Peptidomimetic design of unusual turns by incorporating flexible and rigid ω-amino acids simultaneously

Article abstract of DOI:10.1016/j.molstruc.2009.10.029

The tripeptides Boc-Gly-Aib-m-ABA-OMe (I), Boc-βAla-Aib-m-ABA-OMe (II) and Boc-γAbu-Aib-m-ABA-OMe (III) (Aib: α-aminoisobutyric acid, βAla: β-alanine, γAbu: γ-aminobutyric acid, m-ABA: meta-aminobenzoic acid) with homologated amino acids at the N-terminus

Full text of DOI:10.1016/j.molstruc.2009.10.029

Journal of Molecular Structure 963 (2010) 160–167
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Peptidomimetic design of unusual turns by incorporating flexible and rigid
x
-amino acids simultaneously
Sudeshna Kar a, Michael G.B. Drew b, Animesh Pramanik a,
*
a Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata, West Bengal 700 009, India
b School of Chemistry, The University of Reading, Whiteknights, Reading RG6 6AD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The tripeptides Boc-Gly-Aib-m-ABA-OMe (I), Boc-bAla-Aib-m-ABA-OMe (II) and Boc-cAbu-Aib-m-ABA-
Received 11 September 2009
Received in revised form 12 October 2009
Accepted 19 October 2009
Available online 22 October 2009
OMe (III) (Aib:
a-aminoisobutyric acid, bAla: b-alanine,
cAbu:
c
-aminobutyric acid, m-ABA: meta-amino-
benzoic acid) with homologated amino acids at the N-terminus, the rigid
c-amino acid m-ABA at the
C-terminus and the helicogenic Aib at the central position have been chosen to create unusual turns. Sin-
gle crystal X-ray diffraction studies, solvent dependent NMR titrations and 2D NMR analysis reveal that
peptides II and III adopt unusual turns of 11- and 12-membered rings stabilized by modified 4 ? 1 type
intramolecular hydrogen bonds. Solution phase studies indicate that peptide I exists in the b-turn confor-
mation stabilized by 10-membered intramolecular hydrogen bonding.
Keywords:
Unusual turn
x
-Amino acids
Ó 2009 Elsevier B.V. All rights reserved.
a-Aminoisobutyric acid
Peptidomimetic design
1. Introduction
of acyclic and cyclic chiral b-amino acids [22–25], and 14-helical
structures in cyclic and linear chiral b-amino acids [26–30]. Het-
The interest in the conformational properties of oligopeptides
formed by -amino acids has been stimulated by the recognition
ero-oligomers of a- and/or c-substituted c-amino acids show 14-
helical conformations in solution as evident from their NMR studies
x
that new classes of folded structures can be formed by homoolig-
omers of backbone homologated amino acids [1,2]. Since these
[31,32]. Seebach’s group has shown the formation of 2.614-helical
structures in crystals and in solution for
c-peptides comprising of
amino acids have extra ACH2A unit(s) compared to
a
-amino acids,
2-, 3-, and 4-substituted chiral -amino acids [33]. In most cases
c
they can modify the geometry of the peptide backbones [3] and
unusual turns are exhibited by substituted chiral residues, which
provide proteolytic resistance to bioactive peptide sequences [4].
are conformationally restricted. Designs of turns with unsubstituted
Several reports of incorporation of
peptides originally composed of
the literature [5]. The -amino acids such as
Abu), a neurotransmitter enzymatically produced [6] in the
x
-amino acids into bioactive
-amino acids are reported in
-aminobutyric acid
bAla and
c
Abu, which are more flexible, have also been reported
a
[17,18]. Peptides incorporating a centrally positioned bAla–
segment have been found to exhibit unusual turns involving e.g.
12-, 14-, 16-, and 19-membered hydrogen bonded rings [14].
cAbu
x
c
(c
mammalian brain [7] and b-alanine (bAla), which occurs widely
in the animal and plant kingdoms [8–12], have been used in pep-
tide design to create unusual foldamers [13–20].
