Tetrapeptidic molecular hydrogels: Self-assembly and Co-aggregation with amyloid fragment Aβ1-40

Article abstract of DOI:10.1002/chem.201302651

A new family of isomeric tetrapeptides containing aromatic and polar amino acid residues that are able to form molecular hydrogels following a smooth change in pH is described. The hydrogels have been studied by spectroscopic and microscopic techniques showing that the peptide primary sequence has an enormous influence on the self-assembly process. In particular, the formation of extended hydrophobic regions and the appearance of π-stacking interactions have been revealed as the driving forces for aggregation. Moreover, the interaction of these compounds with amyloid peptidic fragment Aβ1-40 has been studied and some of them have been shown to act as templates for the aggregation of this peptide into non-β-sheet fibrillar structures. These compounds could potentially be used for the capture of toxic, soluble amyloid oligomers. Copyright

Full text of DOI:10.1002/chem.201302651

DOI: 10.1002/chem.201302651
Full Paper
&
Gelation
Tetrapeptidic Molecular Hydrogels: Self-assembly and
Co-aggregation with Amyloid Fragment Ab1-40
Marta Tena-Solsona, Juan F. Miravet,* and Beatriu Escuder*[a]
Abstract: A new family of isomeric tetrapeptides containing
aromatic and polar amino acid residues that are able to
form molecular hydrogels following a smooth change in pH
is described. The hydrogels have been studied by spectro-
scopic and microscopic techniques showing that the peptide
primary sequence has an enormous influence on the self-as-
sembly process. In particular, the formation of extended hy-
drophobic regions and the appearance of p-stacking interac-
tions have been revealed as the driving forces for aggrega-
tion. Moreover, the interaction of these compounds with
amyloid peptidic fragment Ab1-40 has been studied and
some of them have been shown to act as templates for the
aggregation of this peptide into non-b-sheet fibrillar struc-
tures. These compounds could potentially be used for the
capture of toxic, soluble amyloid oligomers.
Introduction
The self-assembly of peptides into fibrillar aggregates is a cur-
rent hot topic in materials science as well as in biomedicine.[1]
In particular, the aggregation and fibrillization of peptides is at
the core of fatal amyloidogenic diseases such as Alzheimer’s
disease.[2] Important studies on the self-assembly of model
peptides have been carried out to gain an understanding of
the structures of their aggregates as well as to design inhibi-
tors for such an unwanted process. For example, the toxic pep-
tides Ab1-40 and Ab1-42 have been extensively studied and
the relationship between the mechanisms of aggregation and
disease has been a matter of intense discussion.[3] Furthermore,
shorter peptidic fragments found within these sequences have
been studied to assess the role of each residue in the self-as-
sembly and fibrillization.[4] This information has been used in
the design of inhibitors either peptidic[5] or non-peptidic.[6]
In addition, it is well known that the hierarchical organiza-
tion of peptidic fibrils into long fibers and physically cross-
linked fibrillar networks leads to the formation of hydrogels.
Hydrogel formation has been widely studied for proteins and
peptides,[1a,7] and more recently for low-molecular-weight
amino acid based analogues (e.g., di- to pentapeptides, pep-
tide amphiphiles, peptidomimetics).[1b,8] Some of these systems
have shown parallelisms with amyloid b-peptide aggregation
and may serve as simple models for the development of ag-
gregation inhibition strategies.[9]
Scheme 1.
Herein we report on a family of six tetrapeptides (1–6;
Scheme 1) that combine nonpolar aromatic phenylalanine (F)
and polar aspartic acid (D) residues and are able to form hy-
drogels that we have designed foreseeing the parallelism be-
tween the hydrogelation of small molecules and the fibrilliza-
tion of amyloidogenic peptides. Through this sequence we
aimed on one hand to study the role of the hydrophobic/hy-
drophilic balance and disposition of the residues in the fibrilli-
zation and on the other hand to evaluate the potential of
these compounds for the interaction with amyloid Ab1-40 by
screening positively charged Lys residues that seem to play an
important role in amyloid misfolding.
