Conformational and self-assembly studies of helix forming hexapeptides containing two α-amino isobutyric acids

Article abstract of DOI:10.1016/j.tet.2007.11.016

Single crystal X-ray diffraction studies reveal that three hexapeptides with general formula Boc-Ile-Aib-Xx-Ile-Aib-Yy-OMe, where Xx and Yy are Leu in peptide I, Leu and Phe in peptide II, and Phe and Leu in peptide III, respectively, adopt equivalent con

Full text of DOI:10.1016/j.tet.2007.11.016

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 549e558
www.elsevier.com/locate/tet
Conformational and self-assembly studies of helix forming
hexapeptides containing two a-amino isobutyric acids
Anita Dutt a, Michael G.B. Drew b, Animesh Pramanik a,
*
a Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700 009, India
b School of Chemistry, The University of Reading, Whiteknights, Reading RG6 6AD, UK
Received 20 July 2007; received in revised form 16 October 2007; accepted 1 November 2007
Available online 7 November 2007
Abstract
Single crystal X-ray diffraction studies reveal that three hexapeptides with general formula BoceIleeAibeXxeIleeAibeYyeOMe, where
Xx and Yy are Leu in peptide I, Leu and Phe in peptide II, and Phe and Leu in peptide III, respectively, adopt equivalent conformations that can
be described as mixed 310/a-helice with two 4/1 and two 5/1 intramolecular NeH/O]C H-bonds. The peptides do not generate any helix-
terminating Schellman motif despite having Aib at the penultimate position from C-terminus. In the crystalline state, the helices are packed in
head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. The CD studies of the three hexapeptides
in acetonitrile indicate that they are folded in well-developed 310-helical structures. NMR studies of peptide I in CDCl3 also suggest the
formation of a homogeneous 310-helical structure. The field emission scanning electron microscopic (FE-SEM) images of peptide II in the solid
state reveal a non-twisted ribbon-like morphology, which is formed through lateral association of non-twisted filaments.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Peptide; a-Amino isobutyric acid; 310-Helix; a-Helix; Ribbons
1. Introduction
required for stabilization of the a-helix relative to the 310-
helix in Aib-containing peptides is still not unequivocally
established.
There has been considerable discussion in the literature on
the nature of the helical conformations adopted by Aib-rich
sequences.1e4 Both 310- and a-helical conformations have
been characterized depending upon peptide length, sequence,
and Aib content. The 310-helix is a structure stabilized by suc-
cessive 4/1 CO/NH hydrogen bonds, with idealized 4 and
j values of ꢀ60ꢁ and ꢀ30ꢁ characteristic of the right-handed
screw. The a-helix is stabilized by successive 5/1 hydrogen
bonds with idealized 4 and j values of ꢀ55ꢁ and ꢀ45ꢁ for
a right-handed structure. The distinctions between these two
helical types are subtle and involve very small changes in 4
and j values. The conformational interconversion between
310- and a-helix can be easily achieved and has been the
subject of several investigations.5 Although various designed
peptides have been reported the minimal peptide length
Several studies with hexapeptides containing Aib have been
reported in the literature.2 It has been observed that hexapep-
tides with more than two Aib residues (ꢂ50%) generally adopt
well defined 310-helical structures in the crystalline state. Ta-
ble 1, which includes all the reported hexapeptides with two
Aib residues, shows that the first two hexapeptides (entries 1
and 2) exhibit 310-helical conformations, while the next two
hexapeptides (entries 3 and 4) exhibit mixed 310/a-helical
structures with the 310 segment occurring at the N-terminus.3
The notable difference is that unlike the first two hexapeptides,
the second two peptides contain achiral Aib at the penultimate
position from the C-terminus. Although Aib at this position
generally helps to generate the helix-terminating Schellman
motif through the formation of a 6/1 intramolecular hydro-
gen bond,4,6 the third hexapeptide on the list (entry 3) does
not produce any such motif. Another important difference is
that the hexapeptides of mixed 310/a-helical conformations
(entries 3 and 4) contain Pro in their sequences, which is not
* Corresponding author. Tel.: þ91 33 2484 1647; fax: þ91 33 2351 9755.
E-mail address: animesh_in2001@yahoo.co.in (A. Pramanik).
0040-4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.11.016

550
A. Dutt et al. / Tetrahedron 64 (2008) 549e558
Table 1
Crystal structures of hexapeptide helices containing two Aib residues
Entry
Sequence
No. of H-bonds
Structure
Reference
1.
2.
3.
BoceAlaeLeueAibeAlaeLeueAibeOMe$H2O
L-ProeL-LeueAibeAibeL-GlueL-ValeOH
PiveProeProeAibeLeueAibePheeOMe
Molecule A
3 (4/1)
2 (4/1)
310
310
3a
3b
4 (4/1)
3 (5/1)
3 (4/1)
4 (5/1)
4 (4/1)
3 (5/1)
1 (6/1)
2 (4/1)
2 (5/1)
2 (4/1)
2 (5/1)
2 (4/1)
2 (5/1)
Mixed 310/a
3c
Molecule B
4.
BoceProeAibeGlyeLeueAibeLeueOMe
Mixed 310/a (Schellman motif)
3d
5.
6.
7.
BoceIleeAibeLeueIleeAibeLeueOMe (I)
BoceIleeAibeLeueIleeAibePheeOMe (II)
BoceIleeAibePheeIleeAibeLeueOMe (III)
Mixed 310/a
Mixed 310/a
Mixed 310/a
This work
This work
This work
generally observed in regular natural right-handed a-helices.
Nevertheless, these two peptides indicate that hexapeptides
with less than three Aib residues can show some small degree
of a-helical character. Therefore, further studies are necessary
with hexapeptides to assess the preferences between the 310
and the a-helix conformations and also to assess the possibil-
ity of the formation of the Schellman motif.
