The unique crystallographic signature of a β-turn mimic nucleated by N-methylated phenylalanine and Aib as corner residue: Conformational and self-assembly studies

Article abstract of DOI:10.1039/c3ce41448e

The secondary α-amino acid, proline, having high β-turn forming propensity is known to stabilize discrete conformations in peptides. A similar influence is ascribed to N-methylated amino acids with the advantage of the introduction of a side chain functio

Full text of DOI:10.1039/c3ce41448e

CrystEngComm
View Article Online
View Journal | View Issue
PAPER
The unique crystallographic signature of a β-turn
mimic nucleated by N-methylated phenylalanine
and Aib as corner residue: conformational and
self-assembly studies†
Cite this: CrystEngComm, 2013, 15,
10569
Anita Dutt Konar
The secondary α-amino acid, proline, having high β-turn forming propensity is known to stabilize discrete confor-
mations in peptides. A similar influence is ascribed to N-methylated amino acids with the advantage of the intro-
duction of a side chain functionality. In this report our effort lies to modulate the conformational analysis (solid phase:
x-ray diffraction analysis; solution phase: NMR and CD measurements) of two terminally protected synthetic
tripeptides Boc-N-MePhe-Aib-Xx-OMe, where Xx is Phe in peptide I and mABA (mABA: meta amino benzoic acid) in
peptide II, forming β-turns stabilized by 4 → 1 intramolecular hydrogen bonding. Up to now, a significant amount
of work has been dedicated to employing different strategies in stabilizing β-turn mimics. However, to the best of
our knowledge, these novel examples represent a unique crystallographic evidence of a β-turn mimic nucleated by
an N-methylated amino acid (N-methylated phenylalanine) and Aib as corner residues and stabilized by the confor-
mational features of the acyclic tripeptides. The literature documentation further reveals that the aromatic residue
containing the amyloidogenic peptide calcitonin, consists of ribbon like structures, which twist back upon them-
selves to form a tubular ensemble. Interestingly, the FESEM images of peptide II shows a ribbon like morphology,
resulting due to the aggregation of supramolecular helices in higher order self-assembly. Importantly, peptide I
which also produces supramolecular helices does not produce any type of morphology under similar conditions
emphasizing the importance of the location of the aromatic residue in the peptide sequence in generating a particular
morphological structure. This report may open a new avenue in introducing β-turn motifs, replacing proline turns,
in the bioactive conformation of selected peptides. They may also assist in understanding the structure and function
of misfolded disease causing peptides like prion and Alzheimer's amyloid.
Received 20th July 2013,
Accepted 20th September 2013
DOI: 10.1039/c3ce41448e
www.rsc.org/crystengcomm
Introduction
functionality in biological activities. N-Methylated amino acids
bear a similar resemblance to prolines. Replacing L-Pro by
The β-turn is one of the major secondary structural elements
of proteins that plays an important role in folding and is
often implicated as a recognition element in receptor ligand
interactions.1 Also they have the advantage of being very sim-
ple structurally, which facilitates their simulation by means
of mimics.2 Over the past few years considerable effort has
been dedicated to the development of β-turn inducing tem-
plates using different strategies.3 Among which an important
strategy includes to stabilize these motifs by the incorpora-
tion of secondary α-amino acid, prolines, that are known to
have a high β-turn forming propensity.4 But the major draw-
back in the proline residue lies to its constrained cyclic side
chain which prevents the participation of the side chain
N-MeXaa (N-methylated α-amino acid), regains the side chain
functionality which is absolutely lost in the former circum-
stance.5 This side chain could subsequently be used to improve
the activity and selectivity or can act as a pharmacophore in
peptides.6 The incorporation of Nα-methylamino acids into
biologically active peptides changes the chemical and physical
properties and thus provides important information about the
backbone conformation.7 Moreover the methylated amino
acid containing peptides are also potentially useful thera-
peutics since N-methylation has been shown to improve
important pharmacological parameters such as lyphophilicity,
bioavailability, proteolytic stability, conformational rigidity
and duration of action.8
Despite the encouraging bioactivity of methylated amino
acids, they did not attract the deserved attention of chemists
for several years, due to disadvantages encountered in their
application in peptide synthesis. This is because the reactivity
of the secondary amines is very slow, which may be either
Dept of Chemical Sciences, Indian Institute of Science Education and Research,
Bhopal, ITI Gas Rahat Building, Govindpura, Bhopal 462023, India.
E-mail: anita@iiserb.ac.in; Fax: +91 7554092392; Tel: +91 7554056278
† CCDC numbers 951187–951188. For crystallographic data in CIF or other
electronic format see DOI: 10.1039/c3ce41448e
This journal is © The Royal Society of Chemistry 2013
CrystEngComm, 2013, 15, 10569–10578 | 10569

View Article Online
Paper
CrystEngComm
due to racemization in basic conditions or diketopiperizine
formation. Moreover the incorporation of N-methylated amino
acids in peptide sequences is challenging, which is further
followed by the difficult couplings of the preceding amino
acids to the sterically hindered N-methylated site. But the syn-
thesis of methylated derivatives was revolutionized after the
groundbreaking total synthesis of methylated amino acid con-
taining the cyclic peptides, cyclosporine A and omphalotin.9
Kessler and his co-workers are known for their substantial
inputs in studying the conformational and biological beha-
vior of cyclic meptides (meptides: peptides composed of
N-methylated amino acids in sequences).10 Mixed helical
conformations in heptapeptidal meptides have been
reported by Toniolo and his group.11 Meredith et al. have
employed these alkylated amino acids in some parts of
Aβ-amyloids to develop therapeutic agents against amyloid-
osis.12 In a recent report, Arvidsson and his group synthe-
sized hexapeptides with varying extents of N-methylation
based on residues 32–37, of Aβ, to target its C-terminal
region. They also demonstrated that peptides with multiple
N-methylated amide bonds were capable of inhibiting protein
aggregation caused by intermolecular β-sheet aggregation.13
Sewald et al. have elucidated that methylated analogues of
peptaibiotic efrapeptin C exhibit inhibition properties similar
to that of native efrapeptin C.14 Although significant reports
are documented in the literature employing N-methylated amino
acids, the design of β-turn mimetics in acyclic peptidyl
systems remains in a rudimentary stage. In this report our
effort lies to modulate this class of biologically important
amino acid in β-turn design in short acyclic peptides.
several others is noteworthy.16,17 Even though the previously
reported peptides and our peptides form β-turns, our
peptides differ significantly from the earlier reports as for the
first time they contain N-methylated amino acids and Aib as
the corner residues in the β-turn design in short acyclic
peptides. This may open a new avenue in introducing β-turn
motifs, replacing proline turns, in the bioactive conformation
of selected peptides.
