Role of side-chain and chirality of the amino acids on the supramolecular assemblies of dipeptides

Article abstract of DOI:10.1016/j.molstruc.2020.128877

The supramolecular self-association of alternating L/L and L/D amino acid-containing dipeptides, Boc-L-Ile-L-Phe-OMe (1), Boc-L-Ile-D-Phe-OMe (2), Boc-L-Ile-L-Phg-OMe (3, Phg = phenylglycine), and Boc-L-Ile-D-Phg-OMe (4) have been investigated. The presence of extra methylene (-CH₂-) group on the side-chain of C-terminal Phe-of 1 causes a significant deviation of molecular arrangements from that of 3. While the molecules in 1 self-assemble to form a single helix-like architecture, the molecules in 3 self-associate around a water molecule to form a cylinder-like structure in the crystalline state. Alternatively, the observed supramolecular arrangements of 2 and 4 are similar, but deviate from that of 1 and 3, due to chirality difference. Peptides 2 and 4 exhibit a discontinued double helix-like structure in the crystalline state. While 1 and 3 self-associate to form helical ribbon-like and nano tube-like structures, respectively, both 2 and 4 form nano rod-like structures in solution.

Full text of DOI:10.1016/j.molstruc.2020.128877

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Role of Side-Chain and Chirality of the Amino acids on the
Supramolecular Assemblies of Dipeptides
Sayanta Roy , Rajat Subhra Giri , Gobinda Dolai ,
Bhubaneswar Mandal
PII:
S0022-2860(20)31202-3
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128877
MOLSTR 128877
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8 July 2020
9 July 2020
Please cite this article as: Sayanta Roy , Rajat Subhra Giri , Gobinda Dolai , Bhubaneswar Mandal ,
Role of Side-Chain and Chirality of the Amino acids on the Supramolecular Assemblies of Dipeptides,
Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128877
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Highlights
•
2
Presence of extra methylene (-CH -) group in peptide Boc-L-Ile-L-Phe-OMe helps to form a
single helix-like architecture whereas Boc-L-Ile-L-Phg-OMe forms a cylinder-like structure
around the water molecule in the crystalline state.
•
A discontinued double helix-like structure in the crystalline state has shown for both Boc-L-Ile-
L-Phg-OMe (, Phg = phenylglycine), and Boc-L-Ile-D-Phg-OMe.
•
Boc-L-Ile-L-Phe-OMe and Boc-L-Ile-L-Phg-OMe self-associate to form helical ribbon-like and
nano tube-like structures, respectively, both Boc-L-Ile-D-Phe-OMe and Boc-L-Ile-D-Phg-OMe
build nano rod-like structures in solution.

Role of Side-Chain and Chirality of the Amino acids on the
Supramolecular Assemblies of Dipeptides
#
#
Sayanta Roy, Rajat Subhra Giri, Gobinda Dolai and Bhubaneswar Mandal*
Department of Chemistry, Laboratory of Peptide and Amyloid Research, Indian Institute of
Technology Guwahati, Assam-781039, India.
*
Corresponding Author’s email: bmandal@iitg.ac.in
#
These authors contributed equally.
Keywords: Discontinued double helix, hexagonal tube, L/L and D/L amino acid, self-assembly,
and single helix.
Graphical abstract
Abstract
The supramolecular self-association of alternating L/L and L/D amino acid-containing
dipeptides, Boc-L-Ile-L-Phe-OMe (1), Boc-L-Ile-D-Phe-OMe (2), Boc-L-Ile-L-Phg-OMe (3,
Phg = phenylglycine), and Boc-L-Ile-D-Phg-OMe (4) have been investigated. The presence of

