Self-Assembly of Tetraphenylalanine Peptides

Article abstract of DOI:10.1002/chem.201501793

Three different tetraphenylalanine (FFFF) based peptides that differ at the N- and C-termini have been synthesized by using standard procedures to study their ability to form different nanoassemblies under a variety of conditions. The FFFF peptide assembles into nanotubes that show more structural imperfections at the surface than those formed by the diphenylalanine (FF) peptide under the same conditions. Periodic DFT calculations (M06L functional) were used to propose a model that consists of three FFFF molecules defining a ring through head-to-tail NH₃⁺?^-OOC interactions, which in turn stack to produce deformed channels with internal diameters between 12 and 16 ?. Depending on the experimental conditions used for the peptide incubation, N-fluorenylmethoxycarbonyl (Fmoc) protected FFFF self-assembles into a variety of polymorphs: ultra-thin nanoplates, fibrils, and star-like submicrometric aggregates. DFT calculations indicate that Fmoc-FFFF prefers a parallel rather than an antiparallel β-sheet assembly. Finally, coexisting multiple assemblies (up to three) were observed for Fmoc-FFFF-OBzl (OBzl = benzyl ester), which incorporates aromatic protecting groups at the two peptide terminals. This unusual and noticeable feature is attributed to the fact that the assemblies obtained by combining the Fmoc and OBzl groups contained in the peptide are isoenergetic. Variety show! Three different tetraphenylalanine-based peptides that differ at the N- and C-termini have been synthesized by using standard procedures to study their ability to form different nanoassemblies (e.g., nanotubes, see figure) under a variety of conditions.

Full text of DOI:10.1002/chem.201501793

DOI: 10.1002/chem.201501793
Full Paper
&
Nanostructures
Self-Assembly of Tetraphenylalanine Peptides
[a, b]
[c]
[d]
[a, b]
[c]
Enric Mayans,
Gema Ballano, Jordi Casanovas, AngØlica Díaz,
Maria M. PØrez-
[a, b]
[e]
[a, b]
Madrigal,
Francesc Estrany, Jordi Puiggalí,*
Carlos Cativiela,* and
[
a, b]
Carlos Alemµn*
Abstract: Three different tetraphenylalanine (FFFF) based
peptides that differ at the N- and C-termini have been syn-
thesized by using standard procedures to study their ability
to form different nanoassemblies under a variety of condi-
tions. The FFFF peptide assembles into nanotubes that show
more structural imperfections at the surface than those
formed by the diphenylalanine (FF) peptide under the same
conditions. Periodic DFT calculations (M06L functional) were
used to propose a model that consists of three FFFF mole-
the experimental conditions used for the peptide incubation,
N-fluorenylmethoxycarbonyl (Fmoc) protected FFFF self-as-
sembles into a variety of polymorphs: ultra-thin nanoplates,
fibrils, and star-like submicrometric aggregates. DFT calcula-
tions indicate that Fmoc-FFFF prefers a parallel rather than
an antiparallel b-sheet assembly. Finally, coexisting multiple
assemblies (up to three) were observed for Fmoc-FFFF-OBzl
(OBzl = benzyl ester), which incorporates aromatic protect-
ing groups at the two peptide terminals. This unusual and
noticeable feature is attributed to the fact that the assem-
blies obtained by combining the Fmoc and OBzl groups
contained in the peptide are isoenergetic.
+
À
cules defining a ring through head-to-tail NH ··· OOC inter-
3
actions, which in turn stack to produce deformed channels
with internal diameters between 12 and 16 Š. Depending on
Introduction
the self-assembly of aromatic peptides. Typically, the self-as-
sembly takes place in solution and is driven by hydrophobic
[
1]
[2]
Since the pioneering work of Reches and Gazit in 2003, in
which the formation of diphenylalanine (FF) nanotubes in
aqueous solution was discovered, significant efforts have been
made to develop a new generation of biomaterials based on
forces. In addition to tubular nanostructures, the most-stud-
ied FF-based polymorphs are peptide spheres composed of
[
3]
tert-butoxycarbonyl (Boc) FF and peptide fibrils composed of
[
4]
fluorenylmethoxycarbonyl (Fmoc) FF. Reviews show that ef-
[
5]
forts have been essentially focused on small FF peptides,
which constitute the most investigated building block for the
construction of peptide self-assemblies. Amazingly, research
into the self-assembly of triphenylalanine (FFF) and tetrapheny-
lalanine (FFFF) based peptides is very scarce.
[
a] M. Sc. E. Mayans, Dr. A. Díaz, Dr. M. M. PØrez-Madrigal, Prof. J. Puiggalí,
Prof. C. Alemµn
Department of Chemical Engineering, ETSEIB
Universitat Polit›cnica de Catalunya
Av. Diagonal 647, 08028 Barcelona (Spain)
E-mail: jordi.puiggali@upc.edu
Recent studies of FFF-based peptides showed that FFF, Boc-
FFF, and Fmoc-FFF can self-assemble into solid fibrillary plate-
carlos.aleman@upc.edu
[
7]
[8]
[
b] M. Sc. E. Mayans, Dr. A. Díaz, Dr. M. M. PØrez-Madrigal, Prof. J. Puiggalí,
Prof. C. Alemµn
like nanostructures (“nanoplates”), nanospheres, and hydro-
[
9]
gels, respectively. In all cases, p–p stacking interactions be-
tween aromatic rings were found to play a decisive role in the
formation of the supramolecular aggregates. On the other
Center for Research in Nano-Engineering
Universitat Polit›cnica de Catalunya, Campus Sud, Edifici C’
C/Pasqual i Vila s/n, 08028 Barcelona (Spain)
[
9]
[10]
hand, Tamamis et al. and Guo et al.
used atomistic and
[
c] Dr. G. Ballano, Prof. C. Cativiela
Departamento de Química Orgµnica
Instituto de Síntesis Química y Catµlisis HomogØnea-ISQCH
CSIC-Universidad de Zaragoza
C/ Pedro Cerbuna, 12, 50009 Zaragoza (Spain)
E-mail: cativiela@unizar.es
coarse-grained molecular dynamics (MD) simulations, respec-
tively, to study the assembly mechanism and the molecular
basis for the structural features of FFF-based peptide nano-
structures. The authors found that FFF-based peptides sponta-
neously assembled into solid nanospheres and nanorods with
substantial antiparallel b-sheet content.
