Aggregation propensity of amyloidogenic and elastomeric dipeptides constituents

Article abstract of DOI:10.1016/j.tet.2016.07.022

This study demonstrates the self-assembly of N- and C-terminal protected dipeptides Phe–Gly and Pro–Gly which were derived from amyloidogenic and elastomeric peptide sequences. These constituents afforded nanostructured supramolecular ensembles through va

Full text of DOI:10.1016/j.tet.2016.07.022

Accepted Manuscript
Aggregation propensity of amyloidogenic and elastomeric dipeptides constituents†
Vikas Kumar, K. Vijaya Krishna, Shruti Khanna, Khashti Ballabh Joshi
PII:
S0040-4020(16)30645-7
10.1016/j.tet.2016.07.022
TET 27918
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 3 May 2016
Revised Date: 21 June 2016
Accepted Date: 6 July 2016
Please cite this article as: Kumar V, Krishna KV, Khanna S, Joshi KB, Aggregation propensity
of amyloidogenic and elastomeric dipeptides constituents†, Tetrahedron (2016), doi: 10.1016/
j.tet.2016.07.022.
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Aggregation Propensity of Amyloidogenic and
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Elastomeric Dipeptides Constituents†
Vikas Kumara, K. Vijaya Krishnab, Shruti Khannab, and Khashti Ballabh Joshi*, a
a,* Department of Chemistry, School of Chemical Science and Technology, Dr Harisingh Gour Central University Sagar, MP, 470003,
India. E-mail: kbjoshi77@gmail.com; kbjoshi@dhsgsu.ac.in,
bDepartment of Chemistry , Indian Institute of Technology Kanpur, 208016, India

1
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Tetrahedron
journal homepage: www.elsevier.com
Aggregation Propensity of Amyloidogenic and Elastomeric Dipeptides Constituents†
a
Vikas Kumar a, K. Vijaya Krishnab, Shruti Khannab and Khashti Ballabh Joshi
*
,
a.* Department of Chemistry, School of Chemical Science and Technology, Dr Harisingh Gour Central University,Sagar, MP, 470003, India.
E-mail: kbjoshi77@gmail.com; kbjoshi@dhsgsu.ac.in,
b Department of Chemistry, Indian Institute of Technology Kanpur, 208016, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
This study demonstrates the self-assembly of N- and C-terminal protected dipeptides Phe-
Gly and Pro-Gly which were derived from amyloidogenic and elastomeric peptide
sequences. These constituents afforded nanostructured supramolecular ensembles through
various non-covalent interactions in the solid state which can be directly correlated with
their fibrillation event in solution phase. Interestingly microscopic observations revealed
that the amyloidogenic dipeptide constituents assembled into hollow tubular structures
whereas the elastomeric dipeptide constituents assembled into the feather or sheet like
structures.
Available online
Keywords: Peptide, Self assembly, Fibrillation,
Amyloidogenic, Elastomeric
2009 Elsevier Ltd. All rights reserved
.
the nuclear pore complex (NPC) forming the gateway to regulate the
flow of macromolecules between the cell nucleus and the cytoplasm
contain FG peptide repeats which forms major components of
NPC.43-52 Large number of protein molecules can be transported by a
single NPC every minute. About 50 repeat units of FG making
clusters of hydrophobic groups become unfolded during the passage
of protein and RNA through NPC and get converted into three
dimensional cluttered structures with hydrogel-like properties.
Various approaches have applied to explore the nature of FG domain
and it is found that flexible brush like unfolded structures exist and
exhibit entropic elasticity.
