Studies of β-turn opening with model peptides containing non-coded α-amino isobutyric acid

Article abstract of DOI:10.1016/j.tet.2007.07.075

Single crystal X-ray diffraction studies show that among the three terminally protected model tripeptides I-III, Boc-Ile-Aib-Xx-OMe (Xx in peptide I: Val; II: Leu; III: Phe) with a centrally placed non-coded amino acid Aib (Aib: α-amino isobutyric acid), peptide I displays a conformational preference for β-turn, peptide II forms a hydrated β-turn representing the solvent mediated intermediate for the interconversion between β-turn and β-strand and peptide III adopts a completely unfolded β-strand like structure. By varying the steric bulk of the third residue, Xx(3), various conformations related to the structural interconversion between the β-turn and β-strand have been isolated. The peptide conformations in the solution phase have been probed by solvent dependent NMR titration and CD spectroscopy. Morphological studies with scanning electron microscopy (SEM) reveal that among the three peptides only peptide III can form filamentous fibrils in the solid state.

Full text of DOI:10.1016/j.tet.2007.07.075

Tetrahedron 63 (2007) 10282–10289
Studies of b-turn opening with model peptides containing
non-coded a-amino isobutyric acid
Anita Dutt,a Arpita Dutta,a Raju Mondal,a Elinor C. Spencer,b Judith A. K. Howardb
and Animesh Pramanika,
*
aDepartment of Chemistry, University of Calcutta, 92 A. P. C. Road, Kolkata 700 009, India
bDepartment of Chemistry, University of Durham, South Road, Durham DH1 3LE, UK
Received 22 March 2007; revised 10 July 2007; accepted 20 July 2007
Available online 29 July 2007
Abstract—Single crystal X-ray diffraction studies show that among the three terminally protected model tripeptides I–III, Boc-Ile-Aib-Xx–
OMe (Xx in peptide I: Val; II: Leu; III: Phe) with a centrally placed non-coded amino acid Aib (Aib: a-amino isobutyric acid), peptide I
displays a conformational preference for b-turn, peptide II forms a hydrated b-turn representing the solvent mediated intermediate for the
interconversion between b-turn and b-strand and peptide III adopts a completely unfolded b-strand like structure. By varying the steric
bulk of the third residue, Xx(3), various conformations related to the structural interconversion between the b-turn and b-strand have been
isolated. The peptide conformations in the solution phase have been probed by solvent dependent NMR titration and CD spectroscopy.
Morphological studies with scanning electron microscopy (SEM) reveal that among the three peptides only peptide III can form filamentous
fibrils in the solid state.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
namely, solvent polarity and steric interactions. This creates
the opportunity to isolate various conformations related
to the structural interconversion between the b-turn and
b-strand. In the present report, three terminally protected
model tripeptides I–III, Boc-Ile-Aib-Xx–OMe (Xx in
peptide I: Val; II: Leu; III: Phe) with a centrally placed
non-coded amino acid Aib have been chosen where the first
two residues Ile(1)–Aib(2) are kept unchanged (Fig. 1). By
varying the steric bulk of the third residue, Xx(3), various
conformations related to the structural interconversion
between the b-turn and b-strand have been isolated. Gener-
ally conformationally restricted Aib is highly helicogenic
and b-sheet breaker. Therefore tripeptides with a centrally
positioned Aib(2) are found to adopt b-turn structure.8 Inter-
estingly the tripeptides I–III demonstrate conformational
heterogeneity. This work has important implications in
designing biologically active peptides.9 All the peptides
were synthesised using conventional solution phase method-
ology and their solid-state structures are determined by
X-ray diffraction analysis.
It is believed that various hydrated reverse turns can be con-
sidered as folding intermediates that are trapped in the fold-
ing–unfolding process between an a-helix and a b-strand.1
The surprisingly high stability of b-turns in several small
linear peptides suggests that turns are to be expected in the
polypeptide chains under folding conditions where they
have a profound influence on protein folding pathways.
The experimental observation of b-turns in linear peptides
does not itself establish a role for these structures in the
initiation of protein folding. Some studies do, however lend
support to the notion that reverse turns play an important
role in protein folding.2 Although there are studies on the
detection and isolation of a-helix,3 hydrated helix,4 reverse
turns,5 hydrated reverse turns6 and b-strand,7 no attempt
has been made to isolate all the conformations related to
a particular structural interconversion to gain a deeper insight
into the mechanistic aspect of the process.
Therefore we are interested in designing small model
peptides that exhibit b-turns, these peptides will have the po-
tential to unfold under the influence of two important factors
2. Results and discussion
The crystal structure of peptide I reveals a type II b-turn
structure with Ile(1) and Aib(2) occupying the i+1 and i+2
positions, stabilised by an intramolecular hydrogen bond
(Fig. 2). The torsion angles at Ile(1) and Aib(2) were found
Keywords: b-Turn; Hydrated turn; b-Strand; a-Aminoisobutyric acid;
Filamentous fibrils.
