Effect of Solvent Choice on the Self-Assembly Properties of a Diphenylalanine Amphiphile Stabilized by an Ion Pair

Article abstract of DOI:10.1002/cphc.201700180

A diphenylalanine (FF) amphiphile blocked at the C terminus with a benzyl ester (OBzl) and stabilized at the N terminus with a trifluoroacetate (TFA) anion was synthetized and characterized. Aggregation of peptide molecules was studied by considering a pe

Full text of DOI:10.1002/cphc.201700180

Accepted Article
Title: Effect of the solvent choice in the self-assembly properties in a
diphenylalanine amphiphile stabilized by an ion pair
Authors: Enric Mayans, Gema Ballano, Javier Sendros, Merçe Font-
Bardia, J. Lourdes Campos, Jordi Puiggali, Carlos Cativiela,
and Carlos Aleman
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ChemPhysChem
10.1002/cphc.201700180
ARTICLE
Effect of the solvent choice in the self-assembly properties in a
diphenylalanine amphiphile stabilized by an ion pair
Enric Mayans,[a,b] Gema Ballano, Javier Sendros,
[c]
[a,b]
Merçè Font-Bardia,[d,e] J. Lourdes Campos,[a]
Jordi Puiggalí,*[a,b] Carlos Cativiela,* and Carlos Alemán*
[c]
[a,b]
Abstract: A diphenylalanine amphiphile blocked at the C-terminus
with benzyl ester and stabilized at the N-terminus with
formation of diphenylalanine (FF, where F= L-Phe) nanotubes in
a
a
aqueous solution, which is due to the directionality offered by a
combination of hydrogen bonding and repeated phenyl stacking
interactions. In subsequent studies the FF was proved as a
minimal sequence that forms self-assembled peptide
nanoestructures,[2-8] giving place to the development of a new
class of biomaterials that is based on the addition of various N-
and C-terminal capping groups to the aromatic FF or, even, on
chemical modification of the own F residues.
trifluoroacetate (TFA) anion has been synthetized and characterized.
The aggregation of peptide molecules has been studied considering
a peptide solution in an organic solvent and adding pure water, a
KCl solution or another organic solvent as co-solvent. The choice of
the organic solvent and co-solvent, and the solvent:co-solvent ratio
allows tuning the mixture by modulating the polarity, the ionic
strength and the peptide concentration. Differences in the properties
of the media used to dissolve the peptides result in the formation of
different self-assembled microstructures (e.g. fibers, branched-like
structures, plates and spherulites). Furthermore, crystals of TFA·FF-
OBzl have been obtained for X-ray diffraction from aqueous peptide
The
peptide
amphiphile
Fmoc-FF
(Fmoc=
fluorenylmethoxycarbonyl), which form stable gels, is among the
most studied FF-based biomaterials. Thus, Fmoc-FF gels with a
variety of properties have been prepared using different
[
9]
solutions. Results reveal
a
hydrophilic core constituted by
approaches. Gazit and co-workers formed gels by dissolving
Fmoc-FF in an appropriate water-miscible solvent,[
10,11]
while
carboxylate (from TFA), ester and amide groups, which is
surrounded by a hydrophobic crown with ten aromatic rings. This
segregated organization explains the assemblies observed in
different solvent mixtures as a function of the environmental polarity,
ionic strength and peptide concentration.
Ulijn and co-workers used a pH switch approach coupled with
changes in the temperature to yield Fmoc-FF gels with variable
properties (i.e. depending on the rate of pH decrease and the
final pH).[
12-15]
The mixing method was also used by other groups,
showing that the mechanical properties of Fmoc-FF gels depend
on the final ratio of dimethylsulfoxide (DMSO) to water.[
More recently, Adams and co-workers formed Fmoc-FF gels by
dissolving the peptide in an organic solvent (OS) and adding
16,17]
Introduction
water, rheological properties depending on the choice of the
OS.[18] Furthermore, gels formed using acetone were meta-
In their pioneering work, Reches and Gazit[1] demonstrated the
stable and single crystals suitable for X-ray diffraction were
collected. The structure showed a parallel stacking of Fmoc-FF
molecules, neighbouring molecules interacting through hydrogen
bonds and weak offset - interactions.[18] Similarly, studies on
[
a]
E. Mayans, J. Sendros, Dr. J. L. Campos, Prof. Dr. J. Puiggalí and
Prof. Dr. C. Alemán
Departament d’Enginyeria Química
Universitat Politècnica de Catalunya
EEBE, Edifici I.2, C/ Eduard Maristany, 10-14, 08019, Barcelona,
Spain
E-mail: jordi.puiggali@upc.edu and carlos.aleman@upc.edu
E. Mayans, J. Sendros, Prof. Dr. J. Puiggalí and Prof. Dr. C. Alemán
Barcelona Research Center for Multiscale Science and Engineering
Universitat Politècnica de Catalunya
C/ Eduard Maristany, 10-14, 08019, Barcelona, Spain
E-mail: jordi.puiggali@upc.edu and carlos.aleman@upc.edu
Dr. G. Ballano and Prof. Dr. C. Cativiela
Department of Organic Chemistry and Instituto de Síntesis Quimica
y Catalisis Homogenea (ISQCH)
University of Zaragoza–CSIC
Nap-FF
(Nap=
naphthalene)
and
Cbz-FF
(Cbz=
belzyloxycarbonyl) hydrogels evidenced fibrous structures made
of -sheet arrangements, even though fibril dimensions
depended on the aromatic capping group.[ Interestingly, the
Fmoc-FF-OFm peptide, capped with Fmoc and 9-
fluorenylmethyl ester (OFm) at the N- and C-terminal,
19]
[
b]
c]
respectively, exhibited
a
great variety of polymorphic
[
microstructures (e.g. doughnut, stacked-braids, dendritic and
microtubes) depending on the solvents used to promote the self-
assembly.[ It is worth noting that stacking interactions play a
dominant role in such highly aromatic peptide.