Experimental
Abbreviations
Aib, α-aminoisobutyric acid; TFA, trifluoroacetic acid; mABA,
m-amino benzoic acid; Boc, tertiary butyloxy carbonyl;
OMe, methoxy group; DCC, dicyclohexylcarbodiimide; HOBt,
1-hydroxy benzotriazole, HATU, (O-(7-azabenzotriazol-1-yl)-N,
N,N′,N′-tetramethyluronium hexafluorophosphate).
Synthesis of peptides
Peptides I and II containing the Boc group at the N-terminus
and methoxy carbonyls at the C-terminus were synthesized
using conventional solution-phase methodology, with racemisation-
free techniques, followed by a fragment condensation strategy
employing DCC, HATU and 1-hydroxybenzo-triazole (HOBT)
as coupling agents (Scheme 1).18 All the intermediates obtained
were checked for purity by thin layer chromatography (TLC)
on silica gel and used without further purification. All of the
final peptides were purified by column chromatography using
silica gel (100–200 mesh) as the stationary phase and ethyl ace-
tate and petroleum ether mixture as the eluent. The reported
peptides I and II were fully characterised by X-ray crystallography,
NMR and IR measurements.
As a part of the investigation this piece of work presents
the conformational analysis of two terminally protected
acyclic meptides Boc-N-MePhe-Aib-Xx-OMe, where Xx is Phe
in peptide I and mABA (mABA: meta amino benzoic acid) in
peptide II with
a centrally placed non-coded α-amino
Boc-Aib-Phe-OMe (1)
isobutyric acid (Aib) (Fig. 1). The solid state structure was
determined by X-ray diffraction analysis and the solution
phase by solvent dependent NMR titration ROESY and circu-
lar dichroism measurements. Generally, aromatic peptides
possess a tendency to form amyloid like fibrils, the character-
istic factor of various neurodegenerative disease.15 Based on
this concept we chose the third residues of the meptides,
keeping the corner residues unchanged. The idea behind
this was to explore whether the effect of the position of the
aromatic residue in the synthetic peptides, produces any
significant amyloidogenic behavior (in the side chains in
peptide I and backbone in peptide II). In this context, the
significant contribution of Bannerjee and Pramanik and
The PheOMe obtained from its hydrochloride (4.24 g, 19.7 mmol)
was added to an ice-cooled solution of Boc-Aib-OH (2 g, 9.85 mmol)
in 5 ml of DCM. Then DCC (3.04 g, 14.78 mmol) was added to
the cooled mixture, which was stirred for 12 h at room tem-
perature. The progress of the reaction was monitored by TLC.
The residue was then taken into ethyl acetate and the DCU
(N,N-dicyclohexylurea) was filtered off. The organic layer was washed
with 2 M HCl (3 × 100 ml), 1 M sodium carbonate (3 × 100 ml)
and brine (2 × 100 ml), dried over anhydrous sodium sulfate
Fig. 1 A schematic representation of peptides I and II.
Scheme 1 The synthetic strategy of peptides I and II.
10570
|
CrystEngComm, 2013, 15, 10569–10578
This journal is © The Royal Society of Chemistry 2013

View Article Online
CrystEngComm
Paper
and evaporated in vacuo to obtain a white solid. The crude
peptide was used without further purification.
Yield: 3.35 g (8.87 mmol, 90%). 1H NMR (500 MHz,
CDCl3, δ ppm): 1.48 (Boc-MeHs, 9H, s), 1.5 (Aib CβHs, 6H, s),
3.92 (methoxy, 3H, s), 5.67 (Aib NH, 1H, s), 7.87–6.65
(aromatic Hs).
1H, m); 3.70 (–OCH3, 3H, s); 2.76–3.11 (CβHs of NMe-Phe(1) &
Phe(3), 4H, m); 2.64(NMe-Hs of NMe-Phe(1)); 1.52 (CβHs of Aib,
3H, s); 1.1.49 (CβHs of Aib, 3H, s); 1.43 (Boc-CH3s, 9H, s);
1H NMR 125 MHz (CDCl3, δ ppm): 173.72, 172.28, 170.13, 156.44,
138.11, 136.09, 129.08, 128.44, 127.5, 126.45, 81.01, 61.51, 57.17,
53.66, 52.07, 37.58, 33.81, 32.60, 28.24, 25.91, 24.76.
Boc-Aib-mABA-OMe (2)
Boc-NMe-Phe-Aib-m-ABA-OMe (peptide II)
mABAOMe obtained from its hydrochloride (4.5 g, 24.63 mmol)
was added to an ice-cooled solution of Boc-Aib-OH (2.5 g,
12.31 mmol) in 5 ml of DMF. Then DCC (3.8 g, 18.47 mmol;
1,3-dicyclohexylcarbodiimide) was added to the cooled
mixture, which was stirred for 12 h at room temperature. The
progress of the reaction was monitored by TLC. The
residue was then taken into ethyl acetate and the DCU
(N,N-dicyclohexylurea) was filtered off. The organic layer was
washed with 2 M HCl (3 × 100 ml), 1 M sodium carbonate
(3 × 100 ml) and brine (2 × 100 ml), dried over anhydrous
sodium sulfate and evaporated in vacuo to obtain a slightly
brownish solid material. The crude peptide was used without
further purification.
To Boc-Aib-mABA-OMe (1.7 g, 5.05 mmol), trifluoroacetic acid
(3 ml) was added at 0 °C and stirred at room temperature.