* Corresponding author. Tel.: +91 33 2484 1647; fax: +91 33 2351 9755;
e-mail: animesh_in2001@yahoo.co.in
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.07.075

A. Dutt et al. / Tetrahedron 63 (2007) 10282–10289
10283
Table 2. Selected intra and intermolecular hydrogen bonding parameters
O
R
O
ψ3
φ1
ψ1
H
N
φ3
˚
˚
D–H/A
H/A/A
D/A/A
D–H/A/ꢁ
OMe
N
O
N
H
φ2
ψ2
H
Peptide I
Peptide II
N3–H3N/O2
N3–H3N/O1O
O1O–H1O/O2
O1O–H1O/O5i
N1–H1/O1Oii
N1–H1N/O3iii
N2–H2N/O4iv
2.71(2)
2.22(2)
1.92(2)
1.96(2)
2.19(2)
2.09(2)
2.15(2)
3.499(2)
3.032(2)
2.759(2)
2.855(2)
3.002(2)
2.959(2)
2.943(2)
152(2)
176(2)
175(2)
173(2)
176(1)
O
O
Peptide I : R = -CH(CH3)2
Peptide II : R = -CH2CH(CH3)2
Peptide III : R = -CH2Ph
Peptide III
174(2)
164.5(19)
Figure 1. Schematic representation of peptides I–III.
Symmetry codes: (i) 1+y, 1ꢀx, 0.25+z; (ii) 1ꢀy, x, zꢀ0.25; (iii) 1+x, y, z;
(iv) 1+x, y, z.
titrated against polar (CD3)2SO. The changes in the chemi-
cal shifts are presented in Figure 3. The assignment of NH
resonances was quite straightforward. The Ile(1)–NH
appears as doublet in the upfield region (d¼4.99 ppm in
CDCl3) among the three NH resonances resulting from the
urethane functional group (Boc-group). The Aib(2)–NH
appears at d¼6.53 ppm, as a singlet due to lack of an a-hy-
drogen atom. The only remaining doublet at d¼7.08 ppm
was assigned to Val(3)–NH at the C-terminal of the peptide.
The solvent titration shows that by increasing the percentage
of (CD3)2SO in CDCl3 from 0 to 12% (v/v) the net changes
in the chemical shift (Dd) values for Ile(1)–NH, Aib(2)–NH
and Val(3)–NH are 0.55, 0.87 and 0.18 ppm, respectively
(Fig. 3). The order of solvent exposure of the NH groups
is Aib(2)–NH>Ile(1)–NH>Val(3)–NH. The Dd values dem-
onstrate that the Val(3)–NH is solvent shielded, and the other
two NH groups are solvent exposed, which is a characteristic
feature of a b-turn in a structure where the Val(3)–NH is
involved in an intramolecular hydrogen bond to Boc–CO.
The CD spectrum of the peptide at 25 ꢁC in methanol reveals
a clear positive ellipticity in the far-UV region with a distinct
maximum at 203 nm, which indicates the presence of a
b-turn structure (Fig. 4).11
Figure 2. Crystal structure of peptide I with atom numbering scheme.
to be f1: ꢀ62.32, j1: 134.96 and f2: 56.19, j2: 34.73, re-
spectively (Table 1), which are slightly deviated from the
ideal values for a type II b-turn f1: ꢀ60, j1: 120 and f2:
80, j2: 0. As a result a weak intramolecular hydrogen
bond between Boc–CO and Val(3)–NH is observed (H/
˚
O, 2.71 A, Table 2). The steric interaction between the
side chains of Ile(1) and Aib(2) is responsible for an unstable
b-turn structure that is of interest to the present investigation.
Although there are several examples of b-turns with cen-
trally placed Aib in tripeptides,8 we report the first use of
Ile at the N-terminal position of the tripeptide.
It has been suggested that hydrated reverse turns may fun-
ction as intermediates in unfolding of a-helices to give
b-strands and b-sheets.12 b-Sheet-driven amyloidogenesis
is responsible for various neurodegenerative diseases.13
Model studies may help to gain more insight into the mech-
anistic aspect of the structural interconversion between a
b-turn and b-strand. Interestingly, the crystal structure of
In order to investigate the existence of intramolecular hydro-
gen bonding and peptide conformation in solution phase the
solvent dependence of the NH chemical shifts was examined
by NMR titration.10 In this experiment a solution of the pep-
tide in nonpolar CDCl3 (10 mM in 0.5 ml) was gradually
8.0
7.5
Val(3)-NH
Table 1. Selected backbone torsion angles of peptides
Torsion angles
Peptide I
Peptide II
Peptide III
7.0
Aib(2)-NH
a
u0
f1
172.81(14)
ꢀ62.32(19)
134.96(14)
170.95(13)
56.19(19)
178.45(12)
ꢀ85.04(15)
126.87(12)
171.42(11)
54.74(16)
ꢀ175.01(15)
ꢀ58.30(2)
ꢀ44.50(2)
166.65(17)
56.90(2)
6.5
6.0
5.5
j1b
u1
f2
j2c
u2
f3
34.73(19)
44.33(16)
50.90(2)
172.24(16)
ꢀ48.3(2)
171.92(13)
ꢀ64.24(18)
143.44(13)
173.18(12)
ꢀ73.96(17)
ꢀ53.52(16)
j3
141.50(19)
Ile(1)-NH
5.0
a
Torsion angle around the amide C–N bond at the N-terminal of the
peptides.
Torsion angle around the peptide C–N bond between Ile(1) and Aib(2) of
the peptides.
Torsion angle around the peptide C–N bond between Aib(2) and Xx(3) of
the peptides.
b
c
0
2
4
% of (CD3)2SO in CDCl3
6
8
10
12
Figure 3. NMR solvent titration curve for NH protons in peptide I.