In this context, we chose a set of three hexapeptides IeIII,
BoceIleeAibeXxeIleeAibeYyeOMe, where Xx and Yy
are Leu in peptide I, Leu and Phe in peptide II, and Phe
and Leu in peptide III, respectively (Fig. 1). All the peptides
IeIII incorporate achiral Aib residue at the penultimate
position and do not contain the helix-breaking Pro. So far,
the Schellman motif has been characterized crystallographi-
cally only in peptide helices containing at least seven residues
apart from the one hexapeptide (entry 4) included in Table 1.
Therefore, the present study will provide insights into the oc-
currence of the Schellman motif in Aib-containing hexapepti-
des and also the preferred helical conformations. Peptides Ie
III were synthesized using conventional solution phase
methodology (Scheme 1) and their solid-state structures and
crystal packing were determined by X-ray diffraction analysis.
The peptide conformations in the solution phase were probed
by CD and NMR studies.
Apart from the conformational interest, the cylindrical he-
lical structures readily generated in Aib-rich sequences are
likely to find many novel applications in material science. Rel-
evant reports include the synthesis of monodisperse liquid
crystalline peptides based on Aib-containing helices7 and the
formation of self-assembling chiral monolayers of helical
peptides bound to gold surfaces, in which immobilization is
achieved by side chain thioether interaction.8 Aib-containing
helical peptides may be employed to assemble fibrils of vari-
ous morphologies. In this report, field emission scanning
O
O
O
O
H
N
H
N
H
N
X
O
N
H
N
H
N
H
Y
OMe
O
O
O
Figure 1. Schematic representation of peptides IeIII. Peptide I: X¼eCHe
CH2eCHe(CH3)2, Y¼eCHeCH2eCHe(CH3)2; peptide II: X¼eCHe
CH2eCHe(CH3)2, Y¼eCHeCH2ePh; peptide III: X¼eCHeCH2ePh,
Y¼eCHeCH2eCHe(CH3)2.
O
O
H
O
a
N
O
N
H
OMe
O
N
H
CO2H
O
b, c
b, d
O
O
O
H
N
O
H
N
OMe
OMe
O
N
N
H
O
N
H
N
H
H
O
O
O
O
b, e
b, f
b, e
Peptide III
(81%)
Peptide II
(86%)
Peptide I
(83%)
Scheme 1. Reagents and conditions: (a) DCM, HeAibeOMe, DCC, HOBt, 0 ꢁC, 90% yield; (b) MeOH, 2 M, NaOH, 80% yield; (c) DMF, HeLeueOMe, DCC,
HOBt, 0 ꢁC, 90% yield; (d) DMF, HePheeOMe, DCC, HOBt, 0 ꢁC, 95% yield; (e) DMF, BoceIULeOMe, CF3COOH, 8 h, 0 ꢁC; DMF, HeIULeOMe, DCC,
HOBt, 0 ꢁC; (f) DMF, BoceIUFeOMe, CF3COOH, 8 h, 0 ꢁC; DMF, HeIULeOMe, DCC, HOBt, 0 ꢁC.

A. Dutt et al. / Tetrahedron 64 (2008) 549e558
551
electron microscopy (FE-SEM) has been employed to investi-
gate the morphological properties of the peptides in the solid
state.
2. Results and discussion
2.1. Single crystal X-ray diffraction studies
All the three peptides IeIII crystallized with one molecule
in the crystallographic asymmetric unit. Figures 2e4 illustrate
the observed conformations of the peptides and demonstrate
their similarities, while Tables 2 and 3 list the backbone tor-
sion angles and the inter- and intramolecular hydrogen bond-
ing dimensions, respectively. In peptide I the backbone torsion
angles for residues 1, 2, and 5 lie in the helical (310/aR) region
of the Ramachandran map. The 4 and j values at residues 3
[ꢀ98.2(5)ꢁ and 7.9(6)ꢁ] and 4 [ꢀ104.6(5)ꢁ and ꢀ53.5(6)ꢁ] de-
viate significantly from the idealized values of helical (310/aR)
region (Fig. 2, Table 2). Inspection of the hydrogen bonding
parameters listed in Table 3 reveals that the molecule is stabi-
lized by four intramolecular hydrogen bonds. Hexapeptide I
adopts a mixed 310/a-helix conformation with two 4/1 intra-
molecular hydrogen bonds at the N-terminal and two 5/1 at
the C-terminal. The Ile(1)CO is involved in bifurcated intra-
molecular hydrogen bonding with HNeIle(4) (4/1) and
HNeAib(5) (5/1). As a result the backbone torsion angles
(4 and j) at Leu(3) and Ile(4) deviate significantly from the
helical region (310/aR) of the Ramachandran plot (Table 2). In-
terestingly, despite having achiral Aib at the penultimate posi-
tion, peptide I does not generate the helix-terminating
Schellman motif through a 6/1 intramolecular hydrogen
bond at the C-terminal, which is commonly observed in pep-
tide helices, which have lengths of seven residues or greater.
Figure 3. The ORTEP diagram of peptide II including atom numbering
scheme. Thermal ellipsoids are shown at the level of 25% probability. Hydro-
gen bonds are shown as dotted lines.
Peptide II where the last residue, Leu(6), of peptide I has
been replaced by Phe also shows a similar mixed 310/a-helical
conformation without Schellman motif (Fig. 3, Table 2). The
result shows that positioning Phe after the penultimate Aib
in peptide II sequence does not help in Schellman motif for-
mation. Peptide III, which incorporates Phe at the middle of
the sequence also shows similar structural preferences to
peptides I and II (Fig. 4, Table 2).
The crystal packing arrangements of peptides IeIII are
shown in Figure 5 (with side chains). Both peptides I and II
crystallize in space group P21 but there are no hydrogen bonds
between molecules related by the screw axis. Instead, peptides
I and II are packed in head-to-tail fashion through inter-
molecular hydrogen bonds (Table 3) between the Aib(2)e
Figure 2. The ORTEP diagram of peptide I including atom numbering scheme.