2
extra methylene (-CH -) group on the side-chain of C-terminal Phe of 1 causes a significant
deviation of molecular arrangements from that of 3. While the molecules in 1 self-assemble to
form a single helix-like architecture, the molecules in 3 self-associate around a water molecule to
form a cylinder-like structure in the crystalline state. Alternatively, the observed supramolecular
arrangements of 2 and 4 are similar, but deviate from that of 1 and 3, due to chirality difference.
Peptides 2 and 4 exhibit a discontinued double helix-like structure in the crystalline state. While
1
2
and 3 self-associate to form helical ribbon-like and nano tube-like structures, respectively, both
and 4 form nano rod-like structures in solution.
1. Introduction
The naturally occurring amino acids are differentiated based on their side-chain structure. 3D
structure formation of peptides and proteins is mainly governed by the interactions not only
[
1,2]
between the side-chains but also between the side-chain and backbone.
These two
interactions, along with other supramolecular non-covalent interactions, stabilize a peptide or
[
3-8]
protein.
Nowadays, the design and development of various non-proteinogenic amino acid
building blocks are attractive as they play essential roles in the formation of secondary structures
[
9-11]
(
e.g., helix or turn) of the peptide.
The construction of the different type of unnatural amino
acid, e.g., α-Aminoisobutyric acid (Aib) and its higher homologous linear alkyl side-chains (α,α-
diethylglycine (Deg) α,α-di-n-propylglycine (Dpg) and α,α-di-n-butylglycine (Dbg)) and
cycloalkyl side-chains (1-aminocycloalkane-1-carboxylic acids, Ac
atoms in the ring);
n
c, n= 3-12, the no. of carbon
chiral α,α-dialkylated amino acids (e.g., α-methyl phenylalanine, α-
methyl homo-phenylalanine, α-methyl phenylglycine, α-methyl valine, and α-methyl-α-
[
12-17]
[
18-20]
ethylglycine (isovaline));
2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid
[
21]
[22]
[23]
(
TOAC);
gabapentin (Gpn);
is performed artificially by the mimicry of the side-chain and backbone.
gababutin (Gbn),
and α/β/γ cyclic or acyclic amino
[
10,11,24,25]
acids,
Moreover, the steric interaction of the two side-chains in alternating L/D amino acids containing
[
26-28]
peptide dictates backbone orientation, and this is useful to build a cyclic peptide.
al. first developed a cyclic octapeptide from alternating D/L amino acids.
Ghadiri et
Lorenzi et al.
described the supramolecular self-assembly of alternating L/L and D/L amino acids containing
[
29]
[30]
dipeptides.
Recently, we reported the crystal structures of Gly, Phe, and Phg amino acid
[
31,32]
containing tripeptides.
They exhibited different supramolecular architectures, e.g.,
herringbone helical, single helical, and double helical structures due to the presence of
heterochiral amino acids. We also demonstrated the formation of nanorod and nanotube structure
[33]
from alternating L/L and D/L amino acid-containing dipeptides.

Fig. 1. The chemical structures of dipeptides 1, 2, 3, and 4
Herein, we designed and synthesized a set of the N- and C-protected alternating L/L and L/D
amino acid-containing dipeptides Boc-L-Ile-L-Phe-OMe (1), Boc-L-Ile-D-Phe-OMe (2), Boc-L-
Ile-L-Phg-OMe (3), and Boc-L-Ile-D-Phg-OMe (4, Fig. 1) and investigated their supramolecular
self-assemblies. To understand the role of the side-chain on self-assembly, we kept L-Ile at the
N-terminus and either L-Phe or L-Phg (a non-coded amino acid that contains one methylene less
than that of the side-chain of Phe) at the C terminus. The C-terminal amino acids were
exchanged with their D-isomers to examine the effect of chirality.
2. Experimental Section
Boc protected amino acid, and deprotected amino acids, 2-nitrobenzenesulfonyl chloride and
Oxyma were purchased from GL Biochem (Shanghai). N, N-Diisopropylethylamine (DIPEA),
acetonitrile (HPLC grade), dichloromethane (extra pure grade) Citric acid, and Sodium
bicarbonate were purchased from Spectrochem (India). o-NosylOXY was prepared following a
[34]
previously established protocol in our laboratory.
.1 Peptide synthesis
Boc-protected amino acid (1 equiv), o-NosylOXY (1 equiv) as a coupling reagent,
2
[
35-37]
Hünig's
base, DIPEA (1 equiv) in dichloromethane (DCM) was taken in an RB. Methyl ester of the next
amino acid (1.2 equiv) neutralized by DIPEA (1.2 equiv) in DCM was added to the reaction
mixture after 5 min stirring for preactivation. The reaction mixture was kept with stirring at room
temperature for 4-5 h. After completion of the reaction (monitored by TLC), 50 mL DCM was
added to it. The organic portion was washed with 10% citric acid solution followed by saturated
3 2 4
NaHCO solution, three times in each case. The organic layer was dried over anhydrous Na SO ,