[
d] Dr. J. Casanovas
Department of Chemistry, Escola Polit›cnica Superior
Universitat de Lleida, C/Jaume II no 69, 25001 Lleida (Spain)
Investigations of FFFF-based peptides have focused on pep-
tide–polymer conjugates. More specifically, Castelletto and
[
e] Dr. F. Estrany
Department of Chemical Engineering, EUETIB
Universitat Polit›cnica de Catalunya
Comte d’Urgell 187, 08036 Barcelona (Spain)
[11]
Hamley studied amphiphilic FFFF-polyethylene glycol (PEG)
conjugates. Results indicated that hydrophobic association into
irregular aggregates occurs at low concentration, whereas
well-developed b sheets only occur at higher concentration.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501793.
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Furthermore, drying the FFFF-PEG solutions resulted in crystal-
lization of PEG without disruption to the local b-sheet structure
defined by the peptide block. The independent organization of
the two counterparts of the FFFF-PEG amphiphile was ex-
plained at the microscopic level by using atomistic MD simula-
[
12]
[13]
tions. On the other hand, Tzokova et al. used click chemis-
try to prepare poly(ethylene oxide) [PEO] conjugated to FFFF
with an ethyl group at the C-terminus (FFFF-OEt). The length
of PEO was found to play a crucial role in the assembly. For
shorter PEO blocks, PEO-FFFF-OEt nanotubes were formed by
self-assembly of anti-parallel b-sheets, stabilized by surround-
[
1]
ing PEO chains. Entanglement between adjacent nanotubes
resulted in the formation of soft hydrogels. In contrast, p–p
stacking interactions were prevalent for conjugates with
longer PEO lengths giving rise to fibers and worm-like micel-
[13b]
les.
Self-assembled short-peptide-based materials (including FF
peptides) have potential biomedical and biotechnological ap-
plications, for example in general powerful new therapies for
[14]
regenerative medicine, to fabricate stable drug-delivery sys-
Figure 1. Scheme of the coupling reactions used to obtain FFFF, Fmoc-FFFF,
[15]
tems with proteolytic resistance, to prepare modern nanode-
and Fmoc-FFFF-OBzl. i) Boc-F-OH/benzyl bromide, THF; ii) CF
3
COOH (TFA)/
[
16]
CH Cl 1:1; iii) 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC)/1-hy-
vices, and to fabricate ultrasensitive and selective sensors for
2
2
[17]
droxy-7-azabenzotriazole (HOBt), N-methylmorpholine (NMM; to maintain
pH 8); iv) H /Pd, MeOH; v) FmocCl, CH CN.
the detection of toxins. Despite of the importance of this
new kind of material, the self-assembly of FFFF-based peptides
that are not conjugated to polymer chains remains unstudied.
In this work, we synthesized three FFFF-based peptides to ex-
plore the formation of ordered self-assembled nanostructures.
The results were compared with those reported for shorter FF-
and FFF-analogues under similar conditions. In particular, the
three peptides studied in this work were FFFF, Fmoc-FFFF, and
Fmoc-FFFF-OBzl (OBzl=benzyl ester; Scheme 1). Theoretical
2
3
with either a Boc or Fmoc amino protecting group. A general
procedure for the coupling reactions is given in Figure 1.
FFFF nanotubes
Optical microscopy and AFM analyses of all tested conditions
evidenced that FFFF self-assembles into relatively short and
well-defined tubes of submicrometric thickness at 48C in hexa-
fluoroisopropanol/ethanol (HFIP/EtOH) solutions with peptide
À1
concentrations ranging from 1–2 mgmL
(Figure 2a). The
average diameter of these tubes (estimated by AFM) is (0.58Æ
.12) mm, whereas the length is typically less than 100 mm.
0
Both the length and abundance of these tubes increases no-
ticeably when the EtOH cosolvent is replaced by water, which
is reflected in the SEM images and optical micrographs dis-
played in Figure 2b. In this case, the thickness of the tubular
structures ranges from ꢀ50 to ꢀ700 nm, and the tube was
several hundreds of mm long. Interestingly, tubes obtained in
HFIP/water at room temperature displayed abundant defects
along the tube surface (Figure 2c), which represents a signifi-
cant difference with respect to the tubes obtained at 48C. Fur-
thermore, the thickness of the tubes is remarkably variable,
which is reflected by the high standard deviation of the aver-
age diameter, (0.48Æ0.37) mm. A DMF/water mixture also pro-
motes the formation of tubular structures at 48C similar to
those achieved in HFIP/EtOH and HFIP/water when the peptide
Scheme 1. Chemical structure of the FFFF-based peptides examined.
calculations for FFFF and Fmoc-FFFF aggregates were per-
formed to get a deeper understanding of the molecular inter-
actions involved in the self-assembly process.
Results and Discussion
À1
concentration ranges from 0.1–0.5 mgmL (Figures S1a and
Peptide synthesis
b in the Supporting Information). The abundance of these
tubes was found to depend on the peptide concentration,
with metastable tube-like assemblies being observed at very
low concentrations (Figure S1c in the Supporting Information).
The preparation of FFFF-based peptides was carried out by fol-
lowing standard procedures for peptide synthesis in solution
starting from the corresponding phenylalanine (F) derivative
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Figure 3. a) Low- and high-magnification SEM micrographs of tubes ob-
À1
tained at 48C for FF dissolved at a final concentration of 0.5 mgmL in 1:9
Figure 2. a) AFM image and optical micrograph (inset) of the nanotubes ob-
tained at 48C for FFFF dissolved at a final concentration of 1 mgmL in 1:4
HFIP/water. b) SEM micrographs and c) 3D and 2D AFM images (inset: phase
À1
image) of tubes obtained at 48C for FF dissolved at a final concentration of
À1
HFIP/EtOH. b) SEM and optical microscopy (inset) images of nanotubes ob-
0.3 mgmL in 3:47 HFIP/EtOH.
À1
tained at 48C for FFFF dissolved at a final concentration of 0.5 mgmL in
1
:9 HFIP/water. c) SEM, optical microscopy (inset), and AFM images of nano-
tubes obtained at 258C for FFFF dissolved at a final concentration of
À1
0
.5 mgmL in 1:9 HFIP/water. d) Optical micrograph obtained by using
at both 258C and 48C present a smooth surface free of de-
fects, thus they display microscopic structural differences com-
pared with FFFF. The definition of FF tubes improves upon re-
placement of water by EtOH, which is evidenced in the micro-
graphs recorded for the nanostructures derived from dilute
peptide HFIP/EtOH solutions at 48C (Figure 3b). The root
mean square (RMS) roughness and average diameter of these
tubes was determined by AFM (Figure 3c) to be (40.5Æ
5.2) nm and (0.86Æ0.09) mm, respectively. Furthermore, optical
microscopy indicated that the tortuosity of the tubes increased
with decreasing peptide concentration (Figure S2b in the Sup-
porting Information). The tubes obtained at a peptide concen-
cross-polarized light illumination of the fibers obtained at 48C for FFFF dis-
solved at a final concentration of 0.3 mgmL in 3:47 DMF/water. e) SEM
and optical microscopy (inset) images of tubes obtained at 48C for FFFF dis-
solved at a final concentration of 1 mgmL in 1:4 DMF/water.