Results and Discussion
[a] M. Tena-Solsona, Dr. J. F. Miravet, Dr. B. Escuder
Departament de Quꢀmica Inorgꢁnica i Orgꢁnica
Universitat Jaume I, 12071 Castellꢂ (Spain)
Fax: (+34)964728214
Compounds 1–6 were prepared by solution peptide synthesis
and contain two phenylalanine and two aspartic acid residues
in different positions (see the Supporting Information for de-
tails). The chain termini in these compounds were blocked to
avoid additional ionization. The benzyloxycarbonyl (Z) protect-
ing group was used at the N terminus as an additional aromat-
E-mail: miravet@uji.es
escuder@uji.es
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201302651.
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ic fragment that may assist the aggregation through p-stack-
ing interactions.[10]
Aggregation studies
Compounds 1–6 are highly insoluble in water and either it is
not possible to dissolve them by heating at concentrations in
the millimolar range or they precipitate after cooling the hot
solutions. However, hydrogels could be prepared after dissolu-
tion in basic media by deprotonation of the D residues and
subsequent acidification with HCl (aqueous solution or vapors).
In this way, white opaque hydrogels could be prepared. How-
ever, it was found that the high local concentration of HCl
upon acidification of the samples in many cases led to hetero-
geneous aggregation and the formation of large particles lead-
ing to poor reproducibility.
To obtain homogeneous transparent hydrogels, solutions of
d-gluconolactone (GL) were used as pH-tuning media. As re-
ported by Adams et al., the hydrolysis of GL into gluconic acid
in basic aqueous solution may be used to smoothly lower the
pH and produce a homogeneous change in pH in the entire
sample.[11] In a typical experiment, the hydrogelator was dis-
solved in basic media (Na2CO3) and a weighed amount of GL
was added to this solution. The hydrolysis of GL starts immedi-
ately, lowering the pH with the concomitant protonation of
the aspartate residues of the tetrapeptides and the formation
of hydrogels with different macroscopic aspects, as shown in
Figures 1–3 (see the Supporting Information for details). The
final pH of the samples was in the range of 5.5–6.5 in all cases,
which indicates an aggregation-induced pKa shift of the car-
boxylic acids, which are expected to have pKa values of about
4.5.[12]
Figure 1. TEM (left) and SEM (right) micrographs of xerogels of compounds
1–3 (top to bottom) prepared from hydrogels at their mgc. Insets right:
Macroscopic aspects of the gels.
this case the microscopic network appears less crowded than
in previous compounds. As can be seen in Figure 3, TEM re-
vealed the presence of fibers of several micrometers in length
and around 10 nm in width. These fibers co-exist in two differ-
ent shapes, some as loosely curled right-handed helices rang-
ing between 200 and 600 nm in pitch and others with a left-
handed helical shape with a much shorter pitch of 50 nm. We
believe that the latter are formed by the assembly of two fi-
brils of the former type.
The minimum gel concentrations (mgc) observed by using
this methodology are collected in Table 1. The best hydrogela-
Table 1. Hydrogelation behavior and minimum gel concentrations (mgc)
for compounds 1–6 at 258C.
