Formation of supramolecular single and double helix-like structures from designed tripeptides

Article abstract of DOI:10.1039/c9ce01168d

The conformation and self-assembly of N- and C-protected tripeptides, Boc-Gly-l-Phg-d-Phe-OMe (1, Phg: phenylglycine) and Boc-Gly-l-Phg-d-Phg-OMe (2), have been investigated. The effect of the insertion of two non-coded amino acid residues, l-Phg/d-Phe and l-Phg/d-Phg, consecutively on the structure of two tripeptides has been investigated. The single crystal X-ray diffraction analysis of 1 and 2 suggested that both peptides adopted an anti-parallel β-sheet structure but they further self-assembled to form supramolecular single helix and double helix-like architectures, respectively, by various non-covalent interactions in the crystalline state. To the best of our knowledge, this is the first crystallographic report where alternating d/l unnatural amino acid containing small tripeptides exhibited double helix-like architectures. The conformation of these peptides was examined by 2D NMR, solvent dependent NMR titration, and CD spectroscopic studies in solution. Peptides 1 and 2 self-aggregated to form a bunch of flower-petal-like and flower-like architectures, respectively, in an acetonitrile-water medium under a field emission scanning electron microscope (FESEM).

Full text of DOI:10.1039/c9ce01168d

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DOI: 10.1039/C9CE01168D
Journal Name
ARTICLE
Formation of Supramolecular Single and Double Helix-like
Structures by Designed Tripeptides
Rajat Subhra Giri and Bhubaneswar Mandal*
Received 00th January 20xx,
Accepted 00th January 20xx
The conformation and self-assembly of an N- and C- protected tripeptide, Boc-Gly-L-Phg-D-Phe-OMe (1, Phg:
Phenylglycine) and Boc-Gly-L-Phg-D-Phg-OMe (2) have been investigated. The effect of the insertion of two non-coded
amino acid residues, L-Phg/D-Phe and L-Phg/D-Phg, consecutively on the structure of two tripepetides has been
investigated. The single crystal X-ray diffraction analysis of 1 and 2 suggested that both peptides adopted anti-parallel β-
sheet structure but they further self-assembled to form supramolecular single helix and double helix-like architectures,
respectively, by various non-covalent interactions in the crystalline state. To the best of our knowledge, this is the first
crystallographic report where alternating D/L unnatural amino acid containing small tripeptide exhibited double helix-like
architecture. The conformation of these peptides was examined by 2D NMR, solvent dependent NMR titration, and CD
spectroscopic studies in solution. Peptide 1 and 2 self-aggregated to form a bunch of flower-petals-like and flower-like
architecture, respectively, in an acetonitrile-water medium under field emission scanning electron microscope (FESEM).
DOI: 10.1039/x0xx00000x
www.rsc.org/
reported a most aggregation-prone tripeptide, Pro-Phe-Phe
self-assembled to form a rigid helical-like sheet structure
which was confirmed by SC-XRD experiment.16
Introduction
Formation of various secondary structures, e.g., α-helix, and β-
sheet has an essential role in biological system.1-3 Therefore de
novo design of helix forming peptide moieties can be useful for
biomedical and material chemistry applications. However,
design and development of single helical or double helical
assembly through peptide or peptidomimetic is a challenging
task.4,5
With this inspiration, Banerjee and co-workers reported the
helical assembly of Aib (α-aminoisobutyric acid) containing
tripeptides6 and the double helical assembly of N-terminal β-
alanine containing dipeptides.7 Haldar et al. reported the
formation of supramolecular double helix from N-terminal L-
tyrosine and central Aib (α-aminoisobutyric acid) containing
tripeptides8 as well as from Boc and N,N’-dicyclohexylurea
caped γ-peptide.9 Dutt Konar et al. described the double
helical structures of model Aib containing tripeptides10 and
pyridine
carboxamide
containing
tyrosine
analog
pseudopeptides.11 Görbitz developed double helical structures
from a series of dipeptides belonging to Val-Ala class.12 Gopi et
al. reported α/γ4-hybrid peptide helices13 and artificial β-
double helices from achiral γ-peptides.14 Luis and co-works
reported the formation of columnar helical assemblies from
Val and Phe containing pseudopeptides.15 Recently, Gazit et al.
