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The Role of Molecular Structure of Phenylalanine Peptides on the

Formation of Vertically Aligned Ordered Bionanostructures:
Implications for Sensing Application
Vefa Sahibbeyli, Dilara B. Yildiz, Gözde Papir, Yavuz Dede,* and Gokhan Demirel*
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ABSTRACT: Nanostructured peptide materials in ordered 3-D morphologies are

promising in various applications including sensing, nanomedicine, and energy
harvesting. Despite the significant progress in the fabrication of such materials,
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critical issues such as the role of amino acid sequence or protecting group
anchoring the biomolecular structure on the formation of their ordered 3-D
morphologies still prevent their further applications. In order to investigate the
above points and to establish instructive engineering rules for the fabrication of 3-
D bionanostructures, in this work, different biological molecules with varying numbers of phenylalanine units (single amino acid, H-
Phe-NH2 to tetrapeptide H-Phe-Phe-Phe-Phe-OH) and various protecting groups (i.e., Boc, Fmoc) are studied. The thin films
generated from these molecules are fabricated through a physical vapor deposition technique by engineering the deposition
parameters. The results obtained indicate that H-Phe-Phe-OH, Boc-Phe-Phe-OH, and H-Phe-Phe-Phe-Phe-OH peptides generate
vertically aligned, highly ordered 3-D bionanostructures, whereas H-Phe-NH2, Fmoc-Phe-OH, Fmoc-Phe-Phe-OH, and H-Phe-Phe-
Phe-OH do not yield any distinctly ordered structure under the same deposition condition. Quantum chemical calculations suggest
that while 2-D morphologies are dictated by both weak H-bonding and π−π stacking interactions, clusters that possess H-bonds as
the primary interaction display a 3-D morphology. Our results provide not only a detailed understanding of the role of molecular
structure of phenylalanine based peptides on the formation of ordered 3-D morphologies but also promising possibilities in the field
of sensing and tissue engineering applications.
KEYWORDS: phenylalanine, peptide, self-assembly, vapor-deposition, molecular engineering, morphological engineering

1. INTRODUCTION condition.12 However, due to the harsh experimental

Since the discovery of the peptide nanotube formation via the
self-assembly of diphenylalanine (Phe-Phe) dipeptides by
Reches and Gazit,1 fabrication of such bionanostructures
using various starting materials in liquid medium were
reported.2−5 By adjusting the solution parameters (e.g.,
solvent, pH, and ionic strength), amino acid sequence, and
protecting groups such as fluorenylmethyloxycarbonyl
(Fmoc)6 and tert-butyloxycarbonyl (Boc),5 anchoring to the
molecular structure of peptides, bionanostructures featuring
different morphologies including tube,7 fibril,8 rod,9 plate,9 and
vesicle10 were obtained. Apart from the unique morphological
variations in 2-D, the fabrication of ordered 3-D biomolecular
thin films has also been proposed using different approaches.
The first approach is based on the rapid evaporation of the
organic solvent, which is employed to prepare the peptide
solution, at a relatively high temperature over a siliconized
glass.11 Although the vertically aligned array of peptide
structures can be formed through this way, the loosely
scattered morphology of peptides on the substrate and
incapability for the large-area fabrication is limited to its
further applications. Another approach to achieve ordered 3-D
biomolecular morphology is the solid-phase growth of peptides
at high temperatures using aniline vapor under anhydrous
conditions, the inherent features of fabricated peptide
structures through this technique might be compromised. In
addition to these techniques, vapor deposition of the peptide
molecules on a solid substrate was utilized to fabricate
vertically aligned and ordered 3-D biomolecular films.13−15
Although a large number of reports concerning the
manipulation of peptide self-assembly in liquid medium have
been published in the literature, critical problems related to the
vapor-based fabrication of such materials remain somewhat
unclear. Consequently, the development of novel bionanos-
tructures with unique 3-D morphologies is still an active field
in science and technology. In this context, to understand the
role of the molecular structure of peptides on the formation of
ordered 3-D morphologies and to establish instructive
engineering rules for the vapor-based fabrication of 3-D
Received: February 18, 2020
Accepted: April 23, 2020
Published: April 23, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acsanm.0c00456

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Scheme 1. (A) Structure of Molecules Studied in This Work and (B) Schematic Illustration of the PVD Process for Fabricating
the Peptide Films
Figure 1. Top-view and cross-sectional SEM images of H-Phe-NH2 (a−c) and Fmoc-Phe-OH (e−g) thin films. The θ-2θ XRD patterns of
fabricated H-Phe-NH2 (d) and Fmoc-Phe-OH films (h).
bionanostructures, herein, we studied different biological
molecules with varying numbers of phenylalanine units ranging
from single amino acid (H-Phe-NH2) to tetrapeptide (H-Phe-
Phe-Phe-Phe-OH), and protecting groups (i.e., Boc, Fmoc)
(Scheme 1). We demonstrated that H-Phe-Phe-OH, Boc-Phe-
Phe-OH, and H-Phe-Phe-Phe-Phe-OH molecules lead to
vertically aligned, highly ordered 3-D bionanostructures,
whereas H-Phe-NH2, Fmoc-Phe-OH, Fmoc-Phe-Phe-OH,
and H-Phe-Phe-Phe-OH do not form any distinctly ordered
structure under the same deposition condition. Theoretical
calculations also indicate that while 2-D morphologies are
mainly dictated by both weak H-bonding and π- π stacking
interactions, clusters that possess H-bonds as the primary
interaction display a 3-D morphology. Our results clearly
suggest that the molecular and morphological engineering of
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biomolecules undoubtedly can emerge as promising functional
materials for future sensing and tissue engineering applications.