The removal of the Boc group was monitored by TLC. After
12 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 diethyl ether. The pH
of the aqueous solution was adjusted to 8 with sodium bicar-
bonate and extracted with ethyl acetate. The extracts were
pooled, washed with saturated brine, dried over sodium sul-
phate, and concentrated to a highly viscous liquid that gave a
positive ninhydrin test. This tripeptide free base was added
to a well ice-cooled solution of Boc-NMe-Phe-OH (1 g, 3.6 mmol)
in DMF (4 ml) followed by HATU (2 g, 5.4 mmol) and DIPEA
(3 ml). The reaction mixture was stirred at room temperature
for 12 h. The residue was taken up in ethyl acetate and the
dicyclohexylurea (DCU) was filtered off. The organic layer was
washed with 2 M HCl (3 × 50 ml), a 1 M Na2CO3 solution (3 × 50 ml)
and brine, dried over anhydrous Na2SO4 and evaporated in vacuo,
to yield a white solid. Purification was done using silica gel as
the stationary phase and an ethyl acetate–petroleum ether mix-
ture as the eluent. Single crystals were grown from an acetone–
petroleum ether mixture by slow evaporation and were stable at
room temperature.
1
Yield: 3.31 g (9.85 mmol, 80%). Mp = 143–145 °C; H NMR
(400 MHz, CDCl3, δ ppm): 1.48 (Boc-MeHs, 9H, s), 1.5 (Aib CβHs,
6H, s), 3.92 (methoxy, 3H, s), 5.67 (Aib NH, 1H, s), 7.87–6.65
(m-ABA Hb, Hd, Hc, Ha 4H, m),7.88 (m-ABA NH, 1H, s).
Boc-NMe-Phe-Aib-Phe-OMe (peptide I)
To Boc-Aib-Phe-OMe (1) (2.2 g, 6.05 mmol), trifluoroacetic
acid (3 ml) was added at 0 °C and stirred at room tempera-
ture. The removal of the Boc group was monitored by TLC.
After 12 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 diethyl ether.
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 sulphate, and concentrated to a highly viscous liquid
that gave a positive ninhydrin test. This tripeptide free base
was added to a well ice-cooled solution of Boc-NMe-Phe-OH
(1.3 g, 4.64 mmol) in DMF (4 ml) followed by HATU (2.64,
6.96 mmol) and DIPEA (4.7 ml). The reaction mixture was
stirred at room temperature for 12 h. The residue was taken
up in ethyl acetate. The organic layer was washed with 2 M
HCl (3 × 50 ml), a 1 M Na2CO3 solution (3 × 50 ml) and brine,
dried over anhydrous Na2SO4 and evaporated in vacuo, to yield
a white solid. Purification was done using silica gel as the
stationary phase and an ethyl acetate–petroleum ether
mixture as the eluent. Single crystals were grown from an
acetone–petroleum ether mixture by slow evaporation and were
stable at room temperature.
Yield: 1.35 g (2.7 mmol, 76%). Mp = 108–110 °C; IR (KBr):3450,
1
3338, 1726, 1675, 1441 cm−1; H NMR 500 MHz (CDCl3, δ ppm):
9.25 (m-ABA(3) NH, 1H, s); 8.29 (m-ABA(3) Ha, 1H, s); 8.01
(m-ABA(3) Hd, 1H, d, J = 10 Hz); 7.79 (m-ABA(3) Hb, 1H, d,
J = 10 Hz); 7.41 (m-ABA(3) Hc, 1H, t, J = 5 Hz); 7.23–7.32
(10 phenyl ring protons of NMe-Phe(1)); 6.39 (Aib(2) NH, 1H,
s); 4.20–4.24 (CαH of NMe-Phe(1), 1H, m); 3.99 (–OCH3, 3H, s);
3.41–3.44 (CβH of NMe-Phe(1), 1H, m); 3.16–3.21 (CβH of
NMe-Phe(1) 1H, m); 2.65 (NMe-Hs of NMe-Phe(1)); 1.64
(CβHs of Aib, 6H, s); 1.51 (Boc-CH3s, 9H, s); 1H NMR 125
MHz (CDCl3, δ ppm): 172.74, 170.30, 170.21, 169.16, 166.84,
156.23, 138.78, 137.93, 130.39, 129.20, 128.18, 126.54, 124.31,
120.90, 81.26, 63.52, 57.66, 51.97, 49.74, 34.11, 33.67, 28.21,
26.32, 23.51.
NMR experiments
1
All H NMR and 13C NMR studies were recorded on a Bruker
Avance 500 model spectrometer operating at 500 and
125 MHz respectively. The 2D Experiment was carried out in
CDCl3 on a Bruker equipped with a 5 mm broad band inverse
probe head. The peptide concentrations were in the range
Yield: 1.96 g (3.71 mmol, 80%). Mp = 116 °C; IR (KBr):
1
3468, 3297, 1753, 1654, 1454 cm−1; H NMR 500 MHz (CDCl3,
δ ppm): 7.26–7.30 (phenyl ring protons of NMe-Phe(1) & Phe(3));
7.17 (Phe(3) NH, 1H, d, J = 5 Hz); 6.59 (Aib(2) NH, 1H, s);
4.81–4.82 (CαH of Phe(3), 1H, m); 4.60–4.61 (CαH of NMe-Phe(1),
1
of 5–10 mM in CDCl3 for H NMR and 30–40 mM in CDCl3
for 13C NMR.
This journal is © The Royal Society of Chemistry 2013
CrystEngComm, 2013, 15, 10569–10578 | 10571

View Article Online
Paper
CrystEngComm
Circular dichroism spectroscopy
Solutions of peptides I and II in acetonitrile and methanol
(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 averaging time and a scan speed of
50 nm min−1, using a JASCO spectropolarimeter (J 720 model)
equipped with a 0.1 cm pathlength cuvette. The measure-
ments were taken at 0.5 nm wavelength intervals, 2.0 nm
spectral bandwidth and five sequential scans were recorded
for each sample.
Field emission scanning electron microscopic study (FESEM)
The morphology of peptide II was investigated using an
FESEM microscope (JEOL JSM-6700F). For the study, fibrous
materials (slowly grown from acetone and petroleum ether,
same solvent as that used in crystallization) were dried and
gold coated.