Reverse turns play important roles in stabilizing secondary and
tertiary structures, initiating folding and facilitating intermolecular
meta-Aminobenzoic acid (m-ABA) is considered as a rigid c-ami-
nobutyric acid with an all-trans extended conformation suitable for
promoting a b-sheet-like structures [34–37]. In this paper, we are
interested to explore the possibility of generating unusual turns in
small peptides by inserting both flexible
as well as rigid -amino acid (m-ABA) simultaneously. Therefore we
have chosen peptides Boc-Gly-Aib-m-ABA-OMe (I) (Aib, -amino-
isobutyric acid), Boc-bAla-Aib-m-ABA-OMe (II) and Boc- Abu-Aib-
x-amino acids (bAla, cAbu)
recognition [21]. Generally in a polypeptide chain constituted of
amino acids, the ith C@O is hydrogen bonded to the i + 2 NH to form
-turn (7-membered), i + 3 NH to form a b-turn (10-membered),
and i + 4 NH to form an -turn (13-membered). Insertion of one or
a
-
x
a
c
a
c
a
m-ABA-OMe (III) (Fig. 1). Insertion of one or more extra carbon
more extra carbon atom(s) into the intramolecular hydrogen
bonded, folded structures leads to the creation of unusual turns
and novel helical folds. As for examples helical structures with 12-
membered hydrogen bonded rings have been observed in oligomers
atom(s) in the sequence of peptides II and III has been carried out
by changing the N-terminal Gly of peptide I with bAla and cAbu.
The centrally placed helicogenic Aib is expected to provide the turn
structure in the peptide backbone [38–47]. Peptide I which contains
two a-amino acids and only one x-amino acid, m-ABA at the C-ter-
minus has the highest potentiality among the three peptides to form
* Corresponding author. Fax: +91 33 2351 9755.
a turn-like structure. We wanted to establish whether the insertion
E-mail address: animesh_in2001@yahoo.co.in (A. Pramanik).
0022-2860/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2009.10.029

S. Kar et al. / Journal of Molecular Structure 963 (2010) 160–167
161
O
O
with diethyl ether. The aqueous layer was cooled in ice-bath, neu-
tralized by using 2 N HCl and extracted with ethyl acetate. The sol-
vent was evaporated in vacuo to give a waxy colorless solid. Yield:
2.2 g (85.93%).
H
N
H
N
O
N
H
OMe
O
O
O
PeptideI
O
O
2.1.3. Boc-Gly-Aib-m-ABA-OMe (peptide I)
θ1
φ
ψ
H
N
Peptide 2 (2.2 g, 8.46 mmol) was dissolved in dichloromethane
(DCM, 5 ml). H-mABA-OMe obtained from its hydrochloride
(3.17 g, 16.92 mmol) was added to former solution, followed by
addition of DCC (2.61 g, 12.69 mmol) and HOBT (1.14 g,
8.46 mmol) in ice-cold condition. The reaction mixture was stirred
at room temperature for 2 days. The precipitated DCU was filtered
off. The organic layer was diluted with 30 ml ethyl acetate and
washed with 1 N HCl (3 ꢀ 30 ml), brine, 1 M Na2CO3 solution
(3 ꢀ 30 ml) and then again with brine. The solvent was then dried
over anhydrous Na2SO4 and evaporated in vacuo, to give a colorless
solid compound. Purification was done using silica gel as stationary
phase and ethyl acetate–petroleum ether mixture (1:4) as eluent.
Yield: 2.8 g (84.33%). Mp = 168 °C; IR (KBr): 3352, 3284.6,
O
N
N
H
OMe
H
O
II
Peptide
O
O
θ1
φ
ψ
θ2
H
H
N
O
N
N
H
OMe
O
O
PeptideIII
Fig. 1. Schematic diagram of peptides I–III.
of flexible and rigid
x-amino acids simultaneously as in peptides II
and III can lead to the creation of unusual turns and novel helical
folds through intramolecular hydrogenbonding. There are examples
of constrained cyclic peptides in which substituted benzenes have
been inserted to mimic the turn region of the neurotrophin, a nerve
growth factor [48]. Peptides I–III will be examples of acyclic ana-
logues if they adopt turn structures. All the peptides were prepared
by conventional solution phase synthesis and their conformational
studies have been carried out by single crystal X-ray diffraction
and NMR studies.
