Hydrogen-bonded dimer can mediate supramolecular β-sheet formation and subsequent amyloid-like fibril formation: A model study

Article abstract of DOI:10.1016/j.tet.2004.05.041

FT-IR data of six terminally blocked tripeptides containing Acp (ε-aminocaproic acid) reveals that all of them form supramolecular β-sheets in the solid state. Single crystal X-ray diffraction studies of two peptides not only support this data but also disclose the fact that the supramolecular β-sheet formation is initiated via dimer formation. The Scanning Electron Microscopic images of all peptides exhibit amyloid-like fibrils that show green birefringence after binding with Congo red, which is a characteristic feature of many neurodegenerative disease causing amyloid fibrils.

Full text of DOI:10.1016/j.tet.2004.05.041

Tetrahedron 60 (2004) 5935–5944
Hydrogen-bonded dimer can mediate supramolecular b-sheet
formation and subsequent amyloid-like fibril formation:
a model study
Arijit Banerjee,a Samir Kumar Maji,a Michael G. B. Drew,b Debasish Haldar,a
Apurba Kumar Dasa and Arindam Banerjeea
,
*
aDepartment of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
bDepartment of Chemistry, The University of Reading, Whiteknights, Reading RG6 6AD, UK
Received 26 January 2004; revised 19 April 2004; accepted 13 May 2004
Available online 7 June 2004
Abstract—FT-IR data of six terminally blocked tripeptides containing Acp (1-aminocaproic acid) reveals that all of them form
supramolecular b-sheets in the solid state. Single crystal X-ray diffraction studies of two peptides not only support this data but also disclose
the fact that the supramolecular b-sheet formation is initiated via dimer formation. The Scanning Electron Microscopic images of all peptides
exhibit amyloid-like fibrils that show green birefringence after binding with Congo red, which is a characteristic feature of many
neurodegenerative disease causing amyloid fibrils.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
the pathway(s) of fibril formation and neurotoxicity-
associated with quaternary b-sheet assemblage is extremely
significant for the developments of therapeutics for these
diseases. All previous reports for establishing the path-
way(s) of the quaternary structure formation of amyloid
fibrils are based on methods, such as, CD spectro-
polarimetry, ANS binding fluorescence, different types of
electron microscopic studies (SEM, TEM), Atomic Force
Microscopy, and X-ray fiber-diffraction studies,8 which are
unable to elucidate the details of the quaternary structure
and pathway(s) of fibril formation at the atomic level, unlike
single crystal X-ray diffraction studies. It is, however,
extremely difficult to get a crystal structure of a real
amyloidogenic protein or a protein fragment due to its very
low solubility and noncrystallinity. Although there are an
increasing number of reports concerning the organization of
b-sheets in Alzheimer’s b-amyloid fibrils by solid-state
NMR techniques,9 we are still lacking the knowledge of the
atomic details of the self-assembly mechanism and the
intermediate(s) involved in the amyloid-fibrils formation. It
is therefore, worthwhile to study a small model peptide
containing non-protein amino acids10 (stereochemically
restricted/flexible) whose structure can be determined
using single crystal X-ray diffraction studies as this may
provide insights into how subunit (monomer or dimmer)
self-associate to form b-sheet and amyloid-like fibril at the
atomic resolution.
The design of short model compounds that can form well-
defined dimers and self-associate via non-covalent inter-
actions to form supramolecular b-sheet structures is a highly
emerging area of recent research.1 Dimer systems,
preferably those which give b-sheet structures, are import-
ant as a model for studying b-sheet interactions in proteins.2
b-sheet formation between peptides is generally associated
with the formation of insoluble aggregates of ill-defined
structure. Dimer-mediated supramolecular b-sheet for-
mation is particularly important as this mimics many
neurotoxic amyloid sequences that self-assemble to form
the b-sheet structure via dimerization.3
The molecular basis of fatal, neurodegenerative diseases
like prion protein diseases,4 Alzheimer’s disease2e,5 and
other related diseases6 involves an intermolecular b-sheet
aggregation and subsequent formation of highly ordered
fibrillar structures. Though it is well accepted that the
amyloid fibrils consist of majorly b-sheet structure, the
structure-function relationship of amyloidosis is still not
clear. Moreover, some recent results demonstrate that not
the matured fibril but the intermediates are potent
neurotoxic agents in Alzheimer’s disease.7 So, deciphering
Keywords: Self-assembling peptides; Dimer; Aib; b-Sheet; Amyloid-like
fibrils.
Our previous studies demonstrated that a tripeptide with an
extended backbone conformation11 and a turn forming
*
Corresponding author. Tel.: þ91-33-2473-4971; fax: þ91-33-2473-2805;
e-mail address: bcab@mahendra.iacs.res.in
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.05.041

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A. Banerjee et al. / Tetrahedron 60 (2004) 5935–5944
tripeptide12 self-assemble through various non-covalent
interactions to form highly ordered b-sheet assemblage in
crystals and an amyloid-like fibrillar structure in the solid
state. However, we present here Acp (1-aminocaproic acid)
containing short synthetic tripeptides that form a hydrogen
bonded dimer, which we believe can mediate the supra-
molecular b-sheet aggregation and subsequent amyloid-like
fibril formation.
located pentamethylene unit provides sufficient flexibility13
to the peptide backbone to achieve an extended backbone
conformation, a prerequisite for forming a b-sheet structure.