concentrated under reduced pressure. The obtained solid crude was purified by silica gel column
chromatography using Hexane/EtOAc as eluent in the 17:3 ratio.
Synthesis of peptide 1
To a solution of Boc-L-Ile-OH (300 mg, 1.297 mmol) in DCM (3 mL), DIPEA (167 mg, 1.297
mmol), o-NosylOXY (424 mg, 1.927 mmol) were added. The mixture was stirred for 5 min for
preactivation. After that, H
2
N-L-Phe-OMe (279 mg, 1.556 mmol) was neutralized by DIPEA
(
201mg, 1.556mmol) and was added to the reaction mixture. The resulting mixture was stirred at
room temperature for 4 h. After completion of the reaction (monitored by TLC), the reaction
mixture was worked up. The obtained crude compound was purified by column chromatography
over silica gel using Hexane/EtOAc as eluent. The desired pure peptide was obtained as a white
powder. The purity of the isolated peptide was checked by using an analytical HPLC. It was
1
1
13
characterized by mass spectrometry as well as 1D [ H] ( H and C) NMR spectroscopy
(
Supporting Information, Fig. S1-S4).
1
White solid, (452 mg, 89%), H NMR (CDCl
3
; 600 MHz) δ 0.89-0.88 (6H, m); 1.09-1.06 (1H,
m); 1.44 (9H, s); 1.83-1.75 (2H, m); 3.14-3.09 (2H, m); 3.71 (3H, s); 3.95-3.92 (1H, m); 4.89-
1
3
4
.87 (1H, m); 5.02 (1H, s); 6.35 (1H, s); 7.12-7.10 (2H, d, J = 7.2 Hz); 7.30-7.23 (3H, m). C
NMR (CDCl ; 150 MHz) δ 11.6, 15.6, 24.8, 28.5, 37.4, 38.1, 52.5, 53.2, 59.4, 80.0, 127.3, 128.8,
29.4, 135.8, 155.8, 171.4, 171.8. ESI-MS: calculated [M+H] 393.2391, found m/z 393.2580.
3
+
1
R
HPLC: retention time (t ) = 5.56 min.
Synthesis of peptide 2
To a solution of Boc-L-Ile-OH (300 mg, 1.297 mmol) in DCM (3 mL), DIPEA (167 mg, 1.297
mmol), o-NosylOXY (424 mg, 1.927 mmol) were added. The mixture was stirred for 5 min for
preactivation. After that, H
2
N-D-Phe-OMe (279 mg, 1.556 mmol) was neutralized by DIPEA
(
201mg, 1.556 mmol) and was added to the reaction mixture. The resulting mixture was stirred at
room temperature for 4 h. After completion of the reaction (monitored by TLC), the reaction was
worked up. The obtained crude compound was purified by column chromatography over silica
gel using Hexane/EtOAc as eluent. The desired pure peptide was obtained as a white powder.
The purity of the isolated peptide was checked by using an analytical HPLC. It was characterized
1
1
13
by mass spectrometry as well as 1D [ H] ( H and C) NMR spectroscopy (Supporting
Information, Fig. S5-S8).
1
White solid, (444 mg, 87%), H NMR (CDCl
3
; 600 MHz) δ 0.86-0.82 (6H, m); 1.01-0.99 (1H,
m); 1.43 (9H, s); 1.86-1.71 (2H, m); 3.15-3.06 (2H, m); 3.71 (3H, s); 4.02-4.00 (1H, m); 4.91-
1
3
4
.87 (1H, m); 4.97 (1H, s); 6.44 (1H, s); 7.11-7.10 (2H, d, J = 7.2 Hz); 7.29-7.22 (3H, m). C