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A. Dutt et al. / Tetrahedron 63 (2007) 10282–10289
Peptide I
Peptide II
Peptide III
80
60
40
20
0
Figure 6. Solvent assisted b-turn opening of peptide II as observed in
crystalline state.
and Leu(3)–NH are 0.43, 0.63 and 0.28 ppm, respectively.
On the other hand the CD spectrum of peptide II in methanol
displays a positive ellipticity in the far-UV region with
distinct double maxima at 203 and 218 nm, suggesting the
presence of a turn structure (Fig. 4). Thus in an aqueous
methanolic environment the peptide II preferentially re-
mains in the hydrated turn conformation. This finding is of
particular biological relevance as it has been suggested
that hydrated reverse turns promote helix–coil unfolding.1
190
200
210
220
in nm
230
240
250
Figure 4. CD curves of peptides I–III in methanol (1.5 mM).
the peptide II, Boc-Ile-Aib-Leu–OMe, grown from acetone–
water mixture shows a hydrated open b-turn structure
(Fig. 5). A water mediated hydrogen bond is observed be-
tween Boc–CO and Leu(3)–NH with an open b-turn struc-
ture with Ile(1) and Aib(2) occupying the corner positions
(Table 2). The torsion angles within the Ile(1) and Aib(2)
moieties are f1: ꢀ85.04, j1: 126.87 and f2: 54.74, j2:
44.33, respectively (Table 1), which deviate significantly
from the ideal values. These structural changes correspond
to an open structure. The insertion of water molecules helps
in opening the b-turn, which is further influenced by two
neighbouring water molecules that are hydrogen bonded to
Ile(1)–NH and Leu(3)–CO from that are located on the
external side of the turn region (Fig. 6, Table 2). The inter-
conversion between the b-turn and b-strand requires an aque-
ous environment for the formation of the hydrated b-turn
intermediate.
Generally, short aromatic peptides are found to have the
ability to form b-sheet mediated amyloid fibrils.14 Therefore
we have chosen peptide III, Boc-Ile-Aib-Phe–OMe, which
incorporates a Phe(3) residue, to determine if this peptide
forms a b-turn or b-strand structure. The peptide is found
to adopt a b-strand structure in the solid state (Fig. 8). The
torsion angles within Ile(1) are f1: ꢀ58.3 and j1: ꢀ44.5,
within Aib(2) they are f2: 56.9 and j2: 50.9, and within
Phe(3) they are f3: ꢀ48.3 and j3: 141.5, indicating an
open structure with three ‘kinks’ along the peptide backbone
(Table 1). In the solid state the b-strand structure of peptide
III self-assembles to supramolecular b-sheet through inter-
molecular hydrogen bonding along the crystallographic
a axis (Fig. 9). There are two intermolecular hydrogen bonds
N1–H1/O3 and N2–H2/O4 connecting individual peptide
molecules to form a sheet like structure (Table 2). The C]O
groups of Ile(1) and Aib(2) of one molecule are inter-linked
with NH groups of Ile(1) and Aib(2) of another neighbouring
molecule, respectively, through intermolecular hydrogen
bonding. This type of supramolecular b-sheet mediated
self-assembly of small peptide may create the possibility of
The solvent dependence of the NH chemical shifts that was
demonstrated in CDCl3–(CD3)2SO titration experiment
indicates that all three –NH groups of peptide II are solvent
exposed indicating an open structure (Fig. 7). The net change
in the chemical shift (Dd) values for Ile(1)–NH, Aib(2)–NH
8.0
7.5
Leu(3)-NH
7.0
Aib(2)-NH
6.5
6.0
5.5
5.0
Ile(1)-NH
8
0
2
4
6
10
12
% of (CD3)2SO in CDCl3
Figure 5. Crystal structure of peptide II with atom numbering scheme.
Figure 7. NMR solvent titration curve for NH protons in peptide II.

A. Dutt et al. / Tetrahedron 63 (2007) 10282–10289
10285
dried fibrous materials of peptide III grown slowly from ace-
tone clearly demonstrate that the aggregates in the solid state
are bunches of filamentous fibrils, resembling the amyloid
fibrils (Fig. 10). It is noteworthy that the other two peptides
I and II, which adopt the turn structure, do not produce any
such fibrous material under similar conditions.
The NMR CDCl3–(CD3)2SO titration experiment implies
that all three NH groups of peptide III are solvent exposed
(Fig. 11), the Dd values for Ile(1)–NH, Aib(2)–NH and
Phe(3)–NH are 0.37, 0.56 and 0.38 ppm, respectively, indi-
cating a b-strand like conformation. Interestingly, a positive
ellipticity with two maxima at 205 and 220 nm is observed in
the CD spectra of the peptide in methanol (Fig. 4), indicating
a significant amount of turn structure. Actually in an aqueous
environment peptide III exists as a mixture of hydrated turn
and b-strand conformation. The gradual opening of b-turn
structure from peptide I to III is quite evident from the
changes in the pattern of CD spectra (Fig. 4). The increasing
flattening of the CD curves indicates greater population of
the open structure.
Figure 8. Crystal structure of peptide III with atom numbering scheme.