Introduction
The fundamental topological property of proteins and polypeptides is
the formation of three dimensional architectures that are used to the
greatest advantage by nature for molecular recognition1-6 and its
functions in biological systems. To understand the primary principles
which direct the architecture of such motifs is a necessity for probing
the origin of many physical, biophysical, and biomedical phenomena
related to specific diseases.7,8 The common supramolecular motifs,
helical and sheet like arrangements are found in all functional
proteins. The challenging area of research in the ground of ordered
supramolecular peptide assemblies by means of short peptides has
been much focused in recent years.9, 10
In this context, proteins and polypeptides, which are molecular
determinants of elasticity, are highly demanded. Elastomeric proteins
are well known for their robustness and elasticity and are present in a
wide range of living organisms.11-17 Disorderness in their structures
and associated hydration are important features of the elastomeric
proteins.18-21 Nucleoporins,22-26 Tropoelastin,27,28 Spider Silk29-35 and
Resilin36-39 are few examples of natural disordered proteins with
elastomeric function. These elastomeric proteins are rich in amino
acid sequences mainly; Phenylalanine (F), Proline (P) and Glycine
(G), however they all are widely varying in their amino-acid content
along with the dipeptide Phe-Gly and Pro-Gly repeat.40-42 For
example Nucleoporins; which are the constituent building blocks of,
Figure 1. Molecular structure of protected and deprotected dipeptides, Top:
dipeptides with L-amino acid, and Bottom: with D-amino acid.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x

2
Tetrahedron
Apart from FG, PG also often appears in the sequence of
to see the nature of non-covalent interactions, both in solution
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disordered proteins like spider silk. Both Proline and Glycine
contribute to disorder the protein but differently.53-64 Proline which
contain cyclic five membered side chain and known for its rigid
structure, is stiff and highly restricted, disturbing the secondary
structure formation of protein. Whereas Glycine, is known for its
flexibility and lacking any side chain whose order is entropically
unfavourable. However the composition of Proline and Glycine
affect their amyloid formation tendency and elasticity.65-67 For
example the elastic nature of silk fiber was shown to correlate
with high glycine content by measurements from circular
dichroism spectroscopy. The main constituent of amyloidogenic
peptide Aβ42 is Phe-Phe recognition motif and by replacing the
one constituent of this motif with other non aromatic amino acid
showed lower aggregation propensity compare to higher-order
self assembled structures of Phe-Phe.68 Amyloid fibrils are
associated with tissue degenerative diseases whereas elastin
provides extensible tissues including skin and arteries. Though
self organization of these proteins into fibrils is common
phenomenon associated with both the systems but the physical
properties and energy contribution for other biomedical
applications of these systems are poorly resolved.
and solid state73-75 and to understand how subtle change of a
dipeptide sequence can alter the self-assembly and the nano-
structure formation.
We started this work by correlating the solid state structure
of protected dipeptides with solution-phase aggregation.
Crystals of protected dipeptides 1, 2 and
diffraction studies, were grown by careful, time dependent slow
evaporation of solvent methanol/cyclohexane (~70:30).
However our several attempts fail to grow the crystals of
5 suitable for X-ray
conjugate
6
and deprotected D and L dipeptides.76 It was found
that all three protected dipeptides crystallize in orthorhombic
space group P21 21 21 (No. 19) (Fig. 2). Crystallographic
signature of 1, 2 and 5 displayed the difference in solid state
conformation,77-80 which perhaps is the basis of significant
difference in ordered self-assembly. A tabulated comparison of
torsional angles of
that the difference in solid state conformation. Most of the
torsional angles, φ1 ψ1 φ2 and ψ2 values of the constituent
amino acids residues for the compound fall within the
polyproline II (PP-II) structural77 region, however for
compound the torsional angles φ1 ψ1 φ2 and ψ2 values (table
1 (as representative for 5 also) and 2 reveal
,
,
1
2
,
,
1) of the constituent amino acids residues fall only in the
Collagen fiber region of the Ramachandran plot.81
Table -1 Selected torsional angles of compound
t-Boc-Phe-Gly-OMe t-Boc-Pro-Gly-Ome
Torsion Angles Value Torsion Angles Value
1 and 2.
ω1
φ1
ψ1
ω2
φ2
171.2 (2)
-88.9 (3)
163.7(2)
176.2 (3)
-122.1(3)
-15.6 (4)
ω1
φ1
ψ1
ω2
φ2
-12.8 (4)
-51.6 (4)
142.8 (3)
170.5 (3)
-64.4 (4)
154.8 (3)
ψ2
ψ2
A time-dependent aggregative propensity of 1 and 2 was
evaluated by dark field optical microscopic imaging at 63X
magnification. Fresh solution of 1 (3 mM) in 50% methanol-water,
did not reveal any significant and noticeable self-assembly under
optical microscope, but long straight tubular fibers were evident after
2 days of incubation at ambient temperature (Fig. 3a). A continued
Figure 2. Solid state structure of (a) 1, (b) 2 and (c) 5 (hydrogen atoms are
omitted for the sake of clarity). Color code; blue, nitrogen; red, oxygen; gray,
carbon.