Thermal ellipsoids are shown at the level of 25% probability. Hydrogen bonds
are shown as dotted lines.
Figure 4. The ORTEP diagram of peptide III including atom numbering
scheme. Thermal ellipsoids are shown at the level of 25% probability. Hydro-
gen bonds are shown as dotted lines.

552
A. Dutt et al. / Tetrahedron 64 (2008) 549e558
Table 2
Selected backbone torsions (ꢁ) of peptides IeIII
Residues
4
j
u
Peptide I
Ile(1)
ꢀ50.4(6)
ꢀ39.7(6)
ꢀ179.8(4)
ꢀ178.3(4)
ꢀ173.2(4)
171.8(4)
Aib(2)
Leu(3)
Ile(4)
ꢀ50.2(6)
ꢀ98.2(5)
ꢀ104.6(5)
ꢀ57.2(6)
ꢀ129.0(5)
ꢀ41.1(6)
7.9(6)
ꢀ53.5(6)
ꢀ43.0(5)
ꢀ179.8(5)
Aib(5)
Leu(6)
172.5(4)
ꢀ167.4(5)
Peptide II
Ile(1)
ꢀ48.1(7)
ꢀ49.7(6)
ꢀ90.1(6)
ꢀ76.1(6)
ꢀ63.5(6)
ꢀ132.9(5)
ꢀ43.2(6)
ꢀ47.2(6)
ꢀ11.2(7)
ꢀ45.3(6)
ꢀ39.5(6)
ꢀ96.9(7)
ꢀ179.2(4)
ꢀ173.3(4)
ꢀ169.0(5)
169.9(4)
Aib(2)
Leu(3)
Ile(4)
Aib(5)
Phe(6)
171.5(5)
ꢀ174.1(5)
Peptide III
Ile(1)
ꢀ56.8(4)
ꢀ51.8(5)
ꢀ92.0(4)
ꢀ81.7(4)
ꢀ52.6(5)
ꢀ117.4(4)
ꢀ34.3(4)
ꢀ44.4(5)
0.7(5)
ꢀ175.1(3)
ꢀ174.5(3)
ꢀ172.7(3)
161.8(3)
Aib(2)
Phe(3)
Ile(4)
ꢀ52.2(4)
ꢀ50.3(4)
148.3(5)
Aib(5)
Leu(6)
176.9(3)
ꢀ165.4(3)
Table 3
Hydrogen bonding parameters of peptides IeIII
Figure 5. Packing diagrams of peptides IeIII showing the formation of supra-
molecular helices. Intermolecular hydrogen bonds are indicated by dotted
lines. Peptides I and II show one hydrogen bond between molecules related
by translational symmetry along the a axis while peptide III shows two hydro-
gen bonds between adjacent molecules around the 31 screw axis.
DeH/A
H/A (A)
D/A (A)
DeH/A (ꢁ)
˚
˚
Peptide I
Intramolecular
BoceCO/HNeLeu(3)
Ile(1)eCO/HNeIle(4)
Ile(1)eCO/HNeAib(5)
Aib(2)eCO/HNeLeu(6)
2.22
2.12
2.10
2.57
2.95(1)
2.93(1)
2.91(1)
3.21(1)
143
156
157
133
contrast with I and II the molecule forms two intermolecular
hydrogen bonds between BoceNH/OCeAib(5) and Aib(2)e
NH/OCeIle(4) related by the 31 screw axis.
Intermolecular
Aib(2)eNH/OCeIle(4)a
2.06
2.90(1)
165
2.2. Solution conformational analysis
Peptide II
Intramolecular
Complete 1H NMR assignments of peptide I were obtained
using a combination of 2D COSY and ROESY methods in
CDCl3. In order to investigate the existence of intramolecular
hydrogen bonding and peptide conformation in the solution
phase, the solvent dependence of the NH chemical shifts
was examined by NMR titration.4a,9 In this experiment, a solu-
tion of the peptide in non-polar CDCl3 (10 mM in 0.5 ml) was
gradually titrated against polar (CD3)2SO. The changes in the
chemical shifts are presented in Figure 6. The solvent titration
experiment shows that by increasing the percentage of
(CD3)2SO in CDCl3 from 0 to 14% (v/v) the net changes in
the chemical shift (Dd) values for Ile(1)eNH, Aib(2)eNH,
Leu(3)eNH, Ile(4)eNH, Aib(5)eNH, and Leu(6)eNH are
0.82, 0.50, 0.10, 0.17, 0.08, and 0.25 ppm, respectively.
From the Dd values, it is evident that Ile(1) and Aib(2) NH
are solvent exposed, while the remaining NH groups at
Leu(3), Ile(4), Aib(5) ,and Leu(6) are solvent shielded, indi-
cating their involvement in intramolecular hydrogen bonding.
These observations are consistent with the presence of the
310-helical conformation in solution because earlier literature
suggests that the solvent exposure of only two N-terminal
BoceCO/HNeLeu(3)
Ile(1)eCO/HNeIle(4)
Ile(1)eCO/HNeAib(5)
Aib(2)eCO/HNePhe(6)
2.38
2.45
2.19
2.27
3.09(1)
3.07(1)
3.05(1)
2.94(1)
141
130
176
135
Intermolecular
Aib(2)eNH/OCeIle(4)a
2.09
2.93(1)
161
Peptide III
Intramolecular
BoceCO/HNePhe(3)
Ile(1)eCO/HNeIle(4)
Ile(1)eCO/HNeAib(5)
Aib(2)eCO/HNeLeu(6)
2.44
2.27
2.08
2.30
3.15(1)
2.95(1)
2.94(1)
3.05(1)
140
136
172
145
Intermolecular
BoceNH/OCeAib(5)b
Aib(2)eNH/OCeIle(4)b
a
2.20
2.15
3.00
3.00
159
167
Symmetry elements: xꢀ1, y, z.
b
Symmetry elements: xþy, ꢀx, zꢀ1/3.