2973.1, 2930.7, 1719.8, 1676.3, 1541.5 cmꢁ1 1H NMR 300 MHz
;
(CDCl3, d ppm): 9.16 (m-ABA(3)-NH, 1H, s), 8.23 (Ha m-ABA(3),
1H, s), 7.97(Hd m-ABA(3), 1H, d, J = 7.8 Hz), 7.75 (Hb m-ABA(3),
1H, d, J = 7.9 Hz), 7.37 (Hc m-ABA(3), 1H, m), 6.60 (Aib(2)-NH, 1H,
a
s), 5.34 (Gly(1)-NH, 1H, s), 4.30 (C H of Gly(1), 2H, t, J = 6.6 Hz),
3.89 (AOCH3, 3H, s), 1.62 and 1.25 (CbH of Aib(2), 6H, s), 1.46
(Boc-CH3s, 9H, s); 13C NMR 75 MHz (CDCl3, d ppm): 172.48,
169.61, 166.87, 138.69, 130.89, 130.63, 128.85, 126.34, 125.07,
124.53, 128.85, 126.34, 125.07, 124.53, 121.03, 81.14, 58.08,
52.06, 28.18, 25.50; Anal. Calcd for C19H27N3O6 (393.43): C,
57.99; H, 6.91; N, 10.68%; Found: C, 57.91; H, 6.84; N, 10.60%.
2. Experimental
2.1. Synthesis of the peptides
2.1.4. Boc-bAla-Aib-OMe (3)
Peptide 3 was synthesized following the same procedure as that
of peptide 1, starting with Boc-bAla-OH (2.0 g, 10.57 mmol). Yield:
2.6 g (85.52%).
The peptides were synthesized by conventional solution phase
methods. The tert-butyloxycarbonyl (Boc) group was used for N-
terminal protection and the C-terminus was protected as methyl
ester. Couplings were mediated by N,N0-dicyclohexylcarbodiimide
(DCC)/1-hydroxybenzotriazol (HOBT) [49]. All intermediates were
characterized by thin layer chromatography on silica gel and used
without further purification. Final peptides were purified by col-
umn chromatography using silica gel (100–200 mesh) as the sta-
tionary phase. Ethyl acetate and petroleum ether mixture was
used as the eluent. The peptides II and III were fully characterized
by X-ray crystallography and NMR studies and peptide I with NMR
spectroscopy.
2.1.5. Boc-bAla-Aib-OH (4)
Peptide 4 was synthesized following the same procedure as that
of peptide 2. Yield: 2.1 g (85.02%).
2.1.6. Boc-bAla-Aib-m-ABA-OMe (peptide II)
Synthesis and purification of peptide II were done following the
same procedure as in the case of peptide I starting with peptide 4.
Single crystals of peptide II were grown from CHCl3–petroleum
ether by slow evaporation and were stable at room temperature.
Yield: 2.7 g (86.53%). Mp = 108 °C; IR (KBr): 3357.9, 3322.6,
2981.9, 1710.3, 1668.5, 1532.2 cmꢁ1 1H NMR 300 MHz (CDCl3, d
;
2.1.1. Boc-Gly-Aib-OMe (1)
Boc-Gly-OH (2.0 g, 11.42 mmol) was dissolved in a mixture of
dichloromethane (DCM, 6 ml) and dimethylformamide (DMF,
5 ml). H-Aib-OMe obtained from its hydrochloride (3.5 g,
22.84 mmol) was added to the former solution followed by addi-
tion of DCC (3.52 g, 17.13 mmol) and HOBT (1.54 g, 11.42 mmol)
in ice-cold condition. The reaction mixture was stirred at room
temperature for 1 day. The precipitated N,N0-dicyclohexylurea
(DCU) was filtered off. The organic layer was diluted with 30 ml
ethyl acetate and washed with 1 N HCl (3 ꢀ 30 ml), brine, 1 M
Na2CO3 solution (3 ꢀ 30 ml) and then again with brine. The solvent
was dried over anhydrous Na2SO4 and evaporated in vacuo to give a
waxy colorless solid. Yield: 2.7 g (86.26%).
ppm): 9.32 (m-ABA(3)-NH, 1H, s), 8.14 (Ha m-ABA(3), 1H, s), 7.77
(Hd m-ABA(3), 1H, d, J = 8.1 Hz), 7.75 (Hb m-ABA(3), 1H, d,
J = 7.9 Hz), 7.37 (Hc m-ABA(3), 1H, m), 6.4 (Aib(2)-NH, 1H, s), 5.10
(bAla(1)-NH, 1H, s), 3.90 (AOCH3, 3H, s), 3.39–3.45 (CbH of bAla(1),
a
2H, m), 2.35–2.49 (C H of bAla(1), 2H, m), 1.62 (CbH of Aib(2), 6H,
s), 1.42 (Boc-CH3s, 9H, s); 13C NMR 75 MHz (CDCl3, d ppm): 173.02,
172.45, 166.80, 156.42, 138.53, 130.78, 128.94, 125.16, 124.61,
121.04, 79.83, 58.57, 52.11, 37.48, 36.96, 28.34, 25.36; Anal. Calcd
for C20H29N3O6 (407.47): C, 58.90; H, 7.17; N, 10.31%; Found: C,
58.82; H, 7.08; N, 10.25%.