Fig. 1 Peptide synthesis in solution and chemical structures of tripeptides
1 and 2
Phenylglycine (Phg) is a non-proteinogenic amino acid, which
is very similar to Phenylalanine (Phe, proteinogenic) but one
methylene (-CH2-) group less in the side chain than that of Phe.
Interestingly, the steric and electronic effect of the phenyl ring
in Phg influences the conformation, reactivity, and physical
properties of the peptides.17 Phg containing peptides are used
as an antibiotic, e.g., virginiamycin S,17 streptogramin B17,18
and pristinamycin I.17,18 Therefore, to develop new drugs and
biologically active materials, Phg is useful. Furthermore, long
polypeptides of regularly alternating D/L amino acids usually
form channel or cavity through the formation of β-helix, e.g.,
Department of Chemistry, Laboratory of Peptide and Amyloid Research, Indian
Institute of Technology Guwahati, Assam- 781039, India.
E-mail: bmandal@iitg.ernet.in
† Electronic Supplementary Information (ESI) available: [2D NMR, titration data
and plot, crystallographic data, Hirshfeld data, HPLC profiles, characterization
spectra for all the synthesized peptides. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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View Article O1nlinine
Gramicidin A, Feglymycin, and Ramoplanin. They are used as color, Fig. 2a) in the asymmetric unit. The conformation of
antibiotic.19-21
Keeping in mind the advantages and structural features of Phg conformation. The torsion angles are listed in Table 1.
and importance of alternating D/L amino acids in Molecule and of were interconnected through
sequence,17-21 we designed and synthesized both N- and C- intermolecular H-bond to form an antiparallel β-sheet
protected tripeptides, Boc-Gly-L-Phg-D-Phe-OMe ( ) and Boc- arrangement along a direction (Fig. 2b). Each subunit of was
Gly-L-Phg-D-Phg-OMe ( ) (Fig. 1). further stabilized by two C-H…π (2.83 and 2.75 Ǻ between
Recently, we reported the crystal structures of two tripeptides, aromatic H of L-Phg of ) and three C-
and sp2 C of L-Phg of
Boc-Gly-L-Phe-L-Phe-OMe and Boc-Gly-L-Phg-L-Phe-OMe. They H…O (2.57 Ǻ between βH of D-Phe of and urethane carbonyl
adopted rare novel open turn and parallel β-sheet oxygen of ; 2.70 Ǻ between aromatic H of D-Phe of and
structures, respectively.22 The structural differences observed urethane carbonyl oxygen of
; 2.43 Ǻ between αH of Gly of
between above two reported peptides were probably due to and amide oxygen of L-Phg of ) interactions (Fig. 2c). It was
the crystalline state showed that it had extended backbone
DOI: 10.1039/C9CE01168D
a
A
B
1
1
1
2
B
A
A
a
B
B
A
B
A
the presence of the non-proteinogenic amino acid L- further self-assembled in higher-order packing along
phenylglycine. Therefore in the present study, we kept the N- crystallographic a-direction (Fig. 2d). The structure was formed
terminal dipeptide glycylphenylglycine (-Gly-L-Phg-) unaltered through the interaction of C-H…π (average CH-π distance 2.83
but changed the next C-terminal amino acid to D-Phe (
D-Phg ( ), respectively, and wanted to observe the effect of aromatic π of D-Phe of
the reversion of chirality. (average CH…O distance 2.70 Ǻ, the aromatic C-H of D-Phe of
Thus, we investigated the conformation and self-assembly of that particular molecule and the C=O of Boc of another
the above-mentioned alternating D/L peptides in the solid molecule). Similarly, two molecules were stabilized by
state as well as in a solution. Interestingly, SC-XRD revealed similar interactions with a nearby molecule (Fig. 2f). The
that formed an antiparallel β-sheet structure which further backbone orientation of two consecutive molecule of
1
) and Ǻ, the aromatic C-H of D-Phe of one particular
B molecule and
2
A
molecule, Fig. 2e) and C-H…O
B
A
B
A
1
B
1
self-assembled through non-covalent interactions to form along b axis appeared as a helix-like stretch (Fig. 2g). The
helix-like architecture in the crystalline state. Whereas, molecular packing along b direction also exhibited a helix-like
peptide
2 adopted an antiparallel β-sheet structure which structure (Fig. 2f).