8.0
7.5
Phe(3)-NH
7.0
Aib(2)-NH
6.5
6.0
5.5
Ile(1)-NH
5.0
0
2
4
% of (CD3)2SO in CDCl3
6
8
10
12
Figure 11. NMR solvent titration curve for NH protons in peptide III.
Figure 9. Packing diagram of peptide III along the crystallographic a direc-
tion showing intermolecular hydrogen bonding in the b-sheet structure (side
chains and hydrogen atoms are omitted for clarity).
3. Conclusions
A comprehensive understanding of the molecular basis of
b-turn formation and opening in small tripeptides is essential
for designing small bioactive peptides. By selecting three
fibrillogenesis in the solid state. Interestingly the field emis-
sion scanning electron microscopic (FESEM) images of the
Figure 10. FESEM of peptide III (a) showing the formation of filamentous fibrils in the solid state; (b) the same at the higher resolution.

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A. Dutt et al. / Tetrahedron 63 (2007) 10282–10289
model peptides in which the first two residues are kept
unchanged, and where the only perturbation introduced
into the structure was variation in the steric bulk of the third
residue, we are able to open up the b-turn structure. As a re-
sult, it was possible for various conformations related to the
structural interconversion between b-turn and b-strand to be
isolated. The crystal structure of the hydrated b-turn in
peptide II provides an insight into the mechanism of inter-
conversion between b-turns and b-strands, which could be
a way of modulating b-sheet mediated amyloidogenesis.
Generally, in nature, mutation within the protein causes
misfolding by disrupting the local turn conformations.15
The formation of b-strand like structure in peptide III where
the third residue is a bulky Phe(3) group, suggests that steric
factor plays an important role in regulating the transition
between b-turn and b-strand structures. Therefore the results
establish that even tripeptides containing a centrally posi-
tioned Aib(2) can display structural diversities in the back-
bone depending upon the co-operative steric interactions
amongst the amino acid residues.
hydrochloride was added, followed by DCC (5.17 g,
25.10 mmol). The reaction mixture was stirred at room
temperature for 3 days. The precipitated dicyclohexylurea
(DCU) was filtered and diluted with ethyl acetate. The
organic layer was washed with excess of water, 1 M HCl
(3ꢂ50 ml), 1 M Na2CO3 solution (3ꢂ50 ml) and again
with water. The solvent was then dried over anhydrous
Na2SO4 and evaporated in vacuo, giving a light yellow solid.
Yield: 7.45 g (90.0%). IR (KBr): 3307, 1656 cmꢀ1; 1H NMR
300 MHz (CDCl3, d ppm): 6.62 (Aib NH, 1H, s); 5.07 (Ile
NH, 1H, d, J¼7.2 Hz); 3.84–3.89 (CaH of Ile, 1H, m);
3.68 (OCH3, 3H, s); 1.91 (CbH of Ile, 1H, m); 1.49 (CbHs
of Aib, 6H, s); 1.45 (Boc–CH3s, 9H, s); 1.21–1.23 (CgHs
of Ile, 3H, m); 0.91–0.97 (CgHs and CdHs of Ile, 5H, m).
Anal. Calcd for C16H30N2O5 (330.42): C, 58.15; H, 9.15;
N, 8.48. Found: C, 58.22; H, 9.20; N, 8.39.
4.1.3. Boc-Ile-Aib–OH (2). Compound
1
(6.0 g,
18.18 mmol) was dissolved 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 the
methanol was evaporated. The residue obtained was diluted
with water and washed with diethylether. The aqueous layer
was cooled in ice and neutralised by 2 M HCl and extracted
with ethyl acetate. The solvent was evaporated in vacuo to
give a light yellow waxy solid.
4. Experimental
4.1. Synthesis of peptides
The peptides I–III were synthesised by conventional solu-
tion phase procedures.16 The t-butyloxycarbonyl and methyl
ester groups were used for amino and carboxyl protections,
respectively, and dicyclohexylcarbodiimide (DCC), 1-hy-
droxybenzotriazole (HOBT) as coupling agents. Methyl
ester hydrochlorides of Aib, Val, Leu and Phe were prepared
by the thionyl chloride–methanol procedure. All the inter-
mediates obtained were checked for purity 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 (100–200 mesh)
as the stationary phase and ethyl acetate and petroleum ether
mixture as the eluent. The reported peptides I–III were fully
characterised by X-ray crystallography, NMR and mass
spectrometry.
Yield: 4.57 g (80.0%). IR (KBr): 3314, 1666 cmꢀ1; 1H NMR
300 MHz (CDCl3, d ppm): 7.19 (Aib NH, 1H, s); 5.57 (Ile
NH, 1H, br s); 4.31 (CaH of Ile, 1H, m); 1.60–1.80 (CbH
of Ile, 1H, m); 1.57 (CbHs of Aib, 6H, s); 1.45 (Boc–
CH3s, 9H, s); 1.21–1.40 (CgHs of Ile, 3H, m); 0.92–0.98
(CgHs and CdHs of Ile, 5H, m). Anal. Calcd for
C15H28N2O5 (316.39): C, 56.93; H, 8.92; N, 8.86. Found:
C, 56.80; H, 8.84; N, 8.75.