NMR (CDCl
3
; 150 MHz) δ 11.6, 15.6, 24.4, 28.4, 37.3, 38.1, 52.5, 53.1, 59.3, 80.1, 127.3, 128.8,
+
1
29.3, 135.9, 155.9, 171.4, 171.0. ESI-MS: calculated [M+H] 393.2391, found m/z 393.2578.
R
HPLC: retention time (t ) = 5.62 min.
Synthesis of peptide 3
To a solution of Boc-L-Ile-OH (400 mg, 1.729 mmol) in DCM (4 mL), DIPEA (223 mg, 1.729
mmol), o-NosylOXY (565 mg, 1.729 mmol) were added. The mixture was stirred for 5 min for
preactivation. After that, H
2
N-L-Phg-OMe (343 mg, 2.074 mmol) was neutralized by DIPEA
(
268 mg, 2.074 mmol) and was added to the reaction mixture. The resulting mixture was stirred
at room temperature for 5 h. After completion of the reaction (monitored by TLC), the reaction
mixture was worked up. The obtained crude compound was purified by column chromatography
over silica gel using Hexane/EtOAc as eluent. The desired pure peptide was obtained as a white
powder. The purity of the isolated peptide was checked by using an analytical HPLC. It was
1
1
13
characterized by mass spectrometry as well as 1D [ H] ( H and C) NMR spectroscopy
(
Supporting Information, Fig. S9-S12).
1
White solid, (578 mg, 88%), H NMR (CDCl
3
; 600 MHz) δ 0.95-0.94 (3H, d, J = 6.6 Hz); 0.91-
0
.88 (3H, t, J = 7.2 Hz, J = 6.6 Hz); 1.17-1.12 (1H, m); 1.42 (9H, s); 1.78-1.73 (2H, m); 3.72
1
3
(
3H, s); 4.01-3.99 (1H, m); 5.04 (1H, s); 5.54-5.53 (1H, m); 6.96 (1H, s); 7.35-7.33 (5H, m). C
NMR (CDCl ; 150 MHz) δ 11.5, 15.7, 24.9, 28.4, 37.4, 52.9, 56.7, 59.3, 80.1, 127.4, 128.7,
29.1, 136.4, 155.9, 171.1, 171.2. ESI-MS: calculated [M+H] 379.2235, found m/z 379.2412.
3
+
1
R
HPLC: retention time (t ) = 5.13 min.
Synthesis of peptide 4
To a solution of Boc-L-Ile-OH (400 mg, 1.729 mmol) in DCM (4 mL), DIPEA (223 mg, 1.729
mmol), o-NosylOXY (565 mg, 1.729 mmol) were added. The mixture was stirred for 5 min for
2
preactivation. After that, H N-D-Phg-OMe (343 mg, 2.074 mmol) was neutralized by DIPEA
(
268 mg, 2.074 mmol) and was added to the reaction mixture. The resulting mixture was stirred
at room temperature for 5 h. After completion of the reaction (monitored by TLC), the reaction
was worked up. The obtained crude compound was purified by column chromatography over
silica gel using Hexane/EtOAc as eluent. The desired pure peptide was obtained as a white
powder. The purity of the isolated peptide was checked by using an analytical HPLC. It was
1
1
13
characterized by mass spectrometry as well as 1D [ H] ( H and C) NMR spectroscopy
(
Supporting Information, Fig. S13-S16).
1
White solid, (570 mg, 87%), H NMR (CDCl
3
; 600 MHz) δ 0.87 (6H, brs); 1.08-1.07 (1H, m);
1
.43 (9H, s); 1.88-1.84 (2H, m); 3.72 (3H, s); 4.05 (1H, s); 5.04 (1H, s); 5.55-5.54 (1H, m); 7.04