Crystal data collection and structure determination
Colorless crystals suitable for diffraction were chosen for
X-ray structural analysis, and intensity data were collected
on a Brüker APEX-II CCD diffractometer using a graphite
monochromated MoKα radiation source (α = 0.71073 Å) at
298 K. The data collection was performed using the φ and ω
scan. The structures were solved using direct methods
followed by full matrix least square refinements against F2
(all data HKLF 4 format) using SHELXTL.19,20 A multi-scan
absorption corrections, based on equivalent reflections were
applied to the data. Anisotropic refinement was used for all
non-hydrogen atoms. Hydrogen atoms were placed in appropriate
calculated positions.
Fig. 2 The crystal structure of (a) peptide I and (b) peptide II with an atom numbering
scheme. The hydrogen bonds are shown as dotted lines, (color code: carbon, grey;
nitrogen, blue; oxygen, red; hydrogen, light bluish grey).
Table 1 Selected backbone torsions (°) of peptides I and II
Results and discussion
Solid state structures
Peptide I
Peptide II
(ω0)a
(φ1)b
(ψ1)c
(ω1)a
(φ2)b
(ψ2)c
(ω2)a
−168.0(2)
−54.9(2)
−41.6(2)
−174.1(2)
−59.4(2)
−26.2(2)
−175.0(2)
177.0(5)
55.6(6)
31.5(7)
177.9(5)
54.3(7)
33.9(7)
175.0(5)
Single crystals suitable for X-ray diffraction analysis were
obtained from an acetone–petroleum ether mixture of
both peptides I and II by a slow evaporation method. The
crystal structures of peptides I and II reveal a type III and
III′ β-turn conformation respectively, with NMePhe-Ala(1)
and Aib(2) occupying the i + 1 and i + 2 positions stabi-
lized by an intramolecular hydrogen bond (Fig. 2). The ideal-
a
Torsion angle around the amide C–N bond from the N-terminal of
b
the peptides. Torsion angle around the peptide C–N bond between
NMePhe(1) and Aib(2) of the peptides. Torsion angle around the
peptide C–N bond between Aib(2) and Phe(3) of the peptides in peptide I
and Aib(2) and mABA(3) in peptide II.
c
ized torsion angles for a type III β-turn are, ϕi+1 = −60°, ψi+1
=
−30°, ϕi+2 = −60°, ψi+2 = −30° and for a type III′ β-turn, are ϕi+1
= 60°, ψi+1 = 30°, ϕi+2 = 60°, ψi+2 = 0° (Table 1).21 In peptides
I and II, these torsion angles are found to deviate apprecia-
bly ((ϕi+1 (−54.9(2)/55.6(6)°), ψi+1 (−41.6(2)/31.5(7)°), ϕi+2
(−59.4(2)/54.3(7)°) and ψi+2 (−26.2(2)/33.9(7)°) (Table 1)
from the ideal values. As a consequence, a weak intra-
molecular hydrogen bond between Boc-CO and Phe(3)-NH
in peptide I and Boc-CO and mABA(3)-NH in peptide II is
observed (peptide I: H4⋯O2, 2.161(1) Å; N4⋯O2, 2.971(1) Å;
N4–H4⋯O2, 156.78(2)°; peptide II: H2⋯O3, 2.181(1)Å;
N2⋯O3, 2.996(1) Å; N2–H2⋯O3, 153.92(3)°); (Table 2).17
The crystal data of both the peptides are reported in
Table 2 Selected intra- and intermolecular hydrogen bonding parameters
D–H⋯A
H⋯A/Å
D⋯A/Å
D–H⋯A/°
Peptide I
Peptide II
N4–H4⋯O2
N3–H3⋯O5a
N2–H2⋯O3
N4–H4⋯O5a
2.161(1)
2.104(1)
2.181(1)
2.232(1)
2.971(1)
2.889(1)
2.996(1)
2.939(1)
156.78(3)
151.46(3)
153.92(3)
137.15(2)
a
Symmetry codes: x, y, z.
10572
|
CrystEngComm, 2013, 15, 10569–10578
This journal is © The Royal Society of Chemistry 2013

View Article Online
CrystEngComm
Paper
Table 3. Platon analysis revealed very large solved accessible
voids (32.3% of the unit cell) for peptide II.
In the solid state, the β-turn subunits of both peptides I
and II are regularly inter-linked via intermolecular hydrogen
bonds between the Aib(2) NH moiety of one molecule and
the Aib(2) CO group of another molecule (peptide I: N3–H3–O5,
2.104(1) Å, N3⋯O5, 2.889(1) Å, x, y, z, N3–H3–O5, 151.46(3)°,
Peptide II: N4–H4–O5, 2.232(1) Å, N4⋯O5, 2.939(1) Å, x, y, z,
N4–H4–O5, 137.15(2)°), to create a supramolecular helix along
the crystallographic c axis (Fig. 3). The hydrogen bonding
parameters are listed in Table 2. Another interesting feature is
that the peptides are stacked over each other in an antiparallel
fashion so that a supramolecular helix is formed utilizing
intermolecular hydrogen bonds (Fig. 3).
In the literature several groups have reported β-turns in syn-
thetic peptides which display various supramolecular architec-
tures on self-assembly.16,17 But in every case it was observed
that the β-turns with hydrophobic amino acids and Aib at the
N-terminus form a supramolecular β-sheet architecture on
self-assembly. N-methylated phenylalanine is extremely hydro-
phobic in nature. Inspite of this fact, it forms a supramolecular
helical architecture in higher order self-assembly. This may
be due to the contribution of N-methylated phenylalanine in
the N-terminal position, that inhibits β-sheet aggregation by
binding to one face of the peptide but is unable to hydrogen
bond on the other face, because of the N-methyl group replacing
a backbone NH in phenylalanine.
Fig. 3 The formation of the supramolecular helical assembly of a) peptide I and
b) peptide II as observed along the crystallographic c axis in the wireframe model,
(color code: carbon, grey; nitrogen, blue; oxygen, red; hydrogen, light bluish grey).