Our first synthesized peptide 1, having a centrally
positioned flexible Gly residue fails to provide single
crystals. The two residues Aib(2) and Leu(3) for peptide 2
and Aib(2) and Phe(3) for peptide 3, containing hydro-
phobic groups in their side chains help to increase the
crystallinity of these peptides and facilitate the association
of each dimer, apart from their hydrogen bonding
functionalities, through the interactions between the iso-
butyl side chain of Leu for peptide 2 and side chain Phe/Phe
interaction for peptide 3. In peptide 3, a CH· · ·p
interaction14 [the contact of a C(sp2)–H group of the
phenyl ring of molecule B of one asymmetric unit to the p
cloud of the phenyl ring of molecule A of the neighboring
2. Result and discussion
The schematic presentations of all terminally protected
tripeptides are shown in Figure 1. Here we have used an 1-
aminocaproic acid residue (Acp) in which the two end
moieties, NH and CvO can provide hydrogen bonding
functionalities that serve to form intermolecular hydrogen
bonds among the individual strands while the centrally
˚
asymmetric unit along crystallographic a axis is 2.74 A]
assists each dimer to bind together along the a direction. It is
a well documented fact that p–p interactions have a
significant role in amyloid aggregation.15 For peptides 4, 5
and 6 we have used Val, D-Val, and Ile respectively,
replacing the terminal Leu residue from peptide 2. All
peptides were studied using FT-IR, NMR, scanning electron
microscopy and optical microscopy.
2.1. Solid state FT-IR study
Preliminary information on the conformational preferences
of all peptides were obtained from FT-IR studies.16 The
most informative frequency ranges are (i) 3500–
3200 cm21, corresponding to the N–H stretching vibrations
of the peptide and N-protecting urethane groups, and (ii)
1800–1600 cm21, corresponding to the CvO stretching
vibrations of the peptide, urethane, and ester groups.
Important IR data of all these reported compounds are
listed in Table 1. In the 3500–3200 cm21 region (Fig. 2), an
intense band corresponding to strongly hydrogen bonded
NH groups has been observed at 3375–3275 cm21 for each
of the reported peptides. In the 1800–1600 cm21 region,
several bands have been observed (Fig. 2). The bands at
1783–1740 and 1737–1610 cm21 (Fig. 2) are assigned to
the stretching of the CvO groups of free methyl ester
moieties and hydrogen-bonded urethane groups respect-
ively. The observed strong and medium bands at 1733–
1690 cm21 are attributed to the stretching of the CvO
groups of hydrogen bonded esters and/or free urethanes.16a,
d–f
The strong bands at 1627–1650 cm21 are assigned to
the CvO groups of hydrogen bonded peptide moieties. The
bands observed at 1627–1650 and 3227–3286 cm21 are
typical of a fully developed b-sheet conformation.16 For
peptides 2 and 3 strong bands are observed at nearly
Figure 1. Schematic representation of peptides 1, 2, 3, 4, 5 and 6.
Table 1. Infrared (IR) absorption frequencies (cm21) for all reported peptides in solid state (on KBr pellet)
Peptide
NH stretch
CvO stretch
Boc-Acp-Gly-Leu-OMe (1)
Boc-Acp-Aib-Leu-OMe (2)
Boc-Acp-Aib-Phe-OMe (3)
Boc-Acp-Aib-Val-OMe (4)
Boc-Acp-Aib-DVal-OMe (5)
Boc-Acp-Aib-Ile-OMe (6)
3307(st)
3315(st)
1745(m),1730(st),1681(m), 1651(st), 1645(sd)
1707(w), 1685(st), 1660(st)
1735(m), 1687(st), 1660(st), 1651(st)
1651–1633(st)
1712(m), 1701(st), 1681(v.w.), 1660(st)
1735(st), 1687(vs), 1666(st), 1649(st)
3371(st), 3336(st), 3317(m), 3273(st)
3354(st), 3288(st)
3354(st), 3290(st)
3373(st), 3340(st), 3269(st)
st¼strong, w¼weak, v.w¼very weak, m¼medium, sd¼shoulder, v.s¼very strong.

A. Banerjee et al. / Tetrahedron 60 (2004) 5935–5944
5937
molecular dimer of two different conformers (Table 2 for
peptide 2 and Table 3 for peptide 3). There is no intra-
molecular hydrogen bond in molecules A and B for peptides
2 and 3. In all cases, the Acp residue adopts an extended
backbone conformation, while the centrally located Aib
residue adopts a helical conformation, a very distinctive
feature of the Aib residue.17 However, the terminal residues,
Leu for peptide 2 and Phe for peptide 3, adopt extended
backbone conformations. This provides an overall extended
backbone structure with a small kink at the central place
(due to the presence of a helix forming Aib residue) for each
conformer of the molecular dimers of both peptides 2 and 3.