À1
À1
The latter structures are considered to be FFFF aggregates at
the early stages of the nanotube-formation process.
Microscopy using cross-polarized light illumination showed
birefringence for the FFFF fibers, independent of both the sol-
vent and the peptide concentration. This feature, which is illus-
trated in Figure 2d for assemblies obtained in DMF/water, indi-
cates that the peptides retain the same orientation along the
whole assembly. On the other hand, the tubes tend to aggre-
gate in poorly defined clusters when the peptide concentra-
tion in DMF/water is ꢁ1 mgmLÀ1 (Figure 2e).
À1
tration of 2 mgmL were completely straight, whereas those
derived at lower concentrations showed sinuosity.
On the other hand, tube-like structures organized in dendrit-
ic branches similar to those obtained in HFIP/water were also
FF tubular nanostructures are typically obtained by using
peptide concentrations significantly higher than those used in
obtained in DMF/water at 48C for peptide concentrations of
À1
1–2 mgmL
(Figure S3a in the Supporting Information),
[
1,2,5–7]
this work for FFFF.
To provide a comparison between the
whereas DMSO/water at 48C promoted the crystallization of FF
two systems, FF tubes were prepared from dilute solutions
analogous to those tested for FFFF. Very well defined FF nano-
tubes that grew following an architecture recalling dendritic
hyperbranched structures were obtained in HFIP/water at 48C
(Figure S3b in the Supporting Information). The crystal struc-
[
18]
ture of FF was reported by Gçrbitz
who found that this
small peptide crystallizes with hydrogen-bonded head-to-tail
chains, which form hydrophilic channels (nanotubes) embed-
ded in a hydrophobic matrix created by the peptide side
chains. Indeed, the sequence formed by two consecutive F res-
idues was proposed to be an attractive membrane-channel
model due to the substantial size of the hydrophilic channels.
Interestingly, the conformation adopted by this dipeptide in
(
Figure 3a). This hyperbranched-like organization transforms
into a spherulitic-nucleus-like shape when the temperature in-
creases from 48C to 258C (Figure S2a in the Supporting Infor-
mation). Despite this morphological difference, high-resolution
SEM micrographs clearly indicated that the FF tubes obtained
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the solid state was recently identified as a minimum-energy
[17]
conformation in a theoretical study. It is worth noting that
[18]
the crystallization conditions reported by Gçrbitz (i.e., fast
À1
evaporation of an aqueous solution of FF (2.5 mgmL ; 3 mL)
at 808C) were different from the conditions used in this work
to obtain the monocrystals displayed in Figure S3b (see the
Supporting Information). Unfortunately, FFFF monocrystals
were not identified in any of the tested conditions.
To compare the molecular organization of FFFF and FF pep-
tides in the nanotubular assemblies, DFT calculations were car-
ried out by using the M06L functional combined with the 6-
3
1G(d) basis set. This functional is able to account for the inter-
[18]
actions between aromatic groups. Gçrbitz reported that FF
nanotubes are formed by six peptide units that define cyclic
hexamers, which are stacked to produce narrow channels with
a van der Waals diameter of ꢀ10 Š and a tube diameter of
ꢀ
24 Š. The oppositely charged amino- and carboxylate groups
of neighboring FF molecules, which interact by forming head-
+
À
to-tail NH3 ··· OOC hydrogen bonds, surround the inner core
of the tubes. All the side chains appear to emanate from the
channel core to form the hydrophobic surface. This arrange-
ment is advantageous because it provides appropriate sites for
anchoring polar molecules to the peptide matrix, which is
characterized by numerous phenyl units from the FF side
[18]
chains. Furthermore, Gçrbitz found that the channel core is
filled with nine crystallographic water molecules with occupan-
cies ranging from 0.38 to 0.15.
In our approach, the crystallographic coordinates of the six
FF molecules defining the hydrophilic turn were used to con-
struct the explicit fragment of the model structure, and one-di-
mensional periodic boundary conditions (PBC) were applied
perpendicularly to the average plane of the turn (c-axis) to
create the three-dimensional nanotube. PBC-M06L/6-31G(d)
geometry optimizations were performed considering the fol-
lowing starting structures:
Figure 4. a) An FF nanotube derived from PBC-M06L/6-31G(d) calculations
for a system with six explicit peptide molecules forming a turn. Left: top
+
À
3
view of the six explicit FF molecules with head-to-tail NH ··· OOC hydro-
gen-bond distances (dH–O [Š]) displayed. Middle: side view of the nanotube;
the c-axis is represented by the red line. Right: details showing the interpep-
tide hydrogen bonds (dH–O [Š]) and p–p stacking (dp–p [Š]) interactions be-
tween FF molecules located at consecutive turns. b) Top view of the FF
nanotube with six explicit water molecules (first layer) filling the hydrophilic
core derived from PBC-M06L/6-31G(d) calculations. Details of the hydrogen
bonds formed between a water molecule and the head and the tail of differ-
ent FF molecules are displayed (distances in Š). c) Top view of the FF nano-
tube with twelve explicit water molecules (first and second layers) filling the
hydrophilic core derived from PBC-M06L/6-31G(d) calculations. Details of the
hydrogen-bonded network of water molecules surrounding the hydrophilic
core are displayed.
1
2
) the constructed nanotube (i.e., 258 explicit atoms).
) the nanotube with six explicit water molecules, each inter-
acting with a charged amino or carboxylate group and
forming the first water layer that fills the hydrophilic core
(
i.e., 276 explicit atoms).
3
) the structure constructed in 2) with six additional explicit
water molecules defining a second water layer that inter-
acts with the first layer (i.e., 294 explicit atoms). The posi-
tion of the water molecules used in 2) and 3) were selected
3) p–p stacking interactions between the phenyl groups of FF
molecules located at different turns; the separation be-
tween the stacked rings (dp–p)=5.32 Š.
[18]
according to Gçrbitz’s observations for FF.
The optimized geometries reflected that, as expected, the
On the other hand, the nanotube and channel diameters are
[
18]
nanotube is stable with or without water molecules filling the
channel (Figure 4a). This stability was attributed to the three
types of interpeptide interactions detected in the tube:
in excellent agreement with those described by Gçrbitz (i.e.,
ꢀ24 and ꢀ10 Š, respectively).
The conformational parameters for the FF molecules in the
three optimized structures are compared in Table S1 (see the
Supporting Information) with those reported for the crystalline
+
À
1
2
) head-to-tail NH3 ··· OOC hydrogen bonds between adja-
cent FF molecules of the same turn; the NÀH···O distances
range from 1.65–1.84 Š.