4.1.4. Boc-Ile-Aib-Val–OMe (peptide I). Compound 2
(1.0 g, 3.16 mmol) was dissolved in DMF (3 ml). Val–OMe
(0.42 g, 3.16 mmol) obtained from its hydrochloride was
added followed by DCC (0.65 g, 3.16 mmol). The reaction
mixture was stirred at room temperature for 5 days. The
precipitateddicyclohexylurea (DCU) was filteredand diluted
with ethyl acetate. The organic layer was washed with excess
of water, 1 M HCl (3ꢂ30 ml), 1 M Na2CO3 solution
(3ꢂ30 ml) and again with water. The solvent was then dried
over anhydrous Na2SO4 and evaporated in vacuo, giving a
light yellow solid. Purification was done using silica gel as
stationary phase and ethyl acetate–petroleum ether mixture
as the eluent. Single crystals were grown from acetone–water
mixture (90:10) by slow evaporation and were stable at room
temperature.
4.1.1. Boc-Ile–OH. The amino acid isoleucine (5 g,
38.16 mmol) was suspended in a 1:1 dioxane–water mixture
and Boc–N3 (5.92 ml, 38.16 mmol) was added to it. The mix-
ture was stirred at room temperature maintaining the pH in
the alkaline range with 4 M NaOH. After 24 h, the solution
was diluted and extracted with ether. The aqueous layer
was cooled in an icebath, acidified with 2 M HCl and
extracted with ethyl acetate. The organic layer was washed
with excess of water and dried over anhydrous Na2SO4 and
evaporated in vacuo producing a white solid.
1
Yield: 8.37 g (95%); H NMR 300 MHz (CDCl3, d ppm):
5.17 (Ile NH, 1H, d, J¼8.4 Hz); 4.27–4.31 (CaH of Ile, 1H,
m); 1.91 (CbH of Ile, 1H, m); 1.45 (Boc–CH3s, 9H, s);
1.21–1.23 (CgHs of Ile, 3H, m); 0.91–0.97 (CgHs and CdHs
of Ile, 5H, m). Anal. Calcd for C11H21N1O4 (231.28): C,
57.12; H, 9.15; N, 6.05. Found: C, 57.25; H, 9.21; N, 6.14.
Yield: 1.22 g (90.0%). Mp¼109–110 ꢁC; IR (KBr): 3423,
1
3367, 3301, 1732, 1703, 1658 cmꢀ1; H NMR 300 MHz
(CDCl3, d ppm): 7.09 (Val NH, 1H, d, J¼8.4 Hz); 6.58
(Aib NH, 1H, s); 5.03 (Ile NH, 1H, d, J¼7.8 Hz); 4.48–
4.53 (CaH of Val, 1H, m); 3.90–3.91 (CaH of Ile, 1H, m);
3.73 (–OCH3, 3H, s); 2.12–2.24 (CbH of Val, 2H, m);
1.87–1.92 (CbH of Ile, 1H, m); 1.56 (CbHs of Aib, 6H, s);
1.45 (Boc–CH3s, 9H, s); 1.07–1.21 (CgHs of Ile, 5H,
m); 0.90–0.96 (CgHs of Val and CdHs of Ile, 9H, m);
4.1.2. Boc-Ile-Aib–OMe (1). Boc-Ile–OH (5.8 g,
25.10 mmol) was dissolved in dimethylformamide (DMF;
8 ml). Aib–OMe (2.94 g, 25.10 mmol) obtained from its

A. Dutt et al. / Tetrahedron 63 (2007) 10282–10289
10287
13C NMR 75 MHz (CDCl3, d ppm): 172.99, 172.27, 171.53,
155.88, 80.15, 59.83, 57.60, 57.44, 51.99, 36.94, 31.14,
30.86, 28.26, 25.64, 24.79, 18.95, 17.72, 15.57, 11.35.
Anal. Calcd for C22H41N3O6 (443.57): C, 59.57; H, 9.32;
N, 9.48. Found: C, 59.52; H, 9.28; N, 9.52; HR-MS
(M+Na+)¼451.86, Mcalcd (M+Na)+¼452.54.
13C NMR 75 MHz (CDCl3, d ppm): 173.75, 171.85,
171.18, 155.89,135.96, 129.28, 128.46, 127.02, 80.14,
59.64, 57.27, 53.50, 52.09, 37.87, 36.90, 28.27, 25.08,
24.89, 24.79, 15.52, 11.37. Anal. Calcd for C25H39N3O6
(477.56): C, 62.86; H, 8.23; N, 8.80. Found: C, 62.83; H,
9.19; N, 8.75; HR-MS (M+Na+)¼499.99, Mcalcd
(M+Na)+¼500.56.
4.1.5. Boc-Ile-Aib-Leu–OMe (peptide II). Compound 2
(1.0 g, 3.16 mmol) was dissolved in DMF (3 ml). Leu–
OMe (0.46 g, 3.16 mmol) obtained from its hydrochloride
was added followed by DCC (0.65 g, 3.16 mmol). The reac-
tion mixture was stirred at room temperature for 5 days. The
precipitated dicyclohexylurea(DCU) was filtered and diluted
with ethyl acetate. The organic layer was washed with excess
of water, 1 M HCl (3ꢂ30 ml), 1 M Na2CO3 solution
(3ꢂ30 ml) and again with water. The solvent was then dried
over anhydrous Na2SO4 and evaporated in vacuo, giving
a light yellow solid. Purification was done using silica gel
as stationary phase and ethyl acetate–petroleum ether mix-
ture as the eluent. Single crystals were grown from ace-
tone–water mixture (90:10) by slow evaporation and were
stable at room temperature.