NH moiety of one molecule with the Ile(4)CO moiety of
another molecule (symmetry element xꢀ1, y, z) to create
supramolecular helical structures along the crystallographic
axis a. Peptide III crystallizes in space group P31 and by
NH groups is indicative of
a continuous 310-helical

A. Dutt et al. / Tetrahedron 64 (2008) 549e558
553
7.5
7.0
6.5
6.0
5.5
5.0
iþ1) pattern, indicative of a helical conformation in solution.
Moreover, the ROESY spectrum clearly shows a long range
connectivity da N (i, iþ2) [Ile(1)4Leu(3); Leu(3)4Aib(5);
Ile(4)4Leu(6)] (Fig. 7), diagnostic of a 310-helical conforma-
tion.11 In an a-helix the da N (i, iþ2) would be approximately
˚
4.5 A, whereas in the 310-helix the same separation is reduced
Leu(3)
Ile(4)
Leu(6)
Aib(5)
Aib(2)
Ile(1)
˚
to 3.7 A. Therefore, only in a 310-helix can one expect to detect
the da N (i, iþ2) crosspeaks, although these will be of low inten-
sity.11 In this peptide the only da N (i, iþ4) detectable NOE is that
relating the Ile(1)CaH proton to the Aib(5) NH proton. Since this
interaction is not observed in Figure 7, it is reasonable to assume
that the structure adopted by peptide I is a 310 type.
The conformations of the peptides IeIII were probed
further in solution phase by far-UV CD measurements in ace-
tonitrile. The CD patterns for peptides IeIII are presented in
Figure 8. Peptides I and III show a strong negative band cen-
tered at 204 nm, which is followed by a weak negative shoul-
der at about 225 nm. Generally in the right-handed a-helix the
intensities of the two negative maxima at 208 nm (parallel
component of the p/p* transition) and 222 nm (n/p*
transition) are very close.12 It is known that in the 310-helix
the n/p* transition is expected to exhibit a drastically re-
duced intensity with respect to that of the p/p* transition
and tends to undergo a modest blue shift.13 Therefore, peptides
0
2
4
6
8
10
12
14
% of d6 DMSO in CDCl3
Figure 6. NMR solvent titration curve for NH protons in peptide I.
conformation.9a,10 We are unable to characterize the solvent
perturbation experiment of peptides II and III due to partial
overlapping of the NH signals with the aromatic protons.
Further attempts to characterize the conformation in CDCl3
using NOEs reveal that peptide I exhibits most of the NN(i,
Figure 7. Section of the ROESY spectrum of peptide I in CDCl3 (peptide concn: 1ꢃ10ꢀ2 M). The [da N (i, iþ2), where i¼1, 3, and 4] crosspeaks typical of a 310
-
helix are indicated (full arrow). The area in the spectrum where the undetected [da N(i, iþ4), where i¼1] crosspeak typical of an a-helix was expected is also
marked (dashed arrow).

554
A. Dutt et al. / Tetrahedron 64 (2008) 549e558
20
0
-20
-40
-60
-80
-100
Peptide III
Peptide II
Peptide I
CH3CN
200
210
220
230
240
250
λ (nm)
Figure 8. CD curves of peptides IeIII in acetonitrile (1.5 mM).
I and III adopt 310-helical conformations in acetonitrile. Pep-
tide II shows a weak positive shoulder at about 225 nm instead
of a weak negative shoulder as it is observed in peptides I
and III (Fig. 8), which may be due to some aggregation in
acetonitrile. Nevertheless, the CD data strongly favor the
conclusion that the three peptides IeIII are folded into well-
developed homogeneous 310-helical conformations in solution.
2.3. Morphological studies
It has been suggested that not only b-sheets but also helices
have a significant role in amyloid fibril formation where the
a-helices are stacked along the fibril axis.14 Goldsbury et al.
have suggested that for human amylin, a-helices may have
a role in highly ordered self-aggregated amyloid plaque for-
mation.15 It is established that in the amyloidogenic peptide
calcitonin ribbon-like structures twist back upon themselves
to afford hollow tube-like assemblies.16 Recently, several
reports show that peptide based supramolecular helices also
produce fibrillar structures through self-assembly.17 Therefore,
we became interested in exploring the possibility of fibril for-
mation in peptides IeIII. Field emission scanning electron mi-
croscopic (FE-SEM) images (10,000 times magnified) of the
dried fibrous material of peptide II grown slowly from acetone
clearly demonstrate that the aggregates in the solid state are
bunches of non-twisted ribbon-like structures (Fig. 9a and
9b). It was observed that peptides I and III do not produce
any fibrous material under similar conditions. Peptide II
exhibits higher ordered self-assembly to generate ribbon-like
structures. The supramolecular helices of II generated from
primary building block helix (Fig. 5) aggregate through
non-covalent interactions to form non-twisted filaments. The
filaments can lead to ribbon-like assemblies through lateral as-
sociation. Interestingly, the SEM images of II clearly demon-
strate that the ribbon-like structures are formed through lateral
association of non-twisted filaments (Fig. 9a and 9b). The reg-
ular parallel alignment of phenyl rings on the surface of supra-
molecular helices of peptide II (Fig. 5) probably helps in
Figure 9. (a) FE-SEM image (ꢃ10,000) of peptide II showing the formation of
non-twisted ribbon-like structures. (b) The enlarged SEM image clearly dem-
onstrates that the ribbons are formed through lateral association of non-twisted
filaments.
higher ordered self-assembly through non-covalent interac-
tions, which is absent in peptides I and III. The quasi-crystal-
line structures of flat non-twisted morphology may be useful
for specific nano-applications. Various attempts to fabricate
these types of structures using amyloid proteins, peptides,
and viruses have been reported.18
3. Conclusions
In peptides with lower Aib content (<50%), conforma-
tional heterogeneity is a common phenomenon. The present
attempt to characterize the preferred conformations of the
three designed hexapeptides containing Aib at positions 2
and 5 establishes that in the solid state they adopt mixed
310/a-helical conformations and in the solution phase well-
developed homogeneous 310 helical conformations. Because
of considerable conformational flexibility, they attain different
structures in different environments through conformational

A. Dutt et al. / Tetrahedron 64 (2008) 549e558
555
transitions. It is reasonable to assume that hexapeptides with
less than two Aib residues may facilitate a-helical conforma-
tions. The present study also reveals that hexapeptides cannot
generate the helix-terminating Schellman motif unlike longer
peptides when the achiral Aib is placed at the penultimate
position from the C-terminus. In the crystalline state, the heli-
ces are packed in head-to-tail fashion through intermolecular
hydrogen bonds to create supramolecular helical structures.