20]
50009 Zaragoza, Spain
E-mail: cativiela@unizar.es
Dr. M. Font-Bardia
Departament de Mineralogia, Cristallografia i Depòsits Minerals
Universitat de Barcelona
Facultat de Geologia, c/Martí i Franquès, E-08028, Spain
Dr. M. Font-Bardia
Unitat de Difracció de Raigs X, Centre Científic y Tecnològic
Universitat de Barcelona
C/Solé i Sabarís 1, Barcelona E-08028, Spain
An alternative approach is the chemical modification of the F
residues. Reches and Gazit[5] explored the self-assembly of FF-
[
d]
e]
based dipeptides in which the phenyl side chains were modified
by halogen atoms, additional phenyl groups or with nitro
substitutions. These homo-aromatic dipeptide motifs formed
tubular, spherical and fibrillary structures in the nanoscale, in
some cases nanocrystals and 2D nanoplates being also
detected. These results proved that the properties of FF-based
biomaterials can be properly tailored by engineering the F
residue.
[
Supporting information for this article is given via a link at the end of
the document
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ChemPhysChem
10.1002/cphc.201700180
ARTICLE
Another investigated strategy was the co-assembly of FF-based Results and Discussion
biomaterials with other molecules bearing aromatic groups.[21-24]
This approach, which may provide intermolecular transfer
Results presented in this work correspond to the conditions in
which repetitive, stable and structured morphologies were
observed. More specifically, assemblies have been required to
fulfill the following conditions: i) to present a clearly defined
morphology; ii) to be systematically observed when the same
conditions are used in different and independent experiments;
and iii) to remain formed upon manipulation for optical
microscopy, SEM and/or AFM observations).
mechanisms,[
25,26]
was applied to the Npm-FF (Npm=
naphthoxymethyl) donor / dansyl acceptor system.[21] Peptide
fibres based partly on aromatic stacking interactions with the
dansyl component intercalated within this structure exhibited a
redshift in the fluorescence emission and the corresponding
quenching of the emission associated with the donor species.[21]
Besides, hydrogels derived from the co-assembly of Fmoc-FF
[
22]
and Fmoc-diglycine (Fmoc-GG) or Fmoc-Arg-Gly-Asp (Fmoc-
RGD)[
23]
showed higher elastic moduli than Fmoc-FF alone,
Peptide synthesis and Preparation of initial TFA·FF-OBzl
solutions
The synthesis of TFA·FF-OBzl was carried out following the
procedure provided in Figure 1.
while the combination of Fmoc-FF with Fmoc-Lys (Fmoc-K),
Fmoc-Ser (Fmoc-S) or Fmoc-Asp (Fmoc-D) resulted in
significant changes in the rheological properties and fiber
morphology.[
24]
As the main aim of this study is to investigate the influence of
both the polarity of the medium and the peptide concentration in
the assembly of TFA·FF-OBzl a two-step procedure was used.
Firstly, concentrated (5.0 mg/mL) stock solutions were prepared
using solvent able to completely dissolve the peptide. For this
purpose, four solvents with very different polarities were
selected: hexafluoroisopropanol (HFIP), dimethylformamide
(DMF), DMSO and milli-Q water. The dielectric constants of
such solvents are: = 16.7 (HFIP), 37.2 (DMF), 46.7 (DMSO)
and 78.5 (water). Secondly, the peptide concentration and the
polarity of the medium were altered by direct addition of a co-
solvent to the stock solution. In addition, of the above mentioned
Besides, solvent-induced structural transitions have been
[27]
examined by different authors. Li and co-workers reported the
transition of an organogel obtained by self-assembly of FF in
toluene into a lower-like microcrystal merely by introduced
ethanol as co-solvent. Huang et al.[28] reported the structural
transition of self-assembled FF from microtubes to nanofibers by
introducing acetonitrile as co-solvent in water phase.
Mumaraswamy et al.[29] found that the dimensions of FF
nanotubes are strongly influenced not only by the temperature
and pH but by the ionic strength of the solution. Mba and co-
workers[30] synthetized two organogelators based on a pyrene
moiety liked to FF, which formed spherical aggregates and
entangled fibrillary networks in acetonitrile and o-
solvents, both methanol (MeOH, = 32.6), chloroform (CHCl , =
3
4.7), which are not able to completely dissolve the peptide, were
considered as co-solvent. This procedure allowed varying the
peptide concentration in the prepared solvent : co-solvent
mixtures between 0.05 to 4.8 mg/mL. Both the final peptide
concentration and the chemical nature of the mixture will be
provided for each discussed structure.
[
31]
dichlorobenzene, respectively. Wang et al. used FF to prove
that a trace amount of solvents can be a predominant factor to
tune peptides self-assembly. More specifically, these authors
showed that the addition very small amounts of solvents can be
used to force solvent-bridged hydrogen bonds, which is a crucial
interaction in directing fiber formation.
