs, 2H, m), 2.17–2.22 (d-Ava(1) CaHs, 2H, m), 1.60–
1.71 (d-Ava(1) Cb and CgHs, 4H, m), 1.53 (Aib(2) CbHs,
6H, s), 1.44 (Boc-CH3s, 9H, s); mass spectral (ESI) data
(MCNa)CZ339.2, McalcdZ316; (found: C, 56.93; H, 8.82;
N, 8.92 C15H28N2O5 (316) requires C, 56.96; H, 8.86; N,
8.86%).
4. Experimental
4.1. Synthesis of peptides
All peptides were synthesized by conventional solution
phase methods by 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. Deprotections were performed using the
saponification method. Couplings were mediated by
4.1.3. Boc-d-Ava(1)-Aib(2)-OH 7. To a sample of 6
(6.64 g, 21 mmol), MeOH (75 mL) and 2 M NaOH
(30 mL) were added and the progress of saponification

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A. Banerjee et al. / Tetrahedron 61 (2005) 5906–5914
was monitored by thin layer chromatography (TLC). The
reaction mixture was stirred. After 10 h. methanol was
removed in vacuo, the residue was dissolved in water
(50 mL), and washed with diethyl ether (2!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!50 mL). The
extracts were pooled, dried over anhydrous sodium sulfate,
and evaporated in vacuo to yield compound 7 as a white
solid. YieldZ6.08 g (20.13 mmol, 95.86%); mp 122–
124 8C; dH (300 MHz, CD3SOCD3) 12.18 (–COOH, 1H,
br), 7.89 (Aib(2) NH, 1H, s), 6.71 (d-Ava(1) NH, 1H, t, JZ
8.7 Hz), 3.89–3.97 (d-Ava(1) CdHs, 2H, m), 1.52–1.61 (d-
Ava(1) CaHs, 2H, m), 1.28–1.35 (d-Ava(1) Cb and CgHs,
4H, m), 1.13 (Boc-CH3, 9H, s), 0.81 (Aib (2) CbHs, 6H, s);
mass spectral (ESI) data (MCNa)CZ325.2, McalcdZ302;
(found: C, 55.65; H, 8.58; N, 9.31. C14H26N2O5 (302)
requires C, 55.63; H, 8.61; N, 9.27%).
yield white solid of 2. Purification was done by silica gel
column using ethyl acetate as eluent. YieldZ1.5 g
(3.6 mmol, 72%); mp 110–112 8C; dH (300 MHz, CDCl3)
7.13 (Val(3) NH, 1H, d, JZ8.1 Hz), 6.10 (Aib(2) NH, 1H,
s), 4.70 (d-Ava(1) NH, 1H, br), 4.48–4.52 (Val(3) CaH, 1H,
m), 3.73 (–OCH3, 1H, s), 3.15–3.11 (d-Ava(1) CdHs, 2H,
m), 2.25–2.21 (d-Ava(1) CaHs, 2H, m), 2.18–2.16 (Val
CbH, 1H, m), 1.63–1.71 (d-Ava(1) Cb and CgHs, 4H, m and
1H, m), 1.43 (Boc-CH3, 9H, s), 1.25 (Aib(2) CbHs, 6H, s),
0.96–0.88 (Val(3) CgHs, 6H, m); Mass spectral (ESI) data
(MCH)CZ416.4, McalcdZ415; [a]2D5 C17.4 (c 2.16,
CHCl3); (found: C, 57.87; H, 8.95; N, 10.07; C20H37N3O6
(415) requires C, 57.83; H, 8.92; N, 10.12%).
4.1.6. Boc-d-Ava(1)-Aib(2)-Phe(3)-OMe 3. Boc-d-Ava(1)-
Aib(2)-OH (1.51 g, 5 mmol) in DMF (10 mL) was cooled in
an ice-water bath and H-Phe-OMe was isolated from the
corresponding methyl ester hydrochloride (2.16 g,
10 mmol) by neutralization, subsequent extraction with
ethyl acetate and concentration to 7 mL and it was added to
the reaction mixture, followed immediately by DCC
(1.03 g, 5 mmol) and of HOBt (0.68 g, 5 mmol). The
reaction mixture was stirred for three days. The residue
was taken in ethyl acetate (60 mL) and the DCU was filtered
off. The organic layer was washed with 2 M HCl (3!
50 mL), brine (2!50 mL), 1 M sodium carbonate (3!
50 mL), brine (2!50 mL), dried over anhydrous sodium
sulfate and evaporated in vacuo to yield 3 as white solid.
