Conformational studies of γ-turn in pseudopeptides containing α-amino acid and conformationally constrained meta amino benzoic acid/meta nitro aniline

Article abstract of DOI:10.1016/j.molstruc.2012.11.001

Reverse turns (commonly β-turns and γ-turns), a common motif in proteins and peptides, have attracted attention due to their relevance in a wide variety of biological processes. In an attempt to artificially imitate and stabilize these turns in short acyc

Full text of DOI:10.1016/j.molstruc.2012.11.001

Journal of Molecular Structure 1036 (2013) 350–360
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Conformational studies of c-turn in pseudopeptides containing a-amino acid
and conformationally constrained meta amino benzoic acid/meta nitro aniline
⇑
Anita Dutt Konar
Department of Chemical Sciences, Indian Institute of Science Education and Research, Bhopal, ITI Gas Rahat Building, Govindpura, Bhopal 462 023, India
h i g h l i g h t s
"
"
"
"
Pseudopeptides I & II with appropriate N & C terminal protecting groups adopt
c
-turn conformation.
-turn in solution.
c-turn stabilization.
Pseudopeptides III–V displaying extended conformation in solid state forms
c
Importance of steric interactions amongst amino acid residues is crucial for
The work may open a new avenue in introducing c-turn within the bioactive conformation of peptides.
a r t i c l e i n f o
a b s t r a c t
Article history:
Reverse turns (commonly b-turns and c-turns), a common motif in proteins and peptides, have attracted
attention due to their relevance in a wide variety of biological processes. In an attempt to artificially imi-
tate and stabilize these turns in short acyclic peptides, a series of N-terminally protected pseudopeptides
comprising of an a-amino acid and conformationally constrained meta amino benzoic acid (mABA)/meta
nitro aniline (mNA) (peptides I–VI) have been synthesized. The molecules were well characterized by var-
ious spectroscopic techniques and subjected to a systematic conformational analysis. Our experimental
results reveal that only pseudopeptides I and II with methyl as the sidechain, tertiary butyloxy carbonyl
Received 15 September 2012
Received in revised form 1 November 2012
Accepted 1 November 2012
Available online 23 November 2012
Keywords:
Pseudopeptides
as the N-terminal protecting group and (mABA)/(mNA) at the C-terminus adopt
solid state as well as in solution. Even slight modification of any of the stated conditions donot support
the formation of this -turn architecture in the solid state. Interestingly, the peptides III–V which displays
extended conformation in solid state forms -turn structure in solution. Thus this result reflects the
c-turn conformations in
c-Turn
Alanine
c
Protecting groups
m-Amino benzoic acid
mNA
c
importance of co-operative steric interactions amongst various amino acid residues in stabilizing a par-
ticular conformation in peptides in different phases (solid and solution). This report may open a new ave-
nue in introducing
c
-turn motifs within the bioactive conformation of selected peptides.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
roles in various biological processes. Structural studies have
revealed that naturally occurring peptides such as vasopressin
and the related desmopressin, bradykinin, and angiotensin II,
that function as hormones or neurotransmitters, or have other
In proteins,
c-turn consist of three amino acids, which may
or may not be stabilized by an intramolecular hydrogen bond
between the C@O of the first residue (i) and the NH of the
regulatory roles in organisms, adopt
[4–7]. The -turn present in vitronectin has been reported to
contribute to the specific recognition by integrin receptor
vb3 playing a role in tumor cell adhesion angiogenesis, and
osteoporosis [8,9]. -Turn mimetic inhibitor forms complex with
c-turn conformations
third residue (i + 2), forming
a
seven-membered ring [1,2].
c
Depending on whether the side chain of the residue i + 1 is
in an equatorial or axial orientation on the seven-membered
a
ring,
giving rise to a kink in the chain or a direction change [3].
Although less recurrent than b-turn, -turns play important
c
-turns are classified as inverse or classical, respectively
c
HIV-I protease which provides information about structure–
activity relationship and helps in rational drug design [10]. It
c
Abbreviations: mABA, m-amino benzoic acid; mNA, m-nitro aniline; Boc, tertiary butyloxy carbonyl; OMe, methoxy carbonyl; DCC, dicyclohexylcarbodiimide; HOBt, 1-
hydroxy benzotriazole.
⇑
Tel.: +91 7554056278; fax: +91 7554092392.