In this paper we use the solvent mixing method (i.e. dissolving
the peptide in OS and adding water or another OS as co-
solvent) to examine the self-assembly of a new FF-based
amphiphile. In this new compound, hereafter denoted TFA·FF-
OBzl (Scheme 1), the C-terminus is capped with a benzyl ester
1
2 R = Bzl
3 R = Bzl
(
OBzl) group and the protonated amino group is stabilized
forming an ion pair with trifluoroacetate (TFA). Accordingly,
aromatic interactions are expected to be weaker than on Fmoc-
FF, Nap-FF and Fmoc-FF-OFm, while the dominant role played
by intermolecular electrostatic interactions in FF is expected to
decrease considerably because of the stability provided by the
TFA.
TFA·FF-OBzl
Figure 1. Scheme of the reactions used to obtain TFA·FF-OBzl. Reagents and
conditions: (a) EDC, HOBt, DIPEA CH2Cl2, 0ºC 30 min, room temperature 24
h; (b) TFA, CH2Cl2, room temperature 1 h.
For the formation of the assembled structures, 10 or 20 μL
aliquots of the prepared peptide solutions were placed on
microscope coverslips or glass slides (glass sample holders)
and kept at room temperature (21 ºC) or inside a cold chamber
Scheme 1. Chemical structure of TFA·FF-OBzl
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ChemPhysChem
10.1002/cphc.201700180
ARTICLE
(
4 ºC) until dryness. The humidity was kept constant in both
spherulite and the branches are made of ultra-thin plates that,
despite resembling lamellar crystal structures, are obtained
through the hierarchical assembly of nanowires. When the
peptide concentration decreases to 2.0 mg/mL and,
consequently, the polarity and ionic strength of the 4:6
DMSO:50mM-KCl(aq) mixture increase, branches become
worsen defined and less abundant (Figure S3), even though
self-assembly characteristics are similar to those described
above for the concentrated peptide solution. Moreover, these
supramolecular structures coexist with randomly distributed
micrometric crystals of oval shape (Figure 3d). Finally, when the
peptide concentration is reduced, as for example in 0.25 mg/mL
1:19 DMSO:50mM-KCl(aq), spherulitic-like microstructures
surrounded by large salt crystals are observed (Figure 3e).
According to the micrographs displayed in Figure 3c-e and S3,
the combination of a polar OS (i.e. = 46.2 for DMSO) with an
aqueous salt solution, KCl(aq), results in a hierarchical assembly
of the amphiphilic peptide under study, even though this
tendency becomes less pronounced with decreasing amount of
DMSO (i.e. higher ionic strength). This feature should be
associated to the influence of solvent molecules and salt ions on
the balance between peptide-peptide and peptide-solvent
interactions.
laboratories at 50%. It is worth noting that no thermal treatment
was applied to improve the solubility of the peptide or to
accelerate the evaporation of the solvents. In spite of the huge
number of conditions examined, the structures obtained from all
them were carefully examined by optical microscopy. However,
only those structures that fulfill the requirements described
above (i.e. well-defined morphology and reproducibility) were
subsequently studied by SEM and AFM for discussion in this
work.
Aqueous environment
Dissolution of TFA·FF-OBzl into 2.0 mg/mL 4:6 HFIP:water
directed the self-assembly process towards peptide nanofibers
with a diameter () of 250 nm that align and pack forming well-
defined microfibers of up to 10 m (Figure 2a). At the same
time, such microfibers form very dense aggregates with a spike-
like morphology. When the polarity of the mixture increases and
the peptide concentration decreases in 0.1 mg/mL 1:49
HFIP:water, the density of aggregated microfibers decreases,
whereas a very porous mesh of randomly oriented (bundled)
fibers coexists coating partially the spike-like supramolecular
structure (Figure 2b). These bundled fibers exhibit very different
diameters (i.e. from 100 nm to 1 m) and do not present any
kind of imperfection, as it can be observed in the corresponding
SEM and AFM images. The mesh is replaced by small needle-
like crystals emerging from the spike-like microstructures in 1.0
mg/mL 1:4 DMF:water peptide solutions (Figure 2c). However, a
remarkable difference is that such spikes are not formed by long
aligned nanofibers, as observed in Figure 2a for the 2.0 mg/mL
4:6 HFIP:water mixture, but by relatively short and sometimes
broken interconnected nanofibers. These results suggest that
the structure of the nanofibers, as well as their supramolecular
organization (i.e. the hierarchical self-assembly of TFA·FF-OBzl),
change with both the polarity of the solvent mixture and the
concentration of the peptide.
The addition of diluted KCl aqueous solutions (50 mM) to the
HFIP, DMF or DMSO peptide solutions caused drastic
morphological changes, which mainly consists in the apparition
of branched-like structures and, in some cases, ultra-thin plates.
Thus, poorly defined micrometric branched-like architectures
(
Figure 3a), which coexist with peptide microfibers (Figure S1a),
grow from 4.8 mg/mL 24:1 HFIP:50mM-KCl(aq) peptide
solutions. However, microfibers coated with salt and abundant
defects (Figure S1b) are the only structures observed upon
reduce the peptide concentration to 1.0 mg/mL. Besides,
branched-like structures (Figure 3b), coexisting with disordered
microfibers agglomerates (Figure S2), are obtained in 1.0
mg/mL 1:4 DMF:50mM-KCl(aq). The branching is much better
defined than in Figure 3a, suggesting that this class of
architecture is promoted by enhancing both the polarity of the
mixture and the ionic force.