further self-associated to form supramolecular double helix- From Table S1 (ESI), it was observed that the torsion angles of
like architecture. To the best of our knowledge, this is the first previously reported consecutive L/L amino acids containing
crystallographic report where alternating D/L unnatural amino tripeptide, Boc-Gly-L-Phg-L-Phe-OMe22 were different from
acid containing small tripeptide exhibited double helix-like consecutive D/L amino acid containing tripeptide Boc-Gly-L-
architecture.
At first, peptides were synthesized by using standard different in the crystalline state, as expected.
conventional coupling method (Fig. 1) in solution23,24 and On the other hand, peptide
exhibited one molecule in the
purified by column chromatography and then characterized by asymmetric unit, and they were interconnected to form
mass spectrometry and NMR spectroscopy. antiparallel β-sheet through intermolecular H-bonding along a
The conformation and self-assembly of the synthesized axis (Fig. 3b). Each subunit of was further stabilized by two C-
Phg-D-Phe-OMe (1). As a result, their conformations were also
2
2
peptides were studied by single crystal X-ray diffraction H…π (2.76 and 2.73 Ǻ) and two C-H…O (2.61 and 2.45 Ǻ)
experiments in the solid state. Colorless block-shaped crystal interactions (Fig. 3c). Interestingly, it self-assembled to form
(Fig. S1, ESI) suitable for X-ray diffraction were matured from supramolecular double helix-like architecture with a helical
the acetonitrile-water medium by slow evaporation at room pitch of 18.53 Ǻ in higher-order packing along the b axis (Fig.
temperature. The obtained triclinic crystal of
crystal of contained space group P1 and C2, respectively. H…π interaction with average distance (CH-π) 2.79 Ǻ and C-
Peptide
1 and monoclinic 3d and 3e). This double helix was further stabilized through C-
2
1
showed two molecules (
A
in orange and
B
in green H…O (2.40 Ǻ) interactions.
Table 1: The backbone torsion angles (deg) of
1
and
2
Torsion
angles
Boc-Gly-L-Phg-D-Phe-OMe (1)
Boc-Gly-L-Phg-D-Phg-OMe (2)
C5-N1-C6-C7= 172.9(3)
Molecule A
Molecule B

C5-N1-C6-C7= -59.8(4)
C30-N4-C31-C32= 155.7(3)


C7-N2-C8-C15= -141.4(3)
C15-N3-C16-C24= 141.1(3)
N1-C6-C7-N2= 150.1(3)
N2-C8-C15-N3= 112.7(3)
N3-C16-C24-O6= 8.0(5)
O1-C5-N1-C6= 165.2(3)
C6-C7-N2-C8= -179.9(3)
C8-C15-N3-C16= -175.4(3)
C32-N5-C33-C40= -128.7(3)
C40-N6-C41-C49= 133.6(3)
N4-C31-C32-N5= -143.2(3)
N5-C33-C40-N6= 140.9(3)
N6-C41-C49-O12= -109.7(3)
O7-C30-N4-C31= 175(3)
C7-N2-C8-C15= -138.0(3)
C15-N3-C16-C23= 142.3(3)
N1-C6-C7-N2= -175.1(3)
N2-C8-C15-N3= 141.3(3)
N3-C16-C23-O25= 18.1(4)
O1-C5-N1-C6= -166.4(3)
C6-C7-N2-C8= 179.4(3)
C8-C15-N3-C16= -175.3(3)






C31-C32-N5-C33= -174.4(3)
C33-C40-N6-C41= 172(3)
2 | J. Name., 2012, 00, 1-3
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Fig. 2 (a) The ORTEP diagram with ellipsoid of 30% probability, (b) antiparallel β-sheet along a axis, (c) non-covalent interactions, (d) higher order assembly along a axis, (e) two A
molecules are held by one B molecule, (f) two B molecules are held by one A molecule, (g) a partial spacefill representation of helical species of B molecules, and (f) spacefill
representation of helix-like structure in molecular packing along b axis, of peptide 1
Fig. 3 (a)The ORTEP diagram with ellipsoid of 30% probability, (b) antiparallel β-sheet along a axis, (c) non-covalent interactions, (d) double helix-like structure in higher order along
the b axis, and (e) spacefill representation of double helix-like structure along the b axis, of peptide 2
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DOI: 10.1039/C9CE01168D
between Cα and ortho hydrogens of L-Phg, and it causes
Table 2: Crystal Parameters and Refinement Data of
1 and 2
backbone extension. Moreover, in the C-terminus, both
1 and
Parameters
Boc-Gly-L-Phg-D-Phe-
OMe (
Boc-Gly-L-Phg-D-Phg-
OMe (
2
contained D-Phe and D-Phg, respectively. The consecutive
1
)
2)
existence of L and D amino acid forces their side chains to
flank in the same direction, which caused more backbone
elongation than the L/L amino acid containing peptides. The
backbone elongation is more in
terminal D-Phg (the aromatic ring is directly attached to Cα)
than (Fig. 4c and 4d).