1
3
(
5
3
3
1H, s); 7.36-7.32 (5H, m). C NMR (CDCl ; 150 MHz) δ 11.6, 15.7, 24.7, 28.4, 37.4, 53.0,
+
6.6, 59.2, 80.2, 127.4, 128.7, 129.1, 136.4, 156.0, 171.2, 171.3. ESI-MS: calculated [M+H]
79.2235, found m/z 379.2411. HPLC: retention time (t ) = 5.15 min.
R
2
.2 High-performance liquid chromatography (HPLC)
The analytical HPLC, Agilent 1260 Infinity II system with a C18 Agilent column (4.6 x 100 mm,
-1
4
µm) at a flow rate 1 mLmin with a linear gradient 5-100% acetonitrile over 5 min and total
run time 10 min was used to check the purity of those peptides.
Solvent system: water (solvent A) and acetonitrile (solvent B). The wavelength of UV detector:
2
14 and 254 nm.
2
.3 Mass spectrometry
Mass spectrometry experiment was performed on Agilent-Q-TOF 6500 instrument, and the data
analysis was carried on with Mass hunter workstation software.
2
.4 Nuclear magnetic resonance (NMR) spectroscopy
1
13
H and C NMR spectra of all those purified peptides were recorded on a Bruker Ascend 600
3 3
MHz instrument at 298 K using CDCl solvent. Chemical shifts were referenced to CDCl at δ =
1
13
7
.26 ppm ( H NMR) and δ = 77.23 ( C NMR).
2
.5 Single-crystal X-ray diffraction (SC-XRD)
The SC-XRD experiment was carried out an Oxford SuperNova diffractometer (Agilent
Technologies) using MoKα radiation. Data refinement was done by CrysAlisPro. The refinement
[
38]
was performed by SHELXTL software
using full-matrix least-square calculations. All non-
hydrogen atoms were refined anisotropically.
2
.6 Sample preparation
.55 mM of peptide 1, and 2 and 2.64 mM of peptide 3, and 4 were prepared in four different
2
Eppendorf vials (2 mL) by adding 1 mL 50% acetonitrile-water solution. Then the preparation of
a 1 mL (1.5 mM) solution of each peptide was done from the stock solutions. Then these
o
solutions (1.5 mM) were incubated for 4 days at 37 C. Sample for CD and FE-SEM was
prepared on that day.
2
.7 Circular dichroism (CD)
The CD experiment was performed on the JASCO J-1500 instrument, and the spectra were
recorded from 190 nm to 260 nm wavelength using 1 mm pathlength and 1 nm bandwidth. The
mean residue molar ellipticity was calculated using the following equation:
2
-1
6

(deg. cm .dmol ) = Ellipticity (mdeg). 10 / Pathlength (mm). Protein] (μM). N

2
.8 Field emission scanning electron microscopy (FE-SEM)
The 4-day old incubated samples (1.5 mM) were dropped cast on Al-foil and dried them
overnight inside the desiccator. A SIGMA-300 (ZEISS) instrument was used to capture the FE-
SEM images.
3. Results and discussion
[34]
At first, we synthesized four dipeptides by using the conventional coupling method in solution
Scheme 1) and then characterized by mass spectrometry and NMR spectroscopy.
(
Scheme 1. Peptide synthesis in solution
To obtain detailed information on molecular arrangements, we carried out single-crystal X-ray
diffraction (SC-XRD) experiments of these peptides. Colorless block-shaped single crystals of 1,
2
, 3, and 4 were obtained by the slow evaporation of their acetonitrile-water solutions at room
temperature. Peptides 1 and 3 showed an orthorhombic (P 21 21 21) and a hexagonal (P 63)
space group, respectively, whereas 2 and 4 exhibited a monoclinic (P 21) space group in the
crystalline state. They also showed one molecule in the asymmetric unit. The obtained backbone
torsion angles (Table 1) of all these peptides suggested that they adopted a parallel β-sheet
structure.
The subunits of 1 were interconnected through intermolecular H-bond (N1-H1…O2 and N2-
H2…O3) to form a parallel β-sheet structure (Fig. 2b). The subunits were further self-associated
to build a single helix-like architecture along the a-axis in higher-order packing (Fig. 2c).
Table 1. List of torsion angles (deg)
Peptides