It has been shown that peptide II can form ribbon-like struc-
tures that are generated through lateral association of non-
twisted filaments. These quasi-crystalline structures of flat
non-twisted morphology may find useful applications in nano-
technology. The results indicate that synthetic Aib-containing
peptide helices can form highly ordered self-aggregated
amyloid plaque like human amylin.15
C21H39N3O6 (429.54): C, 58.72; H, 9.15; N, 9.78. Found: C,
58.55; H, 8.98; N, 9.58.
4.1.2. BoceIleeAibePheeOH 2
BoceIleeAibePheeOMe20 (0.6 g, 1.26 mmol) was dis-
solved in methanol (15 ml) and 2 M NaOH (8 ml) was added.
The reaction mixture was stirred at room temperature for 2
days. The progress of the reaction was monitored by TLC. Af-
ter completion of the reaction, methanol was evaporated. The
residue obtained was diluted with water and washed with di-
ethylether. The aqueous layer was cooled in ice, neutralized
by 2 M HCl, and extracted with ethyl acetate. The solvent
was evaporated in vacuo to give a white solid.
Yield: 0.52 g (89.0%). Mp¼108e110 ꢁC; IR (KBr): 3415,
3314, 1666, 1523 cmꢀ1 1H NMR (300 MHz, DMSO-d6,
;
d ppm): 7.87 (Aib(2) NH, 1H, s), 7.49 (Phe(3) NH, 1H, d,
J¼7.5 Hz), 7.18e7.28 (phenyl ring protons, 5H, m), 6.76
(Ile(1) NH, 1H, d, J¼7.5 Hz), 4.40e4.42 (CaH of Phe(3),
1H, m), 3.73e3.78 (CaH of Ile(1), 1H, m), 2.89e3.06 (CbHs
of Phe(3), 2H, m), 1.66e1.68 (CbHs of Ile(1), 1H, m), 1.37
(BoceCH3s, 9H, s), 1.28 (CbHs of Aib(2), 6H, s), 1.05e
1.10 (CgHs of Ile(1), 2H, m), 0.80e0.88 (CgHs and CdHs of
Ile(1), 6H, m). Anal. Calcd for C24H37N3O6 (463.56): C,
62.17; H, 8.04; N, 9.06. Found: C, 62.02; H, 7.89; N, 9.24.
4. Experimental
4.1. Synthesis of peptides
Peptides IeIII were synthesized by conventional solution
phase procedures using a racemization free, fragment condensa-
tion strategy (Scheme 1).19 The tert-butyloxycarbonyl and
methyl ester groups were used for amino and carboxyl protec-
tions, respectively, and dicyclohexylcarbodiimide (DCC) and
1-hydroxybenzotriazole (HOBT) as coupling agents. Deprotec-
tions were performed using trifluoroacetic acid or saponifica-
tion, respectively. Methyl ester hydrochlorides of Aib, Leu,
and Phe were prepared by the thionyl chlorideemethanol
procedure. All the intermediates obtained were checked for pu-
rity by thin layer chromatography (TLC) on silica gel and used
without further purification. All the final peptides were purified
by column chromatography using silica gel (100e200 mesh) as
the stationary phase and ethyl acetate and petroleum ether mix-
ture as the eluent. The reported peptides IeIII were fully char-
acterized by X-ray crystallography and NMR studies.
4.1.3. BoceIleeAibeLeueIleeAibeLeueOMe (peptide I)
To BoceIleeAibeLeueOMe20 (0.52 g, 1.17 mmol), tri-
fluoroacetic acid (5 ml) was added at 0 ꢁC and stirred at
room temperature. The removal of the Boc group was moni-
tored by TLC. After 8 h, the trifluoroacetic acid was removed
under reduced pressure to afford the crude trifluoroacetate salt.
The residue was taken up in water and washed with diethy-
lether. The pH of the aqueous solution was adjusted to 8
with sodium bicarbonate and extracted with ethyl acetate.
The extracts were pooled, washed with saturated brine, dried
over sodium sulfate, and concentrated to a highly viscous liq-
uid that gave a positive ninhydrin test. This tripeptide free base
was added to a well ice-cooled solution of 1 (0.50 g,
1.17 mmol) in DMF (4 ml) followed by DCC (0.26 g,
1.28 mmol) and HOBt (0.17 g, 1.28 mmol). The reaction mix-
ture was stirred at room temperature for 4 days. The residue
was taken up in ethyl acetate and dicyclohexylurea (DCU)
was filtered off. The organic layer was washed with 2 M
HCl (3ꢃ50 ml), 1 M Na2CO3 solution (3ꢃ50 ml), and brine,
dried over anhydrous Na2SO4, and evaporated in vacuo to
yield a white solid. Purification was done using silica gel as
stationary phase and ethyl acetateepetroleum ether mixture
as the eluent. Single crystals were grown from acetoneepetro-
leum ether mixture by slow evaporation and were stable at
room temperature.
4.1.1. BoceIleeAibeLeueOH 1
BoceIleeAibeLeueOMe20 (1.1 g, 2.48 mmol) was dis-
solved in methanol (15 ml) and 2 M NaOH (10 ml) was added.