4.2. FTIR spectroscopy
IR spectra were examined in Perkin–Elmer-782 model spec-
trophotometer. The solid-state FTIR measurements were
performed using the KBr disk technique.
4.3. NMR experiments
All 1H NMR and 13C NMR studies were recorded on
a Bruker Avance 300 model spectrometer operating at
300, 75 MHz, respectively. The peptide concentrations
1
were in the range 1–10 mM in CDCl3 for H NMR and
30–40 mM in CDCl3 for 13C NMR. Solvent titration exper-
iments were carried out at a concentration of 10 mM in
CDCl3 with gradual addition of d6-DMSO from 0 to 12%
v/v approximately.
Yield: 1.27 g (90.0%). Mp¼123–124 ꢁC; IR (KBr): 3390,
3314, 1715, 1666 cmꢀ1 1H NMR 300 MHz (CDCl3,
;
d ppm): 7.05 (Leu NH, 1H, d, J¼7.8 Hz); 6.61 (Aib NH,
1H, s); 5.05 (Ile NH, 1H, d, J¼7.2 Hz); 4.53–4.60 (CaH of
Leu, 1H, m); 3.87–3.90 (CaH of Ile, 1H, m); 3.72 (–OCH3,
3H, s); 1.87–1.89 (CbHs of Ile and Leu, 3H, m); 1.55
(CbHs of Aib, 6H, s); 1.45 (Boc–CH3s, 9H, s); 1.12–1.16
(CgHs of Ile, 2H, m); 0.92–0.93 (CdHs of Ile and Leu, 9H,
m); 13C NMR 75 MHz (CDCl3, d ppm): 173.93, 173.23,
171.37, 155.88, 80.16, 59.81, 57.40, 52.09, 50.97, 41.28,
36.91, 28.25, 25.40, 24.81, 24.78, 24.75, 22.76, 21.83,
15.50, 11.33. Anal. Calcd for C22H41N3O6 (443.57): C,
59.57; H, 9.32; N, 9.48. Found: C, 59.53; H, 9.29; N, 9.53;
HR-MS (M+Na+)¼466.06, Mcalcd (M+Na)+¼466.57.
4.4. Mass spectrometry
Mass spectra were recorded on a HEWLETT PACKARD
Series 1100MSD and Micromass Qtof Micro YA263 mass
spectrometers by positive mode electrospray ionisation.
4.5. Circular dichroism spectroscopy
Methanolic solution of peptides I–III (1.5 mM as final con-
centration) was used for obtaining the spectra. Far-UV CD
measurements were recorded at 25 ꢁC with 0.5 s averaging
time, a scan speed of 50 nm/min, using a JASCO spectropo-
larimeter (J 720 model) equipped with a 0.1 cm pathlength
cuvette. The measurements were taken at 0.2 nm wavelength
intervals, 2.0 nm spectral bandwidth and five sequential
scans were recorded for each sample.
4.1.6. Boc-Ile-Aib-Phe–OMe (peptide III). Compound 2
(1.0 g, 3.16 mmol) was dissolved in DMF (3 ml). Phe–
OMe (0.57 g, 3.16 mmol) obtained from its hydrochloride
was added followed by DCC (0.66 g, 3.16 mmol). The reac-
tion mixture was stirred at room temperature for 5 days. The
precipitated dicyclohexylurea(DCU) was filtered and diluted
with ethyl acetate. The organic layer was washed with
excess of water, 1 M HCl (3ꢂ30 ml), 1 M Na2CO3 solution
(3ꢂ30 ml), and again with water. The solvent was then dried
over anhydrous Na2SO4 and evaporated in vacuo, giving
a light yellow solid. Purification was done using silica gel
as stationary phase and ethyl acetate–petroleum ether mix-
ture as the eluent. Single crystals were grown from ace-
tone–water mixture (90:10) by slow evaporation and were
stable at room temperature.
4.6. Field emission scanning electron microscopic study
Morphology of peptide III was investigated using field
emission scanning electron microscope (FESEM). For the
study, fibrous materials (slowly grown from acetone) were
dried and gold coated. The micrograph was taken in a
FESEM apparatus (JEOL JSM-6700F).
4.7. Single crystal X-ray diffraction study
All data were collected with graphite monochromated X-ra-
˚
diation (l¼0.71073 A) on a Bruker SMART diffractometer.
Yield: 1.43 g (95.0%). Mp¼114–115 ꢁC; IR (KBr): 3394,
Data processing was performed with standard Bruker soft-
ware.17 Structure solutions were by direct methods. Refine-
ment was on F2 using full-matrix least-squares techniques.