Peptide 1 C5-N1-C6-C11
-96.8(6)
N1-C6-C11-N2
= 99.6(5)
C11-N2-C12-C13 N2-C12-C13-O5 O1-C5-N1-C6
= -124.8(5) = 32.5(6) = 179.0(4)
C6-C11-N2-C12
= 179.0(4)
=
Peptide 2 C5-N1-C6-C11
-123.5(4)
N1-C6-C11-N2
= 111.0(4)
C11-N2-C12-C13 N2-C12-C13-O5 O1-C5-N1-C6
= 150.9(4) = 176.9(4) = 178.3(4)
C6-C11-N2-C12
= -171.3(3)
=

Peptide 3 C5-N1-C6-C11
-114.5(7)
N1-C6-C11-N2
= 122.7(7)
C11-N2-C12-C13 N2-C12-C13-O5 O1-C5-N1-C6
= -150.3(7) = 169.1(6) = -178.2(6)
C6-C11-N2-C12
= 178.2(6)
=
Peptide 4 C5-N1-C6-C11
-118.9(5)
N1-C6-C11-N2
= 110.3(5)
C11-N2-C12-C13 N2-C12-C13-O5 O1-C5-N1-C6
= 148.2(5) = -175.6(5) = 176.2(5)
C6-C11-N2-C12
= -178.4(5)
=
Fig. 2. (a) The ORTEP diagram with an ellipsoid of 30% probability, (b) parallel β-sheet structure along c-axis and
c) single helix-like architecture along a-axis in the higher-order packing, of peptide 1
(
Similarly, 2 adopted a parallel β-sheet structure through intermolecular H-bonding interactions
N1-H1…O2 and N2-H2…O3) along the a-axis (Fig. 3b). In higher-order packing, the
(
molecules are further self-organized to form a discontinued double-stranded helix-like
architecture along the b-axis (Fig. 3c). The strands are stabilized through C-H…O and C-H…π
(
C=O) interactions (Fig. 3c). Interestingly, for the presence of D-Phe instead of L-Phe in C-
terminus of 2, the backbone becomes extended, and their packing pattern differs.

Fig. 3. (a) The ORTEP diagram with an ellipsoid of 30% probability, (b) a parallel β-sheet along a-axis, and (c) a
discontinued double-stranded helix-like architecture in the higher-order assembly along b-axis of peptide 2
Fig. 4. (a) The ORTEP diagram with an ellipsoid of 30% probability, (b) a parallel β-sheet along b-axis, and (c) a
hexagonal hollow tube-like arrangement around a water molecule in the higher-order assembly along c-axis of
peptide 3
On the other hand, 3 exhibited a parallel β-sheet structure (Fig. 4b) through intermolecular H-
bonding interactions (N1-H1…O2 and N2-H2…O3) along the b-axis in the solid-state. The six
molecules are self-organized, such a way that their phenyl rings of the side chain of C-terminal
L-Phg interconnected together through van der Waals interactions around a water molecule to
form a hexagonal tube-like architecture along the c-axis in higher-order packing (Fig. 4c).