Each dimer then self-assembles via intermolecular hydro-
gen bonds forming an extended parallel b-sheet assemblage
in both peptides 2 (Fig. 4(a)) and 3 (Fig. 4(b)). These b-
sheets are themselves regularly stacked via van der Waals
interactions to form a more complex quaternary structure
(Fig. 5 for peptide 2 and Fig. 6 for peptide 3). The hydrogen
bonding parameters of peptides 2 and 3 are listed in Tables 2
and 3. There are three intermolecular hydrogen bonds
N14A–H14A· · ·O15B, N7A–H7A· · ·O8B and N4A–
H4A· · ·O51B for peptide 2 and three (N1A–H1A· · ·O2B,
N4A–H4A· · ·O5B, N11B–H11B· · ·O12A) for peptide 3
that are responsible for connecting the individual molecules
in the asymmetric unit to form and stabilize the dimer.
There are three other intermolecular hydrogen bonds
N4B–H4B· · ·O51A, N7B–H7B· · ·O8A and N14B–
H14B· · ·O15A for peptide 2 and two intermolecular
hydrogen bonds N11A–H11A· · ·O12B and N4B–
H4B· · ·O2A for peptide 3 which are involved in connecting
the individual dimer to form the parallel b-sheet structure.
Crystal data for these two peptides are detailed in Table 4.
This data confirms our initial insight from the FT-IR data
that these peptides have a sheet-like structure in their solid
state. These crystal structures also support the fact that
Figure 2. FT-IR spectra at the region 3200–3500 cm21 (a) and 1500–
1750 cm21 (b) of peptides 1, 2, 3, 4, 5 and 6 in solid state.
1650 cm21 region. For peptide 1, a strong band at
1651 cm21 with a shoulder at 1645 cm21 has been observed
in the solid state. All these data suggest an intermolecularly
hydrogen bonded b-sheet structure for peptide 1 in the solid
state. Similarly, FT-IR data corresponding to peptides 4, 5
and 6 (Table 1) are indicative of the b-sheet-like structure in
the solid state. It is suggested from the solid state FT-IR data
that the solid state structure of all the reported peptides are
similar and that they form intermolecularly hydrogen
bonded b-sheet-like structures.
2.2. Single crystal X-ray diffraction study
Among all peptides, only peptides 2 and 3 give a single
crystal suitable for X-ray diffraction analysis. ORTEP
diagrams with the atom numbering schemes are provided
in Figure 3(a) and in Figure 3(b) for peptides 2 and 3,
respectively. Interestingly, both Figures 3(a) and (b) show
that there are two molecules (named A and B) in the
asymmetric unit for both peptides and they are joined
together by intermolecular hydrogen bonds to form a stable
Figure 3. (a) The ORTEP diagram of peptide 2 and (b) the ORTEP diagram
of peptide 3 with the atomic numbering scheme showing the dimer
formation. Ellipsoids at 20% probability. The dimer formation via three
intermolecular bonds is shown as dotted lines.

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A. Banerjee et al. / Tetrahedron 60 (2004) 5935–5944
Table 2. Characteristics of peptide 2 (Boc-Acp-Aib-Leu-OMe) in molecules A and B
(a) Selected torsional anglesa (8) of peptide 2
Residue
Acp(1)
Molecule
A
f
86.9
2102.7
59.2
258.3
284.5
2117.8
c
v
u1
176.9
270.4
—
—
—
u2
176.6
2158.0
—
u3
173.5
179.5
—
—
—
u4
56.7
145.4
—
—
—
113.1
127.9
46.7
250.2
7.3
159.8
2173.6
178.4
174.7
2178.6
171.1
2177.1
B
A
B
A
B
Aib(2)
Leu(3)
—
—
—
—
—
—
(b) Intermolecular hydrogen bonding parameters of peptide 2
˚
H· · ·A (A)
˚
D· · ·A (A)
D–H· · ·A
D–H· · ·A (8)
N4A–H4A· · ·O51Bb
N4B–H4B· · ·O51A
N7A–H7A· · ·O8Bb
N14A–H14A· · ·O15Bb
N14B–H14B· · ·O15Ac
2.26
2.44
2.20
2.11
2.12
2.990
3.183
3.041
2.928
2.956
143
144
166
159
136
a
The torsion angles for rotation about the bonds of peptide backbone: (f, c, v); Torsions in the main chain in the N-terminal Acp residue about Ca–Cb, Cb–
Cg, Cg–Cd and Cd–C1: u4 to u1, respectively.
Symmetry equivalent x, 1þy, z.
b
c
Symmetry equivalent 1þx, 21þy, z.
Table 3. Characteristics of peptide 3 (Boc-Acp-Aib-Phe-OMe) in molecules A and B
(a) Selected torsional anglesa (8) of peptide 3
Residue
Acp(1)
Molecule
A
f
95.2
c
2117.8
2106.4
245.7
241.5
94.0
v
u1
2146.1
2179.4
—
u2
2172.3
2173.7
—
u3
266.0
64.9
—
—
—
u4
263.6
175.1
—
—
—
2177.1
2178.5
2173.6
2176.3
175.9
B
A
B
A
B
2144.9
254.6
254.4
299.0
2148.8
Aib(2)
Phe(3)
—
—
—
—
—
—
167.9
2180.0
—
—
(b) Intermolecular hydrogen bonding parameters of peptide 3
˚
H· · ·A (A)
˚
D· · ·A (A)
D–H· · ·A
D–H· · ·A (8)
N1A–H1A· · ·O2Bb
N4A–H4A· · ·O5Bb
N4B–H4B· · ·O2A
N11A–H11A· · ·O12B
N11B–H11B· · ·O12Ac
2.08
2.12
2.12
2.16
2.27
2.90
2.97
2.87
3.01
3.08
161
171
144
169
157
a
The torsion angles for rotation about the bonds of peptide backbone: f, c, v; torsions in the main chain in the N-terminal Acp residue about Ca–Cb, Cb–Cg,
Cg–Cd and Cd–C1: u4 to u1, respectively.