Results and Discussions
Based on the above literature information, it is possible that
elastomeric and amyloidogenic peptides can be differentiated by
applying simple chemical and physical changes in shortest
peptides viz dipeptides. In order to understand the fundamental
behaviour of the self assembly of amyloid or elastin like protein
aggregates, we designed a set of D and L- dipeptides sequences
constituted by FG and PG having physico-chemical properties
compatible with those of the hydrophobic domains of amyloid
fibrils, as well as elastin respectively. Since our growing interest
in peptide self assembly and its application in biomedical field,69-
72
encouraged us to study widely the assembling behaviour of all
the protected and deprotected dipeptides. Therefore we have
strategically designed (Fig. 1) the two model dipeptides; where
the second amino acid, i.e. glycine residue was intact in both
peptides but the alteration in the first amino acid viz
phenylalanine and proline. To understand the fundamental
mechanism of these peptides in relation to self assembly we wish
Figure 3. Optical micrographs of self-assembled morphology by 1, (a) after
36 h, (b) 42 h, (c) 48 h, incubation (d) Optical micrographs of self-assembled
morphology of fresh sample of 2 in dark field condition.
follow up confirmed that 42 hours incubation time period changed
the dimensions of tubular structures. The diameter of these tubular

3
structures increases from 0.6 micron to 1.0 micron and length
increased from 1 micron to several micrometers. The lumen of
tubular fiber visualized, hints hollowness of these tubular fiber from
rod like assembly (Fig. 5E, F).85-89 The AFM observations are well
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corresponded with OM data of figure 4 and further confirmed that
both D- and L- isomers perhaps followed the same energetic during
the formation of tubular structures.
inside.
However,
no
solvent
molecules
have
been
crystallographically detected inside the cavity. Amorphous
aggregates and well developed straight hollow tubular fibers were
observed on incubation of 1 up to 48 hours at ambient temperature
(Fig. 3c). However the protected compound 2 showed long straight
fibers at fresh condition only (Fig. 3d).
To understand the nature of time dependent self assembly, 1 mM
solution of conjugate 1, as representative molecule, and 10 ꢀM
fluorescein solutions were co-incubated at ambient temperature, in
50% methanol/water solvent. This stock solution was equally divided
into six portions and transferred into six separate eppendorf tubes. A
20 ꢀL aliquot of fresh and the aged solution of different time interval
viz 0, 8, 16, 24, 32 and 40 h from the different respective tubes were
loaded on a glass slide and dried at room temperature followed by
imaging under a fluorescence optical microscope.
Interestingly, instantaneous formation and appearance of
spherical structures were observed at time 0 h which were organized
into a compact colony of spherical structures from time 8 h to 32 h
and finally transformed into fibrillar/tubular structures after 40 h of
incubation (Fig. 4). This time dependent study reveals that
formations of tubular structures are thermodynamically stable. When
considering the transformation of spherical aggregates to defined
tubular structures, thermodynamic factors should also be invoked.
Figure 5. AFM micrographs of conversion of spherical aggregates of 5 into
rod like structures; at (A) 0 h, (B) 12 h, (C) 36 h, and (D) 48 h incubation,
(E) formation of robust rod like assembly and (F) corresponding 3D structure.
Figure 4. Florescence optical micrographs of conversion of spherical
aggregates of 1 into tubular structures; at (A) 0 h, (B) 8 h, (C) 16 h, (D) 24 h,
(E) 32 h and (F) 40 h incubation. Scale bar 20 µm
To check the self assembly of all the protected dipeptide
conjugate, we have used 3 days incubated samples of 1, 2, 5 and 6.