2. EXPERIMENTAL SECTION
Fabrication of Samples. All materials, H-Phe-NH2, Fmoc-Phe-
OH, H-Phe-Phe-OH, Fmoc-Phe-Phe-OH, Boc-Phe-Phe-OH, H-Phe-
Phe-Phe-OH, and H-Phe-Phe-Phe-Phe-OH were purchased from
Bachem AG (Switzerland) and used as received. Vapor deposition of
all starting materials was carried out on the silicon wafer (001
crystallographic orientation and 1−10 Ω resistivity) substrates (MTI,
U.S.). In a typical fabrication, the substrates were first cleaned with a
piranha solution for 30 min and dried under nitrogen gas flow. Before
the deposition, precleaned silicon substrates were also treated with
oxygen plasma at low pressure for 15 min. The deposition of selected
molecules was carried out in a modified physical vapor deposition
(PVD) system (NANOVAK HV) with an ultrafast deposition rate
(>40 nm/s) and a 90° deposition angle for varying deposition times
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Figure 2. Top-view and cross-sectional SEM images of H-Phe-Phe-OH (a−c), Fmoc-Phe-Phe-OH (e-g), and Boc-Phe-Phe-OH (i−k) thin films.
The θ-2θ XRD patterns of fabricated H-Phe-Phe-OH (d), Fmoc-Phe-Phe-OH (h), and Boc-Phe-Phe-OH (l) films.
(5−10 min) under a fixed base pressure (∼10−6 Torr). After
deposition, all fabricated film morphologies were characterized by a
scanning electron microscope (FEI QUANTA 400F Field Emission
SEM) at an acceleration voltage of 10 kV. The density of
nanostructures fabricated on the surfaces was calculated from SEM
images using a freeware ImageJ software. The molecular packing of
fabricated films was also analyzed using a Rigaku Ultima-IV X-ray
diffractometer. The diffracted intensity of Cu Kα radiation (40 kV and
30 mA) was investigated over 2θ values ranging from 2.5 to 65°at a
scan rate of 1°/min.
Computational Details. All amino acid geometries from cluster
sizes 1 to 4 were optimized using UFF16 force field. The single point
energy calculations were also performed using density functional
theory17−19 with the unrestricted formalism of ωB97XD20 functional
and 6-31G(d,p)21 basis set as implemented in Gaussian0922 software.
3. RESULTS AND DISCUSSION
We first deposited thin films using H-Phe-NH2 amino acid
without any protecting group via PVD on Si substrates. The
deposition parameters, however, were engineered to have a
short source-substrate distance of ∼5−7 cm, deposition angle
of 90°, a low substrate temperature of ∼30−40 °C, and
ultrafast deposition rate of >40 nm/s to facilitate uniform and
reproducible growth of the material.23,24 Figure 1a−c reveal
the SEM images of fabricated samples. It is found that H-Phe-
NH2 molecules in the deposition lead to the formation of the
interconnected two-dimensional (2-D) plate-like grains with
lateral sizes of 5.3 ± 0.8 μm with a thickness of 0.2 ± 0.1 μm.
Similar 2-D highly interconnected ribbon-like structures have
also been observed when Fmoc-Phe-OH amino acids were
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deposited under the same fabrication condition (Figure 1e−g).
In the presence of the Fmoc protecting group, Phe amino acids
might have assembled to form ribbon-like morphologies
instead of plates due to the strong π-stacking interaction
between Fmoc moieties.
In order to enlighten the microstructural features of the
deposited films, X-ray diffraction (XRD) measurements were
performed. As shown in Figure 1d, the strong diffraction peaks
at 2θ = 6.45° and 24.6° were identified for H-Phe-NH2 films.
These values correspond to a periodicity (d-spacing) of 13.7
and 3.6 Å, respectively and suggest that the Phe amino acids
without Fmoc protecting group grow along the out-of-plane
direction indicating close π−π stacking between phenyl rings.
Additionally, a relatively weaker low-angle secondary diffrac-
tion peak at 6.45° (d-spacing = 13.7 Å) corresponds that H-
Phe-NH2 molecules are also standing upright on the surface. In
light of these results, it is reasonable to conclude that the
combination of π−π stacking and two-dimensional hydrogen-
bond networks between H-Phe-NH2 molecules drive the
formation of 2-D films.25,26 Quantum chemical calculations
also support this view (vide infra). The Fmoc-Phe-OH thin
films were also analyzed by XRD. However, no strong
diffraction peaks were identified contrary to H-Phe-NH2
films (Figure 1h). The weak and broad XRD peak of Fmoc-
Phe-OH films at 25.3° corresponds to a d-spacing of 3.5 Å.