E-mail address: anita@iiserb.ac.in
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.11.001

A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
351
is also proposed that in proteins, hydrated
helix-coil unfolding [11].
c
-turns promote
tides comprising of alternating
a
-aminoxy acids and
a
-amino acids
[19]. Martinez and his co-workers has made substantial inputs in
designing -turn mimetics by incorporating 2-alkyl 2-carboxy-
Various investigations suggest that
c-turns seldom exist in
c
short, natural peptides. Initially it was believed that short acyclic
peptides do not have the ability to adopt any preferential confor-
mation. Spectroscopic studies indicate that the conformational
space of tripeptides is more restricted than originally thought so
that structures of limited stability can be formed [12]. Small pep-
tides which are biomedically relevant as protease inhibitors, as
azetidine residue in the i + 1 position of simply modified peptides
and showed that these non-protenogenic amino acids possess a
tremendous ability to nucleate
apparent that various governing factor dictating the overall nucle-
ation of -turn formation remains still in its infancy.
To gain further insight regarding the design of -turns in small
c-turns [20,21]. Therefore, it is
c
c
taste receptors and for enzyme regulation, should maintain
conformation to show the activity [13–17]. Therefore designing
and stabilizing -turn structure in small acyclic peptides is chal-
c
-turn
acyclic peptides and contribution of various residues in the induc-
tion of this motif, we have synthesized pseudopeptides, I–VI
(Table 1, Entry a–f), comprising of an a-amino acid and
c
lenging and has been of great interest recently.
conformationally biased meta amino benzoic acid (mABA)/meta ni-
tro aniline (mNA), with an all trans extended configuration. The
idea was to introduce some conformational constraint on the adja-
cent peptide bond incorporating mABA/mNA which may compel
the carbonyl at the ith position and NH of the i + 2th amino acid
Jimenez et al. have demonstrated the use of D-c3Dip (N0methyl-
2,2-diphenyl-1-amino cyclopropane carboxamide), when coupled
with acetyl L-Pro to constitute the first unequivocal evidence of
the ability of a cyclopropane
a-amino acid to adopt a c-turn dispo-
sition in a non-propitious environment [18]. Y. D. Wu and his
to come closer in the same direction and attain the
c-turn confor-
group has made a seminal contribution in inducing c-turns in pep-
mation. Keeping this view in mind, our design aims in performing a
Table 1
List of pseudopeptides (Entry a–k) with torsion angles (°) of the residues at i + 1.
Entry
Peptides
u1 (°)
75.0
ꢀ79.0
ꢀ105.9(2)
107.7(3)
ꢀ157.3(1)
–
145.3(2)
ꢀ85.4(2)
101.66
62.6
ꢀ65.2
ꢀ139.2
155.8
w1 (°)
Ref.
Idealized classical
c
-turn
ꢀ64.0
[1–3]
[1–3]
Idealized inverse
c
-turn
69.0
a.
b.
c.
d.
e.
f.
Boc-L-Ala-mNAa (I)
95.7(2)
This work
This work
This work
This work
This work
This work
[28]
Boc-D-Ala-mNAa (II)
ꢀ95.0(3)
160.1(1)
–
ꢀ79.5(2)
ꢀ160.8(1)
ꢀ96.31
ꢀ130.6
ꢀ179.6
142.8
177.6
140.2
–
143.11
ꢀ165.30
ꢀ165.27
176.31
ꢀ179.62
Piv-L-Ala-mNAa (III)
Fmoc-L-Ala-mABA-OMeb (IV)
Boc-L-b-cyano-Ala-mABA-OMeb (V)
Boc-Me-Gly-mABA-OMeb (VI)
Boc-L-Ala-mABA-OMeb
Boc-Gly-mABA-OMeb (Mol.A)
(Mol. B)
g.
h.
[29]
i.
j.
k.
l.
m.
n.
o.
p.
q.
Boc-b-Ala-mABA-OMeb
[22]
[22]
[30]
[30]
[31]
[31]
[31]
[32]
[32]
Boc-
c
-Aba-mABA-OMeb
Boc-b-Ala-mNAa
136.4
–
Boc-
c
-Aba-mNAa
Boc-Gly-pNAc
ꢀ60.70
Boc-b-Ala-pNAc
Boc-Pro-pNAc
95.24
55.13
Boc-Pro-mNAa
Boc-Pro-mABA-OMeb
ꢀ68.02
76.58
Aib =
a
aminoisobutyric acid, b-Ala = b-Alanine,
c
-Aba =
c-amino butyric acid.
a
mNA = meta nitro aniline.
b
c
mABA-OMe = meta amino benzoic acid.
pNA = para nitro aniline.