Compounds 2 and 4 present slightly higher mgc values (4.5
and 4 mm, respectively) and form translucent hydrogels (see
Figures 1 and 2). TEM revealed that the hydrogel of 2 is com-
posed of a mixture of thin tapes of around 5–10 nm in width
and wider ones of around 40–50 nm. Larger tapes of more
than 100 nm in width were also observed by SEM. In this case,
the heterogeneity in sizes provokes the scattering of light and
a translucent macroscopic aspect. On the other hand, the hy-
drogels formed by compound 4 consist of a network of thin fi-
brils of about 8 nm in width that co-exist with larger left-
handed helical fibers. These nanohelices are formed after the
assembly of several fibrils (see Figure 2, inset) and, although
they show different pitches, the pitch-to-width ratio is constant
and equal to 5. In this case, self-assembly follows a hierarchical
pathway in which the chiral molecular information is not clear-
ly transferred to the small fibrils but emerges after the associa-
tion of several of them into higher-order helical nanofibers. Fi-
nally, compound 5 forms opaque hydrogels with an mgc of
7.5 mm. At the microscopic level, these hydrogels are formed
by a mixture of fibrils of around 10 nm in width and large
Compound
Hydrogel[a]
mgc [mm]
1
2
3
4
5
6
TpG
TlG
TpG
TlG
OG
TpG
1.5
4.5
2.5
4.0
7.5
3.0
[a] Key: TpG, transparent gel; TlG, translucent gel; OG, opaque gel.
tor is compound 1 (1.5 mm) followed by 3 (2.5 mm), both
forming transparent gels. TEM revealed that these hydrogels
are formed by networks of thin fibrils with widths of about 5–
8 nm and lengths in the range of micrometers (Figure 1). The
networks are highly uniform and the fibers are physically
cross-linked with few defects and branching. A regular mesh
size is clearly observed in both cases by SEM. Compound 6
also forms transparent hydrogels with an mgc of 3 mm, but in
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Structural studies
The supramolecular structures of
the aggregates were studied by
FTIR and CD spectroscopy. The
hydrogels were filtered and
washed several times with water
to remove nonaggregated com-
pounds (GL and salts), and IR
spectra were recorded after
drying (see the Supporting Infor-
mation). All the compounds
present an intense amide I
stretching band in the range
1634–1641 cmÀ1 assignable to
a b-sheet structure as the major
feature of these aggregates.[13]
However, this band is at the
upper limit for this kind of sec-
ondary structure, which suggests
that the sheets may be twisted
or distorted from the ideal
planar arrangement. In addition,
less intense broad shoulders are
observed at higher wavelengths.
These bands, which should cor-
respond to vibrations related to
Figure 2. TEM (top) and SEM (bottom) micrographs of hydrogels of compound 4 prepared at its mgc. Inset right
bottom: Macroscopic aspect of the gel.
carboxylic
acids
(1700–
1730 cmÀ1) and to minor secon-
dary structure components of
amide groups (random coil or
turns), were difficult to assign di-
rectly and were analyzed by
using a curve-fitting procedure
(JASCO FTIR Curve Fitting soft-
ware, see the Supporting Infor-
mation for details). The position
of the carboxylic acid vibration
band is quite sensitive to the
strength of the hydrogen bonds
involving this functional group.
For example, non-hydrogen-
bonded carboxylic acid mono-
mers typically present a band at
around 1730 cmÀ1, whereas this
band is shifted to lower wave-
numbers in hydrogen-bonded
dimers (ca. 1720 cmÀ1 for acyclic
dimers, ca. 1700 cmÀ1 for cyclic
dimers).[14] In the current case,
two carboxylic groups are pres-
ent in each molecule and may
be located in environments with
different hydrogen-bonding fea-
Figure 3. TEM (left) and SEM (right) micrographs of hydrogels of compounds 5 (top) and 6 (down) prepared at
their mgc. Insets right: Macroscopic aspects of the gels.
tapes of a few hundreds of nm in width. Closer inspection re-
vealed that these tapes are formed by the merging of tens of
fibrils in the direction of the tape.