Symmetry equivalents: x21, y, z.
Symmetry equivalents: xþ1, y, z.
b
c
supramolecular b-sheet formation is mediated through
dimerization for the representative peptides 2 and 3.
these reported peptides with amyloid fibrils has been studied
using a scanning electron microscope (SEM). The SEM
images of peptides 2, 3 and 6 (Figs. 8, 9 and 12,
respectively) of the dried fibrous material grown from
ethyl acetate/methanol–water clearly demonstrate that the
aggregate in the solid state is a bunch of long small
2.3. Morphological study
The morphological similarity of the b-sheet assemblage of
Figure 4. (a) Packing diagram of the peptide 2 in the b projection and (b)
packing of peptide 3 along a projection illustrating intermolecular hydrogen
bonding in solid state and the formation of continuous b-sheets columns.
Hydrogen bonds are shown as dotted lines. Only hydrogen bonded H atoms
are shown for clarity.
Figure 5. The packing of peptide 2 dictating the packing of individual
columns via van der Waals’ interactions along the crystallographic c axis
into higher order b-sheet structure in crystal. Dotted lines are indicated for
hydrogen bonds.

A. Banerjee et al. / Tetrahedron 60 (2004) 5935–5944
5939
Figure 9. Typical SEM image of dried fibrous material of peptide 3
obtained from methanol–water solution.
Figure 6. The crystal packing of peptide 3 showing the formation of a
higher order b-sheet structure along the crystallographic c axis via van der
Waals’ interactions between the individual columns. Hydrogen bonds are
shown as dotted lines.
Table 4. Crystallographic data for peptide 2 and peptide 3
Peptide 2
Peptide 3
Formula
C22H41N3O6
443.68
Ethyl acetate
Triclinic
293
C25H39N3O6
477.59
Methanol–water
Monoclinic
293
Formule weight
Crystallizing solvent
Crystal system
Temperature (K)
Space group
P1
P21
˚
a (A)
9.354(14)
9.844(14)
15.72(2)
79.378(10)
76.445(10)
87.222(10)
1383(3)
2
10.386(12)
9.538(12)
27.71(3)
90
93.985(10)
90
2738.44
4
1.158
˚
b (A)
˚
c (A)
a (8)
b (8)
g (8)
Figure 10. Typical SEM image of dried material of peptide 4 from
methanol–water solution showing intertwined helical filaments.
3)
˚
U (A )
Z
Dcalcd (g cm23
)
1.065
˚
l (A)
R1
wR2
0.71.73
0.0674
0.2224
0.71073
0.0866
0.2070
Figure 11. Typical SEM image of dried peptide 5 showing intertwined
helical filaments obtained from methanol–water solution having reverse
handedness to that of peptide 4.
Figure 7. (a) Typical SEM image of peptide 1 taken from dried material
growing from methanol–water, showing fibrillar network. (b) SEM image
of peptide 1 in higher magnification showing intertwined helical filaments.
filaments, resembling amyloid fibrils. Morphologies of
peptides 1, 4 and 5 are the most remarkable. Figure 7(a)
shows fibrillar network of peptide 1, growing from
methanol–water solvent system. Figure 7(b) shows higher
magnification of the SEM picture of the peptide suggesting
an intertwined helical nature of the fibrils. Figures 10 and 11
exhibit the SEM images of peptides 4 and 5 respectively
from methanol–water. All these three (Figs. 7(b), 10 and
11) indicate that peptides 1, 4 and 5 form inter-twined
helical filaments, a special characteristic of many neurode-
generative disease causing amyloid fibrils.18 Interestingly,
the handedness of the helicity of these fibrils obtained from
the two enantiomeric peptides (peptide 4 and peptide 5) is
reverse. This is presumably due to the presence of the amino
acid residues with opposite chirality (L-Val and D-Val) in
those peptides.
Figure 8. The SEM image of dried fibrous material of peptide 2 obtained
from ethyl acetate solution.

5940
A. Banerjee et al. / Tetrahedron 60 (2004) 5935–5944
X-ray diffraction studies. The morphological resemblance
of these peptides with Alzheimer’s-associated Ab peptides
and other neurotoxic amyloid peptides indicates that the
fibril model with parallel b-sheet assemblage20 may be
useful for exploring the structure-function relationship of
amyloidogenic peptides and proteins.
4. Experimental
4.1. Synthesis of peptides
Figure 12. Typical SEM image of peptide 6. Fibrils were grown from
methanol–water solvent system.