1.
2.
3.
4.
5.
6.
Fig. 1. Schematic representations of Peptides I–VI.

352
A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
systematic conformational analysis of
c
-turn formation in pseudo-
purification. The final peptides were purified by column chroma-
peptides PRO-Xaa-Yaa (PRO; protecting group at the N-terminus,
Xaa; amino acid, Yaa: mABA/mNA) by replacement of various
parameters in the sequences like configuration of the N-terminal
amino acid, different protecting groups and different N-terminal
amino acids. Importantly this investigation will provide more
information about the creation of structural diversities in the pep-
tide backbone depending upon the co-operative steric interactions
amongst the side chains of amino acid residues (Fig. 1).
tography using silica gel (100–200 mesh) as the stationary phase
and ethyl acetate and petroleum ether mixture as the eluent.
2.1.1. Boc-L-Ala-m-NA (I)
Boc-L-Ala-OH 0.95 g (5 mmol) was dissolved in dimethylform-
amide (DMF; 3 mL). 0.68 g (5 mmol) of mNA was added followed
by DCC (1.0 g, 5 mmol). The reaction mixture was stirred at room
temperature for 3 days. The precipitated dicyclohexylurea (DCU)
was filtered and diluted with ethyl acetate (80 mL). The organic
layer was washed with excess of water, 1 N HCl (3 ꢁ 30 mL), 1 M
Na2CO3 solution (3 ꢁ 30 mL) and again with water. The solvent
was then dried over anhydrous Na2SO4 and evaporated in vacuo,
giving a brown solid. Yield: 1.39 g (90%). Single crystals were
grown from a methanol–water mixture by slow evaporation and
were stable at room temperature. Mp = 162–163 °C; LR – MS:
2. Experimental section
2.1. Synthesis of peptides
The dipeptides were synthesised by conventional solution
phase methodology [22]. The t-butyloxycarbonyl and methyl ester
groups were used for amino and carboxyl protections and dicyclo-
hexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBT) as cou-
pling agents. Methyl ester hydrochloride of m-ABA were
prepared by the thionyl chloride-methanol procedure [23]. All
the intermediates obtained were checked for purity by thin layer
chromatography (TLC) on silica gel and used without further
C
14H19N3O5
[M + H]+ = 310.14,
[M + Na]+ = 332.12,
Mcalcd
C
14H19N3O5 [M + H]+ = 310, [M + Na]+ = 332; 1H NMR (400 MHz,
CDCl3, d ppm): 1.47 (CbHs of Ala, 3H, m); 1.49 (Boc-CH3s, 9H, s);
a
4.46–4.48 (C Hs of Ala, 1H, m); 5.44–5.46 (Ala-NH, 1H, d,
J = 8 Hz); 7.30–7.34 ((Hc (m-ABA), 1H, m); 7.70 (Hd (m-ABA), 1H,
d, J = 8 Hz); 7.80 (Hb (m-ABA), 1H, d, J = 8 Hz); 8.38–8.39 (Ha (m-
ABA), 1H, m); 9.46 (m-ABA NH, 1H, s); 13C NMR (100 MHz CDCl3,
Fig. 2. Crystal structure of peptide (a) I, (b) II, (c) III, (e) V, (f) VI with atom numbering scheme.

A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
353
Table 2
Selected torsion angles (°) of peptides I–III, and V–VI.
Residues
u1
w1
x0
u2
w2
x1
h1
178.4(2)
ꢀ178.6(3)
ꢀ179.2(1)
14.3(3)
h2
Peptide I
Peptide II
Peptide III
Peptide V
Peptide VI
ꢀ105.9(2)
107.7(3)
ꢀ157.3(1)
145.3(2)
ꢀ85.4(2)
95.7(2)
178.3(1)
ꢀ34.7(3)
ꢀ0.5(3)
1.2(5)
ꢀ4.1(2)
ꢀ0.3(4)
11.1(2)
ꢀ175.4(2)
175.2(3)
ꢀ178.0(1)
ꢀ177.4(2)
175.8(2)
ꢀ178.7(2)
179.3(3)
179.0(1)
ꢀ177.1(2)
178.3(2)
ꢀ95.0(3)
ꢀ179.3(3)
ꢀ179.0(1)
168.7(2)
171.3(1)
34.9(5)
172.1(1)
14.2(4)
160.1(1)
ꢀ79.5(2)
ꢀ160.8(1)
ꢀ163.8(2)
ꢀ180.0(1)
Table 3
Hydrogen bond parameters for peptides I–III, V and VI.