tures depending on the sequence. Moreover, the formation of
hydrophobic pockets may also have an influence on the
strength of the hydrogen bonds.[15] Compound 1 with the al-
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ternating sequence Z-FDFD shows a single broad band cen-
tered at 1701 cmÀ1, which suggests that both D residues are in
a strongly hydrogen-bonding environment. In contrast, com-
pound 2 with the reverse alternating sequence Z-DFDF shows
two bands of similar strength centered at 1733 and 1709 cmÀ1
,
which suggests two different carboxylic acid components, one
in a non-hydrogen-bonded environment leading to the band
with the higher wavenumber and the other in a medium-
strength hydrogen-bonding region. Compound 3 with the
block sequence Z-FFDD shows a single broad band centered
at 1724 cmÀ1, which indicates that both D residues are in-
volved in weak hydrogen bonds. The strength of the hydrogen
bonding is increased for compound 4 with the reverse block
sequence Z-DDFF; two different bands are observed, a strong
one at 1722 cmÀ1 and a weaker one at 1704 cmÀ1. For com-
pound 5 with the sequence Z-FDDF, two bands are observed
at 1712 and 1703 cmÀ1, which again suggests a strongly hydro-
gen-bonded environment for both D residues. Finally, for com-
pound 6 with the sequence Z-DFFD, the major band appears
at 1722 cmÀ1, which suggests a poor hydrogen-bonding envi-
ronment. All the compounds present a weak band between
1684 and 1693 cmÀ1, which is usually indicative of an antiparal-
lel b-sheet secondary structure although there is some contro-
versy about this assignment.[13]
CD spectra of compounds 1–6 were recorded in solution
(0.75 mm) and as hydrogels at their respective mgc.[16] As can
be seen in Figure 4A, in solution these compounds do not
show a defined secondary structure by circular dichroism. In
principle, random coil structures are suggested, however, the
presence of several aromatic chromophores in the molecule
could lead to a mixing of excitons and alteration of the spec-
tral shape. Remarkably, aggregation into hydrogels produced
noticeable changes in the CD spectra for some of the com-
pounds (Figure 4B), most significantly for compounds 1 and 4,
which show an intense negative band centered at 228 nm re-
vealing an important conformational change on going from
solution to the gel phase and a reorganization of the chromo-
phores completely different to the rest of the compounds.
These two compounds, with no clear similarities in their pri-
mary sequence (1: FDFD; 4: DDFF), self-assemble into highly
CD-active supramolecular structures. The rest of the hydrogels
are less CD active. Compound 5 presents a broad negative
band of lower intensity between 230 and 250 nm related to
the aromatic side-groups, which would be involved in the con-
formational change on going from solution to the hydrogel. In
the case of compound 3, the spectrum shows a negative band
at 222 nm and a positive lobe at 211 nm, which can be as-
signed to a typical b-sheet structure. Compounds 2 and 6 are
much less active, probably due to the cancellation of excitons,
and present small negative bands at 235 and 230 nm, respec-
tively.
Figure 4. Mean-residue molar ellipticity of compounds 1–6 in solution
(0.75 mm, A) and as hydrogels at their respective mgc (B).
1–6 in solution (0.75 mm) as well as in the gel state (see the
Supporting Information). In the former case, we aimed to
study the effect of the sequence on the fluorescence of the
phenylalanine residues. As can be seen in Table 2, all the com-
pounds except 5 show an emission peak at 284–285 nm,
which indicates isolated aromatic fragments. However, com-
pound 5 presents a peak at 288 nm and this redshift of 4 nm
can be ascribed to an intramolecular interaction between the
aromatic fragments. This effect requires the folding of the mol-
Table 2. Fluorescence studies of solutions (0.75 mm) and hydrogels (mgc)
of compounds 1–6.[a]
Compound lem at
lem at mgc Aggregation Additional peak at
0.75 mm [nm] [nm]
induced
redshift
[nm]
460 nm
1
2
3
4
5
6
285
284
284
285
288
284
293
296
295
297
302
295
8
12
11
12
14
11
no
yes
weak
yes
yes
Fluorescence spectroscopy was used to investigate the envi-
ronment of the phenylalanine residues. It has been reported
that the involvement of the aromatic fragment in p-stacking
interactions can be monitored by the shift of the emission
peak at 284 nm characteristic of this amino acid residue.[17] In
the current case we studied the fluorescence of compounds
no
[a] lex =260 nm.