The reaction mixture was stirred at room temperature for 2
days. The progress of the reaction was monitored by TLC.
After completion of the reaction, methanol was evaporated.
The residue obtained was diluted with water and washed
with diethylether. The aqueous layer was cooled in ice and
neutralized by 2 M HCl and extracted with ethyl acetate.
The solvent was evaporated in vacuo to give a white solid.
Yield: 0.96 g (90.0%). Mp¼118e120 ꢁC; IR (KBr): 3419,
3315, 1667, 1523 cmꢀ1 1H NMR (300 MHz, DMSO-d6,
;
d ppm): 7.90 (Aib(2) NH, 1H, s), 7.37 (Leu(3) NH, 1H, d,
J¼7.8 Hz), 6.76 (Ile(1) NH, 1H, d, J¼7.5 Hz), 4.22e4.24
(CaH of Leu(3), 1H, m), 3.73e3.77 (CaH of Ile(1), 1H, m),
1.55e1.80 (CbHs of Ile(1), Leu(3); CgHs of Leu(3); 4H, m),
1.38 (BoceCH3s, 9H, s), 1.35 (CbHs of Aib, 6H, s), 1.06e
1.11 (CgHs of Ile(1), 2H, m), 0.85e0.87 (CgHs of Ile(1),
CdHs of Ile(1), Leu(3), 12H, m). Anal. Calcd for
Yield: 0.73 g (83.0%). Mp¼178e180 ꢁC; [a]52809 ꢀ8.1 (c
0.10 g/100 ml, CH3OH); IR (KBr): 3421, 3337, 1738,
1668 cmꢀ1
;
1H NMR (500 MHz, CDCl3, d ppm): 7.45
(Leu(3) NH, 1H, d, J¼8.0 Hz), 7.20 (Ile(4) NH, 1H, d,
J¼6.0 Hz), 7.12 (Leu(6) NH, 1H, d, J¼8.5 Hz), 6.92 (Aib(5)
NH, 1H, s), 6.69 (Aib(2) NH, 1H, s), 5.22 (Ile(1) NH, 1H,
d, J¼2.5 Hz), 4.54e4.58 (CaH of Leu(6), 1H, m), 4.29e

556
A. Dutt et al. / Tetrahedron 64 (2008) 549e558
4.32 (CaH of Ile(4), 1H, m), 4.18e4.21 (CaH of Leu(3), 1H,
m), 3.73e3.75 (CaH of Ile(1), 1H, m), 3.68 (eOCH3, 3H,
s), 1.59e2.15 (CbHs of Ile(1), Leu(3), Ile(4), and Leu(6),
6H, m), 1.56 (CbHs of Aib(5), 6H, s), 1.53 (CbHs of Aib(2),
6H, s), 1.48 (BoceCH3s, 9H, s), 1.25e1.30 (CgHs of Ile(1),
Leu(3), Ile(4), and Leu(6), 6H, m), 0.87e1.0 (CgHs of Ile(1)
and Ile(4); CdHs of Ile(1), Ile(4), Leu(3), and Leu(6), 24H,
m); 13C NMR (75 MHz, CDCl3, d ppm): 174.86, 173.57,
173.31, 171.69, 171.03, 157.12, 81.32, 61.20, 59.29, 57.23,
56.69, 54.02, 51.87, 50.91, 40.99, 40.08, 36.14, 35.69,
30.87, 28.13, 27.83, 25.74, 25.47, 25.17, 24.95, 24.58,
23.28, 23.15, 22.97, 20.71, 15.78, 15.46, 11.70, 11.42. Anal.
Calcd for C38H70N6O9 (754.98): C, 60.45; H, 9.34; N,
11.13. Found: C, 60.31; H, 9.23; N, 11.01.
at room temperature for 8 h. The work up of the free base was
done as in the preparation of peptide I. The tripeptide free base
was added to a well ice-cooled solution of 2 (0.55 g,
1.19 mmol) in DMF (4 ml) followed by DCC (0.27 g,
1.31 mmol) and HOBt (0.16 g, 1.31 mmol). After 4 days, the
reaction mixture was worked up as usual to afford the peptide
as a white solid. Purification was done using silica gel as
stationary phase and ethyl acetateepetroleum ether mixture
as the eluent. Single crystals were grown from acetoneepetro-
leum ether mixture by slow evaporation and were stable at
room temperature.
Yield: 0.67 g (81.0%). Mp¼192e194 ꢁC; [a]52809 ꢀ16.6
(c 0.10 g/100 ml, CH3OH); IR (KBr): 3421, 3366, 1738,
1671 cmꢀ1
;
1H NMR (300 MHz, CDCl3, d ppm): 7.48
(Phe(3) NH, 1H, d, J¼8.4 Hz), 7.20e7.31 (Ile(4) NH, phenyl
ring protons 6H, m), 7.07 (Leu(6) NH, 1H, d, J¼8.1 Hz), 6.88
(Aib(5) NH, 1H, s), 6.57 (Aib(2) NH, 1H, s), 5.14 (Ile(1) NH,
1H, d, J¼3.9 Hz), 4.53e4.66 (CaH of Phe(6), 1H, m), 4.43e
4.46 (CaHs of Ile(4), 1H, m), 3.71e3.73 (CaH of Leu(3), 1H,
m), 3.67 (eOCH3, 3H, s), 3.45e3.52 (CaHs of Ile(1), 1H, m),
3.0e3.1 (CbHs of Phe(3), 2H, m), 1.60e1.90 (CbHs of Ile(1),
Ile(4), and Leu(6), 4H, m), 1.55 (CbHs of Aib(5), 6H, s), 1.48
(CbHs of Aib(2), 6H, s), 1.41 (BoceCH3s, 9H, s), 1.27e1.29
(CgHs of Ile(1), Ile(4) and Leu(6), 5H, m), 0.87e0.97 (CgHs
of Ile(1), Ile(4); CdHs of Ile(1), Ile(4), and Leu(6), 18H, m);
13C NMR (75 MHz, CDCl3, d ppm): 174.80, 174.65, 173.25,
172.23, 172.21, 170.84, 157.00, 137.13, 128.47, 128.19,
126.64, 81.03, 60.87, 59.31, 57.151, 56.61, 55.48, 51.83,
50.81, 41.08, 36.53, 35.81, 28.06, 27.11, 25.42, 25.28,
24.58, 24.51, 23.10, 22.83, 21.66, 15.76, 15.41, 11.56,
11.20. Anal. Calcd for C41H68N6O9 (789.00): C, 62.41; H,
8.68; N, 10.65. Found: C, 62.30; H, 8.65; N, 10.55.