2.1.4. Fmoc-L-Ala-mABA-OMe (IV)
The peptide IV was synthesised following the similar procedure
as that of peptide I. Yield: 1.07 g (75%). Mp = 165–167 °C; LR – MS:
D-H—A
H—A(Å)
D—A(Å)
D-H—A(°)
C
26H24N2O5 [M + H]+ = 445.18, Mcalcd C26H24N2O5 [M + H]+ = 445;
Peptide I
1H NMR (400 MHz, CDCl3, d ppm): 1.46–1.47 (CbHs of Ala, 3H, d,
J = 8 Hz), 3.82 (Methoxy, 3H, s), 4.15 (Fmoc, Fulvene, 1H, m); 4.42
N3-H3A—O5i
N2-H1A—O5ii
2.16
2.11
2.98
2.93
160.73
171.86
a
(Fmoc-benzylic H, 1H, m), 4.52 (C Hs of Ala, 1H, m), 5.84 (Ala-
NH, 1H, m), 7.30–7.70 (Fmoc and mABA aromatic H’s, 7H, m);
8.09 (Ha (m-ABA), 1H, s); 8.92 (m-ABA NH, 1H, s).
Peptide II
N3-H3—O4i
N2-H9—O3ii
2.28
2.18
2.96
2.99
156.96
161.00
Peptide III
N2-H2A—O4iii
2.04
2.85
171.36
2.1.5. Boc-b-cyano-Ala-mABA-OMe (V)
Peptide V
The peptide V was synthesised starting from Boc-Asn [24] fol-
lowing the similar procedure as that of peptide I. Yield: 1.51 g
(90%). Single crystals were grown from a methanol water mixture
by slow evaporation and were stable at room temperature.
Mp = 150–153 °C; Yield: 1.2 g (80%). Mp = 160–162 °C; LR – MS:
N1-H1A—N2iv
N3-H2A—O4ii
2.11
2.04
3.06
2.85
162.86
171.36
Peptide VI
N2-H1A—O2v
2.11
3.03
164.93
Symmetry codes:
C
17H21N3O5 [M + H]+ = 348.36, Mcalcd C17H21N3O5 [M + H]+ =
a
x ꢀ 1, y, z.
348.36; 1H NMR 400 MHz (CDCl3, d ppm): 1.38 (Boc-MeHs, 9H,
b
x + 1, y, z.
s), 2.19–2.21 (b-CN-Ala CbHs, 2H, m), 3.80 (Methoxy, 3H, s), 4.58
(b-CN-Ala C H, 1H, m), 4.89 (b-CN-Ala NH, 1H, m), 7.66–8.13
c
ꢀx + 2, ꢀy, ꢀz + 1.
a
d
ꢀx + 1, y + 1/2, ꢀz + 1.
e
ꢀx + 1, y ꢀ 1/2, ꢀz + 1/2.
(mABA aromatic H’s, 4H, m)), 9.96 (mABA NH, 1H, s).
2.1.6. Boc-MeGly-mABA-OMe (VI)
d ppm): 17.43, 28.12, 51.52, 80.75, 99.01, 114.29, 117.59, 125.24,
The peptide VI was synthesised following the similar procedure
as that of peptide I. Yield: 1.40 g (90%). Single crystals were grown
from a methanol water mixture by slow evaporation and were sta-
ble at room temperature. Mp = 123–124 °C; LR – MS: C16H22N2O5
128.88, 139.16, 148.04, 156.25,171.51.