Solution conformational analysis
gradually titrated against polar (CD3)2SO. The changes in the
chemical shifts are presented in Fig. 4 and Table 4. The solvent
titration experiment shows that by increasing the percentage
of (CD3)2SO in CDCl3 from 0 to 11% (v/v) the net changes in
the chemical shift (Δδ) values for Aib(2)-NH, Phe(3)-NH for
peptide I and Aib(2)-NH, mABA(3)-NH for Peptide II are 0.03, 0.06,
0.17, and 0.04 ppm respectively. From the Δδ values it is evident
that in peptides I and II both Aib(2)-NH and Phe(3)/mABA(3)-NH
are solvent shielded, indicating their involvement in intramole-
cular hydrogen bonding (Δδ < 0.2 ppm; intramolecularly
hydrogen bonded NH; Δδ > 0.2 ppm; intermolecularly hydro-
gen bonded NH).17c Table 4 illustrates the Δδ values for all of
the NHs of peptides I and II. Several intra and interresidue
NOEs for peptides I and II are listed in Fig. 5 and Table 5.
Furthermore the ROESY spectrum of peptides I and II in
CDCl3 reveals three important NOEs, namely Aib(2)-NH ↔
Phe(3)-NH, NMe-Phe(1)-CαH ↔ Aib(2)-NH, Aib(2)-CβHs ↔
Phe(3)-NH for peptide I and Aib(2)-NH ↔ mABA(3)-NH,
NMe-Phe(1) Me Hs ↔ Aib(2)-NH, NMe-Phe(1)-CαH ↔ Aib(2)-NH,
Aib(2)-CβHs ↔ mABA(3)-NH for peptide II, respectively.23
This data and the solvent shielded nature of Phe(3)-NH for
peptide I and mABA(3)-NH for peptide II are supportive of a
10 membered intramolecular hydrogen bond involving CO
from the Boc group and Phe(3)-NH/mABA(3)-NH for peptides
I and II respectively in CDCl3. Interestingly, the presence of
NMe-Phe(1)-N-Me Hs ↔ Aib(2)-NH, and also NMe-Phe(1)-CαH ↔
Aib(2)-NH correlation in CDCl3 along with the solvent shielded
Complete 1H NMR assignments of peptide I and II were obtained
using a combination of 2D COSY and ROESY methods in CDCl3.
In order to investigate the existence of the intramolecular
hydrogen bonding and the peptide conformation in the solu-
tion phase, the solvent dependence of the NH chemical shifts
was examined by NMR titration.22 In this experiment a solu-
tion of the peptide in nonpolar CDCl3 (10 mM in 0.5 ml) was
Table 3 The crystallographic refinement details for peptides I and II
Peptide I
Peptide II
Chemical formula
Formula weight(g)
Crystal system
Space group
a(Å)
b(Å)
c(Å)
α(°)
C29H39N3O6
525.63
Monoclinic
P21
10.923(4)
10.889(4)
12.814(4)
90
C27H35N3O6
497.58
Monoclinic
P21
12.575(4)
10.973(3)
13.526(5)
90
105.577(9)
90
1797.9(11)
0.919
0.065
2
4370
2292
1.056
0.0993
0.2502
β(°)
102.503(17)
90
1488.0(9)
1.173
γ(°)
V(Å3)
Dcalc(g cm−1
)
μ(cm−1
)
0.082
Z
2
5386
4850
1.067
0.0448
0.1235
Collected reflections
Unique reflections
Goodness-of-fit (GOF) on F2
R1[I > 2σ(I)]
wR2[I > 2σ(I)]
This journal is © The Royal Society of Chemistry 2013
CrystEngComm, 2013, 15, 10569–10578 | 10573

View Article Online
Paper
CrystEngComm
proline (unlike other amino acid residues) and compels the
NH of i + 2th position to arrive at an orientation for the forma-
tion of this hydrogen bond.
The conformation of peptides I and II was probed further
in solution phase by far-UV CD measurements in two solvents
of different polarity, acetonitrile (low polarity) and methanol
(high polarity), and the results are presented in Fig. 6. The
two peptides show similar CD patterns with a broad band at
225–240 nm along with a distinct minima for peptide I: 216 nm
peptide II: 217 nm and a distinct maxima for peptide I: 202 nm
peptide II: 203 nm. This signature was reported as a contri-
bution of type III β-turn structures in solution.24 Interes-
tingly the similar characteristic CD signature observed in both
solvents reveals that the propensity for the conformational
preferences of β-turns is retained in both the solvents of dif-
ferent polarity with the appearance of few shoulders with
increased polarity. The variation of the conformers or different
contributions of the aromatic units (Phe residue) present in
the side chain or in the peptide backbone results in different
degrees of molar ellipticities in different solvents with the
appearance of some unordered structures with a change in the
solvent polarity.
Nevertheless, in addition to the NMR experiment, the CD
data too strongly favor the conclusion that peptides I and II
adopt a β-turn conformation in solution.17c
Generally, turn formation (smallest unit of a helix) is
driven by favorable enthalpy and entropy changes.25a In a
less polar solvent (acetonitrile), the turn nature is more
pronounced with respect to the turn nature in a more polar
solvent (methanol). The enhancement of this turn propen-
sity in a less polar solvent (acetonitrile) is attributed to the
creation of a hydrophobic environment, stripping of the
polar molecules from the surface of peptides. This allows
the molecule to retain the inherent hydrogen bond intact,
thereby displays increased turn forming propensity. Thus
from the thermodynamic point of view, it can be stated that
in acetonitrile as the turn forming propensity <w> increases
greatly, inspite of some unfavorable enthalpy changes, it
decreases the free energy formation of the turn unit and
hence makes the propagation of the turn spontaneous and
very favorable (ΔG° = ΔH° − RT ln <w>, where <w>; turn
forming propensity, ΔG°; net free energy change during the
turn formation, ΔH°; net enthalpy change during the turn
formation R; the universal constant T; the temperature of
the experiment).25 On the other hand in polar solvents
like methanol, there is always competition between the
skeleton amide bonds of the peptides to be involved in
intermolecular hydrogen bonding with the solvent, which
to some extent, partially disrupts the original structure of
the peptide. So we observe reduced molar ellipticities (<w>
is quite low) along with some additional shoulders. Even
though in methanol some favorable enthalpy changes
occur, yet it is not sufficient enough to predominate over
the entropy effect. Therefore peptide molecules show less
turn forming propensity here. Thus in both the solvents,
peptides I and II behave consistently.