The solutions of these samples were spread over the surfaces of
freshly cleaved mica followed by AFM imaging. The image analysis
revealed that protected dipeptide constituted by D and L isomers of
phenylalanine i.e. 1 (Fig. 6A) and 5 (Fig.5, 6C) are forming similar
robust structures which are almost similar in morphology whereas
the compound 2 giving robust feather or sheet like structures which
are composed of small fibers and 6 (Fig. 6D) assembled into flexible
thin fibers as compared to 2 (Fig. 6B). Therefore we did not observe
any major morphological change in the self assembly of 1 and 5
however, our observations reveal that protected dipeptide 2 and 6
show little variation in their self assembly process, which could
perhaps be due to the amorphous nature of 6 as compared to 2 and
hence did not get crystallize.
The typical condition for the formation of crystals of conjugate
1, 2 and 5 are time dependent slow evaporation at ambient
temperature and the identical applied conditions also accelerate the
transformation of spherical aggregates into tubular structures
observed under optical microscope.82-84 It has been noted that the
prolonged incubation at ambient temperature promoted the ordering
of random aggregates into ordered one and therefore commensurate
with energetic factors which supported the growth of random
aggregates in a particular directions.
Further to check the self assembly process of D-isomer, 5,
which was only differing in its chirality, we prepared a fresh solution
of 5 and imaged under AFM. The Freshly prepared solution of 5
revealed uniformly distributed punctuated structures over the mica
surface (Fig. 5A), while incubating it for 12 h the solution of 5
We further investigated the time dependent self assembly process
of the deprotected analogues of these protected compounds i.e. 3, 4,
7 and 8 (Fig.7). The freshly prepared 1 mM solution of all these
deprotected compounds in 50% methanol-water self assembled into
defined morphology. The compound 3 shows flexible fiber formation
while 7 assembled into straight rod like morphology. In contrast to
resulted prefibrillar aggregates (Fig. 5B) confirming
a time
dependent fibrillation event too. At 36 h of incubation these
aggregates get partially converted into fibrillar/tubular structures,
where the association of spherical aggregates clearly visualized,
which is further confirmed by its 3D image (Fig. 5C, D). The
prolonged incubation of this solution leads to the formation of robust

4
Tetrahedron
protein. All compounds have one flexible amino acid glycine which
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is covalently linked with the phenylalanine and proline, and known
for its trivial hydrophilicity which may increase the solution state
intermolecular or intramolecular hydrogen bonding. If the N and C-
terminus of these peptides are free the non-covalent interactions will
get enhanced hence assembled rapidly. In case of the dipeptides
which contain Phe residues will show slightly different behavior due
to the intervention of aromatic side chain in the mixture of polar
organic and inorganic solvents (50% aqueous methanol). Therefore
AFM microscopic investigations of protected and deprotected
dipeptides reveal that aggregation propensity of all these conjugates
behave differently. Further to understand the nature of the fibrous
morphology obtained by AFM we have recorded the PXRD of fibers
and compared with X-Ray pattern from crystallography. The solution
of peptides samples deposited over the glass surface followed by
drying produced thin film of thick fibers. The observations from
PXRD pattern reveal that mostly all the fibers are amorphous in
nature (data not shown).
Therefore at this stage to understand the solution state self-
assembly process of these conjugates and its correlation with solid
state structures and assembly,
we have taken 1 and 2 as
1
representative molecules (Fig. 8). We did the H NMR titration in
various ratios of CH3OH in CDCl3 (0-40%) to explain the effect of
possible non covalent interactions via the respective participation of
amido –NH, aromatic, Phe-α and Pro-α key protons in the self-
assembly process.90, 91 The downfield chemical shifts of the amide
protons of 1 and 2 and upfield chemical shifts of aromatic, Phe-α and
Pro- α of compound 1 and 2 proton(s) respectively was evident
through an incremental addition of CH3OH in CDCl3. A marked
movement of key proton resonances is suggestive of the crucial role
of intermolecular π stacking and hydrogen bonding interactions in
the supramolecular ensemble of 1 and 2 in solution state (Fig. 8).