[M + H]+ = 323.16,
[M + Na]+ = 345.14,
Mcalcd
C16H22N2O5
2.1.2. Boc-D-Ala-m-NA (II)
[M + H]+ = 323, [M + Na]+ = 345; 1H NMR (400 MHz, CDCl3,
d
The peptide II was synthesised following the similar procedure
as that of peptide I. Yield: 1.24 g (80%). Single crystals were grown
from a methanol water mixture by slow evaporation and were sta-
ble at room temperature. Mp = 157–158 °C; LR – MS: C14H19N3O5
ppm): 1.49 (Boc-CH3s, 9H, s), 3.02 (NMe’s 3H, s), 3.89 (Methoxy,
a
3H, s), 4.0 (C Hs of Gly, 1H, s), 7.35–7.36 ((Hb (m-ABA), 1H, m),
7.74–7.75 (Hc (m-ABA), 1H, m), 7.80–7.81 (Hd (m-ABA), 1H, m);
8.05 (Ha (m-ABA), 1H, s), 8.72 (m-ABA NH, 1H, br); 13C NMR
(100 MHz CDCl3, d ppm): 28.31, 35.69, 51.85, 53.71, 81.06,
120.37, 124.01, 125.37, 128.02, 129.08, 130.11, 137.66, 165.99,
168.11.
[M + H]+ = 310.14,
[M + Na]+ = 332.12,
Mcalcd
C14H19N3O5
[M + H]+ = 310, [M + Na]+ = 332; 1H NMR (400 MHz, CDCl3, d ppm):
1.46–1.47 (CbHs of Ala, 3H, m); 1.49 (Boc-CH3s, 9H, s); 4.44 (C Hs
a
of D-Ala, 1H, m); 5.35 (D-Ala-NH, 1H, d, J = 8 Hz); 7.33–7.36 ((Hc
(m-ABA), 1H, m); 7.71–7.72 (Hd (m-ABA), 1H, m); 7.83 (Hb (m-
ABA), 1H, d, J = 8 Hz); 8.39 (Ha (m-ABA), 1H, s); 9.39 (m-ABA NH,
1H, s); 13C NMR 100 MHz (CDCl3, d ppm): 16.86, 27.82, 50.30,
80.74, 113.96, 117.61, 124.59, 128.88, 138.87, 147.73, 155.92,
171.18.
3. Results and discussion
3.1. Solid state structures of peptides
Colorless crystal, suitable for X-ray diffraction analysis was ob-
tained from methanol–water solution of peptides I–III, V and VI by
slow evaporation method. The crystal structure of peptide I, Boc-
Ala-mNA reveal that the molecule adopts a folded conformation
corresponding to a slightly distorted inverse c-turn structure with
L-Ala occupying the i + 1 position (Fig. 2a). Interestingly, the crystal
structure of peptide II, that contains D-Ala at the N-terminal posi-
tion of peptide I exhibits a classical c-turn almost unnoticed in acy-
clic peptides. Although there are some examples of constrained
cyclic peptides by inserting o-substituted benzenes to mimic the
turn regions of neurotrophin, a nerve growth factor, peptides I
and II present two novel examples where conformationally re-
stricted nitro gamma amino moieties has been incorporated in
2.1.3. Piv-L-Ala-m-NA (III)
The peptide III was synthesised following the similar procedure
as that of peptide I. Yield: 1.44 g (85%). Single crystals were grown
from a methanol water mixture by slow evaporation and were sta-
ble at room temperature. Mp = 158–160 °C; LR – MS: C14H19N3O4
[M + H]+ = 294.32, Mcalcd C14H19N3O4 [M + H]+ = 294; 1H NMR
(400 MHz, CDCl3, d ppm): 1.14 (Piv-CH3s, 9H, s); 1.35–1.36 (CbHs
a
of Ala, 3H, m); 4.54 (C H of Ala, 1H, br); 6.89–6.90 (Ala-NH, 1H,
m); 7.38–7.84 (Hd, Hc, Hb (m-ABA), 3H, m); 8.53 (Ha (m-ABA),
1H, s); 10.21 (m-ABA NH, 1H, s); 13C NMR 100 MHz (CDCl3, d ppm):
18.20, 27.61, 39.36, 49.54, 113.83, 117.12, 124.67, 129.49, 139.47,
147.92, 172.05, 177.75.
the c-turn region of acyclic dipeptides [25]. The idealized torsion

354
A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
10
8
10
mNA (2) NH
mNA (2) NH
Peptide II
8
6
Peptide I
6
D-Ala(1) NH
4
Ala (1) NH
0
2
4
0
2
6
% of d6-DMSO added in CDCl3
% of d6-DMSO added
(a)
(b)
0
9
8
7
9
8
7
6
m
NA(2) NH
m
ABA(2) NH
Peptide III
Peptide IV
Ala(1) NH
Ala(1) NH
0
3
0
3
6
% of d6-DMSO added in CDCl3
% of d6-DMSO added in CDCl3
(c)
(d)
10
8
m
ABA(2) NH
Peptide V
β
-CN-Ala(1) NH
6
0
3
% of d6-DMSO added in CDCl3
(e)
Fig. 3. NMR solvent titration curve for NH protons in peptide I–V (a–e) respectively.
angle values for an inverse
c
-turn are
u
i = ꢀ79.0°,
w
i = 69.0° and
protecting groups fails to display the conformational preference for
-turn. It was not possible to grow single crystals of peptide IV.