Formula
Fw
Crystal
system
Space group
a/Å
C25 H31 N3 O6
469.53
Triclinic
C24 H29 N3 O6
455.50
monoclinic
2 for the presence of C-
P 1
C 2
18.5349(8)
8.8497(3)
14.3748(5)
90
91.092(4)
90
2357.45(15)
4
1
8.3294(6)
9.6074(7)
16.4130(11)
86.861(5)
84.758(5)
74.072(6)
1257.12(16)
2
b/Å
c/Å
α/o
β/o
γ/o
V/Å3
Z
Dc/g cm-3
μ Mo
Kα/mm-1
F000
1.240
0.089
1.283
0.093
500.0
968.0
T/K
θ max.
100.00(10)
28.823
100.00(10)
28.815
Total no. of
reflections
Independent
reflections
Observed
reflections
Parameters
refined
9785
5011
6889
6025
621
4110
3677
302
R1, I > 2σ(I)
0.0544
0.0546
wR2, I > 2σ(I)
GOF (F2)
0.1522
0.787
0.1562
0.817
CCDC No.
1874425
1896847
Fig.4 Comparison of the molecular structures between L/L amino acid containing
tripeptides (a) Boc-Gly-L-Phe-L-Phe-OMe (b) Boc-Gly-L-Phg-L-Phe-OMe and L/D
amino acid containing tripeptides (c) Boc-Gly-L-Phg-D-Phe-OMe (c) Boc-Gly-L-
Phg-D-Phg-OMe.
Two subunits formed a dimer connected by two-fold rotational
symmetry. No water molecule or solvent was observed inside
the double helix. Interestingly, long chain polypeptides having
alternating D/L amino acids usually exhibit β-helix
conformation.25,26 The observed torsion angles of
1 and 2 were
Next, we performed CD experiments to know the
conformation of these peptides in solution. After seven days,
the solutions (1.5 mM) were diluted to 300 µM (to avoid high
voltage in CD), and from them 400 µL of each were taken
separately in a quartz cuvette, and CD was measured spanning
the wavelength range between 190-260 nm (1 mm path length
quite similar to the β-helix peptides (Table S1). All H-bond
distances, bond angles, and crystallographic data are displayed
in Table S2 (ESI) and Table 2.
Next, we compared the molecular structures of our previously
reported L/L amino acid containing peptides Boc-Gly-L-Phe-L-
22
and 1 nm bandwidth). The CD profile of peptide
1 exhibited a
Phe-OMe (
A
) and Boc-Gly-L-Phg-L-Phe-OMe (
B
)
and the
positive Cotton effect at 202 nm and a negative Cotton effect
at 194 nm, indicating the presence of a turn-like structure (Fig.
newly designed alternating L/D amino acid containing peptides
Boc-Gly-L-Phg-D-Phe-OMe ( ) and Boc-Gly-L-Phg-D-Phg-OMe
). In , phenyl ring is connected through a methylene group,
1
5) in solution.27,28 Whereas peptide
2 showed a negative
(2
A
Cotton effect at 223 nm and a positive Cotton effect at 198
nm, indicating the presence of a β-sheet conformation.29
Then, we performed the solvent dependent NMR titration
studies to understand the existence of intra- or inter-
molecular H-bond in these peptides. These experiments were
carried out by adding d6-DMSO to the peptide solution in non-
polar CDCl3. The obtained solvent titration curve indicated that
increasing the percentage of d6-DMSO in CDCl3 solution (v/v)
i.e. the ring is not directly attached to Cα of the central L-Phe.