Figure 6. AFM micrographs of 3 days aged samples of L and D conjugates of
1, 2, 5 and 6. (A) 3 days aged sample of 1 showing tubular structures, (B)
sample of 2 showing sheet like structures, (C) sample of 5 also showing
robust tubular like structures and (D) sample of 6 showing long thin fibers.
this fresh sample of proline containing compounds 4 and 8 rapidly
assembled into extensive thin fibers perhaps due to the increase in
non-covelent interaction compare to protected one and hence can
work as gelants upon changing solvents. Such type of different
behavior in self-assembling morphology among these compounds is
due to the presence of hydrophobic groups such as t-Boc and OMe
and variation of two amino acids specifically phenylalanine an
1
Figure 8. H NMR titration experiments of representative compound 1 (top)
and 2 (bottom) in the presence of increasing amount of CH3OH. The titration
experiments were performed in the CDCl3 solvents. The remarkable change
in the key protons were observed and correlated with solid state structures.
Further these noncovalent and other short contacts were clearly
visualized in the solid state structures of these molecules and
supported by literature too. For example the …C-H…π interaction
Figure 7. AFM micrographs of fresh samples of deprotected L and D
conjugates 3, 4, 7 and 8. Figure depicts: (A) thin flexible fibrilar structures of
3, (B) formation of extensive branch like structures from 4, (C) robust tubular
like structures of 7 and (D) extensive fibrillation event in 8.
t-
was found in the solid state structure of 1 where the …C-H of Boc
group was directly showing the interaction with aromatic ring of
another molecule in the same unit cell. We have further investigated
this behavior with the help of H NMR titration experiment where
aromatic side chain substituent and proline which is also known for
its structure breaking role and rigidity in various peptides77 and
1

5
upon incremental addition of the polar solvent, CH OH, showed up
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3
t-
field shift for Boc protons and aromatic protons (Fig. 9). The solid
state structure (Fig. 2b) of the compound 2 also supports these
observations in which the inter and intramolecular hydrogen bonding
distances range from 2.07 Å – 2.84 Å and other short contacts which
are from 2.36 Å- 3.33 Å stabilizes the crystal packing. These factors
are perhaps responsible for the rapid self-assembly of 2 (Fig. 6B) and
it’s deprotected conjugate 8 in solution state too. These observations
reveal that upon simple side chain modification one can transform
the nature of self-assembly which will be useful for several
nanotechnological applications.
Figure 10. Solid state structure of (a) Boc-Phe-Gly-methyl ester “1” arranged
in a columnar fashion (b) Displaced π-π stacking (non bonded hydrogen’s are
omitted for the sake of clarity), (c) AFM micrograph of solution state self-
assembly of 1.
with a strong C-H...π interactions between the hydrogen from
tert-butyl group and phenylalnine aromatic ring along with
other non covalent interactions. The elongation of crystal lattice
directly correlates with AFM microscopic data where we have
seen the straight fiber formation (Fig. 10c, 5C). Hydrogen
bonding distances which are ranging from 2.05 Å-2.16 Å in
1
Figure 9. H NMR titration experiments of compound 1 in the presence of
increasing amount of CH3OH. The figure depicts that the remarkable
downfield shift in t-Boc and Phe aromatic protons noted and correlated well
with solid state structure of 1.
solid state structure of
1 also help in columnar packing.
The challenging area of research in the ground of ordered
supramolecular peptide assemblies by means of short peptides
has been much focused in recent years.93, 94 The construction of
well-defined three dimensional hollow nanotubular assemblies by
cyclic D and L-oligopeptides was advent of nanotechnology in
past few decades and was contributed by Ghadiri and co-
workers.95, 96 It is known that synthetic tripeptide and terminally
protected acyclic tripeptides self assembled into beeta-sheet type
helical structures97 or into polydispersed nanorods.98 We also
Other than circular dichroism (CD) spectroscopy, FTIR
spectroscopy has also been used to evaluate protein secondary
structure. The specific and qualitative information can also be
obtained by using FTIR spectroscopy, a larger group of peptides and
protein features can be classified especially relating specific Amide I
and Amide II bands to secondary structure. By comparing the
intensity ratios of these two bands we can easily determine the
amount of secondary structures before and after the incubation of
peptides.99-101 Therefore we recorded the FTIR spectra of two
representative compounds to access information about the secondary
structures in self-assembled peptide nanostructures described here.