Purification was done by silica gel column (100–200 mesh)
using ethyl acetate as eluent. YieldZ1.67 g (3.6 mmol,
72%); mp 117–119 8C; dH (300 MHz, CDCl3) 7.11–7.33
(Ph ring protons of Phe(3), 5H, m), 6.88 (Phe(3) NH, 1H, d,
JZ7.26 Hz), 6.05 (Aib(2) NH, 1H, s), 4.86–4.80 (Phe(3)
CaH, 1H, m), 4.70 (d-Ava(1) NH, 1H, m), 3.73 (–OCH3,
3H, s), 3.21–3.06 (Phe(3) CbHs, 2H, m and d-Ava(1) CdHs,
2H, m), 2.20–2.09 (d-Ava(1) CaHs, 2H, m), 1.66–1.59
(d-Ava(1) Cb and CgHs, 4H, m), 1.49 (Aib(2) Cb Hs, 6H, s),
1.43 (Boc-CH3, 9H, s); mass spectral (ESI) data (MC
Na)CZ486.2, McalcdZ463; [a]2D5.1 C36.91 (c 2.25,
CHCl3); (found: C, 62.27; H, 8.12; N, 9.02. C24H37N3O6
(463) requires C, 62.20; H, 7.99; N, 9.07%).
4.1.4. Boc-d-Ava(1)-Aib(2)-Leu(3)-OMe 1. Boc-d-Ava-
Aib-OH 7 (1.51 g, 5 mmol) was dissolved in DMF (10 mL)
in an ice-water bath. H-Leu-OMe was isolated from methyl
ester hydrochloride (1.82 g, 10 mmol) by neutralization,
subsequent extraction with ethyl acetate and concentration
to 7 mL and it was added to the reaction mixture, followed
immediately by dicyclohexylcarbodiimide (DCC) (1.03 g,
5 mmol) and HOBt (0.68 g, 5 mmol). The reaction mixture
was allowed to come to room temperature and stirred for
72 h. The residue was taken in ethyl acetate (30 mL), and
dicyclohexylurea (DCU) was filtered off. The organic layer
was washed with 2 M HCl (3!30 mL), brine (2!30 mL),
then 1 M sodium carbonate (3!30 mL) and brine (2!
30 mL) and dried over anhydrous sodium sulfate and
evaporated in vacuo to yield 1 (1.59 g) in form of white
solid. Purification was done by silica gel column (100–200
mesh) using ethyl acetate as eluent.
YieldZ1.59 g (3.7 mmol, 74%); mp 114–116 8C; dH
(300 MHz, CDCl3) 7.07 (Leu(3) NH, 1H, d, JZ7.5 Hz),
6.18 (Aib(2) NH, 1H, s), 4.76 (d-Ava(1) NH, 1H, t, JZ
7.15 Hz), 4.60–4.53 (Leu(3) CaH, 1H, m), 3.73 (-OCH3, 3H,
s), 3.14–3.12 (d-Ava(1) CdHs, 2H, m), 2.25–2.20 (d-Ava(1)
Ca Hs, 2H, m), 1.86–1.82 (Leu(3) CbHs, 2H, m), 1.68–1.62
(d-Ava(1) Cb Hs and CgHs, 4H, m), 1.58 (Aib(2) CbHs, 3H,
s), 1.56 (Aib(2) CbHs, 3H, s), 1.54–1.49 (Leu(3) CgH 1H,
m), 1.44 (Boc-CH3, 9H, s), 0.96–0.91 (Leu(3) CdHs, 6H,
4.1.7. Boc-d-Ava(1)-Aib(2)-Ile(3)-OMe 4. Boc-d-Ava-
Aib-OH (1.51 g, 5 mmol) was dissolved in DMF (10 mL)
in an ice-water bath. H-Ile-OMe was isolated from methyl
ester hydrochloride (1.82 g, 10 mmol) by neutralization,
subsequent extraction with ethyl acetate and concentration
to 7 mL and it was added to the reaction mixture, followed
immediately by dicyclohexylcarbodiimide (DCC) (1.03 g,
5 mmol) and HOBt (0.68 g, 5 mmol). The reaction mixture
was allowed to come to room temperature and stirred for
72 h. The residue was taken in ethyl acetate (30 mL), and
dicyclohexylurea (DCU) was filtered off. The organic layer
was washed with 2 M HCl (3!30 mL), brine (2!30 mL),
then 1 M sodium carbonate (3!30 mL) and brine (2!