Interestingly, incorporation of an unnatural amino acid L-Phg instead of L-Phe in the C-terminus
of 3, deviated the crystal packing arrangement from 1 significantly.
Peptide 4 adopted a parallel β-sheet structure (Fig. 5b) similar to 2. Here also molecules are self-
organized to construct a discontinued double-stranded helix-like architecture along the b-axis
(
Fig. 5c). The strands are stabilized through C-H…π (C=O) interactions (Fig. 5c).
Fig. 5. (a) The ORTEP diagram with an ellipsoid of 30% probability, (b) a parallel β-sheet along a-axis, and (c) a
discontinued double-stranded helix-like architecture in the higher-order assembly along b-axis of peptide 4
Next, we compared the molecular structures of these peptides. Although 3 is the analog of 1
(
only differ by one side-chain methylene group at the C-terminal) but the obtained backbone
structures are different (Fig. 6a and 6c). The bond angles of N2-C12-C13 () of peptides 1 and 3
o
o
are 110.7(4) and 108.5(6) , respectively. In 1, the aromatic ring is connected through a
methylene group to the Cα in Phe; therefore, no significant steric repulsion is observed between
3
ortho protons and Cα (C12) proton (Fig. 6a). As a result, the sp carbon (C12) is flexible, and the
backbone is less elongated. On the other hand, the phenyl ring is directly attached with Cα (C12)
in Phg (Fig. 6c) of 3, which causes a steric repulsion between the ortho protons of the aromatic
ring and the Cα (C12) proton and NH of Phg (N2H). As a result, the backbone of 3 becomes
more extended than that of 1.

Interestingly, in the presence of alternating L/D amino acids in the peptide sequence, the
obtained backbone orientation of 2 and 4 appeared similar (Fig. 6b and 6d). Such similarity is
probably due to the presence of significant steric repulsions between two side-chains, flanking in
the same direction.
Fig. 6. Comparison of the molecular structures of alternating L/L and L/D amino acid-containing peptides
[31,32]
A similar effect was observed in our previously reported tripeptide structures.
Boc-Gly-L-
Phe-L-Phe-OMe adopted a rare open turn structure, but Boc-Gly-L-Phg-L-Phe-OMe adopted a
backbone extended structure. Moreover, due to the presence of alternating D/L amino acids
(
steric repulsion between two flanking side-chains, in the same direction) the tripeptides, Boc-
Gly-L-Phg-D-Phe-OMe and Boc-Gly-L-Phg-D-Phg-OMe, adopted different orientations than
[
31,32]
alternating L/L amino acid-containing peptides.
All crystallographic data and hydrogen
bonds are listed in Table 2 and Table S2 (Supporting Information).
To understand the conformations of the ensemble of these peptides in solution, we prepared 1.5
o
mM of each peptide solution in 50% acetonitrile-water, incubated for four days at 37 C, diluted
to make 375 µM solution to avoid the high voltage, and performed CD experiments. Peptide 1
(
(
Fig. 7, cyan curve) exhibited two positive Cotton effect at 217 nm and 195 nm, reverse of 2
Fig. 7, yellow curve) which exhibited negative Cotton effect at 218 nm and 196 nm.
Table 2. Crystal parameters and refinement data of 1, 2, 3 and 4
Parameters
Peptide (1)
Peptide (2)
Peptide (3)
Peptide (4)
Formula
Fw
C
21
H
32
N
2
O
5
C
21
H
32
N
2
O
5
3(C20
H
29
N
2
O ), H
5
2
O
20 30 2 5
C H N O
392.49
392.49
1150.37
378.46