As a result, the Cα became flexible and backbone oriented to
form an open turn (Fig. 4a) structure.22 Whereas in
B, a steric
repulsion arises between Cα proton and aromatic ortho proton
of the central L-Phg, therefore the backbone was extended,
and it adopted parallel β-sheet (Fig. 4b) structure.22
Peptides 1 and 2 also contained L-Phg at the central position of
their sequence. Therefore, similar steric repulsion is present
from 0 to 20 % for
1 and 0 to 12% for 2, a monotonic
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downfield chemical shift of NHs was observed. The net change Next, to understand the aggregation behavior of these
View Article Online
DOI: 10.1039/C9CE01168D
of chemical shifts (Δδ(NH)) were 0.43 (Gly NH), 0.48 (L-Phg peptides, we checked their morphology. Both, optical
NH), and 0.23 ppm (D-Phe NH) for
and 0.38 (Gly NH), 0.35 (L-Phg NH), and 0.57 ppm (D-Phg NH) associated in solution to form highly organized a bunch of
for , respectively (ESI, Table S6, and Fig S14), indicating all flower-petals-like (Fig. 6) and a flower-like architecture (Fig. 6),
NHs were involved in intermolecular H-bonding interactions,22 respectively. Then we performed TEM for getting more detail
1 (ESI, Table S4, and Fig S6), microscope and FESEM images indicated that 1 and 2 self-
2
which was also supported by the SC-XRD experiment.
information on the morphology of the self-associated
peptides, and rod-like microcrystals of various sizes were
observed (Fig. 6). These results indicated that the bunch of
flower-like structures which were obtained from FESEM might
have been constructed from the self-association of the rod-like
microcrystal flakes which were observed under TEM. The rod-
like microcrystals, in turn, may have been formed by the
accumulation of the single helix and double helix-like structure
that was observed by SC-XRD experiments.
Experimental
General Information.
All unprotected and Boc N-protected amino acids, Oxyma and
2-nitrobenzenesulfonyl chloride were obtained from GL
Biochem (China). N, N-Diisopropylethylamine (DIPEA),
dichloromethane (extra pure grade), acetonitrile (HPLC grade)
and trifluoroacetic acid (TFA) were collected from
Spectrochem (India). Citric acid, sodium bicarbonate (NaHCO3),
ethyl acetate, hexane, and DMSO-d6 were obtained from
Merck (India). CDCl3 was purchased from Sigma Aldrich.
Fig.5 CD spectra of peptide 1 (orange curve) and peptide 2 (green curve) in 30%
acetonitrile-water using concentration 300 µM.
Next, the 2D NOESY experiment was performed by using CDCl3
with mixing time 0.60 sec to investigate the existence of inter-
residual NOEs in the molecule. The two crucial inter-residual
NOE interactions, namely NH Phg ↔ Hα Gly and NH Phe ↔ Hα
Phg and others NOEs e.g. Hα Phg ↔ aromatic protons of Phg,
NH Phe ↔ meta aromatic protons of Phe, NH Phe ↔
aromatic protons of Phg were observed (ESI, Fig. S9),
indicating turn like structure present in the solution which was
also supported by CD experiment. The results of the CD and
NMR experiments corroborated well with the structures
observed by SC-XRD. However, the conformation of the
peptides usually changes in the solution. On the other hand,
Peptide synthesis
At first, N-terminal t-butyloxycarbonyl (Boc) protected amino
acid (1 equiv), coupling reagent, o-NosylOXY (1 equiv), Hünig's
base DIPEA (1 equiv) in dichloromethane (DCM) were taken in
a 50 mL round-bottom flask (RB) and the reaction mixture
stirred with magnetic stirrer bar for 5 min for preactivation. At
the same time methyl ester of the next amino acid (1.2 equiv)
with DIPEA (1.2 equiv) in DCM was taken in a 25 mL beaker for
neutralization. Then, this slightly basic solution was added
dropwise to the above reaction vessel and kept stirring for 4-5
h at room temperature. The progress of the reaction was
monitored by thin layer chromatography (TLC). After
completion of the reaction, the reaction mixture was diluted
with DCM and washed with a 10% citric acid solution followed
by the saturated NaHCO3 solution 3 times, in each case. Then,
the organic layer was dried over anhydrous Na2SO4, and the
decanted solvent was evaporated under reduce pressure to
obtain solid both N- and C- protected dipeptide. Next, this
dipeptide was taken in a 50 mL RB and the cleavage cocktail
TFA:DCM (90:10) was added onto it. After 3 h reaction, the
TFA was evaporated under reduce pressure followed by
passing N2 over it and again neutralized it by adding DIPEA.