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
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 final compounds were fully characterized by
2.4. Congo red binding studies
Air-dried drops of the solution of all these peptides were
stained with a physiological dye Congo red. This led to the
green birefringence with cross polarizers that is character-
istic of amyloid fibrils when investigated microscopically.19
Figure 13 is the representative picture of peptide 3 stained
with Congo red, and exhibit distinct green birefringence
under polarized light.
1
300 MHz H NMR spectroscopy, mass spectrometry and
elemental analysis. All chiral amino acids are of ‘L’
configuration unless otherwise it is mentioned.
4.1.1. Boc-Acp-OH (7). A solution of 1-aminocaproic acid
(9.84 g, 75 mmol) in a mixture of dioxan (150 mL), water
(75 mL) and 1 M NaOH (75 mL) was stirred and cooled in
an ice-water bath. Di-tert-butylpyrocarbonate (18 g,
82.5 mmol) was added and stirring was continued at room
temperature for 6 h. Then the solution was concentrated in
vacuo to about 70–80 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 in vacuo. The pure
material was obtained as waxy solid.
Figure 13. Congo red stained peptide 3 fibrils observed through crossed
polarizers showing green-gold birefringence, a characteristic feature of
amyloid fibrils.
3. Conclusion
FT-IR data of these peptides (peptides 1, 2, 3, 4, 5 and 6)
reveal that, all the reported peptides self-associate to form
supramolecular b-sheet structure via intermolecular
hydrogen bonds, in which intermolecular hydrogen bonds
are present between the peptide linkages. Crystal structures
of two peptides reveal that they form hydrogen bonded
dimers that gives supramolecular b-sheets on self-assembly.
Several previous reports suggest that many neurotoxic
amyloidogenic peptide sequences can self-associate to form
a quaternary b-sheet structure and ultimately form fibrils via
dimerization.3 Characterization of the peptide dimer at
atomic resolution which self-assembles to form b-sheets is
particularly important for better understanding of the
pathway(s) and kinetics of amyloid fibril formation. We
have established that quaternary b-sheet structure formation
and amyloid-like fibril formation can be mediated by the
dimerization of synthetic peptide molecules having
extended backbone conformations using single crystal
Yield¼14.56 g (63 mmol, 84%). Elemental analysis calcd
(%) for C11H21N1O4 (231): C, 57.14; H, 9.09; N, 6.06.
Found: C, 57.37; H, 7.47; N, 6.24.
4.1.2. Boc-Acp(1)-Gly(2)-OCH2Ph (8). Boc-Acp-OH
(2.31 g, 10 mmol) was dissolved in a mixture of dichloro-
methane (DCM) (20 mL) in an ice-water bath. H-Gly-
OCH2Ph was isolated from the corresponding benzyl ester
p-toluene sulfonate (6.74 g, 20 mmol) by neutralization,
subsequent extraction with ethyl acetate and concentration
(10 mL) and this was added to the reaction mixture,
followed immediately by di-cyclohexylcarbodiimide
(DCC) (7.42 g, 36 mmol). The reaction mixture was
allowed to come to room temperature and stirred for 48 h.
DCM was evaporated, residue was taken in ethyl acetate
(60 mL), and dicyclohexylurea (DCU) was filtered off. The
organic layer was washed with 2 M HCl (3£50 mL), brine,
then 1 M sodium carbonate (3£50 mL) and brine

A. Banerjee et al. / Tetrahedron 60 (2004) 5935–5944
5941
(2£50 mL) and dried over anhydrous sodium sulfate, and
evaporated in vacuo to yield 8 as a waxy solid.
Leu(3), 6H, m). Mass spectral data MþNaþ¼438,
Mcalcd¼415. Elemental analysis calcd (%) for C20H37N3O6
(415): C, 57.83; H, 8.92; N, 10.12. Found: C, 57.94; H, 8.63;
N, 10.32.
Yield¼2.8 g, (7.4 mmol, 74%). 1H NMR (CDCl3,
300 MHz, d ppm): 7.29–7.30 (Ph, 5H, m); 6.11 (Gly(2)
NH, 1H, t, J¼6.0 Hz); 5.19 (–benzyl CH2, 2H, s); 4.61
(Acp(1) NH, 1H, t, J¼5.2 Hz); 4.07 (CaH of Gly, 1H, t,
J¼8.2 Hz); 3.09–3.11 (C1Hs of Acp(1), 2H, m); 2.22–2.34
(CaHs of Acp(1), 2H, m); 1.61–1.71 (CbHs and CgHs of
Acp(1), 4H, m); 1.30–1.35 (CdH3 of Acp(1), 2H, m); 1.44
(Boc-CH3s, 9H, s). Elemental analysis calcd (%) for
C20H30N2O5 (378): C, 63.49; H, 7.94; N, 7.41. Found: C,
63.65; H, 7.62; N, 7.25.
4.1.5. Boc-Acp(1)-Aib(2)-OMe (10). Boc-Acp-OH (9.25 g,
40 mmol) was dissolved in dichloromethane (DCM)
(50 mL) in an ice-water bath. H-Aib-OMe was isolated
from the corresponding methyl ester hydrochloride
(12.29 g, 80 mmol) by neutralization, subsequent extraction
with ethyl acetate and concentration to 15 mL and this was
added to the reaction mixture, followed immediately by
dicyclohexylcarbodiimide (DCC) (8.24 g, 40 mmol). The
reaction mixture was allowed to come to room temperature
and stirred for 48 h. DCM was evaporated, residue was
taken in ethyl acetate (60 mL), and dicyclohexylurea (DCU)
was filtered off. The organic layer was washed with 2 M
HCl (3£50 mL), brine, 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-
Acp(1)-Aib(2)-OMe (10.04 g, 30.4 mmol, 76%) as a white
solid.