2.1.7. Boc-cAbu-Aib-OMe (5)
Peptide 5 was synthesized following the same procedure as that
2.1.2. Boc-Gly-Aib-OH (2)
of peptide 1, starting with Boc-cAbu-OH (2.0 g, 9.84 mmol). Yield:
Peptide 1 (2.7 g, 9.85 mmol) was dissolved in methanol (20 ml)
and 2 N NaOH (10 ml) was added to it. The reaction mixture was
stirred for 1 day at room temperature. The progress of reaction
was monitored by TLC. After completion of reaction the methanol
was evaporated. The residue was diluted with water and washed
2.6 g (87.54%).
2.1.8. Boc-cAbu-Aib-OH (6)
Peptide 6 was synthesized following the same procedure as that
of peptide 2. Yield: 2.2 g (89.06%).

162
S. Kar et al. / Journal of Molecular Structure 963 (2010) 160–167
2.1.9. Boc-
c
Abu-Aib-m-ABA-OMe (peptide III)
a = 9.677(1) Å, b = 24.207(4) Å, c = 9.953(1) Å, b = 104.03(1)°,
V = 2261.8(5) Å3. Diffraction data for the two peptides II and III,
were obtained with MoKa radiation at 150 K using the Oxford Dif-
Synthesis and purification of peptide III were done following
the same procedure as in the case of peptide I starting with peptide
6. Single crystals of peptide III were grown from CHCl3–petroleum
ether by slow evaporation and were stable at room temperature.
Yield: 2.8 g (87.22%). Mp = 134 °C; IR (KBr): 3345.1, 3288.5,
fraction 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 pro-
gram [51]. The structures were solved and refined using direct
methods with the SHELXS-97 programs [52]. The non-hydrogen
atoms were refined with anisotropic thermal parameters. The
hydrogen atoms bonded to carbon were included in geometric
positions and given thermal parameters equivalent to 1.2 times
(or 1.5 times for methyl hydrogen atoms) those of the atom to
which they were attached. Crystallographic details have been
deposited at the Cambridge Crystallographic Data Centre, reference
CCDC Nos. are 742973 and 742974.
2979.5, 1675, 1536.1 cmꢁ1 1H NMR 300 MHz (CDCl3, d ppm):
;
9.78 (m-ABA(3)-NH, 1H, s), 8.19 (Ha m-ABA(3), 1H, s), 7.92 (Hd
m-ABA(3), 1H, d, J = 8.1 Hz), 7.73 (Hb m-ABA(3), 1H, d, J = 7.8 Hz),
7.35 (Hc m-ABA(3), 1H, t, J = 7.95), 6.49 (Aib(2)-NH, 1H, s), 4.84
c
(
c
c
c
Abu(1)-NH, 1H, t, J = 6 Hz), 3.88 (AOCH3, 3H, s), 3.13 (C H of
Abu(1), 2H, m), 2.25(C H of c
Abu(1), 2H, m), 1.79 (CbH of
a
Abu(1), 2H, m), 1.61 (CbH of Aib(2), 6H, s), 1.48 (Boc-CH3s, 9H,
s); 13C NMR 75 MHz (CDCl3, d ppm): 173.01, 172.90, 166.90,
157.26, 139.09, 130.65, 128.80, 124.76, 124.46, 120.88, 79.90,
58.13, 52.00, 33.22, 28.39, 26.95, 25.41; Anal. Calcd for
C21H31N3O6 (421.48): C, 59.83; H, 7.41; N, 9.97%; Found: C,
59.76; H, 7.32; N, 9.88%.