Then this Boc deprotected dipeptide was coupled with next
Boc-N-amino acid, using the procedure as mentioned earlier to
get the tripeptide. The desired product was purified by silica
gel column chromatography using ethyl acetate-hexane
solvent system.
no effective NOE interactions were observed for
S17).
2 (ESI, Fig.
Fig. 6 Optical microscopic, FESEM and TEM images of self-associated tripeptides (1a),
(1b) and (1c) of 1 and (2a), (2b) and (2c) of 2, respectively, in 30% acetonitrile-water
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Synthesis of peptide 1
High-performance liquid chromatography (HPLC)View Article Online
DOI: 10.1039/C9CE01168D
At first, 500 mg (1.992 mmol) of Boc-L-Phg-OH was dissolved The purity of column purified compound was further checked
in 10 mL DCM, and then 651 mg (1.992 mmol) of o-NosylOXY by analytical HPLC, Waters 600E system using a C18 thermo
and 257 mg (1.992 mmol) of DIPEA was added to it and kept scientific column at a flow rate of 1 mLmin-1, linear gradient of
stirring for 5 min for preactivation. After that, 428 mg (2.390 5-100% CH3CN over 8 minutes in a total run time of 20 min.
mmol) of NH2-D-Phe-OMe was neutralized by 308 mg (2.390 Binary solvent system, solvent A (H2O) and solvent B (CH3CN)
mmol) of DIPEA and added to the above reaction vessel and were used. A UV detector, contain dual wavelength at 214 and
stirred for 4 h at room temperature. After completion of the 254 nm was used.
reaction, work up the reaction mixture and the obtained solid
dipeptide, Boc-L-Phg-D-Phe-OMe was treated with TFA to Mass spectrometry
remove the Boc protecting group. Then, the free amine of Mass spectrum of the peptide was recorded from Agilent-Q-
H2N-L-Phg-D-Phe-OMe was coupled with Boc-Gly-OH for 4 h. TOF 6500 instrument in ESI positive mode and equipped with
After purification by column chromatography, we obtained a Mass hunter workstation software.
white solid of Boc-Gly-L-Phg-D-Phe-OMe (1). The purity of the
peptides was confirmed using analytical HPLC. The isolated Nuclear magnetic resonance (NMR) spectroscopy
peptides were characterized by mass spectrometry as well as All NMR spectra were obtained from Bruker Ascend 600 and
1D [1H] (1H and 13C) and 2D (COSY, TOCSY, and NOESY) NMR 400 MHz instrument at 298 K using CDCl3 solvent. 1D [1H]
spectroscopy (Fig. S2-S9).
White solid; mp 171-173 C
spectra and 2D [1H, 1H] TOCSY (Total Correlation
o
.
1H NMR (CDCl3; 400 MHz) δ 1.43 Spectroscopy), 2D [1H, 1H] COSY (Correlated Spectroscopy)
(9H, s); 2.97-2.95 (2H, d, J = 6 Hz); 3.70 (3H, s); 3.81 (2H, s); were recorded with NS = 16 scans and 2D [1H, 1H] NOESY
4.90-4.85 (1H, q, J = 6 Hz, J = 8 Hz); 5.24 (1H, brs); 5.50 (1H, (Nuclear Overhauser Enhancement Spectroscopy) was
brs); 6.55 (1H, brs); 6.68-6.66 (2H, d, J = 7.2 Hz), 7.07-7.03 (2H, recorded with NS = 32 scans. Chemical shifts were referenced
t, J = 7.2 Hz); 7.14-7.11 (1H, t, J = 7.2 Hz); 7.33-7.29 (5H, m); to CDCl3 at δ = 7.26 ppm and δ = 77.23 in H NMR and 13C
1
7.41 (1H, brs). 13C NMR (CDCl3; 100 MHz) δ 28.4, 37.8, 44.3, NMR, respectively.