4.1.4. BoceIleeAibeLeueIleeAibePheeOMe
(peptide II)
Peptide BoceIleeAibePheeOMe20 (0.5 g, 1.05 mmol)
was dissolved in trifluoroacetic acid (5 ml) at 0 ꢁC and stirred
at room temperature for 8 h. The work up of the free base was
done as in the preparation of peptide I. The tripeptide free base
was added to a well ice-cooled solution of 1 (0.45 g,
1.05 mmol) in DMF (4 ml) followed by DCC (0.24 g,
1.15 mmol) and HOBt (0.16 g, 1.15 mmol). After 4 days, the
reaction mixture was worked up as reported in the case of
peptide I to afford the peptide as a white solid. Purification
was done using silica gel as stationary phase and ethyl
acetateepetroleum ether mixture as the eluent. Single crystals
were grown from acetoneepetroleum ether mixture by slow
evaporation and were stable at room temperature.
Yield: 0.71 g (86.0%). Mp¼186e188 ꢁC; [a]52809 ꢀ16.4 (c
0.10 g/100 ml, CH3OH); IR (KBr): 3421, 3366, 1738,
1671 cmꢀ1
;
1H NMR (300 MHz, CDCl3, d ppm): 7.48
(Leu(3) NH, 1H, d, J¼7.5 Hz), 7.38 (Ile(4) NH, 1H, d,
J¼8.1 Hz), 7.17e7.26 (Phe(6) NH, phenyl ring protons 6H,
m), 6.96 (Aib(5) NH, 1H, s), 6.69 (Aib(2) NH, 1H, s), 5.22
(Ile(1) NH, 1H, br s), 4.72e4.75 (CaH of Phe(6), 1H, m),
4.25e4.29 (CaH of Ile(4), 1H, m), 3.75e3.76 (CaH of
Leu(3), 1H, m), 3.70 (eOCH3, 3H, s), 3.64e3.66 (CaH of
Ile(1), 1H, m), 3.12e3.13 (CbHs of Phe(6), 2H, m), 1.60e
1.88 (CbHs of Ile(1), Ile(4), and Leu(3), 4H, m), 1.54 (CbHs
of Aib(5), 6H, s), 1.53 (CbHs of Aib(2), 6H, s), 1.49 (Boce
CH3s, 9H, s), 1.24e1.33 (CgHs of Ile(1), Leu(3), Ile(4), 5H,
m), 0.85e0.97 (CgHs of Ile(1) and Ile(4); CdHs of Ile(1),
Ile(4), and Leu(3), 18H, m); 13C NMR (75 MHz, CDCl3,
d ppm): 174.69, 174.63, 173.53, 172.09, 171.38, 171.09,
157.05, 137.13, 129.34, 128.20, 126.49, 81.41, 60.98, 59.37,
57.21, 56.68, 54.03, 51.95, 40.11, 38.00, 36.30, 35.78,
28.12, 27.81, 26.07, 25.60, 24.97, 24.93, 24.61, 23.35,
23.13, 20.77, 15.74, 15.47, 11.65, 11.46. Anal. Calcd for
C41H68N6O9 (789.00): C, 62.41; H, 8.68; N, 10.65. Found:
C, 62.26; H, 8.52; N, 10.67.
4.2. FTIR spectroscopy
IR spectra were examined using a Perkin Elmer-782 model
spectrophotometer. The solid-state FTIR measurements were
performed using the KBr disk technique.
4.3. NMR experiments
1
All H and 13C NMR studies were recorded on a Bruker
Avance 300 model spectrometer operating at 300 and
75 MHz, respectively. The 2D experiment was carried out in
CDCl3 on a Bruker DRX 500 MHz equipped with a 5 mm
broadband inverse probe head. The peptide concentrations
were in the range 5e10 mM in CDCl3 for 1H NMR and
30e40 mM in CDCl3 for 13C NMR.
4.4. Circular dichroism spectroscopy
Solutions of peptides IeIII in acetonitrile (1.5 mM as final
concentration) were used for obtaining the spectra. Far-UV
CD measurements were recorded at 25 ꢁC with a 0.5 s averag-
ing time, a scan speed of 50 nm/min, using a JASCO spectro-
polarimeter (J 720 model) equipped with a 0.1 cm path length
cuvette. The measurements were taken at 0.2 nm wavelength
4.1.5. BoceIleeAibePheeIleeAibeLeueOMe
(peptide III)
Peptide BoceIleeAibeLeueOMe (0.53 g, 1.19 mmol)
was dissolved in trifluoroacetic acid (5 ml) at 0 ꢁC and stirred

A. Dutt et al. / Tetrahedron 64 (2008) 549e558
557
Table 4
Crystallographic refinement details for peptides IeIII
No. 32-190/2006 (SR)]. We thank EPSRC and the University
of Reading, UK for funds for Oxford Diffraction X-Calibur
CCD diffractometer. The use of NMR facility at Bose Insti-
tute, Kolkata is acknowledged.