We have now taken the FTIR of fresh and incubated (fibers)
samples. Interestingly the observations from FTIR reveal that there is
change in secondary structures after the incubation of samples. We
have taken the solution of fresh samples of protected FG and PG
peptides in appropriate amount as a representative molecules
followed by lyophilization. When the solvent was removed
completely the samples were ready for IR measurement. The FTIR
spectra showing the differences in main functional group
absorbances when compared with FTIR spectra of corresponding
fresh samples. The changes in the Amide I, Amide II, aromatic and
aliphatic regions of these peptides giving hint that there is a
significant change in the secondary structures (ESI) which is well
corresponded with AFM micrographs where we observed the
formation of well defined aggregates or fibers.102
Based on all the above information, we decided to construct a
structural model to understand the emergence of distinct
morphological structures by changing side chain substituent’s in
simple dipeptides. Therefore we have taken 1 as representative
molecule. Model figure depicts (Fig. 10) that in solid state the
molecules of 1 arranged in a columnar fashion when viewed along
“a” axis (Fig. 10). The elongation of crystal packing which is mainly
stabilized by hydrogen bonding and displaced π-π stacking92 and
showed the distance between two aromatic rings i.e. 5.39 Å along
Figure 11. Depicts that (A, B) SEM images of conjugate 1 showing the
hollowness of self assembled structures, (C, D) fluorescence optical
microscopic images further confirming the cavity from the inner side.

6
Tetrahedron
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observed that terminaly protected elastomeric and amyloidogenic
Peptide synthesis - All N-Boc-protected and de-protected
dipeptides sequences assembled into nanotubes or nanorods like
structures. Our observations reveal that the D and L phenylalanine
containing dipeptides produced well defined ordered structures
compare to D and L proline one. We were able to confirm the
nanostructures of phe containing dipeptide 1 is forming tubular
structures, where the inside cavity is clearly visible under SEM (Fig.
11A, B). This observation was further supported by the
peptide conjugates were synthesized by simple solution phase
fragment condensation methodologies using t-Boc chemistry
and in the presence of HOBt. All he compounds were
characterized well and satisfactory analytical spectroscopic
results were also obtained for all the samples.
Atomic Force Microscopy (AFM) – Neat and co-incubated
sample (at 37 C for 0-5 days in methanol/water) solution of all
o
following experiments. The 40h pre incubated sample of 1 stained
with fluorescein dye was, instantaneously analyzed90 under
fluorescence microscope using FITC filter in the dark field. These
nanostructure appeared fluorescent green under the microscope and a
dark lumen was observed at both the end of the tubular structures
(Fig. 11C). The microscopic image of single tubular structures was
further confirming these observations (Fig. 11D) where both the ends
of the tubular structures are dark. Fluorescence detection is possible
only when the dye is showing interaction with peptide molecules and
since the structures are hollow from inside, therefore perhaps peptide
cannot provide any active site at the void for dye interaction and
therefore appearance of dark lumen under fluorescence optical
microscope, especially at both the end of nanotubes.
the peptide conjugates were imaged with an atomic force
microscope. The samples were placed on freshly cleaved
muscovite mica surfaces and the sample-coated substrates were
dried at dust free space under 60W lamp for 12h followed by
high vacuum drying and subsequently examined under atomic
force microscope (AFM).
(INNOVA, ICON Analytical
Equipment, Bruker, Sophisticated Instrument Center-Dr.
Harisingh Gour Central University, Sagar-M.P.) operating
under the Acoustic AC mode (AAC or Tapping mode), with the
aid of a cantilever (NSC 12(c) from MikroMasch, Silicon
Nitride Tip) by NanoDrive™ version 8 software. The force
constant was 0.6 N/m, while the resonant frequency was 94-136
kHz. The images were taken in air at room temperature, with
the scan speed of 1.5-2.2 lines/sec. The data analysis was done
using of Nanoscope Analysis Software.