Fig. 4 NMR solvent titration for the NH protons in a) peptide I and b) peptide II.
Table 4 The characteristic 1H parameters of peptides I and II
δ valuesa (ppm)
Residues
NH/NMe
CαH
CβH
Δδb
Peptide I
NMe-Phe(1)
Aib(2)
2.64
6.59
7.17
4.60–4.61
—
4.81–4.82
2.76–3.11
1.49, 1.52
2.76–3.11
—
0.03
0.06
Phe(3)
a
b
Chemical shifts of proton resonances in CDCl3.
Δδ is the
chemical shift for NH protons in CDCl3 and 11% d6-DMSO.
Peptide II
NMe-Phe(1)
Aib(2)
mABA(3)
2.65
6.39
9.25
4.20–4.24
—
—
3.41–3.44
1.64
—
—
0.17
0.04
nature of Aib(2)-NH is indicative of the presence of a bifur-
cated intramolecular hydrogen bond involving the Boc-CO
and Aib(2)-NH for peptides I and II respectively. The word
bifurcated has been used since the Boc-CO acts as an acceptor
for both the H-bonds (Aib(2)-NH and Phe(3)-NH/mABA(3)-NH
respectively for peptides I and II in solution).17h This uncon-
ventional bifurcated hydrogen bond absent in the solid state
may be attributed to the N-MePhe present at the N-terminus
(i + 1th position) which itself adopts a cis amide bond like
10574
|
CrystEngComm, 2013, 15, 10569–10578
This journal is © The Royal Society of Chemistry 2013

View Article Online
CrystEngComm
Paper
Fig. 5 Schematic representation of the proposed β-turn in a) peptide I and b) peptide II. The hydrogen bonds are shown as dotted lines. Observed NOEs are highlighted by
double edged arrows in CDCl3 (500 MHz). Characteristic NOEs are marked in brown.
Morphological studies
human amylin, the role of α-helices in the highly ordered self-
A thorough literature survey reveals that not only β-sheets but
aggregated amyloid plaque formation has been demonstrated
by Goldsbury and his co-workers.27 The amyloidogenic peptide
also helices play a significant role in amyloid fibrillation.26 In
This journal is © The Royal Society of Chemistry 2013
CrystEngComm, 2013, 15, 10569–10578 | 10575

View Article Online
Paper
CrystEngComm
Table 5 Important intraresidue NOEs for peptides I and II in CDCl3 (characteristic NOEs are marked in bold)
Peptide I
1) Aib(2)-NH ↔ Phe(3)-NH
9) NMe-Phe(1)-CαH ↔ NMe-Phe(1)-Me Hs
2) NMe-Phe(1)-Me Hs ↔ Aib(2)-NH
3) NMe-Phe(1)-CαH ↔ Aib(2)-NH
4) Aib(2)-CβHs ↔ Phe(3)-NH
10) Aib(2)-CβHs ↔ Aib(2)-NH
11) Phe(3)-CβHs ↔ Phe(3)-NH
12) Phe(3)-CβHs ↔ aromatic H's of Phe(3)
13) NMe-Phe(1)-CαH ↔ aromatic H's of NMe-Phe(1)
14) Phe(3)-CαH ↔ aromatic H's of Phe(1)
15) NMe-Phe(1)-Me Hs ↔ Aib(2)-CβHs
5) NMe-Phe(1)-CαH ↔ NMe-Phe(1)-CβHs
6) Phe(3)-CαH ↔ Phe(3)-CβHs
7) a Phe(3)-CαH ↔ Phe(3)-NH
8) NMe-Phe(1)-CαH ↔ aromatic H's of NMe-Phe(1)
Peptide II
1) Aib(2)-NH ↔ mABA(3)-NH
7) mABA(3)-Hc ↔ mABA(3)-Hd
2) NMe-Phe(1)-Me Hs ↔ Aib(2)-NH
3) NMe-Phe(1)-CαH ↔ Aib(2)-NH
4) Aib(2)-CβHs ↔ mABA(3)-NH
5) NMe-Phe(1)-CαH ↔ NMe-Phe(1)-CβHs
6) a mABA(3)-Hc ↔ mABA(3)-Hb
8) Aib(2)-NH ↔ mABA(3)-Ha
9) NMe-Phe(1)-CβH ↔ aromatic H's of NMe-Phe(1)
10) Aib(2)-CβHs ↔ Aib(2)-NH
11) NMe-Phe(1)-Me Hs ↔ NMe-Phe(1)-CαH
calcitonin is known to possess ribbon-like structures that twist
back upon themselves to afford hollow tube-like assemblies.28
Therefore, our interest lied to explore the possibility of
any type of morphological materials from the designed
meptides. Interestingly, the field emission scanning electron
Fig. 7 (a) An FE-SEM image of peptide II (X 3000) showing the formation of non-
twisted ribbon-like structures. (b) The enlarged SEM image clearly demonstrates that
the ribbons are formed through lateral association of non-twisted filaments.
Fig. 6 CD curves of peptides I and II in a) acetonitrile and b) methanol (1.5 mM).
10576
|
CrystEngComm, 2013, 15, 10569–10578
This journal is © The Royal Society of Chemistry 2013

View Article Online
CrystEngComm
Paper
Fig. 8 A schematic representation of formation of ribbons from turn structures through molecular self-assembly.
microscopic (FE-SEM) images (30 000 times magnified) of the
dried fibrous material of peptide II grown slowly from ace-
tone and petroleum ether clearly demonstrate that the aggre-
gates in the solid state are bunches of non-twisted ribbon-like
structures formed due to higher order self-assembly (Fig. 7). It
was observed that peptide I does not produce any such ribbon
like morphology under similar conditions. The supramolecu-
lar helices of peptide II generated from a primary building
block type III′ β-turn (Fig. 7 and 8) aggregate through
intermolecular H-bonds between the peptide linkages of adja-
cent molecules to form non-twisted filaments. These non-
twisted filaments (supramolecular helicates) through lateral
association stabilized by weak interactions (hydrophobic and
van der Waals interactions) further lead to a ribbon-like mor-
phology (length: >50 μm; breadth: 0.5 μm) in hierarchical
self-assembly. The balance between the weak interactions
involved in regular parallel alignment of one aromatic
nucleus on the surface of the supramolecular helix and the
other at the peptide backbone probably helps in the higher
order self-assembly through non-covalent interactions in pep-
tide II. This balance may not be optimum when the two aro-
matic moieties are present in the side chains as in peptide I.