Thus, both the Fmoc groups in the solid-state and the phenyl
rings of Phe resulted in highly interconnected 2-D films due to
strong π−π stacking.
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Figure 3. Top-view and cross-sectional SEM images of H-Phe-Phe-Phe-OH (a−c) and H-Phe-Phe-Phe-Phe-OH (e−g) thin films.
It should be noted that the effect of water on the molecular
self-assembly process was not taken into consideration in our
investigation. We are well aware that the water molecules play
an important role in the molecular self-assembly, especially in
liquid medium, and it is possible that the biological molecules
used in this work contain some residual water. However, our
fabrication process is based on the vapor phase deposition and
we used a thermal heater in this process to evaporate biological
molecules. Therefore, it is plausible to assume that, the water
molecules inside the materials have evaporated before starting
the evaporation of biological molecules, and hence water is
presumably not involved in the mechanism of self-assembly.
Future work will definitely be conducted to understand the
role of water on the vapor phase self-assembly of peptide
molecules in detail.
In the second part of our work, the Phe-Phe dipeptides with
and without protecting groups (i.e., Fmoc and Boc) were used
as starting materials in the deposition. The H-Phe-Phe-OH
dipeptides having no protecting group were first deposited on
Si wafers as mentioned above. The SEM images of the
fabricated films demonstrate the formation of high aspect ratio,
closely packed, and well-aligned nanofibers with a density of
1.3 × 109 nanofibers.cm−2 and an average length of ∼4 μm
(Figure 2a−c). The diameters of these nanofibers, however,
displayed a broad distribution, ranging from 40 to 200 nm.
Interestingly, we could not observe any 3-D ordered
morphology contrary to H-Phe-Phe-OH films, when the
Fmoc-Phe-Phe-OH molecules were employed in the deposi-
tion. The Fmoc-Phe-Phe-OH films only consist of 2-D highly
dense thin films with some fiber-like aggregations (Figure 2e−
g). This finding is somewhat unexpected because, in solution
medium, it is well-documented that the Fmoc-Phe-Phe-OH
assembles into the formation of hydrogels having micro/nano-
fibrillar structures through the dilution of the peptide solution
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in an organic solvent with water or external triggering forces
such as sonication and heating.27−30 Considering that the
strong π−π stacking between the planar Fmoc group at the
molecular termini and other molecular interactions between
Phe-Phe species have pivotal roles during film growth, the
observed morphological differences undoubtedly indicate the
variations in the mechanism of film growth due to varying
growth kinetics/thermodynamics. For the vapor-deposited
Fmoc-Phe-Phe-OH films, the 2-D film morphologies are
most likely resulted from the strong π−π stacking between
the planar Fmoc groups in the peptide molecular structure and
the ultrafast film formation kinetics (<5 s) preventing the
formation of 3-D molecular assemblies. In this part, we finally
fabricated thin films of Phe-Phe having Boc protecting groups
by PVD. Similar to H-Phe-Phe-OH films, vertically aligned,
highly ordered, and closely packed nanofibrillar morphologies
have been observed for Boc-Phe-Phe-OH (Figure 2i−k). The
density and an average length of nanofibers were calculated as
3.4 × 109 nanofibers·cm−2 and ∼1 μm, respectively. The
nanofiber diameters were also in the range of 30−100 nm.
Compared to H-Phe-Phe-OH films, Phe-Phe dipeptides having
Boc protecting groups yielded to a denser (∼3 fold)
nanofibrillar formation albeit with a smaller diameter.
Furthermore, the crystal structures of the fabricated samples
were compared using XRD. The diffraction patterns of the H-
Phe-Phe-OH (Figure 2d), Fmoc-Phe-Phe-OH (Figure 2h),
and Boc-Phe-Phe-OH (Figure 2l) films were considerably
different from the H-Phe-NH2 and Fmoc-Phe-OH counter-
parts. As shown in Figure 2d and l, we observed a strong
diffraction peak at 2θ = 33.2° for both H-Phe-Phe-OH and
Boc-Phe-Phe-OH films corresponding to a very short
periodicity of 2.69 Å. Considering that the typical H-bonding
distance is lower than 3.0 Å,31 it is reasonable to conclude that
both molecules adopt an arrangement in the vertically aligned
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Figure 4. Intermolecular interaction mechanism of peptides. (a) H-Phe-NH2, (b) Fmoc-Phe-OH, (c) Fmoc-Phe-Phe-OH, (d) H-Phe-Phe-Phe-
OH.
3-D film with their long axes on the substrate plane thereby
allowing H-bonding interactions in the out-of-plane direction.