52.6, 53.3, 57.1, 80.3, 127.1, 127.4, 128.6, 129.2, 129.3, 129.4,
135.4, 137.6, 156.2, 169.2, 169.4, 171.7. HRMS (ESI): Single crystal X-ray diffraction (SC-XRD)
calculated [M+H]+ 470.2213, found m/z . 470.4121. HPLC: The single crystal XRD experiment was performed on an
retention time (tR) = 11.9 min. Isolated pure product 689 mg Oxford SuperNova diffractometer (Agilent Technologies) using
(yield: 74% w.r.t. starting meterial Boc-L-Phg).
MoKα radiation. Data collection was carried out by SMART
software. Data refinement and cell reductions were performed
by CrysAlisPro 1.171.38.46 (Rigaku OD, 2015).30 The obtained
Synthesis of peptide 2
At first, 500 mg (1.992 mmol) of Boc-L-Phg-OH was dissolved structures were solved by direct methods implemented in
in 10 mL DCM, and then 651 mg (1.992 mmol) of o-NosylOXY SHELX-2014/7 software.31
and 257 mg (1.992 mmol) of DIPEA was added to it and kept
stirring for 5 min for preactivation. After that, 394 mg (2.390 Sample preparation
mmol) of NH2-D-Phg-OMe was neutralized by 308 mg (2.390 2.13 mM of peptide
1 and 2.19 mM of peptide 2 solutions
mmol) of DIPEA and added to the above reaction vessel and were prepared in an Eppendorf vial (2 mL) by adding 1 mL 30%
stirred for 4 h at room temperature. After completion of the acetonitrile-water mixture. Then, we prepared 1 mL (1.5 mM)
reaction, work up the reaction mixture and the obtained solid solution (stock solution) from the above solution. This stock
o
dipeptide, Boc-L-Phg-D-Phg-OMe was treated with TFA to solution was incubated for 7 days at 37 C. After 7 days, we
remove the Boc protecting group. Then, the free amine of prepared FESEM and Microscopic slide sample.
H2N-L-Phg-D-Phg-OMe was coupled with Boc-Gly-OH for 4 h.
After purification by column chromatography, we obtained a Circular dichroism (CD)
white solid of Boc-Gly-L-Phg-D-Phg-OMe (2). The purity of the CD spectrum was obtained from JASCO J-1500 instrument.
peptides was confirmed using analytical HPLC. The isolated After 7 days, the incubated samples (1.5 mM) were diluted to
peptides were characterized by mass spectrometry as well as 300 µM and from each sample 400 µL was taken in a CD
1D [1H] (1H and 13C) and 2D (COSY, TOCSY, and NOESY) NMR cuvette, and the CD spectrum were recorded from 190 nm to
spectroscopy (Fig. S10-S17).
White solid; mp 168-172 C
260 nm wavelength using 1 mm pathlength and 1 nm
o
.
1H NMR (CDCl3; 600 MHz) δ 1.41 bandwidth. The mean residue molar ellipticity was calculated
(9H, s); 3.70 (3H, s); 3.82 (2H, s); 5.20 (1H, br); 5.52-5.51 (1H, using the following equation:
d, J = 7.2 Hz); 5.66 (1H, brs); 7.12-7.11 (2H, d, J = 6.6 Hz), 7.29-

(deg. cm2 .dmol-1) = Ellipticity (mdeg). 106 / Pathlength (mm).
7.23 (10H, m). 13C NMR (CDCl3; 150 MHz) δ 28.4, 44.4, 53.2, [Protein] (μM). N
56.8, 80.4, 127.1, 127.6, 128.7, 129.2, 135.8, 137.4, 156.1,
169.1, 171.0. HRMS (ESI): calculated [M+H]+ 456.2136, found Optical microscopic images
m/z . 456.2362. HPLC: retention time (tR) = 11.8 min. Isolated 10 µL of 7 days aged solution was drop casted on a
pure product 650 mg (yield: 71% w.r.t. starting meterial Boc-L- microscopic slide and dried it. The bright field image (40X
Phg).