Therefore we are unable to comment on the solid state structure
for classical
c-turn are
u
i = 75.0°,
w
i = ꢀ64.0° respectively [1–3].
c
In peptides I and II, these torsion angles are found to be deviated
as ui (ꢀ105.9(2)/107.7(3)°), wi (95.7(2)/ꢀ95.0(3)°) (Table 2) from
the idealized values. The crystal data of both the peptides are re-
ported in Table 5 [26,27]. Nevertheless, peptide I and II produces
novel c-turn structures (inverse and classical) rarely observed in
the literature.
Peptides III and IV was synthesized by varying the N-terminal
protecting group of peptide I, from Boc to Piv in peptide III and
Fmoc in peptide IV, to study the effects of protecting groups on
and conformational preference of the molecule.
In an effort to examine whether change of the sidechain from
electron donating methyl (peptide I) to electron withdrawing
b-cyano methyl, produces similar conformational resemblance to
that of peptide I, keeping the N-terminal protecting group
unaltered, we designed and synthesized peptide V. The crystal
structure of peptide V is presented in Fig. 3e which reveals a com-
pletely extended structure as is indicated by the backbone torsions
c
-turn nucleation. The solid state structure of peptide III is pre-
sented in Fig. 2c. The torsion angles at Ala (
1 = ꢀ157.3(1),
1 = 160.1(1), (Table 3) residue indicate a fully extended confor-
mation. Thus, it is observed that peptide III, with Piv as N-terminal
at b-cyano-Ala residue (
Next our attempt was to shift the steric bulk from the
to amino nitrogen using Methyl Glycine (Sarcosine) as the N-termi-
u
1 = 145.3(2),
w
1 = ꢀ79.5(2) (Table 3).
u
a
-carbon
w
nal aminoacid in peptide VI, keeping the protecting groups

A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
355
(a)
(b)
(c)
(d)
(e)
Fig. 4. Schematic representation of the proposed c-turn in peptide I–V. The hydrogen bonds are shown as dotted lines. Observed NOEs are highlighted by double edged
arrows in CDCl3 (500 MHz).

356
A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
Fig. 5. ROESY spectrum of the proposed c-turn in peptide I in CDCl3.
Fig. 6. ROESY spectrum of the proposed c-turn in peptide II in CDCl3.
unchanged, and study the conformational preference of the mole-
cule. The crystal structure of peptide VI is presented in Fig. 3f. The
A pseudopeptide Boc-Ala-mABA-OMe, similar to peptide I was
reported by Pramanik and his co-workers in the design of b-sheet
structures [28] (Table 1, Entry g). From the work of the same group
it was demonstrated that changing the N-terminal residue from Ala
torsion angles at Sar residue are
(Table 3) which also indicates a fully extended conformation.
u
1 = ꢀ85.4(2),
w
1 = ꢀ160.8(1)

A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
357
Fig. 7. ROESY spectrum of the proposed c-turn in peptide III in CDCl3.
Fig. 8. ROESY spectrum of the proposed c-turn in peptide IV in CDCl3.

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A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
Table 4
Important interresidue NOEs for peptide I–V in CDCl3.