However, we could not observe a similar diffraction peak for
the Fmoc-Phe-Phe-OH films indicating H-bonding interac-
tions similar to ones identified for the H-Phe-Phe-OH and
Boc-Phe-Phe-OH films (Figure 2h). The Fmoc-Phe-Phe-OH
films only revealed a relatively weak low-angle diffraction peak
at 7.71° (d-spacing = 11.46 Å) suggesting that molecules are
generally standing upright on the surface. As supported by
quantum chemical calculations (vide infra), these results
clearly suggest that H-bonding interactions between peptide
species play an important role during the formation of 3-D
morphologies in the vapor phase. Note that phenylalanine is a
chiral molecule and chirality might play an important role in
the self-assembly process as well. However, note also that,
peptides with fewer sequences (lower than seven residues)
were reported to prefer self-assembling into flat β-sheets
because of their weak molecular chirality.32,33 Additionally, the
amphiphilicity of the biological molecules is another key
parameter for the self-assembly process particularly in the
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solution medium. By controlling of the solution conditions
(e.g., pH, ionic strength, solvent, temperature, etc.), peptide
amphiphiles are self-assembled into a variety of ordered
nanostructures. Although the effect of amphiphilicity of
peptides on their self-assembly process in solution medium
has been well-understood, there is no information in the
literature about the effect of amphiphilicity on the self-
assembly of peptide molecules in the vapor phase. Therefore,
we did not explicitly consider the effect of chirality and
amphiphilicity of the molecules in this work.
In the final part of our work, we further explored the effect of
the length of amino acid sequence on the vapor deposition of
peptides using H-Phe-Phe-Phe-OH and H-Phe-Phe-Phe-Phe-
OH molecules. As stated above, the H-Phe-NH2 amino acid
did not form any ordered 3-D morphologies in the vapor
deposition process, whereas a vertically aligned unique 3-D
nanofibrillar morphology was obtained using H-Phe-Phe-OH
dipeptides. Once the H-Phe-Phe-Phe-OH tripeptides were
employed as a starting material in vapor deposition, similar to
H-Phe-NH2 films, we only obtained highly dense 2-D peptide
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Figure 5. Intermolecular interaction mechanism of peptides. (e) H-Phe-Phe-OH (f) Boc-Phe-Phe-OH, (g) H-Phe-Phe-Phe-Phe-OH.
thin films (Figure 3a−c). However, very interestingly, the
vapor deposition of H-Phe-Phe-Phe-Phe-OH molecules
yielded to a vertically aligned and highly ordered 3-D
nanoribbon-like morphology similar to H-Phe-Phe-OH and
Boc-Phe-Phe-OH films (Figure 3e−g). The density and
average length of nanoribbons were calculated as 8.2 × 108
nanorods·cm−2 and ∼2.0 μm, respectively. The lateral size of
nanoribbons was in the range of 30−150 nm.
In the light of experimental data obtained, it can be surmised
that the strong π−π stacking between aromatic rings in
molecular structures drives the formation of 2-D films in vapor
deposition. In 3-D films, there were slightly weaker
intermolecular interactions and edge-on molecular packing
leads to the observed ordered morphologies. Further support
to these driving forces is obtained via quantum chemical
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calculations with which the molecular structures and
mechanism of intermolecular interactions were studied.
In calculations, the peptides were studied as weakly
interacting clusters of size “n” (n = 1, 2, 3, 4) where “n”
denotes the number of phenylalanine amino acids (a−g in
Figures 4 and 5). Due to computational feasibility and the
energy trends suggesting identical morphologies at n = 3 and 4,
cluster size was not extended beyond 4. For all peptides, the
geometries were fully optimized. Various low energy
conformations at different cluster sizes were also studied (SI
Figures S1−S14). Energies of all the optimized geometries are
given in SI Tables S1−S7 with an emphasis on stabilization
upon the addition of the next amino acid to the cluster. Thus,
our major quantum descriptor throughout the calculations is
the share of a single amino acid within the peptide from the
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total energy gain of the cluster (size = n) with respect to “n”
isolated amino acids, that is, total stabilization of the weakly
interacting cluster per amino acid. For instance, conformation
1 with cluster size 3 optimized for H-Phe-NH2 is at −18 kcal·
mol−1 with respect to three separated H-Phe-NH2 molecules
(SI Table S1). Thus, stabilization per amino acid is −18/3 =
−6 kcal·mol−1. Lowest energy-optimized structures for clusters
of increasing size are given in Figure 4 for peptides with 2-D
morphologies and in Figure 5 for peptides with 3-D
morphologies.
Figure 4a depicts the conformations for H-Phe-NH2 with
increasing cluster size. Upon inclusion of additional H-Phe-
NH2 amino acids, π−π stacking between phenyl rings becomes
easily visible (SI Figure S1). The effect of this stacking
interaction is also captured in the energies (SI Figure S2 and
Table S1) that is, the highest stabilization per amino acid was
calculated for cluster size 4. Various relatively weak H−bonds
are also spotted (SI Figure S15a). The mode of primary
interactions and the associated energy stabilization (SI Figures
S3,S4 and Table S2) for Fmoc-Phe-OH are quite similar to
those of H-Phe-NH2. Phenyl and Fmoc protecting groups
feature π−π stacking interactions with one another (Figure
4b). The π−π stacking among the phenyl groups and the Fmoc
groups also increases upon increasing cluster size. Peptides
comprised of Fmoc-Phe-Phe-OH are again driven by π−π
stacking; however, the stacking is due to interphenyl and inter-
Fmoc interactions (Figure 4c). Due to the larger size of the π-
system of the Fmoc groups, Fmoc-Phe-Phe-OH peptides are
highly stabilized (SI Figures S5−S6 and Table S3).