6 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
2
3
K. A. H. Wildman, D-K. Lee and A. RVaimewaAmrticoleoOrtnhlinye,
Biopolymers, 2002, 64, 246–254.
magnificence) was captured on Nikon Digital Sight DS-U3
microscope.
L. D. Valle, A. Nardi, P. Belvedere, MD. OTIo: 1n0i.1a0n3d9/CL.9CAEl0ib1a16r8dDi,
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Field emission scanning electron microscopy (FESEM)
FESEM samples were prepared by drop casting (10 µL) of 1.5
4
5
S. Mondal and E. Gazit, ChemNanoMat 2016, 2, 323–332.
S. Bera and E. Gazit, Protein & Peptide Letters, 2019, 26, 88-
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mM of peptide
1 and 2 on Al-foil and dried them inside a
6
7
8
9
desiccator. FESEM images were captured using a SIGMA-300
(ZEISS) instrument.
S. Guha, M. G. B. Drew and A. Banerjee, Org. Lett., 2007,
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9,
Transmission electron microscope (TEM)
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TEM images were captured under JEOL (Model: 2100F)
instrument. The 7 days incubated sample (1.5 mM) was
diluted to 100 µM and from this 10 µL of sample was drop-
casted on carbon-coated copper grid followed by adding 2%
uranyl acetate solution (10 µL) and was allowed to float for 1
min and kept inside desiccator.
10 A. Sharma , P. Tiwari, and A. Dutt Konar, J. Mol. Struct., 2018,
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Conclusions
In conclusion, we demonstrated the conformation and
morphology of both N- and C- protected alternating D/L amino
acids containing tripeptides, Boc-Gly-L-Phg-D-Phe-OMe (
Boc-Gly-L-Phg-D-Phg-OMe (
that they exhibited anti-parallel β-sheet structures, but
16 S. Beraꢀ, S. Mondal, B. Xue, L. J. W. Shimonꢀ, Y. Caoꢀ ꢀand E.
Gazit, Nat. Mater. 2019, 18, 503–509.
1) and
2
). The SC-XRD analyses suggested 17 R. S. Al Toma, C. Brie e, M. J. Cryle and R. D. S ssmuth, Nat.
Prod. Rep., 2015, 32, 1207−1235.
1
2
self-
self-
18 Y. Mast and W. Wohlleben, Int. J. Med. Microbiol. 2014, 304
44–50.
,
assembled to form helix-like architecture whereas
associated to form double helix-like structures. Both
supramolecular structures were stabilized by intermolecular H-
bond as well as C-H…O and C-H…π interactions. Although there
was no intramolecular H-bond in these peptides as suggested
19 B. A. Wallace and K. Ravikumar, science, 1988, 241, 182-187.
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22 R. S. Giri and B. Mandal, CrystEngComm, 2019, 21, 236–243.
,
by SC-XRD and solvent dependent NMR studies,
1 exhibited
turn-like structure in solution suggested by CD and 2D NOESY
experiment. They also self-associated to form flower-like
structures in 30% acetonitrile-water. The differences in
molecular arrangements among these peptides are due to the
presence of the methylene group (D-Phe) or absence of
methylene group (D-Phg) at the side chain. The obtained
results may be helpful for the design of supramolecular single
or double helix-like structure using alternating D/L amino
acids.
23 D. Dev, N. B. Palakurthy, K. Thalluri, J. Chandra and B.
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Conflicts of interest
There are no conflicts of interest to declare.
30 CrysAlisPro Oxford Diffraction Ltd. version 1, 2009, 171.
33.34d.
31 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found.
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Acknowledgements
We are thankful to central Instruments facility (CIF),
Department of Chemistry IIT Guwahati for instrumental
facilities and the Department of Biotechnology, Govt. of India,
for financial support (BT/PR16164/NER/95/88/2015).
Notes and references
1
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241.
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DOI: 10.1039/C9CE01168D