Peptide I
Peptide II
Peptide III
Formula
MW
C38H70N6O9
755.00
Monoclinic
C41H68N6O9
789.00
Monoclinic
C41H68N6O9
789.00
Trigonal
Crystal system
Cell dimensions [A ( )]
ꢁ
˚
Supplementary data
a
b
c
9.7556(13)
19.023(5)
11.8045(19)
9.9545(7)
18.5960(16)
11.9741(9)
11.5100(5)
11.5100(5)
28.5496(9)
Supplementary data associated with this article can be
found in the online version, at doi:10.1016/j.tet.2007.11.016.
a
b
g
(90)
91.258(12)
(90)
(90)
91.811(8)
(90)
(90)
(90)
(120)
References and notes
3
˚
U/A
2190.1(7)
1.145
2215.5(3)
1.180
3275.5(2)
1.200
7631
Dcalcd (g cmꢀ3
)
1. (a) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747e6756; (b)
Toniolo, C.; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350e353; (c)
Marshall, G. R.; Hodgkin, E. E.; Langs, D. A.; Smith, G. D.; Zabrocki,
J.; Leplawy, M. T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 487e491; (d)
Karle, I. L.; Flippen-Anderson, J. L.; Uma, K.; Balaram, P. Curr. Sci.
1990, 59, 875e885; (e) Venkatraman, J.; Shankaramma, S. C.; Balaram,
P. Chem. Rev. 2001, 101, 3131e3152; (f) Shamala, N.; Nagaraj, R.;
Balaram, P. Biochem. Biophys. Res. Commun. 1977, 79, 291e297; (g)
Nagaraj, R.; Shamala, N.; Balaram, P. J. Am. Chem. Soc. 1979, 101,
16e20; (h) Toniolo, C.; Benedetti, E. ISI Atlas Sci.: Biochem. 1988, 1,
225e230; (i) Kaul, R.; Balaram, P. Bioorg. Med. Chem. 1999, 7, 105e
117; (j) Aravinda, S.; Shamala, N.; Das, C.; Sriranjini, A.; Karle, I. L.;
Balaram, P. J. Am. Chem. Soc. 2003, 125, 5308e5315; (k) Karle, I. L.;
Flippen-Anderson, J. L.; Uma, K.; Balaram, H.; Balaram, P. Proc. Natl.
Acad. Sci. U.S.A. 1989, 86, 765e769.
Unique reflections
Reflections>2s(I )
R1 (all data)
11,250
3072
11,008
4046
4503
0.2203
0.1464
0.0689
0.1203
0.191,
ꢀ0.206
0.1304
0.1663
0.0696
0.1475
0.339,
ꢀ0.329
0.1118
0.1901
0.0634
0.1558
0.625,
ꢀ0.365
wR2 (all data)
R1 (I>2s(I ))
wR2 (I>2s(I ))
Max, Min elecꢀ. 3
˚
density (e A
)
intervals, 2.0 nm spectral bandwidth, and five sequential scans
were recorded for each sample.
4.5. Field emission scanning electron microscopic study
2. (a) Andreeto, E.; Peggion, C.; Crisma, M.; Toniolo, C. Biopolymers 2006,
84, 490e501; (b) Aubry, A.; Bayeul, D.; Bruckner, H.; Schiemann, N.;
Benedetti, E. J. Pept. Sci. 1998, 4, 502e510; (c) Crisma, M.; Andreetto,
E.; Zotti, M. D.; Moretto, A.; Peggion, C.; Formaggio, F.; Toniolo, C.
J. Pept. Sci. 2007, 13, 190e205; (d) Kokkinidis, M.; Tsernoglou, D.;
Bruckner, H. Biochem. Biophys. Res. Commun. 1986, 136, 870e875.
3. (a) Karle, I. L.; Flippen-Anderson, J. L.; Uma, K.; Balaram, P. Biopoly-
mers 1989, 28, 773e781; (b) Kokkinidis, M.; Banner, D. W.; Tsernoglou,
D.; Bruechner, H. Biochem. Biophys. Res. Commun. 1986, 139, 590e595;
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Uma, M. V.; Shamala, N.; Balaram, P. Biopolymers 1999, 50, 13e22.
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119, 9246e9251.
Morphology of peptide II was investigated using field
emission scanning electron microscope (FE-SEM). For the
study, fibrous materials (slowly grown from acetone) were
dried and gold coated. The micrograph was taken using
a FE-SEM apparatus (JEOL JSM-6700F).
4.6. Single crystal X-ray diffraction study
Crystal data and refinement details are given in Table 4.
Diffraction data for the three peptides IeIII were obtained
with Mo Ka radiation at 150 K using the Oxford Diffraction
X-Calibur CCD System. The crystals were positioned at
50 mm from the CCD; 321 frames were measured with
a counting time of 10 s. Data analyses were carried out with
the Crysalis program.21 The structures were solved using di-
rect methods with the SHELXS-97 program.22 The non-hydro-
gen atoms were refined with anisotropic thermal parameters.
The hydrogen atoms bonded to carbon were included in geo-
metric positions and given thermal parameters equivalent to
1.2 times those of the atom to which they were attached. Crys-
tallographic details have been deposited at the Cambridge
Crystallographic Data centre, reference CCDC 651726e
651728.
5. (a) Aleman, C.; Subirana, J. A.; Perez, J. J. Biopolymers 1994, 32, 621e
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150e160; (f) Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini,
A.; Bavoso, A.; Toniolo, C.; Crisma, M.; Sartore, L. J. Chem. Soc., Perkin
Trans. 2 1990, 1829e1837; (g) Di Blasio, B.; Pavone, V.; Saviano, R.;
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Res. 1994, 7, 55e59.
Acknowledgements
A.D. would like to thank UGC, New Delhi, India for pro-
viding a senior research fellowship. The financial assistance
of UGC, New Delhi is acknowledged [Major Research Project

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A.; Maji, S. K.; Drew, M. G. B.; Haldar, D.; Banerjee, A. Tetrahedron
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dar, D.; Drew, M. G. B.; Banerjee, A. Tetrahedron 2006, 62, 6370e6378;
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