Comparison of Fmoc-Phe-OH and Fmoc-Phe-Phe-OH
shows that the stabilization per molecule is −3.6 kcal·mol−1
for the conformation 1 with cluster size 4 in Fmoc-Phe-OH
molecule, whereas it is −16.7 kcal·mol−1 in Fmoc-Phe-Phe-OH
peptide (Table 1, SI Tables S2, S3 and Figures S4−S6). The
Table 1. Calculated energies for stabilization of each
molecule in the clusters (kcal·mol−1)
cluster size
peptides 1 2 3 4
2-D H-Phe-NH2 0.0 −6.4 −6.0 −7.4
Fmoc-Phe-OH 0.0 −9.9 −0.7 −3.6
Fmoc-Phe-Phe-OH 0.0 −17.2 −12.7 −16.7
H-Phe-Phe-Phe-OH 0.0 −32.6 −35.7 −37.1
3-D H-Phe-Phe-OH 0.0 −18.4 −14.9 −19.5
Boc-Phe-Phe-OH 0.0 −25.8 −7.6 −7.4
H-Phe-Phe-Phe-Phe-OH 0.0 −41.1 −50.1 −49.8
difference between these molecules is the interaction of the
aromatic groups. The π−π stacking is between the phenyl
group and Fmoc protecting group in Fmoc-Phe-OH, whereas
the Fmoc protecting groups and phenyl groups interact with
each other in Fmoc-Phe-Phe-OH. For H-Phe-Phe-Phe-OH,
the only aromatic ring that can undergo π−π stacking is the
phenyl groups (SI Figures S7, S8 and Table S4). Not
surprisingly starting from cluster size 2, π−π stacking
interactions are clearly visible (Figure 4d). Stabilization per
amino acid in cluster size 4 is at a large −37 kcal·mol−1;
however, one should note that the number of phenyl groups in
this amino acid is three.
To sum up, peptides leading to 2-D film morphologies are
mainly featuring π−π stacking interactions and H-bonds are
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relatively long (SI Figure S15a-d). Thus, 2-D film morphology
presumably is a result of π−π stacking interactions. The
stabilization per amino acid in the largest clusters studied is
calculated between −4 and −37 kcal·mol−1; however, the size
and orientation of the aromatic rings vary and hence the large
energy span (Table 1, SI Tables S1−S4). Figure 5 summarizes
the calculations on peptides leading to 3-D film morphologies.
For all peptides considered carbonyl, carboxyl, and amino
groups undergo H-bonding interactions (SI Figures S9−S14).
These H-bonds are relatively shorter (3.2−5.5 Å vs 2.6 to 3.9
Å) than what we have observed for peptides leading to 2-D
film morphologies (SI Figure S15). Energies calculated for
stabilization of each molecule in the clusters of H-Phe-Phe-
OH, Boc-Phe-Phe-OH, and H-Phe-Phe-Phe-Phe-OH are −20,
−7, and −50 kcal·mol−1, respectively, (Table 1, SI Table S5−
S7). The relatively low stabilization calculated for Boc-Phe-
Phe-OH is due to the steric interactions of t-butyl groups
preventing the optimal formation of the H-bonds (SI Figure
S15f).
Among the peptides that include protecting groups, an
interesting feature of the calculated stabilizations is the peptide
interactions when protecting groups are Fmoc and Boc. These
groups have major role for the H-bonding and stacking. The
Boc groups create steric hindrance for stacking of the phenyl
groups and formation of the optimal H-bonding. Thus, a lower
stabilization is calculated. On the other hand, the Fmoc groups
cause strong π−π stacking due to the π-electron density, that is
size of the electron cloud of the Fmoc π−system.
Consequently, augmented stacking in the π−system wins
nonoptimal H-bonding. As shown in Figure 4, the aromatic
groups align due to π−π stacking and 3-D orientation is
realized whenever there are strong H-bonds. Our calculations
thus suggest that stacking is not a result of steric hindrance.
Therefore, coherent with our experimental observations and
in line with the existing data on H-bonding34,35 as well as π−π
stacking36 affecting the morphologies of self-assembly of
nanostructures in the literature, our quantum chemical
calculations capture the key points of the mode of interaction
in peptide clusters and their differences suggesting a potential
utilization of theoretical analyses in realization of peptides with
desired morphological properties.