Crystal system
Space group
a/Å
orthorhombic
P 21 21 21
5.1231(3)
11.1061(14)
40.227(3)
90.00
monoclinic
P 21
hexagonal
P 63
Monoclinic
P 21
14.3440(12)
5.0356(3)
17.1233(17)
90.00
27.5075(16)
27.5075(16)
5.1256(3)
90.00
14.4958(18)
5.0228(5)
16.5927(18)
90.00
b/Å
c/Å
o
α/
o
β/
90.00
113.079(10)
90.00
90.00
114.858(14)
90.00
o
γ/
90.00
120.00
3358.8(5)
2
3
V/Å
2288.8(4)
4
1137.84(18)
2
1096.2(2)
2
Z
-3
c
D /g cm
1.139
1.146
1.138
1.147
-1
α
μ Mo K /mm
0.081
0.081
0.082
0.082
F000
848
424
1238.0
293(2)
25.000
6521
408
T/K
293(2)
293(2)
293(2)
θ max.
Total
28.769
28.620
5081
28.853
4770
no.
of 6475
reflections
Independent
reflections
4593
4159
2784
3691
Observed reflections
Parameters refined
2028
3034
1853
2190
259
259
256
250
R
1
, I > 2σ(I)
0.0665
0.1196
0.935
1973966
0.0629
0.1439
1.066
1973967
0.0696
0.2095
0.849
1973968
0.0729
0.1580
1.057
1973969
wR
2
, I > 2σ(I)
2
GOF (F )
CCDC No.
This is probably due to the presence of alternating reverse chiral amino acids. Similarly, peptide
(Fig. 7, green curve) showed positive Cotton effects at 218 and 198 nm, reverse of 4 (Fig. 7,
3
violet curve), which showed the negative Cotton effect at 219 nm and 200 nm. The careful
observation of the CD profiles indicated that both peptides 1 and 3 formed a mixture of β-turn

[31]
and -sheet structures in solution. Although they adopted a β-sheet structure in the crystalline
state, their structure may partially change in the solution.
Fig. 7. CD spectra of 1 (cyan curve), 2 (yellow curve), 3 (green curve), and 4 (violet curve)
Fig. 8. FE-SEM images of self-aggregated dipeptides (a) 1, (b) 2, (c) 3 and (d) 4 in 50% acetonitrile-water medium
using concentration 1.5 mM
Next, we performed the FE-SEM experiment to understand the morphology of those self-
assembled peptides. Interestingly, the obtained images of 1 and 3 are different, whereas 2 and 4
are quite similar. Peptide 1 formed a helical ribbon-like structure (Fig. 8a) with several
micrometers long; however, 3 formed a nanotube-like (inner diameter ~261.9 nm and outer

diameter ~986.2 nm) structure (Fig. 8c). On the other hand, peptide 2 and 4 self-associated to
form nano rod-like structures (Fig. 8b and 8d).
4. Conclusion
In summary, we investigated the molecular arrangements, self-assembly, and morphology of a
set of alternating L/L and L/D amino acid-containing dipeptides Boc-L-Ile-L-Phe-OMe (1), Boc-
L-Ile-D-Phe-OMe (2), Boc-L-Ile-L-Phg-OMe (3), and Boc-L-Ile-D-Phg-OMe (4). The obtained
results indicated that 1 self-associated to adopt a single helix-like architecture; however, 3 self-
associated around a water molecule to form a cylinder-like structure in the crystalline state.
Interestingly, the alternating L/D amino acid-containing dipeptides 2 and 4 self-assembled to
create a similar type of a discontinued double helix-like structure in the crystalline state. In
solution, 1 and 3 self-aggregated to exhibit a helical ribbon-like and nano tube-like structure,
respectively, whereas both 2 and 4 self-aggregated to show a nano rod-like structure. The
obtained supramolecular self-assembled SC-XRD structure and morphology of 1 and 3 are
different due to their backbone orientation, which is governed by the side-chain-backbone
interaction of C-terminal Phe (peptide 1) and Phg (peptide 3). Such type of comparative study
may be helpful to the design of peptidomimetics and novel materials.
`
Credit Author Statement
BM conceived the project. SR, RSG and GD performed all the works. SR and RSG contributed equally.
RSG and BM analyzed data and wrote the manuscript with the help of all the authors.
Acknowledgement
We thank the central Instruments facility (CIF), IITG, for SC-XRD, FE-SEM, and NMR; DST-
FIST for CD and HRMS; COE FAST for NMR (F. No. 22-3/2016-TS-II/TC); the Department of
Chemistry, IITG for other instrumental facilities; and the Department of Biotechnology, Govt. of
India, for financial support (a twinning program for the North Eastern Region, sanction no.
BT/PR16164/NER/95/88/2015). S.R., R.S.G., and G.D. also thank IIT Guwahati for fellowship.
Conflicts of interest
There are no conflicts of interest to declare.

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