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ecule and should be related to the presence of the DD central
block.
Fluorescence studies were performed also on the hydrogels
at their respective mgc. As can be seen, a redshift of 8 to
14 nm from the solution phase emission band is observed in
all cases, which suggests the presence of strong p-stacking in-
teractions upon aggregation, as reported for related FF con-
taining compounds.[17,18] In addition, for hydrogels 2, 4, and 5,
an additional broad weak band appears at around 460 nm.
Some authors have suggested that this peak corresponds to
the disposition of the aromatic residues in J aggregates.[8,18]
This band is not present in the spectra of compounds 1, 3, and
6 (the more efficient gelators: lowest mgc values and transpar-
ent). On the other hand, the largest redshift (14 nm) corre-
sponds to the least effective gelator 5, which suggests
a strong involvement of aromatic residues in the structural re-
arrangement from solution to the gel state.
Figure 5. Minimized assembly models for compounds 1 (A) and 2 (B). Top
side aromatic (Z, F) and D residues are highlighted in blue and red, respec-
tively. Nonpolar hydrogen atoms have been omitted for clarity.
Wide-angle X-ray diffraction (WAXD) analysis was performed
on the xerogels of compounds 1–6 (see the Supporting Infor-
mation). In general, transparent hydrogels formed from thin fi-
brils are poorly crystalline, showing only a broad background
signal in the cases of 1 and 6, and two broad and weak peaks
at low angles for compound 3. On the other hand, the xerogel
of compound 4 presents a low-angle diffraction peak correlat-
ing to a distance of 32 ꢁ, which may correspond to the size of
an extended molecule, and a second peak correlating to
22.5 ꢁ. Finally, compounds 2 and 5, which are formed from
tapes of different widths, exhibit lamellar packing. In particular,
compound 5, which is the least effective hydrogelator (high
mgc, opaqueness), forms the most crystalline xerogel. It can be
envisaged that crystalline rigid tapes having a low aspect ratio
are less prone to physically cross-linking and a larger amount
of material is required to form the sample-spanning network.
Taking together all the structural information, tentative
models for the assemblies consistent with the experimental
data were constructed by using Macromodel 9.9 (AMBER*
force field and a generalized Born surface area (GB/SA) solvent
simulation for water; see Supporting Information for refer-
ence). After a conformational search, low-energy mo-
lecular structures were used with slight modifications
(i.e., extension of the folded molecule) to construct
the assemblies by using an antiparallel arrangement
of the molecules and then these assemblies were
minimized. The resulting distribution of aromatic resi-
dues in the assemblies was analyzed in detail as it is
known that p–p and hydrophobic interactions are
the driving forces for self-assembly into higher-order
aggregates. For example, compounds 1 and 2, which
possess alternating FD sequences, show alternation
of polar and nonpolar strands on each face of the
modeled sheet (Figure 5). However, the models for
compound 1 present the aromatic Z group close to
an F residue, whereas the aggregates of compound 2
show a more regular distribution of aromatic groups.
These structural differences would have an effect on
into lamellar aggregates, as suggested by WAXD and the pres-
ence of the fluorescence band at 460 nm. Compounds 3 and
4, constructed from blocks of FF and DD, show a scrambling of
aromatic residues on both sides of the sheet and the D resi-
dues appear to be buried among the aromatic residues, there-
by being poorly accessible for hydrogen bonding, in accord-
ance with the high wavenumber of their carboxylic IR bands
(Figure 6). In the case of 3, the presence of a ZFF block may
also influence the intersheet assembly, whereas compound 4
shows slightly higher crystallinity of the aggregates as well as
the additional fluorescence band at 460 nm.