One should not forget, however, that the calculated energy
of interaction within the clusters that is utilized in the
theoretical discussion here cannot be fragmented easily (and
technically correctly) into H-bonding energy and π−π stacking
energy. Although these concepts and observations are quite
useful, the calculated energies (by their very nature) always
include all types of interactions and both strong and the two
types of weak interactions are present. The theoretical
approach used herein basically investigated the experimentally
well identified differences in morphology, that is, there have
been two morphologically distinct groups in our investigations.
These two sets were subjected to structural and energetic
analyses in the computations. The result was presence of both
H-bonding and π−π stacking in the clustering process;
however, π−π stacking was the main mode (source of highest
stabilization) of interaction for 2-D structures and H-bonding
for 3-D morphologies. Thus, although we (and others) are
certainly far from deciphering the fine details of how 2-D and
3-D morphologies can be constructed from atomistic first-
principles calculations or simulations we can point out at a nice
coherence in the experimental and computational data. That is
nature of major/decisive weak interactions for 2-D and 3-D
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peptides are theoretically shown to be of different (H-
bonding/π−π stacking) nature.
4. CONCLUSION
In conclusion, we demonstrated that the molecular structure of
biological molecules has an important role in the fabrication of
3-D ordered morphologies through vapor phase deposition.
The vapor-phase deposition of biomolecules is a solvent free,
environmental friendly, and material-independent method and
offers unique advantages compared with the previously
demonstrated techniques for the fabrication of 3-D vertically
aligned biomolecular thin films. By applying this technique,
undesired impurities, degradation of the underlying layer, and
changes in the mechanical/chemical properties associated with
the use of solvents can be precisely eliminated. Additionally,
large-area fabrication of such ordered biomolecular films is also
possible through vapor-phase deposition. For molecules
studied in this work, an interesting relation depending on
the amino acid sequence and protecting groups anchoring to
the molecular structure of peptides was found. The 2-D films
were obtained for H-Phe-NH2, Fmoc-Phe-OH, Fmoc-Phe-
Phe-OH, and H-Phe-Phe-Phe-OH, whereas H-Phe-Phe-OH,
Boc-Phe-Phe-OH, and H-Phe-Phe-Phe-Phe-OH peptides
generate vertically aligned, highly ordered 3-D bionanostruc-
tures under the same deposition condition. Experimental
investigations along with quantum chemical calculations
suggest that 2-D morphologies are a result of the interplay
between H-bonding and weak π−π stacking interactions;
however, 3-D morphology is due primarily to weak H-bonding.
To fabricate vertically aligned 3-D peptide morphologies, it is
demonstrated that the molecular structure of the peptide
should be carefully designed and must provide H-bonding
interactions that are more pronounced than strong inter-
molecular π−π-stacking. We believe that our results provide
design rules for the fabrication of vertically aligned and ordered
3-D bionanostructured films. Molecular and morphological
engineering of biomolecules is undoubtedly instrumental in
realization of promising functional materials for applications in
sensing and tissue engineering. However, we should frankly
note that it is currently not possible to predict the final
morphology of peptide materials comprised of different amino
acids (other than phenylalanine) or having different sequence
lengths. Along that end detailed studies are on the way in our
laboratories. Future work will extend this approach to other
sequences featuring different functional groups−also including
chiral centersto provide an extented set of building blocks
for the fabrication of functional nanostructured films.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsanm.0c00456.
Additional computational data for H-Phe-NH2, Fmoc-
Phe-OH, Fmoc-Phe-Phe-OH, and H-Phe-Phe-Phe-OH,
H-Phe-Phe-OH, Boc-Phe-Phe-OH, and H-Phe-Phe-Phe-
Phe-OH peptides (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
Yavuz Dede − Department of Chemistry, Faculty of Science,
Gazi University, Ankara 06500, Turkey; Email: dede@
gazi.edu.tr
4312
www.acsanm.org Article
Gokhan Demirel − Department of Chemistry, Faculty of
Science, Gazi University, Ankara 06500, Turkey; orcid.org/
0000-0002-9778-917X; Email: nanobiotechnology@
gmail.com
Authors
Vefa Sahibbeyli − Department of Chemistry, Faculty of Science,
Gazi University, Ankara 06500, Turkey
Dilara B. Yildiz − Department of Chemistry, Faculty of Science,
Gazi University, Ankara 06500, Turkey
Gözde Papir − Department of Chemistry, Faculty of Science,
Gazi University, Ankara 06500, Turkey
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsanm.0c00456
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was partially supported by Gazi University (Grant
No. 05/2015-19). G.D. and Y.D. acknowledge support from
the Turkish Academy of Sciences, Distinguished Young
Scientist Award (TUBA-GEBIP). D.B.Y. thanks TÜ BITAK-̇
̇
BIDEB for a 2210-A scholarship. ULAKBIM-TRGRID is
gratefully acknowledged for high performance computing
resources.
■
†
DEDICATION
Dedicated to the memory of Gözde Papir, who passed away
while pursuing her graduate studies.
■
REFERENCES
(1) Reches, M.; Gazit, E. Casting Metal Nanowires within Discrete
Self-Assembled Peptide Nanotubes. Science 2003, 300, 625−627.