3. Results and discussion
3.1. Peptides conformations in the crystal state
2.2. FT-IR spectroscopy
Although we were able to grow single crystals of peptides II and
III suitable for X-ray diffraction studies, we were not able to gener-
ate single crystals for peptide I. Peptide Boc-bAla-Aib-m-ABA-OMe
(II) crystallizes in the centrosymmetric space group P21/n with two
molecules in the crystallographic asymmetric unit designated as A
and B together with one molecule of solvent chloroform. Both pep-
tide molecules adopt turn structures (Fig. 2). The insertion of one
extra CH2 group into the peptide backbone due to the presence
of b-Ala causes an unusual, modified 4 ? 1 type hydrogen bond be-
tween CO of the Boc group and m-ABA(3)-NH forming 11-mem-
bered hydrogen bonded rings. Since peptide II is achiral the
choice of the signs of the backbone torsion angles is arbitrary
(Table 1). Inspection of the torsion angles shows that molecules
A and B are structurally similar where the centrally placed Aib
IR spectra were examined using a Perkin-Elmer-782 model
spectrophotometer. The solid-state FT-IR measurements were per-
formed using the KBr disk technique.
2.3. NMR experiments
All 1H NMR and 13C NMR studies were recorded on a Bruker
Avance 300 model spectrometer operating at 300, 75 MHz, respec-
tively. The peptide concentrations were 10 mM in CDCl3 for 1H
NMR and 40 mM in CDCl3 for 13C NMR. Solvent titration experi-
ments were carried out at a concentration of 10 mM in CDCl3 with
gradual addition of d6-DMSO from 0% to 10% v/v approximately.
Resonance assignments were done using two-dimensional ROSEY
spectra [50].
adopts helical conformations (/,
w
:
53.3(3)°, 38.3(3)° in A,
angles of bAla of both
59.0(3)°; 36.1(3)° in B) (Table 1). The / and
w
2.4. Crystal data for peptides II and III
molecules are in the semi-extended region. The values of the tor-
sion angles (h1) about the ACH2ACH2A unit of bAla are slightly
higher than the ideal values of gauche conformations, e.g. +60°
and ꢁ60° (Table 1). In all cases the strong intramolecular hydrogen
bonds (N(10)AH(10). . .O(2)@C(2)) characterized by short dis-
tances (2.917(3) Å in A, 2.975(3) Å in B), are well complemented
Peptide II: C20H29N3O6, 0.5CHCl3, M = 467.15, monoclinic, space
group
P21/n,
Z = 8,
a = 12.3329(8) Å,
b = 27.6445(12) Å,
c = 14.6166(5) Å, b = 97.987(4)°, V = 4935.0(4) Å3. Peptide III:
C21H31N3O6, M = 421.49, monoclinic, space group P21/c, Z = 4,
Fig. 2. ORTEP diagrams of peptide II (A and B) with atom numbering scheme. Thermal ellipsoids are shown at the 30% probability. Hydrogen bonds are shown as dotted lines.

S. Kar et al. / Journal of Molecular Structure 963 (2010) 160–167
163
Table 1
Selected backbone torsion angles (°) for peptides II and III.a
Peptide
Residue
/
h1
h2
w
x
IIA
bAla(1)
Aib(2)
92.5(3)
53.3(3)
ꢁ84.5(3)
–
98.9(3)
38.3(3)
169.8(2)
171.9(2)
IIB
III
bAla(1)
Aib(2)
99.7(3)
59.0(3)
ꢁ75.0(3)
–
–
89.6(3)
36.1(3)
166.6(2)
175.2(3)
–
c
Abu(1)
114.2(2)
50.8(2)
ꢁ60.7(2)
ꢁ62.4(2)
142.2(1)
41.8(2)
169.1(1)
ꢁ174.6(1)
Aib(2)
–
–
a
Peptides II and III are achiral and therefore the choice of the signs of the backbone torsion angles is arbitrary.
by the NAH. . .O@C angles of 156° in A, 153° in B to form stable
11-membered unusual turns (Fig. 2 and Table 2). Interestingly a
previous report shows that peptide Boc-bAla-Aib-bAla-OMe where
Table 2
Hydrogen bonds parameters for peptides II and III.
0
0
Type
O. . .HAN/(°)
N. . .O/(AÅ)
H. . .O/(AÅ)
m-ABA of peptide II has been replaced by flexible
x-amino acid
Peptide IIA
Intramolecular
N(10A)AH(10A). . .O(2A)
bAla adopts a fully extended conformation and does not possess
any intramolecular hydrogen bond [38]. The result indicates that
2.917(3)
2.11
156
when the third residue is a flexible
x-amino acid like bAla in the
Intermolecular
above sequence, the centrally placed Aib can not dictate its stereo-
chemical preference fully to produce a turn structure through
cooperative steric interactions amongst the amino acid residues.