The models for 5, which bears a central DD block, indicate
that this compound folds in solution into an amphipathic
structure that clusters the aromatic groups on one side of the
molecule minimizing their exposure to water, in agreement
with the results found in fluorescence studies (band at 288 nm
Figure 6. Minimized assembly models for compounds 3 (A) and 4 (B). Top side aromatic
(Z, F) and D residues are highlighted in blue and red, respectively. Nonpolar hydrogen
their aggregation into fibrils. For example, in the case
of 2, the models point to a more compact packing atoms have been omitted for clarity.
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There is intense debate regarding the aggregation
state of bound ThT when higher concentrations
(above 30 mm) are used relating to the formation of
ThT dimers and other aggregates that produce a de-
crease in fluorescence intensity. In our case, we ob-
served that for compound 1 the fluorescence in-
creased with ThT concentration up to 24 mm and
then a progressive decrease was observed (see the
Supporting Information). As a consequence, we used
a lower concentration (5 mm) to ensure that this
effect does not alter our results. As can be seen in
Figure 8, hydrogels 6, 1, and 3 show a higher affinity
for ThT, followed by 5, with hydrogels 4 and 2 being
the least effective binders of ThT. It can be noted
that compounds 1, 3, and 6, the strongest binders,
present extended aromatic blocks in their aggregates
and a ZF terminal block in the cases of 1 and 3. In
contrast, compounds 2 and 4, which are the poorest
binders, contain an alternating sequence with no aro-
matic blocks. It is also evidenced that compound 5
Figure 7. Minimized assembly models for compounds 5 (A) and 6 (B). Aromatic (Z, F) and
D residues are highlighted in blue and red, respectively. Nonpolar hydrogen atoms have
been omitted for clarity.
and additional broad shoulder at around 400 nm that
is not present for the other compounds, see the Sup-
porting Information). A similar arrangement has been
used to construct a packing model that is consistent
with the lamellar structure of the tapes revealed by
WAXD. As can be seen in Figure 7A, two sheets of
this compound can be assembled into a bilayer with
the aromatic groups closely packed in a hydrophobic
region and the aspartic groups on the outer face
available for hydrogen bonding. This model is in
agreement with the largest shift in the main fluores-
cence band of the aromatic groups (14 nm), the pres-
ence of an additional band at 460 nm, and the low
wavenumbers of the carboxylic IR vibrations ob-
served for this compound. Finally, the models for the
aggregation of compound 6 are similar to those of
compounds 1 and 3, showing segregated blocks of
Figure 8. Fluorescence emission spectra (lex =450 nm) of the hydrogels 1–6 after the ad-
dition of ThT (5 mm, 20 min)
aromatic and acidic groups on both sides of the
sheet leading to low crystallinity (Figure 7B).
Furthermore, the aggregation of compounds 1–6
was tested by the thioflavin T (ThT) binding assay. ThT is a ben-
zothiazole dye that experiences an enhancement of fluores-
cence as a consequence of binding amyloid-like fibrils. This
assay has been extensively used to quantify the degree of fi-
brillar aggregation in amyloidogenic peptides and is based on
the specific enhancement of the emission band at 482 nm
caused by the restriction of the free rotation of the two rings
of ThT upon binding to the amyloid hydrophobic surfaces.[19]
In our studies, we employed this assay to gain information on
the presence of accessible aromatic surfaces in the fibrils. In
a typical experiment the hydrogels were prepared at their
mgc, 20 mL of a stock solution of 0.5 mm ThT in PBS were
added, and their fluorescence spectra were acquired. The final
concentration of ThT was set to 5 mm to avoid uncontrolled ef-
fects observed by some authors at higher concentrations.[18,20]
behaves differently, as commented before. In this case it seems
that the aromatic groups are so closely packed within the ag-
gregates that the access of ThT to the hydrophobic layer could
be quite difficult. These data give us valuable information on
the presence of hydrophobic regions in the aggregates and
are in agreement with the results discussed before: A good
ThT binder should have aromatic blocks and these blocks
should be accessible.