(2) Frederix, P. W. J. M.; Scott, G. G.; Abul-Haija, Y. M.;
Kalafatovic, D.; Charalampos, G. P.; Javid, N.; Hunt, N. T.; Uljin, R.
V.; Tuttle, T. Exploring the Sequence Space for (Tri-)Peptide Self-
Assembly to Design and Discover New Hydrogels. Nat. Chem. 2015,
7, 30−37.
(3) Nguyen, V.; Zhu, R.; Jenkins, K.; Yang, R. Self-Assembly of
Diphenylalanine Peptide with Controlled Polarization for Power
Generation. Nat. Commun. 2016, 7, 13566.
(4) Habibi, N.; Kamaly, N.; Memic, A.; Shafiee, H. Self-Assembled
Peptide-Based Nanostructures: Smart Nanomaterials Toward Tar-
geted Drug Delivery. Nano Today 2016, 11, 41−60.
(5) Ekiz, M. S.; Cinar, G.; Khalily, M. A.; Guler, M. O. Self-
Assembled Peptide Nanostructures for Functional Materials. Nano-
technology 2016, 27, 402002.
(6) Tao, K.; Levin, A.; Adler-Abramovich, L.; Gazit, E. Fmoc-
Modified Amino Acids and Short Peptides: Simple Bio-inspired
Building Blocks for the Fabrication of Functional Materials. Chem.
Soc. Rev. 2016, 45, 3935−3953.
(7) Reches, M.; Gazit, E. Self-Assembly of Peptide Nanotubes and
Amyloid-like Structures by Charged-Termini-Capped Diphenylala-
nine Peptide Analogues. Isr. J. Chem. 2005, 45, 363−371.
(8) Orbach, R.; Mironi-Harpaz, I.; Adler-Abramovich, L.; Mossou,
E.; Mitchell, E. P.; Forsyth, V. T.; Gazit, E.; Seliktar, D. The
Rheological and Structural Properties of Fmoc-Peptide-Based Hydro-
gels: The Effect of Aromatic Molecular Architecture on Self-Assembly
and Physical Characteristics. Langmuir 2012, 28, 2015−2022.
(9) Erdogan, H.; Babur, E.; Yilmaz, M.; Candas, E.; Gordesel, M.;
Dede, Y.; Oren, E. E.; Demirel, G. B.; Ozturk, M. K.; Yavuz, M. S.;
Demirel, G. Morphological Versatility in the Self-Assembly of Val-Ala
and Ala-Val Dipeptides. Langmuir 2015, 31, 7337−7345.
https://dx.doi.org/10.1021/acsanm.0c00456
ACS Appl. Nano Mater. 2020, 3, 4305−4313
ACS Applied Nano Materials www.acsanm.org Article

(10) Demirel, G.; Malvadkar, N.; Demirel, M. C. Control of Protein (27) Williams, R. J.; Smith, A. M.; Collins, R.; Hodson, N.; Das, A.
Adsorption onto Core-Shell Tubular and Vesicular Structures of K.; Ulijn, R. V. Enzyme-Assisted Self-Assembly Under Thermody-
Diphenylalanine/Parylene. Langmuir 2010, 26, 1460−1463. namic Control. Nat. Nanotechnol. 2009, 4, 19−24.
(11) Reches, M.; Gazit, E. Controlled Patterning of Aligned Self- (28) Abbas, M.; Xing, R.; Zhang, N.; Zou, Q.; Yan, X. Antitumor
Assembled Peptide Nanotubes. Nat. Nanotechnol. 2006, 1, 195−200. Photodynamic Therapy Based on Dipeptide Fibrous Hydrogels with
(12) Ryu, J.; Park, C. B. Solid-Phase Growth of Nanostructures from Incorporation of Photosensitive Drugs. ACS Biomater. Sci. Eng. 2018,
Amorphous Peptide Thin Film: Effect of Water Activity and 4, 2046−2052.
Temperature. Chem. Mater. 2008, 20, 4284−4290. (29) Singh, N.; Kumar, M.; Miravet, J. F.; Ulijn, R. V.; Escuder, B.
(13) Adler-Abramovich, L.; Aronov, D.; Beker, P.; Yevnin, M.; Peptide-Based Molecular Hydrogels as Supramolecular Protein
Buzhansky, L.; Rosenman, G.; Gazit, E. Self-Assembled Arrays of Mimics. Chem. - Eur. J. 2017, 23, 981−993.
Peptide Nanotubes by Vapour Deposition. Nat. Nanotechnol. 2009, 4, (30) Erdogan, H.; Yilmaz, M.; Babur, E.; Duman, M.; Aydin, H. M.;
849−854. Demirel, G. Fabrication of Plasmonic Nanorod-Embedded Dipeptide
(14) Vasudev, M. C.; Koerner, H.; Singh, K. M.; Partlow, B. P.; Microspheres via the Freeze-Quenching Method for Near-Infrared
Kaplan, D. L.; Gazit, E.; Bunning, T. J.; Naik, R. R. Vertically Aligned Laser-Triggered Drug-Delivery Applications. Biomacromolecules 2016,
Peptide Nanostructures Using Plasma-Enhanced Chemical Vapor 17, 1788−1794.