Peptide I
Peptide II
(1)
a
a
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Ala(1) CbHs M Ala(1) C Hs
Ala(1) CbHs M Ala(1) C Hs
Ala(1) CbHs M mNA(2) NH
(2)
(3)
(4)
(5)
(6)
Ala(1) CbHs M Ala(1) NH
Ala(1) C Hs M Ala(1) NH
Ala(1) NH M mNA(2) NH
Ala(1) CbHs M mNA(2) NH
Ala(1) NH M mNA(2) NH
Ala(1) CbHs M mNA(2) Ha
a
a
Ala(1) C Hs M Ala(1) NH
Ala(1) NH M mNA(2) NH
mNA(2) Hd M mNA(2) NH
a
Ala(1) C Hs M mNA(2) NH
Peptide III
(1)
(2)
(3)
(4)
Peptide V
(1)
(2)
(3)
(4)
a
a
Ala(1) CbHs M Ala(1) C Hs
b-CN-Ala(1) CbHs M b-CN-Ala(1) C H
Ala(1) CbHs M mNA(2) NH
Ala(1) CbHs M mNA(2) Ha
Ala(1) C H M Ala(1) NH
b-CN-Ala(1) CbHs M b-CN-Ala(1) NH
a
b-CN-Ala(1) C H M mABA(2) NH
a
a
b-CN-Ala(1) C H M b-CN-Ala(1) NH
(5)
(6)
(7)
(8)
Ala(1) NH M mNA(2) NH
mNA(2) Ha M mNA(2) NH
(5)
b-CN-Ala(1) NH M mABA(2) NH
a
Ala(1) C H M mNA(2) NH
Ala(1) CbHs M Ala(1) NH
Peptide IV
(1)
(2)
(3)
(4)
(5)
(6)
(7)
a
Ala(1) CbHs M Ala(1) C Hs
Ala(1) CbHs M mABA(2) NH
mABA(2) Ha M Ala(1) CbHs
a
Ala(1) C H M Ala(1) NH
Ala(1) CbHs M Ala(1) NH
mABA(2) Ha M mABA(2) NH
Ala(1) NH M mABA(2) NH
Fig. 9. ROESY spectrum of the proposed c-turn in peptide V in CDCl3.

A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
359
Table 5
Crystallographic refinement details of Peptide I–III, V and VI (CCDC: I, 876272; II: 876271; III: 876273; V: 876274; VI: 876275).
Peptide I
14H19N3O5
309.32
Triclinic
P1
5.0385(3)
5.6116(3)
13.3741(9)
97.152(4)
91.665(4)
95.767(4)
1
Peptide II
Peptide III
Peptide V
Peptide VI
Formula
C
C14H19N3O5
309.32
Triclinic
P1
5.0685(3)
5.6696(4)
13.5082(8)
97.192(4)
91.116(5)
94.903(5)
1
C14H19N3O4
293.32
Monoclinic
P21/n
6.9562(2)
21.3693(6)
10.5537(3)
90.00
97.752(2)
90.00
C17H21N3O5
347.37
Monoclinic
P21
5.1032(2)
11.6839(4)
14.8422(6)
90.00
98.153(3)
90.00
C16H22N2O5
322.36
Monoclinic
P21/c
15.6945(7)
5.8390(3)
18.1326(8)
90.00
100.295(2)
90.00
Molecular Wt(gm)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
Z
4
2
4
V (Å3)
372.97(4)
1.377
2740
383.53(4)
1.339
2770
1554.46(8)
1.253
4651
876.03(6)
1.317
3693
Density (gm/cm3)
Measured reflns
1.310
3650
R1 > 2
wR2
r(I),
0.0341
0.1027
0.0444
0.1250
0.0551
0.1805
0.0431
0.1406
0.0489
0.1437
to Gly by decreasing the steric bulk of the sidechain displays a
completely extended motif [29] (Table 1, Entry h). Even homologa-
tion of the ACH2A groups in the N-terminal residue of the peptide
supports the formation of extended conformation [22] (Table 1, En-
try i, j). Studies reveal that the C-terminal protecting group have al-
most negligible effect in determining the conformation in small
synthetic peptides [30] (Table 1, Entry k, l). Recent report from
the group of Pramanik displays that pseudopeptides comprising
M mABA(2)NH for peptide V) indicative of
a
reverse turn
conformation in solution(CDCl3) (Figs. 5–9) irrespective of the
configuration of the -amino acid. Moreover the ROESY spectrum
a
a
-
clearly shows a short range connectivity d (i, i + 1) [Ala(1)C
a
N
H M mNA(2)NH] and dbN (i, i + 1) [Ala(1)CbH M mNA(2)NH]
(Figs. 4–9, Table 4), diagnostic of a reverse turn conformation
[34,35]. Since peptides I–V are small acyclic peptides with only
two amino acids, there is no possibility for the presence of a b-turn
conformation. Hence it is reasonable to assume that the structure
of
a and x-amino acids in the i + 1th position and p-nitro ani-
line/mABA in the i + 2th position fails to support the reverse turn
adopted by the peptides I–V in solution is a c-turn. The solution
architecture [31,32] (Table 1, Entry m–q).
conformational analysis of peptide VI is under study.