Interaction of compounds 1–6 with peptide Ab1-40
Compounds 1–6 have shown different abilities to aggregate
into hydrogels and bind to ThT, which suggests a clear se-
quence–properties relationship. These compounds possess
two features that have been shown crucial in amyloid misfold-
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ing and aggregation: Aromatic residues involved in the forma-
tion of large hydrophobic regions and carboxylate side-chains
that may participate in salt bridges. It has been reported that
the neurotoxic forms of the Ab peptide are most probably
based on dimeric structures formed by monomers with an an-
tiparallel b-sheet structure that is stabilized by the formation
of salt bridges between lysine and glutamic or aspartic resi-
dues. Further propagation of these dimers into cross-b-sheets
leads to the formation of oligomers as well as insoluble fi-
brils.[3] On the other hand, there
tration in water. It was observed that some of them main-
tained their tendency to form aggregates in PBS at 378C even
at this low concentration. For example, compound 1 had
formed thin fibrils of around 6 nm even after 6 h of incubation
(Figure 9A), compound 3 had formed a mesh of fibrils after
30 h (Figure SI10A), and compound 5 had formed 2D aggre-
gates after 30 h (Figure 10A). On the other hand, compounds
2, 4, and 6 showed a lesser tendency to aggregate at this con-
centration and only a few objects were observed by TEM (see
is still some controversy as to
whether aromatic interactions
between F residues are necessa-
ry for amyloid aggregation or
the presence of generic hydro-
phobic residues is sufficient to
promote aggregation in different
amyloidogenic peptides.[21] For
the compounds studied here,
not only the presence of F resi-
dues but also their disposition in
the primary structure has a clear
influence on their aggregation
properties. These differences
made us envisage a potential se-
quence-dependent effect on the
interactions of these compounds
with amyloid peptides, that is,
Ab1-40. To investigate this possi-
bility, a qualitative study was un-
dertaken by using TEM and CD.
For this study, samples of Ab1-
Figure 9. TEM micrographs of compound 1 after 6 h (A) and equimolar mixture of Ab1-40 and compound 1 after
6 h (B) and 7 days (C). CD spectra of Ab1-40, compound 1, and their equimolar mixture after 7 days (D).
40 (50 mm), compounds 1–6
(50 mm), and equimolar mixtures
of both in phosphate-buffered
saline (PBS) were incubated at
378C for 7 days. The aggregation
of Ab1-40 can be very sensitive to small environmental
changes and polymorphism has been reported.[22] For this
reason all the experiments were performed under strictly the
same conditions (see the Supporting Information). Samples for
TEM were prepared at different times (6 h, 30 h, 3 days, and
7 days) to determine the kinetics of aggregation. In addition,
CD spectra of all samples were recorded after 7 days of stabili-
zation because no major changes in the solutions of Ab1-40
alone were observed in that time (see below) assuming that
the aggregates are then in local/global minima. As can be
seen in Figure SI8 in the Supporting Information, after 6 h of
incubation of Ab1-40, only a few short fibrils were observed by
TEM, the sample being formed mainly of unstructured materi-
al. After 30 h, the number of short fibrils having a width of
around 20 nm had increased. After 3 days, long fibers with
a dendritic aspect were observed and single fibrils were no
longer detected. These fibers formed an entangled network
after 7 days that formed a foamy precipitate.
the Supporting Information). For example, compound 2
showed few isolated fibrils with widths of around 5 nm and
samples of compound 6 were almost empty. In the case of
compound 4, few aggregates were observed initially and iso-
lated rope-like aggregates were observed after 7 days.