[
18]
structure, and they evidence very close agreement. The in-
fluence of water molecules on these molecular parameters is
practically inexistent. Analysis of the position of the six water
molecules located in the first layer after PBC-M06L/6-31G(d)
À
) NÀH··· OOC hydrogen bonds between FF molecules at dif-
ferent turns; the NÀH···O distance (d )=1.83 Š.
H–O
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geometry optimization indicates that all the molecules, with
the exception of one, interact simultaneously with two differ-
ent FF molecules (Figure 4b). More specifically, each water
tail interactions, which decreases from six in FF to three in
FFFF, has an important effect on the nanotube morphology. In-
termolecular head-to-tail interactions act as strong restraints,
which limit the conformational flexibility of the molecules con-
tained in the turn. In FFFF the number of these restraints
halves and the molecular size doubles with respect to FF,
therefore the conformational flexibility is significantly higher in
the former. This feature is reflected in Table S1 (see the Sup-
porting Information), which presents the average conforma-
tional parameters for the FFFF molecules. Thus, standard devia-
tions are significantly higher than those obtained for FF.
+
molecule interacts with the NH3 head of one peptide mole-
cule to form an NÀH···O ÀH hydrogen bond (OW and HW
W
W
refers to the oxygen atom and a hydrogen atom of water, re-
À
spectively) and with the OOC tail of the adjacent FF molecule
À
at the same turn to form an O ÀH ··· OOC hydrogen bond
W
W
(
Figure 4b, amplified detail). This is fully consistent with the
[
18]
positions of the observed crystallographic water molecules,
which were close and connected to the charged amino and
carboxylate groups. The six water molecules located in the
second layer move from the starting positions to new posi-
tions located between the water molecules of the first layer
As a result of the reduction in the number of restraints, the
hexagonal symmetry observed in the FF model is lost and the
shape of the FFFF tubes becomes relatively irregular with re-
spect to that of the FF dipeptide (i.e., pseudohexagonal sym-
metry). This provokes a reduction in the strength of the inter-
molecular interactions between the FFFF molecules of consec-
(
Figure 4c). As a result of this integration into the first layer,
a complete hydrogen-bonded network of water molecules sur-
rounding the hydrophilic core is obtained (Figure 4c, amplified
detail).
À
utive turns. The dH–O value associated with the NÀH··· OOC hy-
Because of the similarity between FF and FFFF tubes, the
model for FFFF was constructed considering a hexagonal dis-
position of three explicit peptide molecules interacting
drogen bonds increases from 1.83 Š in FF to 2.13 Š in FFFF,
whereas dp–p increases from 5.32 Š in FF to 5.44 Š in FFFF.
However, an additional NÀH···O=C hydrogen bond between
+
À
through head-to-tail NH3 ··· OOC hydrogen bonds (i.e., 249 ex-
plicit atoms). Three different conformations were found to be
compatible with this turn disposition, and these were consid-
ered as starting geometries for PBC-M06L/6-31G(d) calcula-
tions. The most-stable nanotubular arrangement is represented
in Figure 5a. Clearly the reduction in the number of head-to-
two FFFF molecules at different turns is formed with d
=
H–O
1.85 Š. The loss of symmetry also provokes irregularities in the
empty hydrophilic core, the dimension of which cannot be de-
termined by a single parameter (i.e., the diameter in the case
of FF). Accordingly, the dimensions of the central hole in FFFF
nanotubes have been defined by considering two parameters
d and d (Figure 5a; d =15.6 Š, d =12.2 Š). It should be em-
1
2
1
2
phasized that the same conclusions can be reached by consid-
ering the two constructed models of higher energy (not
shown), which only differ from that displayed in Figure 5 by
the conformation of the peptide molecules.
The calculations presented in this section explain the mor-
phological differences between the FF and FFFF tubes. In both
cases, the narrow channels obtained by the stacking of cyclic
arrays of peptide molecules self-associate to produce sheets.
The coiling of such sheets generates nano- and microscale
tubes for the two peptides. However, the perfect hexagonal
symmetry of the FF channels, which is induced by conforma-
tional restraints provoked by regular distribution of the head-
to-tail interactions, promotes perfect hexagonal packing (Fig-
ure 5b). The assembly of these sheets generates tubes that ex-
hibit very homogeneous and regular walls without apparent
surface defects (Figure 3). In contrast, the conformational flexi-
bility of the FFFF units, which is not reduced by head-to-tail in-
teractions, results in the formation of FFFF channels with
poorly defined pseudohexagonal symmetry (Figure 5c). This
provokes the generation of irregular sheets that self-assemble
into tubes with irregular walls and relatively frequent surface
defects (Figure 2).
Figure 5. a) FFFF nanotube derived from PBC-M06L/6-31G(d) calculations on
a system with three explicit peptide molecules forming a turn. Left: top
+
À
3
view of the three explicit FFFF molecules with head-to-tail NH ··· OOC hy-
drogen-bonding distances (dH–O [Š]) displayed. Middle: side view of the
nanotube; the c-axis is represented by the red line. Right: Details showing
interpeptide hydrogen bonds (dH–O [Š]) and p–p stacking (dp–p [Š]) interac-
tions between FFFF molecules located at consecutive turns. Schematic rep-
resentations of the self-assembly of b) FF and c) FFFF units into tubes. FF
and FFFF channels self-associate to form regular and irregular honeycomb-
like arrays, respectively, which give rise to tubes.
Fmoc-FFFF
N-Fmoc-protected FFFF assembles into nanostructures that are
completely different to the nanotubes described for FFFF and
FF. More specifically, in HFIP/water Fmoc-FFFF forms well-de-
fined ultra-thin nanoplates that aggregate in microclusters
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gen bonds involved in the FFFF nanotubes. Indeed, previous
work by several groups demonstrated that the driving force
for the self-assembly of Fmoc-protected peptides is the p–p
[
21]
stacking of aromatic Fmoc units.
The nanoplates and fibrils displayed in Figure 6a have been
associated with the organization of Fmoc-FFFF molecules into
[
6]
b-sheet assemblies. In a previous study of FFF assemblies the
relationship between the nanoplates and sheet assemblies was
detected by spectroscopic methods. In this work, we com-
pared the relative stability of parallel and antiparallel Fmoc-
FFFF b-sheet configurations by two different computational
strategies. In the first strategy, M06L/6-31G(d) calculations
were performed considering small b-sheet models with three
explicit Fmoc-FFFF molecules arranged in a parallel or antipar-
allel fashion. The second approach is based on PBC-M06L/6-
3
1G(d) calculations of b-sheet models constructed from two
explicit Fmoc-FFFF molecules and PBC along the c-axis. Fur-
thermore, four different arrangements that differ in the relative
orientation of the aromatic side chains of adjacent molecules
were constructed for each packing configuration. Therefore,
eight structures were considered in total.