Figure 2. Representative SEM and AFM (height) images of the structures
derived from TFA·FF-OBzl solutions in: (a) 2.0 mg/mL 4:6 HFIP:water at 4 ºC;
(b) 0.1 mg/mL 1:4 HFIP:water at room temperature; and (c) 1.0 mg/mL 1:4
The large influence of the polarity is corroborated in Figure 3c
for the 4.8 mg/mL 24:1 DMSO:50mM-KCl(aq) peptide mixture. In
this case very-well defined branched structures, each one
nucleating from a spherulite, and partially coated with cubic
crystals of salt are abundantly detected. Both the central
DMF:water at room temperature. The shape and hierarchical self-assembly
changes with polarity of the solvents mixture and the peptide concentration.
Red circles in (c) indicate broken nanofibers.
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ChemPhysChem
10.1002/cphc.201700180
ARTICLE
directional) electrostatic interactions associated to the charged
end groups are probably replaced by weak and specific
(
directional) hydrogen bonds after neutralization, affecting the
definition of the assemblies their growing.
It is should be remarked that the branched- and three-like
structures obtained for TFA·FF-OBzl do not resemble the
dendritic structures identified for FF and Fmoc-FFFF-Fmoc.[20]
[32]
Kim and co-workers[
32]
obtained highly ordered multi-
dimensional dendritic nanoarchitectures by self-assembling FF
from an acidic buffer solution. More recently, stable dendritic
structures made of branches growing from nucleated primary
frameworks were observed for Fmoc-FFFF-OFm.[ The fractal
dimension of FF and Fmoc-FFFF-OFm dendrimers was
determined to be 1.7, which evidenced self-similarity and two-
dimensional diffusion controlled growth.[20,32] However, branched
and three-like structures displayed in Figures 3 and S4a do not
exhibit a primary nucleating framework nor a repetitive pattern
for growing of the branches, which are essential to obtain the
characteristic self-similarity of dendritic structures.
20]
Single crystal X-ray structure of TFA·FF-OBzl
X-ray diffractograms were collected for prism-like crystals
obtained by slow evaporation at 80 ºC of a 0.415 mg/mL solution
of TFA·FF-OBzl in milliQ- water. Table S1 summarizes the main
crystallographic data of TFA·FF-OBzl, whereas Table S2 shows
the final atomic parameters (fractional coordinates and thermal
factors) together with the estimated standard deviations.
Geometric parameters are listed in Tables S3 (bond lengths and
angles) and Table S4 (torsional angles).
Conformation of a single TFA·FF-OBzl molecule is shown in
Figure 4a together with labelling of atoms and the corresponding
displacement ellipsoids. It is clear that the molecule adopted a
folded conformation, being the peptide group practically planar
(
(
C10-N1-C1-C2 of -173.3º) and the  (C1-N1-C10-C11) and 
N2-C2-C1-N1 / N1-C10-C11-O3) torsional angles of -86.6º and
Figure 3. Representative SEM and AFM (height) images of the branched
structures obtained at room temperature from TFA·FF-OBzl dissolved in: (a)
127.7º / -54.9º, respectively. It is worth noting that such
conformation does not fit to that expected for a conventional -
strand within a -sheet, which typically exhibits , values
around –135º, +135º. This feature has been attributed to the
formation of an intramolecular - stacking interaction between
the C13-C18 benzyl (Bzl) and C20-C25 phenyl (Ph) rings, which
is indicated by a red arrow in Figure 4a. The dihedral angle
between the Bzl and Ph aromatic groups is 33.6º, while the
distance between the centroids of the two rings is 4.59 Å.
TFA·FF-OBzl crystallizes in an orthorhombic unit cell containing
4 molecules (Figure S5) which are related by binary screw axes
as typical of chiral organic molecules. Figure 4b depicts a
scheme of the molecular packing (b-c projection) with two
neighboring molecules. The existence of a hydrophilic core,
which is formed by the carboxylate (from TFA), ester and amide
groups, is particularly noticeable. This hydrophilic core is
surrounded by a hydrophobic crown (dashed circle) formed by
ten aromatic rings (i.e. 6 phenyl and 4 benzyl rings). Only six of
these rings belong to the two represented molecules whereas
the other four are associated to neighboring ones. Table S5
summarizes the hydrogen bond interactions that can be
considered and that involve ester, amide and carboxylate groups.
4
.8 mg/mL 24:1 HFIP:50mM-KCl(aq); (b) 1.0 mg/mL 1:4 DMF:50mM-KCl(aq);
(c) 4.8 mg/mL 24:1 DMSO:50mM-KCl(aq); (d) 2.0 mg/mL 4:6 DMSO:50mM-
KCl(aq); and (e) 0.25 mg/mL 1:19 DMSO:50mM-KCl(aq).
The formation of branched-like structures is also considerably
affected by the pH. This is reflected in Figure S4, in which the
pH of the corresponding OS : 50mM-KCl(aq) solutions was fixed
at 10.5 by adding 0.5 M NaOH. The poorly defined branched
structures mentioned above for the 4.8 mg/mL 24:1 HFIP:50mM-
KCl(aq) mixture (Figure 2a) results into very well defined tree-
like structures of fibrous nature at basic pH (Figure S4a). In
contrast, the addition of NaOH transforms the spherulite-
nucleated branches observed in Figure 2c into dense bundles of
plates irregularly arranged (Figure S4b). Indeed, some of these
plates resemble deformed microtubes because of their
dimensions. This is reflected by the AFM cross sectional profile
displayed in Figure S4b, which shows that the x- and y-diameter
for one of such elements is 2.3 and 2.0 m, respectively.