The observation of various morphological ensembles resulting
due to the variation in hydrophobicity (such as a variation in
length of the peptide chain, the contribution of side chains
and several other factors) has been observed earlier.29 There-
fore this report further emphasizes the importance of the
hydrophobicity in generating a particular morphology in the
solid state.
N-methylated phenylalanine and Aib as the corner residues
and stabilized by the conformational feature type III and III′
β-turn of the acyclic tripeptides. The peptide conformations
of both the peptides in the solution phase also supports the
existence of the turn conformation in solution (CDCl3). In the
crystalline state, the helices are packed in a head-to-tail fashion
through intermolecular hydrogen bonds to create supramole-
cular helical structures. Interestingly, peptide II forming supra-
molecular helices in higher order self-assembly, forms a
ribbon-like morphology, formed by the lateral association of
non-twisted filaments. Importantly, peptide I which also produces
supramolecular helices does not produce any such type of
structures under similar conditions emphasizing the role of
the position of the aromatic residue in morphology generation
The optimum balance between the weak interactions
involved in the regular alignment of an aromatic nucleus
on the surface of the supramolecular helix and the other at
the peptide backbone dictates a higher order self-assembly
and thereby generating morphological materials. Thus
these assemblies may find useful applications in nano-
technology and assist in understanding the structure activity
relationship of abnormal peptides like prion and Alzheimer's
amyloid. This report may also open a new avenue in introduc-
ing β-turn motifs within the bioactive conformation of selected
peptides with the introduction of side chain functionality
replacing proline turns.
Acknowledgements
Thus these morphological assemblies may find useful appli-
cations in nanoscience and nanotechnology. They may also help
in understanding the structure activity relationship of misfolded
disease causing peptides like prion and Alzheimer's amyloid.
The author wishes to thank DST, New Delhi, India, for financial
support (project no. SR/FT/CS-025/2010) and IISER Bhopal for
providing the infrastructures. The author is very much grateful
to Dr Sanjit Konar for assistance in X-ray crystallography and
valuable advice. She also wishes to thank Dr Aasheesh Srivastava
for academic and administrative support and Dr Deepak Chopra
for helpful suggestions in structure refinement. Mr Amit Adhikary
and Mr Soumava Biswas are sincerely acknowledged for various
measurements for this work.
Conclusions
In summary, this report reflects the unique crystallographic
signature of a β-turn design in acyclic peptides employing
This journal is © The Royal Society of Chemistry 2013
CrystEngComm, 2013, 15, 10569–10578 | 10577

View Article Online
Paper
CrystEngComm
Notes and references
S. K. Maji, W. S. Sheldrick and A. Banerjee, Tetrahedron Lett.,
2002, 43, 2653; (f) D. Haldar, S. K. Maji, M. G. B. Drew,
A. Banerjee and A. Banerjee, Tetrahedron Lett., 2002, 43,
5465S. K. Maji, M. G. B. Drew and A. Banerjee, Chem. Commun.,
2001, 1946.
1 J. D. Tyndall, B. Pfeiffer, G. Abbenante and D. P. Fairlie,
Chem. Rev., 2005, 105, 793–826.
2 (a) G. D. Rose, L. M. Gierasch and J. A. Smith, Adv. Protein
Chem., 1985, 37, 1–109; (b) H. Kessler, Angew. Chem., Int. Ed.
Engl., 1982, 21, 512–523; (c) K. Brickmann, Z. Q. Yunan,
I. Sethon, P. Somfai and J. Kihlberg, Chem.–Eur. J., 1999, 5,
2241–2253.
3 (a) K. S. Kee and S. D. S. Jois, Curr. Pharm. Des., 2003, 9,
1209–1224; (b) A. J. Souers and J. A. Ellman, Tetrahedron,
2001, 57, 7431–7448.
4 (a) C. M. Wilmot and J. M. Thornton, J. Mol. Biol., 1988, 203,
221–232; (b) M. W. MacArthur and J. M. Thornton, J. Mol.
Biol., 1991, 218, 397–412.
5 E. Fischer and W. Lipschitz, Ber. Dtsch. Chem. Ges., 1915, 48,
360–378.
17 (a) A. Dutt, R. Fröhlich and A. Pramanik, Org. Biomol. Chem.,
2005, 3, 661–665; (b) A. Dutt, M. G. B. Drew and A. Pramanik,
Tetrahedron, 2005, 61, 11163–11167; (c) A. Dutt, A. Dutta,
R. Mondal, E. C. Spencer, A. J. K. Howard and A. Pramanik,
Tetrahedron, 2007, 63, 10282–10289; (d) R. Kaur and
R. Kishore, Biopolymers, 2013, 99, 419–426; (e) P. Jana,
S. Maity, S. K. Maity and D. Haldar, Chem. Commun., 2011, 47,
2092–2093; (f) S. K. Maity, S. Maity, P. Jana and D. Haldar,
CrystEngComm, 2012, 14, 4034; (g) A. Dutt Konar, CrystEngComm,
2012, 14, 6689–6694; (h) A. Dutt Konar, J. Mol. Struct., 2013, 1036,
350–360; (i) A. Dutt Konar, CrystEngComm, 2013, 15, 2466–2473;
(j) M. Crisma, G. Valle, C. Toniolo, S. Prasad, R. Balaji Rao
and P. Balaram, Biopolymers, 1995, 35, 1; (k) S. Prasad,
R. Balaji Rao and P. Balaram, Biopolymers, 1995, 35, 11;
(l) S. K. Maji, D. Haldar, M. G. B. Drew, A. Banerjee, A. K. Das
and A. Banerjee, Tetrahedron, 2004, 60, 3251; (m) A. Banerjee,
S. K. Maji, M. G. B. Drew, D. Haldar and A. Banerjee,
Tetrahedron Lett., 2003, 44, 335.