The results derived from the two strategies were fully consis-
tent in terms of both the relative energy and stabilizing inter-
action patterns. The two strategies predicted that the lowest-
energy b-sheet assembly corresponds to a parallel configura-
tion. Thus, DFT calculations with explicit and periodic models
indicated that the most-favored parallel and antiparallel ar-
rangements are separated by an energy gap of 10.0 and
Figure 6. a) Representative SEM and optical microscopy (inset) images of
nanoplates obtained at room temperature from Fmoc-FFFF dissolved at
À1
a final concentration of 0.5 mgmL in 1:9 HFIP/water. b) SEM (left) and AFM
(
right) images of fibril structures obtained at 48C from Fmoc-FFFF dissolved
À1
at a final concentration of 0.3 mgmL in 3:47 HFIP/EtOH. c) SEM micro-
graph of irregular star-like structures obtained at 48C from Fmoc-FFFF dis-
À1
solved at a final concentration of 0.5 mgmL in 1:9 HFIP/EtOH. (d) SEM mi-
crograph of ill-defined nanospherical aggregates obtained at room tempera-
À1
À1
ture from Fmoc-FFFF dissolved at a final concentration of 1 mgmL in 1:4
10.7 kcalmol , respectively. Figures 7a and b depict the most
HFIP/water.
favored parallel and antiparallel periodic models, respectively.
The corresponding models derived from calculations with
three explicit molecules are represented in Figure S4 (see the
Supporting Information).
(
Figure 6a). Replacement of water by EtOH as the cosolvent re-
sults in the formation of peptide fibrils at peptide concentra-
À1
tions <0.5 mgmL
(Figure 6b; average diameter=(535Æ
The intermolecular interactions found in the most-stable ar-
rangement of each b-sheet configuration were almost identical
for the explicit and periodic models. More specifically, the
lowest-energy parallel arrangement (Figure 7a) is stabilized by
p–p interactions involving Fmoc···Fmoc and F···F pairs, in
which the aromatic rings of each pair are disposed cofacially,
and by NÀH···O=C hydrogen bonds (see amplified detail in Fig-
ure 7a). On the other hand, the most-favored antiparallel ar-
rangement (Figure 7b) exhibits p–p stacking interactions in-
volving Fmoc···F pairs, in which the aromatic rings adopt cofa-
cial dispositions.
1
50) nm), whereas irregular star-like structures of submicromet-
ric dimensions (average diameter=(0.96Æ0.25) mm) appear at
higher peptide concentrations (Figure 6c). Poorly defined
nanospherical aggregates were obtained at Fmoc-FFFF con-
À1
centrations of 1 mgmL in HFIP/water (Figure 6d). According-
ly, the polymorphism of Fmoc-FFFF is largely influenced by the
experimental incubation conditions (i.e., concentration and sol-
vents). Polymorphism was also displayed by Fmoc-FF, which
[4]
was found to self-assemble into peptide fibrils similar to
those displayed in Figure 6b for Fmoc-FFFF, and into hydro-
[19]
gels, which were not identified for Fmoc-FFFF. The transfor-
mation between these two polymorphs in DMSO/water mix-
DFT and PBC-DFT calculations indicate that Fmoc-FFFF pre-
fers a parallel b-sheet configuration, which is in agreement
with the model proposed by Nilsson and co-workers for Fmoc-
[20]
tures has been recently examined. Thus, Fmoc-FF assembled
into fibrous networks that formed gels upon addition of water.
Rigid gels were obtained at high water concentrations and
very low peptide concentrations (i.e., 0.01 wt%). The formation
of gels under physiological conditions has been reported for
Fmoc-FFF, which was produced by using an enzymatic reaction
[
22]
F derivatives containing fluorinated phenylalanine residues.
The authors proposed that the stacking of the Fmoc residues
and the exposure of the C-terminus favors the parallel configu-
[
19a]
ration. This model differs from that reported by Smith et al.
[
23]
for Fmoc-FF and by Castelletto et al. for Fmoc-bAH (Fmoc-b-
alanine-histidine), which both are based on antiparallel stack-
ing. More recently, preferred parallel and antiparallel b-sheet
configurations for Fmoc-GRDS and Fmoc-RGDS (G=glycine,
R=arginine, D=aspartic acid, S=serine), respectively, have
[8]
that links Fmoc-F to FF. However, no other polymorph (e.g.,
nanoplates, nanofibrils, or submicrometric star-like aggregates)
has been reported for this N-Fmoc-protected FFF analogue.
These results indicate that the driving force responsible for the
Fmoc-FFFF assemblies is different from the head-to-tail hydro-
[
24]
been proposed. This led us to conclude that the b-sheet ar-
Chem. Eur. J. 2015, 21, 16895 – 16905
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16900 ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Fmoc-FFFF-OBzl
To get more insight about the role of p–p stacking interactions
in the self-assembly of FFFF-based peptides, a new derivative
was prepared by blocking the free carboxylate group of Fmoc-
FFFF with a benzyl ester group. Fmoc-FFFF-OBzl, which has ar-
omatic groups at both the N- and C-termini, exhibited a wide
variety of assemblies depending on the experimental condi-
tions. For example, different polymorphs were identified in
HFIP/water at 48C depending on the peptide concentration (or
on the solvent/cosolvent ratio). Ultrasmall nanoplates (ꢀ80
and ꢀ35 nm length and width, respectively; Figure 8a) were
À1
obtained at very low peptide concentrations (0.05 mgmL ). In
contrast, birefringent irregular agglomerates (Figure 8b) and
three-dimensional clusters of triaxial ellipsoid-like nodules (Fig-
À1
ure 8c) were formed at a peptide concentrations of 2 mgmL
Figure 7. a) Parallel and b) antiparallel Fmoc-FFFF b-sheets derived from
PBC-M06L/6-31G(d) calculations on systems with two explicit peptide mole-
cules. The sheet axis (c) and intermolecular p–p stacking interactions ($)
are depicted. Amphiphilic lateral association of c) four and d) six explicit
Fmoc-FFFF molecules. Dashed circles define the boundaries of the outer-
and inner regions, which contain the polar carboxylate and the aromatic
Fmoc groups, respectively.
rangement of short Fmoc peptides depends on the peptide
length and sequence.