30 mL) and dried over anhydrous sodium sulfate and
evaporated in vacuo to yield 4 as white solid. Purification
was done by silica gel column (100–200 mesh) using ethyl
acetate as eluent. Yield Z1.58 g (3.68 mmol, 73.6 %); mp
108–110 8C; dH (300 MHz, CDCl3) 7.18 (Ile(3) NH, 1H, d,
JZ8.1 Hz), 6.25 (Aib(2) NH, 1H, s), 4.77 (d-Ava(1) NH,
1H, m), 4.62–4.51 (Ile(3) CaH, 1H, m), 3.73 (–OCH3, 3H,
s), 3.19–3.06 (d-Ava(1) CdHs, 2H, m), 2.29–2.21 (d-Ava(1)
m); mass spectral (ESI) data (MCH)CZ430.4, Mcalcd
Z
429; [a]2D4.9 C6.39 (c 2.12, CHCl3); (found: C, 58.78; H,
9.07; N, 9.83 C21H39N3O6 (429) requires C, 58.74; H, 9.1;
N, 9.79%).
4.1.5. Boc-d-Ava(1)-Aib(2)-Val(3)-OMe 2. A sample of 7
(1.51 g, 5 mmol) in DMF (10 mL) was cooled in an ice
water bath. H-Val-OMe was isolated from of the corre-
sponding methyl ester hydrochloride (1.68 g, 10 mmol) by
neutralization, subsequent extraction with ethyl acetate and
concentration to 7 mL and this was added to the reaction
mixture, followed immediately by DCC (1.03 g, 5 mmol)
and HOBt (0.68 g, 5 mmol). The reaction mixture was
stirred for 3 days. The residue was taken in ethyl acetate
(50 mL) and DCU was filtered off. The organic layer was
washed with 2 M HCl (3!50 mL), brine (2!50 mL), 1 M
sodium carbonate (3!50 mL), brine (2!50 mL), dried
over anhydrous sodium sulfate and evaporated in vacuo to

A. Banerjee et al. / Tetrahedron 61 (2005) 5906–5914
5913
CaHs, 2H, m), 1.97–1.88 (Ile CbHs,1H, m), 1.70–1.62 (d-
Ava(1) Cb and CgHs, 4H, m), 1.58 (Aib(2) CbHs, 3H, s),
1.55 (Aib(2) CbHs, 3H, s), 1.44 (Boc-CH3, 9H, s), 1.24–1.11
(Ile(3) CgHs, 2H, m), 0.99–0.90 (Ile(3) Cg and CdHs, 6H,
measured at 28 intervals with a counting time of 2 min.
Data analyses were carried out with the XDS program.22
The structures were solved using direct methods with the
Shelx86 program.23 Non-hydrogen atoms were refined with
anisotropic thermal parameters. The hydrogen atoms
bonded to carbon were included in geometric positions
and given thermal parameters equivalent to 1.2 times those
of the atom to which they were attached. The structures
were refined on F2 using Shelxl.24 Crystallographic data
have been deposited at the Cambridge Crystallographic
Data Center references CCDC 258126, 258127 for peptides
1 and 2.
m); Mass spectral (ESI) data (MCNa)CZ452.2, Mcalcd
Z
429; [a]2D5.3 C21.35 (c 2.04, CHCl3); (found: C, 58.68; H,
9.13; N, 9.76. C21H39N3O6 (429) requires C, 58.74; H, 9.1;
N, 9.79).
4.2. NMR experiments
All NMR studies were carried out on a Bru¨ker DPX
300 MHz spectrometer at 300 K. Peptide concentrations
were in the range 1–10 mM in CDCl3 and CD3SOCD3.
4.8. Mass spectrometry
4.3. FT-IR spectroscopy
Mass spectra were recorded on a Hewlett Packard Series
1100MSD mass spectrometer by positive mode electrospray
ionization.
The FT-IR spectra were taken using Shimadzu (Japan)
model FT-IR spectrophotometer. The solid-state FT-IR
measurements were performed using the KBr disk
technique.
Acknowledgements
4.4. Scanning electron microscopic study
We thank EPSRC and the University of Reading, U.K. for
funds for the Image Plate System. This research is also
supported by a grant from Department of Science and
Technology (DST), India [Project No. SR/S5/OC-29/2003].
Morphologies of all reported tripeptides were investigated
using optical microscopy and scanning electron microscopy
(SEM). For the SEM study, fibrous materials (slowly grown
from methanol–water mixtures) were dried and gold coated.
Then the micrographs were taken in a SEM apparatus (Jeol
Scanning Microscope-JSM-5200).
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