Deposition. Biomacromolecules 2014, 15, 533−540. (31) Gorbitz, C. H. beta Turns, Water Cage Formation and
(15) Demirel, G.; Tamer, U. Isotropic and Anisotropic Dipeptide Hydrogen Bonding in the Structures of L-Valyl-L-Phenylalanine. Acta
Films Based on Gas Phase Deposition. Nanotechnology 2012, 23, Crystallogr., Sect. B: Struct. Sci. 2002, 58, 512−518.
225604. (32) Wang, Y.; Qi, W.; Huang, R.; Yang, X.; Wang, M.; Su, R.; He,
(16) Rappe, A. K.; Casewit, C. J.; Colwell, K. S.; Goddard, W. A.; Z. Rational Design of Chiral Nanostructures from Self-Assembly of a
Skiff, W. M. UFF, A Full Periodic Table Force Field for Molecular Ferrocene-Modified Dipeptide. J. Am. Chem. Soc. 2005, 137, 7869−
Mechanics and Molecular Dynamics Simulations. J. Am. Chem. Soc. 7880.
1992, 114, 10024−10035. (33) Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. Q.;
(17) Becke, A. D. Density-Functional Exchange-Energy Approx- Riekel, C.; Grothe, R.; Eisenberg, D. Structure of the Cross-Beta
imation with Correct Asymptotic Behavior. Phys. Rev. A: At., Mol., Spine of Amyloid-Like Fibrils. Nature 2005, 435, 773−778.
Opt. Phys. 1988, 38, 3098−3100. (34) Lee, O. S.; Stupp, S. I.; Schatz, G. C. Atomistic Molecular
(18) Becke, A. D. Density-Functional Thermochemistry. III. The Dynamics Simulations of Peptide Amphiphile Self-Assembly into
Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648−5652. Cylindrical Nanofibers. J. Am. Chem. Soc. 2011, 133, 3677−3683.
(19) Kohn, W.; Becke, A. D.; Parr, R. G. Density Functional Theory (35) Tsonchev, S.; Troisi, A.; Schatz, G. C.; Ratner, M. A. On the
of Electronic Structure. J. Phys. Chem. 1996, 100, 12974−12980. Structure and Stability of Self-Assembled Zwitterionic Peptide
(20) Chai, J. D.; Head-Gordon, M. Long-Range Corrected Hybrid Amphiphiles: A Theoretical Study. Nano Lett. 2004, 4, 427−431.
Density Functionals with Damped Atom-Atom Dispersion Correc- (36) Fu, C.; Lin, H.-p.; Macleod, J. M.; Krayev, A.; Rosei, F.;
tions. Phys. Chem. Chem. Phys. 2008, 10, 6615−20. Perepichka, D. F. Unravelling the Self-Assembly of Hydrogen Bonded
(21) Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A. Self- NDI Semiconductors in 2D and 3D. Chem. Mater. 2016, 28, 951−
Consistent Molecular Orbital Methods. XX. A Basis Set for 961.
Correlated Wave Functions. J. Chem. Phys. 1980, 72, 650−654.
(22) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson,
G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.;
Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J.
V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.;
Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson,
T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.;
Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Throssell, K.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.;
Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov,
V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.;
Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.;
Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J.
Gaussian, Inc.: Wallingford CT, 2016.
(23) Yilmaz, M.; Ozdemir, M.; Erdogan, H.; Tamer, U.; Sen, U.;
Facchetti, A.; Usta, H.; Demirel, G. Micro-/Nanostructured Highly
Crystalline Organic Semiconductor Films for Surface-Enhanced
Raman Spectroscopy Applications. Adv. Funct. Mater. 2015, 25,
5669−5676.
(24) Yilmaz, M.; Babur, E.; Ozdemir, M.; Gieseking, R. L.; Dede, Y.;
Tamer, U.; Schatz, G. C.; Facchetti, A.; Usta, H.; Demirel, G.
Nanostructured Organic Semiconductor Films for Molecular
Detection with Surface-Enhanced Raman Spectroscopy. Nat. Mater.
2017, 16, 918−924.
(25) Mossou, E.; Teixeira, S. C. M.; Mitchell, E. P.; Mason, S. A.;
Adler-Abramovich, L.; Gazit, E.; Forsyth, V. T. The Self-Assembling
Zwitterionic Form of L-Phenylalanine at Neutral pH. Acta Crystallogr.,
Sect. C: Struct. Chem. 2014, 70, 326−331.
(26) Bera, S.; Mondal, S.; Rencus-Lazar, S.; Gazit, E. Organization of
Amino Acids into Layered Supramolecular Secondary Structures. Acc.
Chem. Res. 2018, 51, 2187−2197.

4313 https://dx.doi.org/10.1021/acsanm.0c00456
ACS Appl. Nano Mater. 2020, 3, 4305−4313