Despite the stability of the b-sheet structure, other assem-
blies based on p–p association of Fmoc groups can be consid-
ered for Fmoc-FFFF. Figures 7c and d show the lateral associa-
tion of four (440 atoms) and six (660 atoms) explicit Fmoc-FFFF
molecules, respectively, after geometry optimization at the
M06L/6-31G(d) level. These amphiphilic organizations, in which
the unprotected C-terminal carboxylate groups are exposed to
the environment, whereas the aromatic Fmoc groups occupy
the inner region, are exclusively stabilized by p–p interactions
in the central core. Such stability increases with the number of
molecules, which is revealed by the average binding energy
experienced by each molecule (four-molecule unit=À12.3, six-
Figure 8. Representative SEM and optical microscopy (inset) images of
a) nanoplates obtained at 48C from Fmoc-FFFF-OBzl dissolved at a final con-
centration of 0.05 mgmL in 1:99 HFIP/water without sonication and b) bi-
À1
refringent irregular agglomerates obtained at 48C after sonication (35 min at
À1
278C) from Fmoc-FFFF-OBzl dissolved at a final concentration of 2 mgmL
in 4:6 HFIP/water. c) Representative SEM (left) and AFM (right) images of tri-
axial ellipsoid-like nodules obtained at 48C without sonication from Fmoc-
À1
molecule unit=À41.5 kcalmol ). This lateral association repre-
À1
FFFF-OBzl dissolved at a final concentration of 2 mgmL in 4:6 HFIP/water.
sents an alternative to the b-sheet organization, which may ex-
plain the existence of assemblies other than nanoplates and fi-
brils.
d), e) SEM micrographs of complex organizations obtained at 48C from
À1
Fmoc-FFFF-OBzl dissolved at a final concentration of 4 mgmL in 4:1 HFIP/
water without sonication. f) Representative SEM and AFM micrographs of
fibril entanglements obtained at room temperature from Fmoc-FFFF-OBzl
À1
dissolved at a final concentration of 0.5 mgmL in 1:9 DMSO/water without
sonication.
Chem. Eur. J. 2015, 21, 16895 – 16905
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
À1
from sonicated and nonsonicated samples, respectively. This
feature indicates that, in this case, sonication facilitates the for-
mation of self-assembled structures. The average dimensions
of the ellipsoid-like nodules (determined from the AFM
images) were a=4.2 mm, b=1.6 mm and c (height)=300 nm
centration (5 mgmL ): 1) micrometric volcano-like structures
that resemble microcontainers, 2) groups of ellipsoid-like nod-
ules, and 3) very well defined tubes, similar to those obtained
for FF and FFFF.
The diameter of a representative volcano-like structure is
ꢀ20 mm, and the diameter of the central hole is ꢀ8 mm (Fig-
ure 9b). The RMS roughness at the surface of the microcon-
tainer sides, with heights between 1.5 and 3.0 mm, is (420Æ
25) nm (Figure 9c. On the other hand, high-resolution SEM
images (Figure 9d) indicate that the ellipsoid-like nodules,
which partially wrap the sides of the microcontainer in Fig-
ure 9b, are similar to those obtained in HFIP/water at 48C (Fig-
ure 8c). This is corroborated by the average dimensions of the
two largest axes (a=5 mm, b=1.5–2.5 mm) determined by
SEM. Detailed inspection of Figure 9d suggests that these ellip-
soid-like nodules are made of stacks of stretched fibers.
The thickness (ꢀ1.9 mm) of the tubes obtained at the condi-
tions described above was determined by SEM (Figure 9e),
and the height (ꢀ490 nm) was measured by AFM (Figure 9e).
The apparent discord between the thickness and the height of
the tubes is attributed to a surface-induced flattening of the
assembly. This surface-induced deformity is significantly larger
than that observed in cylindrical nanosized single molecular
(
assuming aꢁbꢁc) (Figure 8c). These nodules were also ob-
À1
served for higher peptide concentrations (e.g., 4 mgmL )
when the water content is less than the HFIP content (i.e., 4:1
HFIP/water). Amazingly, these particular conditions provoked
restructuring of the nodules into higher organizations, for ex-
ample a feather headdress resembling a bird’s tail (Figure 8d)
or braided microfibers (Figure 8e). On the other hand, compact
fibrillar frameworks (Figure 8 f) were obtained at room temper-
À1
ature for peptide concentrations of 0.3–0.5 mgmL in DMSO/
water. The average dimensions of these fibrils, which form
abundant entanglements (evidenced by high-resolution SEM
micrographs), are 15–30 mm long and 280–420 nm thick. AFM
measurements indicate that the surface roughness of the
fibers is (30Æ5) nm, which increases to (60Æ8) nm in the en-
tangled regions.
Use of HFIP as the solvent led to unusual results at 48C: dif-
ferent types of assemblies coexisted in this sample. These re-
petitive and reproducible observations are illustrated in Fig-
ure 9a, which displays the simultaneous presence of three very
different assemblies achieved at a relatively high peptide con-
[
25]
objects, for example dendronized polymers, but similar to
[
26]
that observed for hyperbranched dendrimers
and tubes
[
27]
made of self-assembled cyclic peptides.
The coexistence of up to three assemblies obtained under
the above-mentioned conditions is attributed to the different
intermolecular p–p stacking interactions that can be achieved
by combining the multiple aromatic groups of Fmoc-FFFF-
OBzl. Thus, the stabilities associated with these different inter-
actions are probably very similar in HFIP, which permits simul-
taneous formation of different types of assemblies at large-
enough peptide concentrations. This assumption was corrobo-
rated by the fact that samples obtained at low peptide con-
centrations in HFIP provided a variety of individual structures
without any clear preference.
Conclusion
We synthesized three FFFF-based peptides with different flank-
ing groups: FFFF, Fmoc-FFFF, and Fmoc-FFFF-OBzl. Unprotect-
+
À
ed FFFF forms head-to-tail NH3 ··· OOC hydrogen bonds,
which are cannot form upon incorporation of the N-Fmoc and
C-OBzl protecting groups, which promotes p–p stacking inter-
actions.
Spontaneous assembly of the FFFF peptide results in the for-
mation of nanotubes that resemble those achieved for FF.
However, detailed microscopy studies reveal that the tubes ob-
tained from FFFF present many surface irregularities and de-
fects that are not present in the structures derived from FF.
Theoretical DFT calculations using periodic conditions indicate
that the intrinsic conformational flexibility of FFFF promotes
the formation of irregular nanotubes that give surface defects
upon packing. In contrast, the conformational flexibility of FF,
which is intrinsically lower than that of FFFF, is restricted by
Figure 9. Assemblies obtained at 258C from Fmoc-FFFF-OBzl dissolved at
a final concentration of 5 mgmL in pure HFIP. a) Representative SEM and
optical microscopy (inset) images showing the three different coexisting as-
semblies. b) SEM micrograph and c) 3D and 2D (inset) AFM height images
showing the dimensions and topography, respectively, of the microcontainer
À1
(
or volcano-like) assembly. d) Representative SEM micrographs of the ag-
glomerates formed by triaxial ellipsoid-like nodules that wrap one of the
slopes of the volcano in a) and b). e) Representative 3D and 2D AFM height
images and SEM micrograph of the tubes. Cross-sectional profile of the zone
marked in the 2D AFM image displaying the height of the tube.