These changes have been attributed to the neutralization of the
peptide by the NaOH. Thus, strong and non-specific (non-
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ChemPhysChem
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ARTICLE
The most relevant intermolecular hydrogen bond, which is
formed along the a direction, involves the N–H (amide) and C=O
assembly of the TFA·FF-OBzl amphiphile. Thus, based on the
morphology of the morphology of the assembled structures and
in the position of the molecules in the crystal structure reported
in this work, as the solvent gradually evaporates, the individual
TFA·FF-OBzl tend to form intermolecular electrostatic and -
stacking interactions, promoting the aggregation of the peptide
molecules and the formation of amphiphile crowns. The
formation of hydrophobic crown in the aggregates is reinforced
by the intramolecular - stacking interactions. These
interactions results in the formation of -sheet structures, which
stack vertically via hydrogen bonds resulting in the lengthening
of the peptide structures. This process is similar to that reported
for the amyloid fibril formation, even though and that case
aggregates are usually formed through electrostatic and
hydrogen bonding interactions (i.e. - stacking interactions
frequently participates in the lengthening of the structures).[
(
ester) groups of molecules with a parallel orientation as
displayed in Figure 4c. It is remarkable that the C=O of amide
+
groups only forms a weak interaction with terminal NH
3
groups.
Finally, a intermolecular  stacking interactions involving C4-
C9 (Ph)···C13-C18 (Bzl) and C4-C9 (Ph)··· C20-C25 (Ph)
aromatic rings are also present, being the the distance between
the centroids around 4.5 Å.
34]
Organic environment
Dissolution of the peptide in a single OS resulted in turbid
solutions with white particles (flocs) in suspension, the
combination of two OSs being necessary to obtain clear
solutions and the subsequent self-assembly processes. Different
levels of organization were obtained depending on both the
polarity of the mixture. Results are summarized in Figure 5.
Figure 4. (a) Scheme of the TFA·FF-OBzl molecule with displacement
ellipsoids drawn at the 50% probability level. H atoms are drawn as circles
with arbitrary radii. (b) View along the c axis showing the packing of two
molecules related by a binary screw axis. Dashed circle points out the
aromatic rings that are disposed around the inner hydrophilic part. H atoms
have been omitted for clarity. (c) Scheme showing the intermolecular
hydrogen bonds established between neighboring chains along the a axis.
Figure 5. Representative SEM and AFM (height) images of the structures
obtained at room temperature from TFA·FF-OBzl dissolved in: a) 4.0 mg/mL
3
DMF:MeOH; (b) 4.0 mg/mL HFIP:MeOH; and (c) 4.0 mg/mL HFIP:CHCl .
Fiber-like organizations evolve towards plate-like organizations with
decreasing polarity of the medium. The dielectric constant of DMF, HFIP,
Peptide···peptide intermolecular electrostatic, hydrogen bonds
and - stacking interactions are known to be an essential
contributor
microarchitectures.
to
the
33]
formation
of
FF
nano-
and
3
MeOH and CHCl is = 37.2, 16.7, 32.6 and 4.7, respectively.
[
According to its crystal structure, the
importance of these intermolecular forces is maintained in
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ChemPhysChem
10.1002/cphc.201700180
ARTICLE
Concentrated peptide solutions ( 4 mg/mL) in polar organic
mixtures, as DMF:MeOH, result in well-defined microfibers (
ranging from 2.5 to 5.5 m) with a smooth surface (Figure 5a),
TFA·FF-OBzl. Thus, the latter exhibits a hydrophobic crown
surrounding a hydrophilic core that contains all the polar groups,
whereas such separation is much less pronounced in Fmoc-FF
because of the steric hindrance induced by the bulky Fmoc
groups.[18] The clear separation between polar groups and
hydrophobic rings in TFA·FF-OBzl facilitates the understanding
of the influence of the solvent on the morphology of self-
assembled aggregates. Thus, the crystal structure of TFA·FF-
OBzl has been considered as representative for understanding
the behavior of amphiphilic FF-based biomaterials in different
environments.
which tend to adopt
a
preferential alignment, crossed
perpendicularly by fibers of smaller diameter ( 0.7-0.9 m).
Microfibers transform into microplates when the polarity of the
environment decreases ( 4 mg/mL HFIP:MeOH), whereas
crossed submicro-fibers change the perpendicular orientation by
a tilted one (45º). Thus, the most remarkable feature caused by
the reduction of the polarity is the branched-like supramolecular
organization of the peptide (Figure 5b). This feature becomes
more pronounced in the least polar environment (4.0 mg/mL
Peptide nano- and microfibers obtained in OS:water
environments with low and very low peptide concentrations (i.e.
mixtures in which the high dielectric constant of water plays a
dominant role because of its high volume ratio with respect to
the OS) has been attributed to an assembly in which
hydrophobic and polar forces are equally important to promote
the longitudinal growing of the fiber, whereas the interactions
between the fiber surface and both the environment and the
glass support are stabilized by polar forces. Accordingly, the
length of the fiber increases because of the favorable
hydrophobic-hydrophobic and polar-polar interactions between
regions of identical nature, while polar groups remain exposed at
the surface of the fibers (Figure 6a). Moreover, this simple
model explains that, for a given OS, the density of aggregated
fibers decreases when the volume ratio of water as co-solvent
increases (i.e. the polarity of the mixture increases). Thus, the
favorable interactions between the peptide polar groups and
water molecules compensate the affinity between the surface
polar groups of different fibers when the concentration of water
in the solvents mixture is high enough.