But replacement of bAla with rigid m-ABA as in peptide II helps
sterically to form turn structure. Peptide II provides the first exam-
ple of the formation of an 11-membered unusual turn stabilized by
an intramolecular hydrogen bond in a tripeptide containing bAla at
the N-terminus. The conformational heterogeneity as it is observed
in case of peptide II (A and B) in the solid state has biological
implications since interconversion between b-turn types have been
observed in HIV protease [53].
N(3A)AH(3A). . .O(9B)a
N(7A)AH(7A). . .O(6B)b
2.852(3)
2.874(3)
2.06
2.21
154
134
Peptide IIB
Intramolecular
N(10B)AH(10B). . .O(2B)
2.975(3)
2.19
153
Intermolecular
N(7B)AH(7B). . .O(9A)c
N(3B)AH(3B). . .O(6A)c
2.837(3)
2.890(3)
2.19
2.04
132
169
Peptide III
Intramolecular
N11AH11. . .O2
3.033(2)
2.25
152
The peptide Boc-cAbu-Aib-m-ABA-OMe (III) crystallizes with
Intermolecular
N3AH3. . .O10d
N8AH8. . .O7e
one molecule in the crystallographic asymmetric unit, again in a
centrosymmetric space group (P21/c). The crystal structure reveals
that it adopts an unusual turn structure stabilized by 12-mem-
bered 4 ? 1 type hydrogen bond between the C(2)@O(2) of the
Boc group and the N(11)AH(11) of m-ABA(3) (Fig. 3). Like peptide
3.003 (2)
2.977(2)
2.18
2.31
160
135
Symmetry elements: a1 ꢁ x, 1 ꢁ y, 1 ꢁ z; ½ ꢁ x, ½ + y, ½ ꢁ z; c1 ꢁ x, 1 ꢁ y, ꢁz;
dx ꢁ 1, y, z; ex, ½ ꢁ y, ½ + z.
b
Fig. 3. ORTEP diagram of peptide III with atom numbering scheme. Thermal ellipsoids are shown at 30% probability. Hydrogen bond is shown as dotted line.

164
S. Kar et al. / Journal of Molecular Structure 963 (2010) 160–167
II, peptide III is also achiral and therefore the choice of the signs of
the backbone torsion angles is arbitrary (Table 1). The centrally
placed Aib is found to adopt a helical conformation with / values
as 50.8(2)°, 41.8(2)°, similar to the values observed in II. The /
10
9
and
region, 114.2(2)°, 142.2(1)°. The torsion angles h1, h2 about the
ACH2ACH2ACH2A unit of Abu are in gauche conformation,
w angles of cAbu in peptide III are found to be in the extended
Gly(1)-NH
Aib(2)-NH
c
ꢁ60.7(2)°, ꢁ62.4(2)°. This disposition facilitates the easy accom-
modation of the CH2 groups into the folded peptide backbone lead-
ing to an unusual 12-membered hydrogen bonded ring in peptide
III. This strong intramolecular hydrogen bond from N(11)AH(11)
to O(2)@C(2) is characterized by the short distance 3.033(2) Å
(N. . .O), well complemented by the NAH. . .O angle of 152° to form
a stable 12-membered ring (Fig. 3 and Table 2). Generation of 12-
membered reverse turns has also been observed previously in syn-
8
m
-ABA(3)-NH
7
6
thetic peptides containing cAbu at the N-terminus [18] and also in
peptides with centrally positioned, sterically constrained dinipe-
cotic acid segments [54,55]. Peptide III provides a unique example
in which a 12-membered turn has been stabilized in a tripeptide in
0
2
4
6
8
10
% of DMSO-d6 inCDCl3(v/v)
spite of having two
x-amino acids in the sequence.