+
À
the strong head-to-tail NH3 ··· OOC hydrogen bonds, which fa-
Chem. Eur. J. 2015, 21, 16895 – 16905
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16902 ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
cilitates the formation of very regular hexagonal channels that
self-associate into homogeneous tubular structures free of sur-
face defects. Calculations also indicated that intermolecular in-
teractions involving peptide molecules at consecutive turns of
the nanotube (i.e., NÀH···O hydrogen bonds and p–p stacking
interactions between aromatic side chains) play a very impor-
tant role in the stability of the assembly.
Preparation of the initial peptide solutions
Peptide solutions (25 or 100 mL) were prepared from 5 mgmL
À1
stock solutions in HFIP, DMSO, or DMF. Milli-Q water or EtOH were
added as cosolvents to reduce the peptide concentration and en-
hance the assembly process. All organic solvents were purchased
from Sigma–Aldrich. The peptide concentrations in the prepared
À1
solutions were 0.05–5 mgmL . Aliquots (10 or 20 mL) were placed
on microscope coverslips and kept at room temperature (258C) or
inside a cold chamber (48C) until dry.
The variety of assemblies increases significantly for Fmoc-
FFFF. Nanoplates, fibrils, star-like aggregates, and ill-defined
nanospheres have all been identified for Fmoc-FFFF depending
on the incubation conditions. The existence of these poly-
morphs suggests that p–p stacking interactions involving
Fmoc groups are more versatile than hydrogen bonds in terms
of molecular self-association. This is consistent with the fact
that p–p interactions are considerably less specific than hydro-
gen bonds, which are severely restricted by the relative dispo-
sition of the NÀH and C=O bonds. DFT calculations with both
explicit and periodic models indicate that Fmoc-FFFF prefers
a parallel rather than an antiparallel b-sheet organization. Fur-
thermore, the ability of Fmoc aromatic groups to form p–p in-
teractions favors the stabilization of other amphiphilic assem-
blies, which is consistent with the experimental observation of
a variety of polymorphs.
Optical microscopy
Optical morphologic observations were performed with a Zeiss Ax-
ioskop 40 microscope. Micrographs were taken with a Zeiss Axios-
Cam MRC5 digital camera.
Scanning electron microscopy
SEM studies were performed in a Focused Ion Beam Zeiss Neon 40
scanning electron microscope operating at 5 kV, equipped with an
energy-dispersive X-ray spectroscopy system. Samples were
mounted on double-sided adhesive carbon discs and sputter-
coated with a thin layer of carbon to prevent sample charging
problems.
Atomic force microscopy
Finally, the coexistence of up to three polymorphs in the
same sample has been observed for Fmoc-FFFF-OBzl. Individu-
al structures were detected for this peptide in HFIP/water and
DMSO/water, however the most-noticeable result corresponds
to the simultaneous presence of microcontainer (or volcano-
like) structures, triaxial ellipsoid-like nodules, and nanotubes in
samples prepared from a high concentration of peptide in
HFIP. This behavior is attributed to the fact that the stability of
the different combinations of intermolecular p–p stacking in-
teractions allowed by the Fmoc and OBzl protecting groups
and the phenyl side chains are similar under the experimental
conditions.
Topographic AFM images were obtained by using either a Dimen-
sion 3100 Nanoman AFM or a Multimode instrument, both from
Veeco (NanoScope IV controller) under ambient conditions in tap-
ping mode. The RMS roughness was determined by using the stat-
istical application of the Nanoscope software and calculates the
average result considering all the values recorded in the topo-
graphic image with exception of the maximum and the minimum.
AFM measurements were performed on various parts of the films,
which gave reproducible images similar to those displayed in this
2
work. Scan-window sizes were 25”25, 15”15, and 5”5 mm .
Theoretical calculations
DFT calculations were performed on two different types of struc-
tures: 1) those in which one-dimensional PBC were applied to an
assembly previously constructed from a given number of explicit
peptide molecules, and 2) those exclusively based on the assembly
of explicit peptide molecules. All DFT calculations were carried out
Experimental Section
Materials
[28]
by using the Gaussian 09 computer package. The geometries of
the different systems investigated were fully optimized by using
Boc- and Fmoc-protected amino acids were supplied by the Poly-
Peptide Group, N-[3-(dimethylamino)-propyl]-N’-ethylcarbodiimide
was purchased from Bachem, and all other reagents for peptide
synthesis were purchased from Sigma–Aldrich.
[29]
the M06L functional, which was developed by Zhao and Truhlar
to account for dispersion, combined with the 6-31G(d) basis set.
PBC were used as implemented in the Gaussian 09 program (i.e.,
periodic systems were simply specified through a translation
[30]
vector to indicate the replication direction). The number of repli-
cated cells was six in all cases, and the k-point mesh (NK point) in
the replicated direction was set to 64, 33, and 21 for FF, FFFF, and
Fmoc-FFFF, respectively. No symmetry constraints were used in the
geometry optimizations. Binding energies (BEs) were corrected
with the basis set superposition error (BSSE) by means of the stan-
dard counterpoise.
Peptide synthesis
Melting points were determined with a Gallenkamp apparatus and
are uncorrected. IR spectra were recorded with a Thermo Nicolet
Avatar 360 FTIR spectrophotometer; n˜ is given for the main ab-
max
1
13
sorption bands. H- and C NMR spectra were recorded with
a Bruker AV-400 instrument at room temperature unless otherwise
indicated; the residual solvent signal is used as an internal stan-
dard, chemical shifts (d) are expressed in ppm, and coupling con-
stants (J) are expressed in Hz. Optical rotations were measured
with a JASCO P-1020 polarimeter. High-resolution mass spectra
were obtained with a Bruker MicrOTOF-Q spectrometer.
General procedure for the peptide coupling
1-Hydroxybenzotriazole (HOBt) (4.40 mmol) was added to a solution
of the appropriately N-protected a-amino acid or peptide carboxyl-
ic acid (4.00 mmol) in CH Cl , and the solution was cooled to 08C
2
2
Chem. Eur. J. 2015, 21, 16895 – 16905
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16903
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
in an ice bath. N-[3-(dimethylamino)-propyl]-N’-ethylcarbodiimide
hydrochloride (4.40 mmol) was added, followed by a solution of
the amino component (4.40 mmol) in CH Cl (obtained after acidic
[2] M. Reches, E. Gazit, Phys. Biol. 2006, 3, S10–19.