3
HFIP:CHCl ), where spherulitic-like structures made of
microplates are frequently identified (Figure 5c).
It should be mentioned that structures obtained using low
peptide concentrations were disordered (i.e. without well-defined
morphology) and poorly reproducible in all cases. As an
hypothesis, this has been attributed to the fact that the
organization of the amphiphilic molecules forming hydrophobic
crowns is difficult in organic solvents, which evaporate faster
than water. Thus, a high amount of peptide molecules are
presumably required to form regular structures with well-defined
morphologies, like those displayed in Figure 5.
Overall, results in OSs indicate that the polarity of the
environment regulates the 3D arrangement of the sheets formed
by TFA·FF-OBzl molecules, controlling the formation of tubes or
plates. On the other hand, comparison of supramolecular
organizations observed in aqueous and OSs reflect two general
trends that are characteristics of the latter environment. Firstly,
the concentration of peptide required to obtain well-defined self-
assembly processes in organic environments is significantly
higher ( 4 mg/mL) than in aqueous solvents. Thus, in organic
environments peptide-solvent interactions are energetically
favored with respect to peptide-peptide interactions. This
suggests that, in water-containing solutions (i.e. those with
higher polarity), attractive interactions between polar groups and
water are far from compensating the repulsive interactions
between such solvent and the aromatic groups of the peptide.
Secondly, the density of supramolecular structures is
considerably higher in water-containing environments than in
OSs, which corroborates our previous hypothesis. Thus, the
interaction of the peptide with the organic solvent is less
repulsive than with water, hindering the self-assembly process in
the former environment.
Influence of the surrounding environment in the assembly
mechanism
The single crystal structure of TFA·FF-Bzl, which was obtained
by slow evaporation from a pure water solution, evidenced the
construction of a network of hydrogen bonds formed by parallel
strands, which also interact through intra- and inter-molecular
stacking interactions (Figure 4). Although the parallel disposition
of the molecules is in agreement with that observed by Adams
and co-workers for single crystals of Fmoc-FF collected from
gels formed in acetone,[18] important differences are detected
between such two structures. These correspond to the drastic
separation between hydrophilic and hydrophobic groups in
Figure 6. Schemes explaining the self-assembly and aggregation of TFA·FF-
OBzl in different conditions considering the hydrophilic (blue) and hydrophobic
(red) regions observed in the corresponding X-ray structure (Figure 4): (a)
fibers; (b) branched fibers; (c) plates; and (d) spherulites.
The enhancement of the ionic strength in OS:water peptide
solutions, which is achieved by replacing the milli-Q water by a
50 mM KCl aqueous solutions as added co-solvent, results in
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ChemPhysChem
10.1002/cphc.201700180
ARTICLE
the formation of microstructures with branched-like architectures
coexisting with continuous (non-branched) fibers. Both the
observation of cubic KCl crystals and the reduction of
continuous fibers with increasing ionic strength are consistent
with the mechanism like that displayed in Figure 6b. According
to this, the formation of salt nanocrystals coating the surface of
peptide fibers nucleates the branches that grow through
favorable KCl···TFA·FF-OBzl electrostatic interactions. The
frequency of this branching process increases with the ionic
strength of the mixture, which explains the fact that the definition
of the branched-like architectures and the presence of non-
branched fibers decrease with increasing volume ratio of KCl
aqueous solution in the OS:co-solvent mixture.
50mM-KCl(aq) or an OS) to a concentrated peptide solution in
HFIP, DMF, DMSO or water. Although polar aqueous
environments tend to promote the growth of fibers, which coexist
with branched-like microstructures when milli-Q water is
replaced by 50mM-KCl(aq), non-polar environments obtained by
mixing two OSs prefer peptide assemblies organized in plates
and spherulites.
X-ray diffractograms collected for TFA·FF-OBzl single crystals
reveal a segregated distribution of hydrophilic and hydrophobic
regions. More specifically, the carboxylate (from TFA), amide
and ester (both from FF-OBzl) groups from a highly polar core
stabilized through hydrogen bonding interactions, which is
ringed by a hydrophobic crown involving 10 aromatic rings. This
unique organization has enabled us to explain the influence of
the solvents mixture properties on the peptide assembly. Thus,
the growing of the peptide structure and the exposition of
hydrophilic or hydrophobic regions is simply determined by the
formation of favorable peptide-solvent interactions at the surface.
Tuning the structure of TFA·FF-OBzl by changing the solvents
used in the mixture is a very attractive feature to expand the
potential utility of peptide assemblies in different fields, for
example as molecular carriers and delivery systems. Thus, both
polar and non-polar compounds could be easily loaded on
TFA·FF-OBzl microstructures by regulating the assembly
through the solvents used in mixture.
Microstructures derived from the combination of two OSs also
depend on the polarity of the mixture. Polar environments with
high peptide concentrations result in the formation of microfibers
by
a mechanism similar to that displayed in Figure 6a,
transforming into microplates when the polarity of the
environment decreases. In order to reduce the access of the
hydrophilic core to the surface, a mechanism like that depicted
in Figure 6c is proposed. In this mechanism, the 2D growing of
the microstructure and the predominant hydrophobic region at
the boundary favor the formation of peptide–solvent interactions.