The molecular packing arrangements of peptides II and III are
shown in Figs. 4 and 5. Each molecule A of peptide II is linked with
three neighbouring B molecules through intermolecular hydrogen
bonding. These interactions involve bAla(B)-NH. . .O@C-bAla(A),
Aib(B)-NH. . .O@C-Aib(A), Aib(B)-C@O. . .HN-bAla(A) and Aib(A)-
NH. . .O@C-bAla(B) (Fig. 4 and Table 2). The same is true for each
B molecule which interacts with three A molecules in a similar
fashion. The molecules of peptide II are interlinked in ac plane to
form two-dimensional sheet-like structure (Fig. 4). Each molecule
of peptide III is linked with four neighbouring molecules through
Fig. 6. NMR solvent titration curves for NH protons in peptide I.
one hydrogen bond to each such as
cAbu-NH. . .O@C-Aib and
Aib-NH. . .O@C- Abu resulting in a two-dimensional sheet-like
c
structure in ac plane (Fig. 5 and Table 2).
3.2. Solution phase conformations
In absence of a crystal structure the solution phase conforma-
tion of the peptide Boc-Gly-Aib-m-ABA-OMe (I) was probed by
Fig. 7. Schematic representation of b-turn structure of peptide I stabilized by 10-
membered intramolecular hydrogen bond.
10
9
β-Ala(1)-NH
Fig. 4. The packing arrangement of peptide II in ac plane. Intermolecular hydrogen
bonds are shown as dotted lines. Hydrogen atoms are omitted for clarity.
Aib(2)-NH
8
m-ABA(3)-NH
7
6
5
0
2
4
6
8
10
% of DMSO-d6 inCDCl3(v/v)
Fig. 5. The packing arrangement of peptide III in the ac plane forming b-sheet-like
structure. Hydrogen bonds are shown as dotted lines. Hydrogen atoms are omitted
for clarity.
Fig. 8. NMR solvent titration curves for NH protons in peptide II.

S. Kar et al. / Journal of Molecular Structure 963 (2010) 160–167
165
Table 3
10
9
Important inter-residue NOEs for peptides II and III.
γ-Abu(1)-NH
Peptide II
Peptide III
Aib(2)-NH
mABA(3)NH M Aib(2)CbH
mABA(3)NH M Aib(2)CbH
m-ABA(3)-NH
Aib(2)CbH M Aib(2)NH
Aib(2)CbH M Aib(2)NH
a
a
Aib(2)NH M bAla(1)C H
Aib(2)NH M
c
c
Abu(1)C H
a
bAla(1)C H M bAla(1)NH
Aib(2)NH M
Abu(1)CbH
8
bAla(1)NH M bAla(1)CbH
c
c
c
Abu(1)C H M
c
c
Abu(1)CbH
a
a
c
Abu(1)C H M
Abu(1)C H
Abu(1)C H
Abu(1)CbH M
c
c
7
0.14 ppm, respectively (Fig. 8), indicating a turn structure where
m-ABA(3)-NH is hydrogen bonded to Boc-CO as it is observed in
the crystal structure (Fig. 2). In a similar experiment the solvent
dependence of the NH chemical shifts, that is demonstrated in this
CDCl3-(CD3)2SO titration experiment of peptide III (Fig. 9), indi-
6
5
cates that
1.02 and 0.71 ppm, respectively, and m-ABA(3)-NH is intramolecu-
larly hydrogen bonded with
cAbu(1)-NH and Aib(2)-NH are free with Dd values as
0
2
4
6
8
10
% of DMSO-d6 inCDCl (v/v)
3
D
d value as ꢁ0.01 ppm, indicating a
turn structure as depicted in the crystal structure (Fig. 3).
Fig. 9. NMR solvent titration curves for NH protons in peptide III.
The formation of unusual turns by peptides II and III in the solu-
tion phase was further confirmed by 2D NMR analysis. Several in-
ter-residue NOEs for peptides II and III are listed in Table 3.
Furthermore, the ROESY spectrum [50] of peptide II in CDCl3
(Fig. 10) reveals two important inter-residue NOEs, namely, mA-
the NMR solvent titration method. In this experiment a solution of
peptide I in nonpolar CDCl3 (10 mM in 0.5 ml) was gradually ti-
trated against polar (CD3)2SO and the changes in the chemical
shifts of NHs were recorded by 1H NMR (Fig. 6) [56–58]. The sol-
vent titration shows that by increasing the percentage of (CD3)2SO
a
BA(3)NH M Aib(2)CbH and Aib(2)NH M bAla(1)C H. This NOE data
and the solvent-shielded nature of mABA(3)-NH are supportive of a
11-membered hydrogen bonded ring involving C@O from the Boc
group and mABA(3)-NH for peptide II in solution. Several inter-res-
idue key NOEs have been found for peptide III in CDCl3 (Fig. 11).