18 M. Bodanszky and A. Bodanszky, The Practice of Peptide
Synthesis, Springer, New York, 1984, pp. 1–282.
19 SADABS and G. M. Sheldrick, "Program for Absorption Correction
of Area Detector Frames", BRUKER AXS Inc., 5465 East Cheryl
Parkway, Madison, WI 53711-USA.
20 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr.,
1990, 46, 467G. M. Sheldrick, SHELXL 97, Program for
Crystal Structure Refinement, University of Göttingen,
Germany, 1997.
21 C. M. Venkatachalam, Biopolymers, 1968, 6, 1425.
22 (a) I. L. Karle, A. Banerjee, S. Bhattacharya and P. Balara,
Biopolymers, 1996, 38, 515–526; (b) A. Banerjee, S. Raghothama
and P. Balaram, J. Chem. Soc., Perkin Trans. 2, 1997, 2087–2094;
(c) K. Wuthrich, NMR of Proteins and Nucleic Acids, Wiley,
New York, NY, 1986, pp. 192–196.
6 J. Chatterjee, F. Rachenmacher and H. Kessler, Angew.
Chem., Int. Ed., 2013, 52, 254–269.
7 (a) B. Vitoux, A. Aubry, M. T. Cung and M. Marraud, Int. J.
Pept. Protein Res., 2009, 27, 617–632; (b) Y. Takeuchi and
G. R. Marshall, J. Am. Chem. Soc., 1998, 120, 5363–5372.
8 (a) D. P. Fairlie, G. Abbenante and D. R. March, Curr. Med.
Chem., 1995, 2, 654–686; (b) W. L. Cody, J. X. He, M. D. Reily,
S. J. Haleen, D. M. Walker, E. L. Reyner, B. H. Stewart and
A. M. Doherty, J. Med. Chem., 1997, 40, 2228–2240.
9 (a) X. Wu, J. L. Stockdill, P. Wang and S. J. Danishefsky,
J. Am. Chem. Soc., 2010, 132, 4098–4100; (b) D. M. Wenger,
Helv. Chim. Acta, 1984, 67, 502–525; (c) B. Thern, J. Rudolph
and G. Jung, Angew. Chem., Int. Ed., 2002, 41, 2307–2309.
10 (a) H. Kessler, J. Med. Chem., 2007, 50, 5878–5881; (b) E. Biron,
J. Chatterjee, O. Ovadia, D. Langenegger, J. Brueggen,
D. Hoyer, H. A. Schmidt, R. Jelinek, C. Gilon, A. Hoffman
and H. Kessler, Angew. Chem., Int. Ed., 2008, 47, 2595–2599;
(c) J. Chatterjee, D. F. Mierke and H. Kessler, Chem.–Eur. J.,
2008, 14, 1508–1517; (d) J. Chatterjee, C. Gilon, A. Hoffman
and H. Kessler, Acc. Chem. Res., 2008, 41, 1331–1342.
11 M. Crisma, W. Bisson, F. Formaggio, B. Q. Broxterman and
C. Toniolo, Biopolymers, 2002, 64, 236–245.
23 S. K. Maji, R. Banerjee, D. Velmurugan, A. Razak, H. K. Fun
and A. Banerjee, J. Org. Chem., 2002, 67, 633–639.
24 M. Krisma, G. D. Fasman, H. Baalram and P. Balaram, Int. J.
Pept. Protein Res., 2009, 23, 411–419.
12 (a) D. J. Gordon and S. C. Meredith, Biochemistry, 2003, 42,
475–485; (b) K. L. Sciaretta, A. Boire, D. J. Gordon and
S. C. Meredith, Biochemistry, 2006, 45, 9485–9495.
13 P. P. Bose, U. Chatterjee, C. Nerelius, T. Govender, T. Norstrom,
A. Gogoll, A. Sandergren, E. Gothelid, J. Johansson and
P. I. Arvidsson, J. Med. Chem., 2009, 52, 8002–8009.
14 A. Dutt Konar, E. Vass, M. Holosi, Z. Majer, G. Gruber,
K. Frese and N. Sewald, Chem. Biodiversity, 2013, 10, 942–951.
15 E. Gazit, FASEB J., 2002, 16, 77–83; M. Reches and E. Gazit,
Amyloid, 2004, 11, 81–89.
25 (a) P. Luo and R. L. Baldwin, Biochemistry, 1997, 36,
8413–8421; (b) P. C. Rakshit,
Chemistry, 6th edition, 2001.
A Text Book of Physical
26 (a) C. Goldsbury, K. Goldie, J. Pellaud, J. Seelig, P. Frey,
S. A. Müller, J. Kistler, G. J. Cooper and U. J. Aebi, J. Struct. Biol.,
2000, 130, 352; (b) A. Dutt, M. G. B. Drew and A. Pramanik,
Tetrahedron, 2008, 64, 549–558.
16 (a) A. Banerjee, S. K. Maji, M. G. B. Drew, D. Haldar,
A. K. Das and A. Banerjee, Tetrahedron, 2004, 60, 5935;
(b) S. K. Maji, D. Haldar, M. G. B. Drew, A. Banerjee, A. K. Das
and A. Banerjee, Tetrahedron, 2004, 60, 3251; (c) S. K. Maji,
D. Haldar, A. Banerjee and A. Banerjee, Tetrahedron, 2002,
58, 8695; (d) S. K. Maji, A. Banerjee, M. G. B. Drew, D. Haldar
and A. Banerjee, Tetrahedron Lett., 2002, 43, 6759; (e) D. Haldar,
27 C. Goldsbury, K. Goldie, J. Pellaud, J. Seelig, P. Frey, S. A. Müller,
J. Kistler, G. J. Cooper and U. Aebi, J. Struct. Biol., 2000,
130, 352.
28 H. H. Bauer, U. Aebi, M. Haner, R. Hermann, M. Muller,
T. Arvinte and H. P. Merkle, J. Struct. Biol., 1995, 115, 1.
29 H. A. Lashuel, S. R. Labrenz, L. Woo, L. C. Serpell and
J. W. Kelly, J. Am. Chem. Soc., 2000, 122, 5262.
10578
|
CrystEngComm, 2013, 15, 10569–10578
This journal is © The Royal Society of Chemistry 2013