[3] a) N. Amdursky, M. Molotskii, E. Gazit, G. Rosenman, Appl. Phys. Lett.
2009, 94, 261907; L. Adler-Abramovich, N. Kol, I. Yanai, D. Barlam, R. Z.
2
2
Shneck, E. Gazit, I. Rousso, Angew. Chem. Int. Ed. 2010, 49, 9939–9942;
Angew. Chem. 2010, 122, 10135–10138.
removal of the protecting group) and N-methylmorpholine (NMM)
4.40 mmol) or N-ethyldiisopropylamine (DIPEA) (4.40 mmol). The
(
[
[
4] N. Amdursky, E. Gazit, G. Rosenman, Adv. Mater. 2010, 22, 2311–2315.
5] a) X. Yan, P. Zhu, J. Li, Chem. Soc. Rev. 2010, 39, 1877–1890; b) E. Gazit,
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reaction mixture was stirred for 1 h at 08C, then at RT for 24 h at
pH 8 (moistened pH paper). The reaction mixture was repeatedly
washed with a 5% aqueous solution of KHSO , a 5% aqueous solu-
4
tion of NaHCO , and water. The organic phase was dried over
3
MgSO and evaporated to dryness. The peptide product was puri-
4
[
[
[
6] P. Tamamis, L. Adler-Abramovich, M. Reches, K. Marshall, P. Sikorski, L.
Serpell, E. Gazit, G. Archontis, Biophys. J. 2009, 96, 5020–5029.
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Togna, F. Bordi, C. Palocci, Soft Matter 2010, 6, 2525–2532.
fied by flash chromatography. A description of all intermediates is
provided in the Supporting Information.
2
0
FFFF: White solid; m.p. 2348C; [a] : +10.1 (c=0.43, AcOH); IR
D
À1
(
nujol) n˜ =3800–3000, 3296, 1734, 1693, 1634 cm ; HRMS (ESI): m/
z calcd for C H N O : 607.2915 [M+H] ; found: 607.2923. (Due to
+
3
6
39
4
5
poor solubility it was impossible to record the NMR spectra in typi-
cal NMR solvents: [D ]DMSO, MeOD , CDCl , CD CN), therefore we
recorded the NMR spectra of the TFA salt.
[9] P. Tamamis, L. Adler-Abramovich, E. Gazit, G. Archontis, in From Compu-
tational Biophysics to Systems Biology 2008 (CBSB08) (Eds.: U. H. E. Hans-
mann, J. H. Meinke, S. Mohanty, W. Nadler, O. Zimmermann), John von
Neumann Institute for Computing (Jülich), NIC Series, Vol. 40, pp. 393–
6
4
3
3
1
TFA·FFFF: H NMR (400 MHz, [D ]DMSO): d=2.72–2.88 (m, 3H),
6
396.
2
.91–3.12 (m, 5H), 3.90–4.00 (m, 1H), 4.46–4.53 (m, 1H), 4.56–4.65
[
[
10] C. Guo, Y. Luo, R. Zhou, G. Wei, Nanoscale 2014, 6, 2800–2811.
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13
(
(
m, 2H), 7.14–7.32 (m, 20H), 8.31–8.76 ppm (m, 3H); C NMR
100 MHz, [D ]DMSO): d=36.70, 37.12, 37.64, 37.74, 53.11, 53.48,
6
[12] D. Zanuy, I. Hamley, C. Alemµn, J. Phys. Chem. B 2011, 115, 8937–8946.
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Tzokova, C. M. Fernyhough, M. F. Butler, S. P. Armes, A. J. Ryan, P. D.
Topham, D. J. Adams, Langmuir 2009, 25, 11082–11089.
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Nune, P. Kumaraswamy, U. Maheswari Krishnan, S. Sethuraman, Curr.
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[15] a) J. J. Panda, A. Mishra, A. Basu, V. S. Chauhan, Biomacromolecules
2008, 9, 2244–2250; b) R. Silva, D. Araujo, E. Silva, R. Ando, W. Alves,
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16] a) S. Cavalli, F. Albericio, A. Kros, Chem. Soc. Rev. 2010, 39, 241–263;
b) A. Lakshmanan, S. Zhang, C. A. Hauser, Trends Biotechnol. 2012, 30,
155–165.
5
1
1
3.65, 53.86, 126.24, 126.34, 126.45, 127.09, 128.03, 128.08, 128.15,
28.22, 128.47, 129.14, 129.23, 129.56, 134.79, 137.38, 137.43,
37.46, 137.63, 168.01, 170.33, 170.65, 170.96, 172.70 ppm.
2
0
Fmoc-FFFF: White solid; m.p. 2388C; [a] : À9.6 (c=0.36, AcOH);
D
[
1
H NMR (400 MHz; [D ]DMSO): d=2.63–2.86 (m, 4H), 2.92–3.14 (m,
6
4
H), 4.06–4.22 (m, 4H), 4.34–4.59 (m, 3H), 7.05–7.42 (m, 24H), 7.53
(
8
d, J=8.8 Hz, 1H), 7.56–7.63 (m, 2H), 7.83–7.91 (m, 2H), 8.04 (d, J=
.0 Hz, 1H), 8.04 (m, 1H), 8.22 ppm (d, J=6.4 Hz, 1H); C NMR
13
(
100 MHz, [D ]DMSO): d=36.96, 37.40, 37.62, 46.53, 53.60, 53.86,
6
5
1
1
3
4.15, 56.03, 65.65, 120.06, 125.24, 125.32, 126.13, 127.06, 127.61,
27.96, 128.00, 129.18, 137.61, 137.69, 138.01, 138.15, 140.63,
[
43.68, 143.78, 155.61, 170.56, 170.60, 171.18 ppm; IR (nujol) n˜ =
À1
283, 1736, 1696, 1641 cm
; HRMS (ESI): m/z calcd for
[
17] a) L. Adler-Abramovich, M. Badihi-Mossberg, E. Gazit, J. Rishpon, Small
+
C H N O Na: 851.3415 [M+Na] ; found: 851.3402.
51
48
4
7
2010, 6, 825–831; b) J. H. Kim, J. Ryu, C. B. Park, Small 2011, 7, 718–
2
0
Fmoc-FFFF-OBzl: White solid; m.p. 1898C; [a] : À13.2 (c=0.35,
722.
D
1
AcOH); H NMR (400 MHz, [D ]DMSO): d=2.64–2.88 (m, 4H), 2.90–
[18] C. H. Gçrbitz, Chem. Eur. J. 2001, 7, 5153–5159.
6
3
2
8
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Keywords: beta sheet · hydrogen bonds · nanotubes ·
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Published online on September 30, 2015
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www.chemeurj.org
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