4. Conclusions
3.2. Solution conformational analysis
In summary, the results display that pseudopeptides I and II
with methyl as the sidechain substituent; mNA, the conformation-
ally constrained nitro gamma amino moiety at the C-terminus and
tertiary butyloxy carbonyl at the N-terminus, forms gamma turn
conformation both in the solid state as well as in solution, irrespec-
In order to examine the existence of reverse turn (b-turn & c-
turn) conformation of peptides I–V in solution phase various
NMR experiments were performed.
Complete 1H NMR assignment of peptides I–V were obtained
using a combination of 2D COSY and ROESY methods in CDCl3. In
order to investigate the existence of intramolecular hydrogen
bonding and peptide conformation in solution phase, the solvent
dependence of the NH chemical shifts were examined by NMR
titration [33]. In this experiment a solution of the peptide in non-
polar CDCl3 (10 mM in 0.5 ml) was gradually titrated against polar
(CD3)2SO. The changes in the chemical shifts are presented in Fig. 3.
The solvent titration experiment shows that by increasing the per-
centage of (CD3)2SO in CDCl3 from 0% to 6% (v/v) the net changes in
tive of the configuration of the
a-amino acid (L or D). Our solid
state systematic conformational analysis further reveals that slight
change in modification of any of the stated conditions donot sup-
port the formation of this c-turn architecture. However this solid
state conformational hypothesis doesnot hold true in solution for
the remaning peptides (III–V) as is revealed by NMR
measurements.
Thus the role of methyl group and methyl derivatives at the N-
terminal residue of a dipeptide containing Ala (irrespective of con-
figuration L or D) coupled with conformationally constrained
the chemical shift (Dd) values for Ala(1)-NH, mNA (2) NH for pep-
tide I, D-Ala(1)-NH, mNA-(2)-NH for peptide II, Ala(1)-NH, mNA-
(2) NH for peptide III, Ala(1)-NH, mABA(2)-NH for peptide IV and
b-CN-Ala(1)-NH, mABA(2)-NH for peptide V are 0.55, 0.66; 0.49,
0.65; 0.05, 0.18; 0.76, 0.50; and 0.73, 0.64 respectively. From the
mABA/mNA is crucial for dictating this overall conformational c-
turn architecture. The importance of co-operative steric interac-
tions in stabilizing a particular secondary motif in peptides is
clearly evident from our systematic studies which may open a
D
d values it is evident that both the NH groups of peptides I–II &
new avenue in introducing c-turn motifs within the bioactive con-
IV–V are solvent exposed, indicating their non-involvement in
intramolecular hydrogen bonding. However these observations
are consistent with the existence of a very weak intramolecular
hydrogen bonding in a small acyclic peptide containing a conform-
ationally constrained system at i + 2 position which on addition of
a strong polar solvent d6-DMSO in a nonpolar environment CDCl3,
gets disrupted. Interestingly the NH groups of peptide III are sol-
vent shielded indicating their involvement in intramolecular
hydrogen bonding in solution.
Further attempts to characterize the reverse turn conformation
in CDCl3 using NOEs reveal that peptides I–V exhibits the
NN(i, i + 1) pattern, i.e. (Ala(1)NH M mNA(2)NH for peptides I–III;
Ala(1)NH M mABA(2)NH for peptide IV and b-CN-Ala(1)NH
formation of selected peptides.The function of other sidechain con-
taining residues at the N-terminus of dipeptide in gamma turn
formation is yet to be explored.
Acknowledgments
The author wishes to thank DST, New Delhi, India, for financial
support (Project No. SR/FT/CS-025/2010) and IISER Bhopal for pro-
viding the infrastructures. The author is very much grateful to Dr.
Sanjit Konar for assistance in X-ray crystallography and valuable
advice. Mr. Amit Adhikary, Mr. Javeed Ahmad Sheikh, Mr. Suresh
Sanda, Mr. Soumyabrata Goswami, Mr Sajal Khatua, Mr. Soumava

360
A. Dutt Konar / Journal of Molecular Structure 1036 (2013) 350–360
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Biswas and Mr. P. Sreenivasulu are sincerely acknowledged for var-
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