4.1.3. Boc-Acp(1)-Gly(2)-OH (9). To a sample of 8 (2.8 g,
7.4 mmol), MeOH (30 mL) and 2 M NaOH (15 mL) 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 in vacuo, the
residue was taken 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
of 9 (1.58 g) as waxy solid.
Yield¼10.04 g (30.4 mmol, 76%). Mp 85–86 8C. 1H NMR
(CDCl3, 300 MHz, d ppm): 6.09 (Aib(2) NH, 1H, s); 4.65
(Acp(1) NH, 1H, t, J¼10.5 Hz); 3.73 (–OCH3, 3H, s);
3.10–3.15 (C1Hs of Acp(1), 2H, m); 2.14–2.19 (CaHs
Acp(1), 2H, m); 1.56–1.68 (CbHs and CgHs of Acp(1), 4H,
m); 1.46–1.51 (CdH3 of Acp(1), 2H, m); 1.53 (CbH3 of
Aib(2), 6H, s); 1.44 (Boc-CH3s, 9H, s). Elemental analysis
calcd (%) for C16H30N2O5 (330): C, 58.18; H, 9.09; N, 8.48.
Found: C, 58.16; H, 9.06; N, 8.52.
Yield¼1.58 g (5.5 mmol, 74%). 1H NMR ((CD3)2SO,
300 MHz, d in ppm): 12.2 (–COOH, 1H, b); 7.05 (Gly(2)
NH, 1H, t, J¼4.5 Hz); 3.56 (CaH of Gly, 2H, t, J¼7.5 Hz);
2.54–2.58 (C1Hs of Acp(1), 2H, m); 1.78–1.81 (CaHs of
Acp(1), 2H, m); 1.11–1.13 (CbHsþCgHs of Acp (1), 4H,
m); 1.10 (Boc-CH3s, 9H, s); 0.82–0.86 (CdHs of Acp(1),
2H, m). Elemental analysis calcd (%) for C13H24N2O5
(288): C, 54.17; H, 8.33; N, 9.72. Found: C, 54.56; H, 8.21;
N, 9.63.
4.1.6. Boc-Acp(1)-Aib(2)-OH (11). To a sample of 10
(10.04 g, 30.4 mmol), MeOH (75 mL) and 2 M NaOH
(30 mL) 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 in vacuo, the residue was taken into water (50 mL),
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 evapor-
ated in vacuo to yield compound 11 (8.38 g) as a white solid.
4.1.4. Boc-Acp(1)-Gly(2)-Leu(3)-OMe (1). Boc-Acp-Gly-
OH (1.44 g, 5 mmol) was dissolved in dichloromethane–
DMF (10 mL) in an ice-water bath. H-Leu-OMe was
isolated from methyl ester hydrochloride (1.82 g,
16 mmol) by neutralization, subsequent extraction with
ethyl acetate and concentration to 7 mL and it was added to
the reaction mixture, followed immediately by dicyclo-
hexylcarbodiimide (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, 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 as waxy
solid. Purification was done by silica gel column (100–200
mesh) using 33% toluene/ethyl acetate as eluent.
Yield¼8.38 g (26.5 mmol, 87.17%). Mp 106–107 8C.1H
NMR ((CD3)2SO, 300 MHz, d in ppm): 11.78 (–COOH,
1H, br); 7.67 (Aib(2)–NH, 1H, s); 4.46 (Acp(1) NH,1H, t,
J¼10.5 Hz); 2.60–2.66 (C1Hs of Acp(1), 2H, m); 1.76–
1.81 (CaHs Acp(1), 2H, m); 1.15–1.23 (CbHs and CgHs of
Acp (1), 4H, m); 1.13 (Boc-CH3s, 9H, s);1.07 (CbHs of
Aib,6H, s); 0.93–1.0 (CdHs of Acp(1), 2H, m). Elemental
analysis calcd (%) for C15H28N2O5 (316): C, 56.96; H, 8.86;
N, 8.86. Found: C, 56.89; H, 8.78; N, 8.92.