in CDCl3 from 0 to 10% the net changes in the chemical shift (
values for Gly(1)-NH, Aib(2)-NH and m-ABA(3)-NH are 0.89, 0.56
and 0.11 ppm, respectively. The d values demonstrate that m-
Dd)
D
The NOEs of mABA(3)NH M Aib(2)CbH and Aib(2)NH M
cAbu(1)-
ABA(3)-NH is solvent shielded, and the other two NH groups are
solvent exposed, which is a characteristic feature of a b-turn struc-
ture where the m-ABA(3)-NH is involved in an intramolecular
hydrogen bond to Boc-CO (Fig. 7). In 1H NMR solvent titration of
a
C H and the solvent-shielded nature of mABA(3)-NH are indicative
of an unusual turn involving a 12-membered hydrogen bonded
ring, with C@O from the Boc group and mABA(3)-NH. Thus, the
crystallographic and NMR data for both peptides II and III are in
mutual agreement.
peptide II the net changes in the chemical shift (
Dd) values for
bAla(1)-NH, Aib(2)-NH and m-ABA(3)-NH are 0.67, 0.99 and
Fig. 10. Partial view of 1H–1H ROESY spectrum of peptide II in CDCl3 (peptide concn: 1 ꢀ 10ꢁ2 M), showing only d N(i, i + 1), where i = 1 and dbN(i, i + 1), where i = 2
a
crosspeaks, diagnostic of turn structure.

166
S. Kar et al. / Journal of Molecular Structure 963 (2010) 160–167
Fig. 11. Partial view of 1H–1H ROESY spectrum of peptide III in CDCl3 (peptide concn: 1 ꢀ 10ꢁ2 M), showing only d N(i, i + 1), where i = 1 and dbN (i, i + 1), where i = 1 and 2,
a
crosspeaks, diagnostic of turn structure.
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4. Conclusions
It has been shown that the incorporation of flexible
acids such as bAla, Abu at the N-terminus and rigid -amino acid
such as m-amino benzoic acid at the C-terminus in acyclic tripep-
tides with centrally placed helicogenic Aib can lead to the forma-
tion of unusual turns of 11- and 12-membered rings stabilized
by modified 4 ? 1 type intramolecular hydrogen bonds. m-amino
x-amino
c
c
benzoic acid, a rigid c-aminobutyric acid, in spite of having high
propensity for b-sheet formation, has been nicely accommodated
into the turn structures generated by peptides I–III. The conforma-
tional heterogeneity as it is observed in case of peptide II with two
isomeric structures provides insights for designing bioactive pep-
tides of conformationally flexible turns. Peptides II and III repre-
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Soc. 119 (1997) 9087.
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sent
a potentially useful way for peptidomimetic design of
unusual turns which will help to develop biologically active pep-
tides resistant to proteolytic hydrolysis.
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Chem. 67 (2002) 633.
[19] A. Sengupta, R.S. Roy, V. Sabareesh, N. Shamala, P. Balaram, Org. Biomol. Chem.
4 (2006) 1166.
Supporting informations
[20] A. Dutt, M.G.B. Drew, A. Pramanik, Org. Biomol. Chem. 3 (2005) 2250.
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[22] D.H. Appella, L.A. Christianson, D.A. Klein, M.R. Richards, D.R. Powell, S.H.
Gellman, J. Am. Chem. Soc. 121 (1999) 7574.
[23] D.H. Appella, L.A. Christianson, D.A. Klein, D.R. Powell, X. Huang, S.H. Gellman,
Nature 387 (1997) 381.
Supplementary data contains 1H NMR of peptides I–III, 1H–1H-
ROESY spectra of peptides II and III and crystallographic data in CIF
formats of peptides II and III.
[24] X. Wang, J.F. Espinosa, S.H. Gellman, J. Am. Chem. Soc. 122 (2000) 4821.
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Acknowledgements
S.K. thanks CSIR, New Delhi, India, for a senior research fellow-
ship (SRF). The financial assistance of UGC, New Delhi is acknowl-
edged [Major Research Project, No. 32-190/2006(SR)]. We
acknowledge the financial assistance of Centre for Research in
Nanoscience & Nanotechnology, University of Calcutta. We thank
EPSRC and the University of Reading, UK for funds for Oxford Dif-
fraction X-Calibur CCD diffractometer.
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