Conclusion
This report clearly demonstrates the formation of a supramolecular
nanostructure in the solid state via hydrophobic interactions and
hydrogen bonding from self-assembling elastomeric and
amyloidogenic dipeptides constituents. The solid state assembly can
directly be correlated with solution state self-assembling
architectures. Further the phenylalnine containing dipetides are
making hollow tubular structures which give us the more insight of
such sophisticated structure of nuclear pore complex and its
tunneling mechanism. The selective permeability of NPC for
different essential biomolecules and minerals and the transportation
mechanism of nuclear membrane can easily be explained by these
observations obtained by such solution phase self assembly of FG
dipeptide. Such supramolecular nanotubular architectures of these
dipeptides can show potential applications in the field of biomedical
and nanotechnology. We have shown the difference between the self
assemblies of the very simple dipetide sequences and which perhaps
is important for the design and construction of new, bioinspired
nano-materials in the future. This report also indicates that not only
cyclic peptides but also shortest acyclic peptides with suitable amino
acid side chain that can form H-bonds with the main chain or side
chain H-bonding group(s) may be used to construct nanochannels
Scanning Electron Microscopy (SEM) - A solution of 20 ꢀL
aliquot of the fresh and aged samples of peptides were dried at
room temperature on a copper stubs and coated with gold.
Scanning electron microscopy images were made using a FEI
QUANTA 200 microscope equipped with a tungsten filament
gun operating at WD 10.6 mm and 20 kV. Concentration of
peptide sample was 1 mM.
Optical Microscopy- Peptide self-assembled structures were
examined under a fluorescent optical microscope (Leica
DM2500M), in a dark field in addition to bright field under 63x.
1-3 mM of the peptide solution was incubated for 0-5 days in
50% methanol/water. 20 µL of this solution was spread on a
glass slide and allowed to dry under petri dish in dust-free place
at room temperature followed by imaging under optical
microscope. For Fluorescein dye staining experiments, 2 days
aged peptide samples were stained by these dye and 10µL of
stained peptide samples were mounted on glass slides and
allowed to dry under Petri dish in dust-free place. The dried
and nanotunnels
.
samples were rinsed with distilled water 3×5s each for removal
of excess and unbounded dye. The excess water was dried and
the samples were analyzed under polarized microscope.
Experimental section
General-
Dichloromethane,
N,
N-dimethylformamide,
NMR titration experiments. NMR titration experiments were
carried out on JEOL-JNM LAMBDA 500 model spectrometer.
1D spectrum was recorded at a peptide concentration of 10
mg/400µL CDCl3, at 298 K. Description of exposed NH groups
and other protons were achieved by titrating a CDCl3 solution
with low concentrations of CH3OH.
methanol and triethylamine were distilled following standard
procedures prior to use. N, N′-dicyclohexylcarbodiimide, N-
hydroxybenzotriazole, L-amino acids, glycine and N-
hydroxysuccinimide were purchased from Spectrochem,
1
Mumbai, India, and used without further purification. H and
13C NMR spectra were recorded on JEOL-JNM LAMBDA 400
model operating at 400 and 100 MHz, respectively. HRMS
mass spectra were recorded at IIT Kanpur, India, on Waters, Q-
Tof Premier Micromass HAB 213 mass spectrometer using
capillary voltage 2.6-3.2 kV.
Fourier Transform Infrared Spectroscopy: Infrared spectra
of peptide samples were recorded using a Bruker Vertex 70
Fourier transform infrared spectroscopy (FTIR) spectrometer
with a resolution 4 cm−1, scan speed 2.5 kHz, and 128 scans co-
addition, in the KBr pellet form. The obtained spectra were

7
smoothened by using the Savintky-Goolay algorithm to
eliminate the noise. Peptide solutions were incubated at 37 °C
followed lyophilization.