Moreover, as the polarity of the environment is not drastically
low, the apparition of irregularities and defects with the
hydrophilic core exposed to the solvent does not represent a
severe thermodynamic penalty for the microstructure generation.
At high peptide concentrations, the affinity between the
hydrophobic surface regions provokes the aggregation of plates,
as is displayed in Figure 6d, giving place to spherulites.
Overall, results displayed in this work combined with the
straightforward models schematized in Figure 6 provide a simple
rational that explains the assembly behavior of the TFA·FF-OBzl
amphiphile. Accordingly, the morphology of microstructures
derived from this peptide can be easily regulated by controlling
both the solvent and the peptide concentration. This versatility
makes TFA·FF-OBzl a very interesting system for applications
that are mainly based on interactions with other chemical
species, as for example drugs. Within this context, TFA·FF-OBzl
microstructures are potential candidates to upload either polar or
non-polar drugs at their surface and, therefore, be used as
versatile carriers and/or delivery systems. Thus, although some
Experimental Section
Peptide synthesis and characterization. Melting points were
determined on a Gallenkamp apparatus and are uncorrected. IR spectra
were registered on a Nicolet Avatar 360 FTIR spectrophotometer; max is
1
13
given for the main absorption bands. H and C NMR spectra were
recorded on Bruker AV-400 or ARX-300 instrument at room
a
temperature, using the residual solvent signal as the internal standard.
Chemical shifts (δ) are expressed in ppm and coupling constants (J) in
Hertz. Optical rotations were measured on a JASCO P-1020 polarimeter.
High-resolution mass spectra were obtained on a Bruker Microtof-Q
spectrometer.
Boc-FF-OBzl (3). To a solution of Boc-F-OH (1) (1.75 g, 6.6 mmol) in
dichloromethane (15 mL) cooled to 0 ºC in an ice bath, was added 1-
hydroxybenzotriazole hydrate (HOBt) (1.01 g, 6.6 mmol) followed by N-
[
3-(dimethylamino)-propyl]-N’-ethylcarbodiimide hydrochloride (EDC·HCl)
peptide amphiphiles have been previously suggested for
delivery,[
35,36]
(1.27 g, 6.6 mmol) and the reaction was stirred for 15 min. Then, a
solution of the amino component H-F-OR (2) (6.0 mmol) [obtained by
addition of N,N-diisopropylethylamine (DIPEA) (1.25 mL, 7.2 mmol) to
the TFA salt of 2] in dichloromethane (5 mL) and additional N,N-
diisopropylethylamine DIPEA (1.15 mL, 6.6 mmol) were added. The
reaction was stirred for 1 h at 0 ºC, then at room temperature for 24 h.
their utility is typically restricted to the loading of
polar or non-polar drugs. However, the adaptability of TFA·FF-
OBzl eliminates the restrictions related with the chemical nature
of the used drugs, giving a new dimension to this application.
The reaction mixture was washed with 5% aqueous solution of NaHCO
3
(
3  15 mL), followed by 5% aqueous solution of KHSO (3  15 mL). The
4
Conclusions
organic phase was dried over anhydrous magnesium sulfate and
evaporated to dryness. The resulting solid was suspended in a diethyl
ether/n-hexane mixture and filtered at reduced pressure to provide 3 as a
white solid.
We have evidenced the remarkable control exerted by the
characteristics of the solvents mixture on the organization of
TFA·FF-OBzl assemblies derived from the addition of a co-
solvent to peptide solution. Thus, the polarity, ionic strength and
peptide concentration in the mixture have been regulated by
adding a selected amount of a given co-solvent (i.e. pure water,
25
D
Yield: 90%. Mp: 180-181 ºC. [α] : -17.7 (c = 0.33, methanol). IR (KBr) :
3332, 1741, 1696, 1681 cm–1. 1H NMR (CDCl
, 400 MHz):  1.32 (s, 9H),
2.923.02 (m, 4H), 4.214.30 (m, 1H), 4.724.76 (m, 1H), 4.86 (bs, 1H),
3
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ChemPhysChem
10.1002/cphc.201700180
ARTICLE
5
5

.02 (s, 2H), 6.20 (d, 1H, J = 7.6 Hz), 6.816.83 (m 2H), 7.077.14 (m,
H), 7.167.21 (m, 5H), 7.267.32 (m, 3H). C NMR (CDCl , 100 MHz):
3
28.36, 38.07, 38.43, 53.44, 55.82, 67.35, 80.32, 127.11, 127.19, 128.65,
at 80 ºC of a 0.415 mg/mL solution of TFA·FF-OBzl in MQ-grade water
and used for X-ray diffraction analysis. The X-ray intensity data were
13
measured on
a D8 Venture system equipped with a multilayer
1
1
5
28.67, 128.70, 128.74, 128.79, 129.41, 129.49, 135.14, 135.61, 136.62,
monochromator and a Cu microfocus (λ = 1.54178 Å).
+
55.39, 170.85, 170.90. HRMS (ESI) C30
H
34
N
2
NaO
5
[M+Na] : calcd.
25.2360, found 525.2375.