All data were corrected for absorption with SADABS.18
All hydrogen atoms are placed at calculated positions, and
have been refined with a riding model. The exception being
the hydrogen atoms bound to nitrogen atoms, and the hydro-
gen atoms of the water molecule in the model of peptide II,
which were all located in the difference Fourier maps and
3319, 1716, 1664 cmꢀ1 1H NMR 300 MHz (CDCl3,
;
d ppm): 7.13–7.26 (phenyl ring protons, 5H, m); 6.88 (Phe
NH, 1H, d, J¼7.2 Hz); 6.55 (Aib NH, 1H, s); 4.99 (Ile NH,
1H, d, J¼7.2 Hz); 4.80–4.87 (CaH of Phe, 1H, m); 3.84–
3.89 (CaH of Ile, 1H, m); 3.72 (–OCH3, 3H, s); 3.06–3.20
(CbHs of Phe, 2H, m); 1.86–1.90 (CbHs of Ile, 1H, m);
1.52 (CbHs of Aib, 6H, s); 1.46 (Boc–CH3s, 9H, s); 1.05–
1.35 (CgHs of Ile, 5H, m); 0.90–0.94 (CdHs of Ile, 3H, m);

10288
A. Dutt et al. / Tetrahedron 63 (2007) 10282–10289
Table 3. Crystallographic refinement details for peptides I–III
Peptide I Peptide II Peptide III
0.28ꢂ0.20ꢂ0.12 0.51ꢂ0.39ꢂ0.12 0.28ꢂ0.27ꢂ0.03
4. (a) Parthasarathy, R.; Chaturvedi, S.; Go, K. Proc. Natl. Acad.
Sci. U.S.A. 1990, 87, 871–875; (b) Karle, I. L. Biopolymers
1996, 40, 157–180; (c) Ramasubbu, N.; Parthasarathy, R.
Biopolymers 1989, 28, 1259–1269; (d) Guha, S.; Drew,
M. G. B.; Pramanik, A. Tetrahedron Lett. 2006, 47, 7951–
7955.
Crystal size
(mm3)
Crystal
colour
Colourless
Colourless
Colourless
Crystal
habit
Block
Block
Plate
5. (a) Richardson, J. S. Adv. Protein Chem. 1981, 34, 167–339; (b)
Rose, G. D.; Gierasch, L. M.; Smith, J. A. Adv. Protein Chem.
1985, 37, 1–105; (c) Wilmot, C. M.; Thornton, J. M. J. Mol.
Biol. 1988, 203, 221–232; (d) Toniolo, C. CRC Crit. Rev.
Biochem. 1980, 9, 1–44; (e) Gunasekharan, K.; Gomathi, L.;
Ramakrishnan, C.; Chandrasekhar, J.; Balaram, P. J. Mol.
Biol. 1998, 284, 1505–1516.
6. (a) Karle, I. L.; Kaul, R.; Rao, R. B.; Raghothama, S.; Balaram,
P. J. Am. Chem. Soc. 1997, 119, 12048–12054; (b) Motta, A.;
Reches, M.; Pappalardo, L.; Andreotti, G.; Gazit, E.
Biochemistry 2005, 44, 14170–14178.
7. (a) Makin, O. S.; Serpell, L. C. J. Mol. Biol. 2004, 335, 1279–
1288; (b) Bond, J. P.; Deverin, S. P.; Inouye, H.; El-Agnaf,
O. M. A.; Teeter, M. M.; Kirschner, D. A. J. Struct. Biol.
2003, 141, 156–170; (c) Sundae, M.; Serpell, L. C.; Bartlam,
M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. F. J. Mol. Biol.
1997, 273, 729–739; (d) Sundae, M.; Blake, C. Adv. Protein
Chem. 1997, 50, 123–159; (e) Damas, A. M.; Saraiva, M. J.
J. Struct. Biol. 2000, 130, 290–299; (f) Dutt, A.; Drew,
M. G. B.; Pramanik, A. Org. Biomol. Chem. 2005, 3, 2250–
2254; (g) Kundu, S. K.; Mazumdar, P. A.; Das, A. K.;
Bertolasi, V.; Pramanik, A. J. Chem Soc., Perkin Trans. 2
2002, 1602–1604.
Chemical
formula
Formula
weight (g)
Crystal
system
C21H39N3O6
C22H41N3O6$H2O C25H39N3O6
429.55
461.59
477.59
Monoclinic
Tetragonal
Triclinic
Space group
Z
P21
2
6.0564(2)
21.34406(1)
9.8971(2)
90.000
107.339(1)
90.000
1221.24(6)
P43
4
P1
1
˚
A (A)
12.0348(5)
12.0348(5)
18.412(1)
90.000
90.000
90.000
2666.8(2)
120(2)
0.085
6.087(1)
9.968(2)
11.413(2)
92.977(4)
100.634(4)
102.505(4)
661.4(2)
120(2)
˚
B (A)
˚
C (A)
a (ꢁ)
b (ꢁ)
g (ꢁ)
3
˚
V (A )
Temperature (K) 120(2)
m (Mo Ka)
)
0.085
7726
4601
0.086
(mmꢀ1
Collected
10,735
5089
6060
4866
reflections
Unique
reflections
No. parameters 293
R(int)
GoF
319
327
0.0215
1.047
0.0329
0.0736
0.0209
1.037
0.0293
0.0645
0.0211
1.055
0.0364
0.0731
R1 [I>2s(I)]
wR2 [I>2s(I)]
8. (a) Dutt, A.; Frohlich, R.; Pramanik, A. Org. Biomol. Chem.
2005, 3, 661–665; (b) Dutt, A.; Drew, M. G. B.; Pramanik,
A. Tetrahedron 2005, 61, 11163–11167; (c) Das, A. K.;
Banerjee, A.; Drew, M. G. B.; Ray, S.; Haldar, D.; Banerjee,
A. Tetrahedron 2005, 61, 5027–5036; (d) Maji, S. K.; Haldar,
D.; Drew, M. G. B.; Banerjee, A.; Das, A. K.; Banerjee, A.