Yield¼1.45 g (3.5 mmol, 70%). Rf (33% toluene/ethyl
1
acetate) 0.68; H NMR (CDCl3, 300 MHz, d ppm): 6.62
4.1.7. Boc-Acp(1)-Aib(2)-Leu(3)-OMe (2). Boc-Acp(1)-
Aib(2)-OH (1.58 g, 5 mmol) in DMF (10 mL) was cooled in
an ice-water bath and H-Leu-OMe was isolated from the
corresponding 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 DCC
(1.03 g, 5 mmol) and HOBt (0.68 g, 5 mmol). The reaction
(Leu(3) NH, 1H, d, J¼9 Hz); 6.60 (Gly(2) NH, 1H, t,
J¼6 Hz); 4.69 (Acp(1) NH, 1H, t, J¼4.8 Hz); 4.57–4.60
(CaH of Leu, 1H, m); 3.96–4.01 (CaHs of Gly, 2H, m); 3.73
(–OCH3, 3H, s); 3.07–3.13 (C1H of Acp, 2H, m); 2.16–
2.21 (CaHs of Acp(1), 2H, m); 1.56–1.71 (CbHs and CgHs
of Acp(1) and Leu(3), 4H, m); 1.50–1.52 (CdHs of Acp(1),
2H, m); 1.44 (Boc-CH3s, 9H, s); 0.93–0.94 (CdHs of

5942
A. Banerjee et al. / Tetrahedron 60 (2004) 5935–5944
mixture was stirred for 3 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,
1 M sodium carbonate (3£50 mL), brine (2£50 mL), dried
over anhydrous sodium sulfate and evaporated in vacuo to
yield white solid (1.64 g, 3.7 mmol). Purification was done
by silica gel column (100–200 mesh) using ethyl acetate as
eluent. Colourless single crystals were grown from ethyl
acetate by slow evaporation.
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, 1 M sodium
carbonate (3£50 mL), brine (2£50 mL), dried over
anhydrous sodium sulfate and evaporated in vacuo to
yield of white solid (1.6 g). Purification was done by silica
gel column using ethyl acetate as eluent.
Yield¼1.6 g (3.73 mmol, 74%). Rf (ethyl acetate) 0.75. Mp
70–71 8C. 1H NMR. (CDCl3, 300 MHz, d ppm): 7.12
(Val(3) NH, 1H, d, J¼9 Hz); 6.03 (Aib(2) NH, 1H, s); 4.66
(Acp(1) NH, 1H, t, J¼6 Hz); 4.48–4.52 (CaH of Val(3),
1H, m); 3.74 (OCH3, 3H, s); 3.07–3.14 (C1Hs of Acp(1),
2H, m); 2.17–2.21 (CaHs of Acp(1), 2H, m and CbH of
Val(3), 1H, m); 1.63–1.71 (CbHs and CgHs of Acp(1), 4H,
m); 1.57, 1.59 (CbHs of Aib(2), 6H, s); 1.47–1.53 (CdHs of
Acp(1), 2H, m); 1.44 (Boc-CH3s, 9H, s); 0.90–0.96 (CgHs
of Val(3), 6H, m). Mass spectral data MþNaþ¼452,
Mcalcd¼429. Elemental analysis calcd for C21H39N3O6
(429): C, 58.74; H, 9.1; N, 9.79. Found: C, 58.60; H, 9.11;
N, 9.71.
Yield¼1.64 g (3.7 mmol, 74%). Rf (ethyl acetate) 0.72. Mp
1
108–109 8C. H NMR (CDCl3, 300 MHz, d ppm): 6.99
(Leu(3) NH, 1H, d, J¼7.74 Hz); 6.05 (Aib(2) NH, 1H, s);
4.69–4.60 (Acp(1) NH, 1H, m); 4.53–4.58 (CaH of Leu(3),
1H, m); 3.73 (OCH3, 3H, s); 3.09–3.12 (C1Hs of Acp(1),
2H, m); 2.16–2.21 (CaHs of Acp(1), 2H, m); 1.62–1.67
(CbHs and CgHs of Acp(1) and Leu(3), 4H, m); 1.57, 1.59
(CbHs of Aib(2), 6H, s); 1.47–1.53 (CdHs of Acp(1), 2H,
m); 1.44 (Boc-CH3s, 9H, s); 0.87–0.94 (Leu(3) of CdHs,
6H, m); MS data MþNaþ¼466, Mcalcd¼443. Elemental
analysis calcd (%) for C22H41N3O6 (443): C, 59.59; H, 9.25;
N, 9.48. Found: C, 59.56; H, 9.11; N, 9.51.
4.1.10. Boc Acp-Aib-D-Val-OMe (5). Boc-Acp-Aib-OH
(1.58 g, 5 mmol) was dissolved in DMF (10 mL) in an ice-
water bath. H-D-Val-OMe was isolated from methyl ester
hydrochloride (1.68 g, 10 mmol) by neutralization, sub-
sequent 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, 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 5 as white solid. Purification was done by
silica gel column (100–200 mesh) using ethyl acetate as
eluent.
4.1.8. Boc-Acp(1)-Aib(2)-Phe(3)-OMe (3). Boc-Acp(1)-
Aib(2)-OH (1.58 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 3 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, 1 M sodium carbonate (3£50 mL), brine (2£50 mL),
dried over anhydrous sodium sulfate and evaporated in
vacuo to yield white solid (1.64 g, 3.7 mmol). Purification
was done by silica gel column (100–200 mesh) using ethyl
acetate as eluent. Colourless single crystals were grown
from methanol–water mixture.
Yield¼1.4 g (3.26 mmol, 65.2%). Rf (ethyl acetate) 0.74.