The frames were integrated with the Bruker SAINT software package
using a narrow-frame algorithm. The integration of the data using an
orthorhombic unit cell yielded a total of 13671 reflections to a maximum θ
angle of 79.25° (0.78 Å resolution), of which 5037 were independent
For Boc deprotection, a solution of the corresponding Boc-protected
compound in dichloromethane was treated with trifluoroacetic acid (TFA-
H; 15 equiv.) and the reaction mixture was stirred at room temperature
for 1 h. After evaporation of the solvent, the residue was suspended in a
diethyl ether/n-hexane mixture and filtered at reduced pressure to
provide the corresponding TFA salt as a white solid in quantitative yield.
(average redundancy 2.714, completeness = 95.0%, Rint = 4.97%, Rsig
=
2
5.67%) and 4499 (89.32%) were greater than 2σ(F ). The final cell
constants of a = 5.8856(3) Å, b = 18.5677(9) Å, c = 23.0370(11) Å,
3
volume = 2517.5(2) Å , are based upon the refinement of the XYZ-
centroids of reflections above 2θ σ(I). Data were corrected for absorption
effects using the multi-scan method (SADABS). The calculated minimum
and maximum transmission coefficients (based on crystal size) are
TFA·FF-OBzl (4). According to the general Boc deprotection procedure,
TFA (2 mL) was added to a solution of 3 (2.0 mmol) in dichloromethane
0.6156 and 0.7461.
(20 mL) to provide the corresponding TFA salt
4 (Figure S6) in
quantitative yield.
The structure was solved and refined using the Bruker SHELXTL
Software Package, using the space group P2 , with Z = 4 for the
. The final anisotropic full-matrix least-squares
refinement on F with 335 variables converged at R1 = 4.26%, for the
observed data and wR2 = 14.04% for all data. The goodness-of-fit was
1.032. The largest peak in the final difference electron density synthesis
2
0
1 1 1
2 2
Mp: 290-292 ºC dec. [α] : 18.2 (c = 0.36, acetic acid). IR (KBr) : 3342,
D
725, 1695, 1662 cm–1. 1H NMR (DMSO, 400 MHz):  2.90 (dd, 1H, J =
4.2 Hz, J = 8.3 Hz), 3.02 (dd, 1H, J = 13.9 Hz, J = 8.1 Hz), 3.083.13 (m,
H), 4.054.13 (m, 1H), 4.634.69 (m, 1H), 5.065.14 (m, 2H), 7.227.38
m, 15H), 8.23 (bs, 2H), 9.15 (d, 1H, J = 7.5 Hz). C NMR (DMSO, 100
MHz):  36.79, 36.99, 53.21, 54.03, 66.37, 111.85, 114.77, 117.68,
formula unit, C27
H
F N O
27 3 2 5
1
1
2
2
13
(
-
3
-
3
was 0.289 e /Å and the largest hole was -0.257 e /Å with an RMS
-
3
deviation of 0.059 e /Å . On the basis of the final model, the calculated
density was 1.363 g/cm and F(000), 1080 e .
1
20.60, 126.80, 127.19, 128.09, 128.20, 128.45, 128.54, 129.18, 129.59,
34.79, 135.60, 136.65, 158.05, 158.40, 158.75, 159.10, 168.43, 170.71.
3
-
1
+
HRMS (ESI) C25
H N
26 2
NaO
3
[M+Na] : calcd. 425.1836, found 425.1821.
Samples preparation. Peptide containing solutions (25 or 100 L) were
prepared from 5 mg/mL stocks using HFIP, DMF, DMSO or milli-Q water
as solvents. The peptide concentration was reduced by adding milli-Q
Acknowledgements
Authors thank supports from MINECO and FEDER (MAT2015-
3
water, MeOH or CHCl , as co-solvent, to a given stock solution. More
69367-R, MAT2015-69547-R and CTQ2013-40855-R) and
specifically, peptide concentrations of 4.8, 4.0, 2.0, 1.0, 0.3, 0.25 and 0.1
mg/mL were obtained using 24:1, 4:1, 4:6, 1:4, 3:47, 1:19 and 1:49
solvent:co-solvent ratios, respectively. On the other hand, the 50mM-
KCl(aq) solution was used as co-solvent to modify the ionic strength.
Finally, 10 or 20 L aliquots were placed on microscope coverslips and
kept at room temperature (25 ºC) or inside a cold chamber (4 ºC) until
dryness. All organic solvents were purchased from Sigma-Aldrich, Fisher
Scientific and Scharlab.
Gobierno de Aragón - Fondo Social Europeo (research group
E40). Support for the research of C.A. was received through the
prize “ICREA Academia” for excellence in research funded by
the Generalitat de Catalunya.
Keywords: Fibers; Hydrophylic core; Nanoplates; Peptide
materials; Stacking interactions
Optical microscopy. Morphological observations were performed using
a Zeiss Axioskop 40 microscope. Micrographs were taken with a Zeiss
AxiosCam MRC5 digital camera.
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Entry for the Table of Contents (Please choose one layout)
ARTICLE
Enric Mayans, Gema Ballano, Javier
Sendros, Merçè Font-Bardia, J. Lourdes
Campos, Jordi Puiggalí,* Carlos
Cativiela,* and Carlos Alemán*
Page No. – Page No.
Effect of the solvent choice in the self-
assembly properties in a
diphenylalanine amphiphile stabilized
by an ion pair
The assembly of a diphenylalanine amphiphile stabilized by a trifluoroacetate ion is
regulated through the properties of solvents mixtures.
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