Tetrahedron 2004, 60, 3251–3259; (e) Banerjee, A.; Maji,
S. K.; Drew, M. G. B.; Haldar, D.; Banerjee, A. Tetrahedron
Lett. 2003, 44, 335–339; (f) Maji, S. K.; Malik, S.; Drew,
M. G. B.; Nandi, A. K.; Banerjee, A. Tetrahedron Lett. 2003,
44, 4103–4107; (g) Maji, S. K.; Drew, M. G. B.; Banerjee, A.
Chem. Commun. 2001, 1946–1947; (h) Banerjee, A.; Maji,
S. K.; Drew, M. G. B.; Haldar, D.; Banerjee, A. Tetrahedron
Lett. 2003, 44, 6741–6744; (i) Haldar, D.; Maji, S. K.;
Sheldrick, W. S.; Banerjee, A. Tetrahedron Lett. 2002, 43,
2653–2656.
9. (a) Bach, A. C.; Espina, J. R.; Jackson, S. A.; Stouten, P. F. W.;
Duke, J. L.; Mousa, S. A.; DeGrado, W. F. J. Am. Chem. Soc.
1996, 118, 293–294; (b) Bisang, C.; Weber, C.; Inglis, J.;
Schiffer, C. A.; Gunsteren, W. F. V.; Jelesarov, I.; Bosshard,
H. R.; Robinson, J. A. J. Am. Chem. Soc. 1995, 117, 7904–7915.
10. (a) Karle, I. L.; Banerjee, A.; Bhattacharya, S.; Balaram, P.
Biopolymers 1996, 38, 515–526; (b) Banerjee, A.;
Raghothama, S.; Balaram, P. J. Chem. Soc. Perkin Trans. 2
1997, 2087–2094.
refined freely. All non-hydrogen atoms were refined aniso-
tropically. Additional crystallographic information can be
found in Table 3.
Cambridge Crystallographic Data Center references CCDC
630721–630723 for peptides I–III, respectively.
Acknowledgements
A.D. would like to thank UGC, New Delhi, India, for provid-
ing a senior research fellowship. A.D. wish to acknowledge
CSIR, New Delhi, India, for a junior research fellowship.
E.C.S. thanks the EPSRC for funding.
References and notes
1. Sundaralingam, M.; Sekharudu, Y. C. Science 1989, 244,
1333–1337.
2. (a) Lewis, P. N.; Momany, F. A.; Scheraga, H. A. Proc. Natl.
Acad. Sci. U.S.A. 1971, 68, 2293–2297; (b) Zimmerman,
S. S.; Scheraga, H. A. Proc. Natl. Acad. Sci. U.S.A. 1977, 74,
4126–4129.
11. Prasad, S.; Rao, R. B.; Balaram, P. Biopolymers 1994, 35,
11–20.
12. Barron, L. D.; Hecht, L.; Blanch, E. W.; Bell, A. F. Prog.
Biophys. Mol. Biol. 2000, 73, 1–49.
3. (a) Pauling, L.; Corey, R. B. Proc. Natl. Acad. Sci. U.S.A. 1951,
37, 235–240; (b) Perutz, M. F. Nature 1951, 167, 1053–1054;
(c) Arnott, S.; Wonacott, A. J. J. Mol. Biol. 1966, 21, 371–
383; (d) Barlow, D. J.; Thornton, J. M. J. Mol. Biol. 1988,
201, 601–619; (e) Toniolo, C.; Benedetti, E. Trends.
Biochem. Sci. 1991, 16, 350–353; (f) Karle, I. L.; Balaram, P.
Biochemistry 1990, 29, 6747–6756.
13. (a) Prusiner, S. B. Science 1997, 278, 245–251; (b) Ng, S. B. L.;
Doig, A. J. Chem. Soc. Rev. 1997, 26, 425–432; (c) Taubes, G.
Science 1996, 271, 1493–1495; (d) Baumiester, R.; Eimer, S.
Angew. Chem., Int. Ed. 1998, 37, 2978–2982; (e) Burkoth,
T. S.; Benzinger, T. L. S.; Urban, V.; Morgan, D. M.;

A. Dutt et al. / Tetrahedron 63 (2007) 10282–10289
10289
Gregory, D. M.; Thiyagarajan, P.; Botto, R. E.; Meredith, S. C.;
Lynn, D. G. J. Am. Chem. Soc. 2000, 122, 7883–7889.
14. (a) Gazit, E. FASEB J 2002, 16, 77–83; (b) Reches, M.; Gazit,
E. Amyloid 2004, 11, 81–89.
15. (a) Wright, P. E.; Dyson, H. J.; Lerner, R. A. Biochemistry
1988, 27, 7167–7175; (b) Yu, M. H.; King, J. J. Biol. Chem.
1988, 263, 1424–1431.
16. Bodanszky, M.; Bodanszky, A. The Practice of Peptide
Synthesis; Springer: New York, NY, 1984; pp 1–282.
17. Bruker. SMART-NT Version 5.0 (Data Collection), SAINT
Version 186.04 (Data Processing), and SHELXTL Version
6.1 (Structure Solution, Refinement); Bruker AXS: Madison,
WI, 1998.
18. Spek, A. L. J. Appl. Cryst. 2003, 36, 7–13.