1
Yield¼1.64 g (3.4 mmol, 68%). Rf (ethyl acetate) 0.65. Mp
92–93 8C. 1H NMR (CDCl3, 300 MHz, d ppm): 7.11–7.35
(phenyl ring protons); 6.84 (Phe(3) NH, 1H, d, J¼7.02 Hz);
5.98 (Aib NH(2), 1H, s); 4.8–4.86 (Acp(1)NH, 1H, m);
4.56–4.58 (CaH of Phe(3), 1H, m); 3.73 (OCH3, 3H, s);
3.21–3.14 (CbHs of Phe (3), 2H, m); 3.06–3.13 (C1Hs of
Acp(1), 2H, m); 2.17–2.12 (CaHs of Acp(1), 2H, m); 1.53–
1.67 (CbHs and CgHs of Acp(1) 4H, m); 1.48, 1.50 (CbHs of
Aib(2), 6H, s); 1.46–1.48 (CdHs of Acp(1), 2H, m); 1.44
(Boc-CH3s, 9H, s); MS data MþNaþþHþ¼501,
Mcalcd¼477. Elemental analysis calcd (%) for C25H39N3O6
(477): C, 62.89; H, 8.18; N, 8.81. Found: C, 62.87; H, 8.17;
N, 8.83.
Mp 86–87 8C. H NMR. (CDCl3, 300 MHz, d ppm): 7.15
(D-Val(3) NH, 1H, d, J¼7.8 Hz); 6.12 (Aib(2) NH, 1H, s);
4.63–4.68 (Acp(1)NH, 1H, t, J¼7.5 Hz); 4.48–4.52
(D-Val(3) CaH, 1H, m); 3.74 (OCH3, 3H, s); 3.08–3.14
(C1Hs of Acp(1), 2H, m); 2.16–2.22 (CaHs of Acp(1), 2H,
m and CbH of D-Val(3), 1H, m); 1.63–1.7 (CbHs and CgHs
of Acp(1), 4H, m); 1.57, 1.59 (CbHs of Aib(2), 6H, s);
1.47–1.52 (CdHs of Acp(1), 2H, m); 1.44 (Boc-CH3s, 9H,
s); 0.9–0.99 (CgHs of D-Val(3), 6H, m). Mass spectral data
MþNaþ¼452, Mcalcd¼429. Elemental analysis calcd for
C21H39N3O6 (429): C, 58.74; H, 9.1; N, 9.79. Found: C,
58.58; H, 9.17; N, 9.83.
4.1.11. Boc Acp-Aib-Ile-OMe (6). Boc-Acp-Aib-OH
(1.58 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, sub-
sequent 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
4.1.9. Boc Acp-Aib-Val-OMe (4). To a sample of 11
(1.58 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

A. Banerjee et al. / Tetrahedron 60 (2004) 5935–5944
5943
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, 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 6 (1.6 g) in form of white solid. Purification
was done by silica gel column (100–200 mesh) using ethyl
acetate as eluent.
crystals were obtained from methanol–water solution by
slow evaporation. Crystal data for peptide 2 and peptide 3
were collected on a Marresearch Image Plate with Mo Ka
radiation. The crystals were positioned at 70 mm from the
image plate. 100 frames were measured at 28 intervals with a
counting time of 2 min. Data analysis was carried out with
the XDS program.22 The structure was 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 equival-
ent to 1.2 times those of the atom to which they were
attached. The structure was refined on F2 using Shelxl.24
Crystallographic data have been deposited at the Cambridge
Crystallographic Data Centre reference CCDC172055 for
peptide 2 and CCDC 211390 for peptide 3.
Yield¼1.6 g (3.6 mmol, 72%). Rf (ethyl acetate) 0.73. Mp
75–76 8C. 1H NMR (CDCl3, 300 MHz d ppm,): 7.14 (Ile(3)
NH, 1H, d, J¼8.3 Hz); 6.18 (Aib NH(2), 1H, s); 4.66
(Acp(1) NH, 1H, t, J¼7.5 Hz); 4.49–4.53 (CaH of Ile(3),
1H, m); 3.70 (OCH3, 3H, s); 3.05–3.11 (C1Hs of Acp(1),
2H, m); 2.15–2.19 (CaHs of Acp(1), 2H, m); 1.87–1.95
(CbHs of Ile(3), 1H, m); 1.57–1.67 (Acp(1) CgHs and
CbHs, 4H, m); 1.53 (CbHs of Aib(2), 3H, s); 1.56 (CbHs of
Aib(2), 3H, s); 1.471.41–1.49 (CdHs of Acp(1), 2H, m and
CgHs of Ile(3), 2H, m); 1.40 (Boc-CH3s, 9H, s); 1.2–136
(CgHs of Ile(3), 3H, m); 0.87–0.92 (CdHs of Ile(3), 6H, m).
Mass spectral data MþNaþ¼466, Mcalcd¼443. Elemental
analysis calcd for C22H41N3O6 (443): C, 59.59; H, 9.25; N,
9.48. Found: C, 59.56; H, 9.11; N, 9.51.
4.7. Mass spectrometry
Mass spectra were recorded on a Hewlett Packard Series
1100MSD mass spectrometer by positive mode electrospray
ionization.
4.2. NMR experiments
Acknowledgements
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.
We thank the EPSRC and the University of Reading, U.K.
for funds for the Image Plate System. This research was also
supported by a grant from the Department of Science and
Technology, India Grant No. (SR/S5/OC-29/2003).
4.3. FT-IR spectroscopy
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.
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