CCD4 X-ray diffraction instrument. The crystal was solved by
ACCEPTED MANUSCRIPT
direct methods and refined by using full-matrix least-squares on
F2 (SHELX97). Crystal data for compound-
: C13H22N2O5: M =
286.32, orthorhombic, space group P21 21 21 (no. 19), a =
4.57Å; b = 14.48 Å; c = 15.65Å, V= 1489.33(3)Å3, Z = 4,
d =
3
Crystal structure refinement details for tert-Boc-L-Phe-Gly-
OMe(1): The compound was crystallized by the slow
evaporation method. Data was collected on a Bruker SMART
CCD4 X-ray diffraction instrument. The crystal was solved by
direct methods and refined by using full-matrix least-squares on
1.28g/cm-3, T = 293(2) K, ꢀ(M0-K) = 0.098 mm-1, 10036
reflections collected, 2134 independent reflection [R(int) =
0.0664], final R1 = 0.078, wR2 = 0.126(all data) [I>2sigma(I)],
R1 = 0.057, wR2 = 0.108 (obs data). The structure was
expanded using Fourier techniques. All other non-hydrogen
atoms were refined anisotropically. Hydrogen atoms are placed
at geometrically idealized positions. The final cycle of full-
matrix least-squares refinement using SHELXL97 converged
with unweighted and weighted agreement factors, R = 0.078
and wR = 0.126 (all data), respectively, and goodness of fit, S =
1.116. The maximum and minimum peaks on the final
difference Fourier map corresponded to 0.387 and -0.269 eÅ-3.
CCDC contains the supplementary crystallographic data for this
paper with a deposition number of CCDC 670518. Copies of
this information can be obtained free of charge on application to
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. [Fax: +44-
F2 (SHELX97). Crystal data for compound-
336.4, orthorhombic, space group P21 21 21 (No. 19), a =
10.62Å; b = 16.68 Å; c = 20.68Å, V= 3667.26(4)Å3, Z = 8,
1: C17H24N2O5: M =
d
=
1.22g/cm-3, T = 293(2) K, ꢀ(M0-K) = 0.090 mm-1, 24535
reflections collected, 5025 independent reflection [R(int) =
0.0724], final R1 = 0.093, wR2 = 0.013(all data) [I>2sigma(I)],
R1 = 0.054, wR2 = 0.0107 (obs data). The structure was
expanded using Fourier techniques. All other non-hydrogen
atoms were refined anisotropically. Hydrogen atoms are placed
at geometrically idealized positions. The final cycle of full-
matrix least-squares refinement using SHELXL97 converged
with unweighted and weighted agreement factors, R1 = 0.093,
wR2 = 0.013(all data), respectively, and goodness of fit, S =
1.049. The maximum and minimum peaks on the final
difference Fourier map corresponded to 0.41 and -0.28 eÅ-3.
CCDC contains the supplementary crystallographic data for this
paper with a deposition number of CCDC 670517. Copies of
this information can be obtained free of charge on application to
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. [Fax: +44-
1223/336-033;
E-mail:
deposit@ccdc.cam.ac
or
www.ccdc.cam.ac.uk.
Acknowledgements
VK thanks NFOBC-UGC-India for pre-doctoral research
fellowship. KVK and SK thank IIT-Kanpur India for SRF.
Authors thank Sophisticated Instrument Centre (SIC)- Dr.
Harisingh Gour Central University Sagar-India for AFM and
SEM facility. Our acknowledgment is incomplete without
thanking Prof. Sandeep Verma, IIT Kanpur for his constant
encouragements, invaluable support and for providing the
research facility. This work is supported by UGC-BSR start-up
scheme from University Grant Commission (UGC), India and
Extramural Research Division, project no. 02(0238)/15/EMR-II
from Council of Scientific and Industrial Research (CSIR) India
to KBJ.
1223/336-033;
E-mail:
deposit@ccdc.cam.ac
or
www.ccdc.cam.ac.uk.
Crystal structure refinement details for tert-Boc-D-Phe-Gly-
OMe(5): The compound was crystallized by the slow
evaporation method. Data was collected on a Bruker SMART
CCD4 X-ray diffraction instrument. The crystal was solved by
direct methods and refined by using full-matrix least-squares on
F2 (SHELX97). Crystal data for compound-
336.4, orthorhombic, space group P21 21 21 (No. 19), a =
5: C17H24N2O5: M =
10.612Å; b = 16.67 Å; c = 20.66 Å, V= 3656.22(4) Å3, Z = 8,
d
= 1.22g/cm-3, T = 293(2) K, ꢀ(M0-K) = 0.090 mm-1, 9225
reflections collected, 5119 independent reflection [R(int) =
Notes and references
0.0724], final R1
= 0.0675, wR2 = 0.1909(all data)
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