Equimolar mixtures of Ab1-40 and compounds 1–6 were
then studied by TEM. It was observed that compounds 2 and
6, which are poorly aggregating, did not have any appreciable
effect on the aspect of amyloid aggregates after 7 days (see
the Supporting Information) and the micrographs are similar
to those of the blank sample of Ab1-40. For the rest of the
compounds, a general increase in the rate of aggregation was
observed and abundant aggregates were observed even after
6 h. Particularly interesting are the cases of compounds 1 and
5. In both cases, with time, the aggregates found in the mix-
tures took the shape of the pure tetrapeptidic compounds,
thin fibrils in the case of 1 (Figure 9) and 2D sheets in the case
of compound 5 (Figure 10). These results point to the co-as-
sembly of both peptides and Ab1-40 ruled by the templating
effect of the tetrapeptide. For mixtures of compounds 3 or 4
On the other hand, compounds 1–6 were also studied at
50 mm, a concentration far below their minimum gel concen-
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Conclusion
We have prepared six isomeric
Z-protected tetrapeptides con-
taining aromatic (F) and polar
(D) residues that are able to
form low-molecular-weight hy-
drogels following
a
smooth
change of pH caused by the in
situ hydrolysis of d-gluconolac-
tone into gluconic acid. We stud-
ied these hydrogels by spectro-
scopic and microscopic tech-
niques and have shown that the
primary sequence of the tetra-
peptides has an enormous influ-
ence on the supramolecular be-
havior of these compounds. In
addition, spectroscopic
and
powder diffraction studies of
these hydrogels revealed a crucial
influence of hydrophobicity and
p-stacking interactions. For the
best hydrogelators, the presence
of segregated blocks of aromatic
Figure 10. TEM micrographs of compound 5 after 30 h (A) and equimolar mixture of Ab1-40 and compound 5
after 30 h (B), 3 days (C), and 7 days (inset to C). CD spectra of Ab1-40, compound 5, and their equimolar mixture
after 7 days (D).
with Ab1-40, neither seems to dominate and no information
could be obtained in this respect (see the Supporting Informa-
tion).
and polar fragments was shown to be a determining factor.
The presence of extended aromatic regions in the aggregates
was also confirmed by the binding of ThT. The binding of
these compounds to amyloid peptide Ab1-40 was also studied
and revealed a remarkable preference for the interaction of
Ab1-40 with compound 1, even at an equimolar concentration,
with a less pronounced effect for compounds 5 and 4. Remark-
ably, compounds 1 and 5 act as seeds for the aggregation of
Ab1-40 and, moreover, have a significant influence on the sec-
ondary structure domains of this peptide, which changed from
the typical cross-b-amyloid structures into a non-b-sheet struc-
ture.
CD spectra of blank solutions as well as of mixtures were re-
corded after 7 days of incubation. The spectrum of the blank
solution of Ab1-40 shows a negative band centered at around
225 nm, zero molar ellipticity at 208 nm, and a positive lobe at
194 nm typical of a b-sheet structure. This spectrum clearly
changed after incubation with some of the tetrapeptides. In
particular, incubation with compound 1 led to the disappear-
ance of the characteristic b-sheet amyloid band at 225 nm and
the appearance of the spectral shape of pure compound
1 with two negative bands at 231 and 205 nm (Figure 9). In
the case of compound 5, the pure tetrapeptide is not very
active towards CD, in contrast to the pure amyloid. However, it
can be seen that the intensity of the CD signal of Ab1-40 de-
creases considerably in the presence of this compound, which
suggests that the secondary structure of Ab1-40 has been
modified in the co-assembly with 5 (Figure 10). A similar effect
was observed for the mixture with compound 4. For the re-
maining compounds (2, 3, and 6), the spectrum of Ab1-40 is
almost unaffected, which suggests a weak interaction between
these peptides and the amyloid, in agreement with the obser-
vations made by TEM. In summary, there is a clear influence of
the amino acid sequence on the interaction of compounds 1–
6 with Ab1-40 and in particular the presence of the Z-FD
moiety at the N terminus in the most effective compounds
1 and 5.
These results are of great interest because the capture of
amyloids to form large aggregates through their interactions
with other molecules has been reported as a strategy to
reduce the quantity of toxic soluble oligomers.[5b,23] Although
there are previous reports suggesting a potential parallelism
between the hydrogelation of small molecules and the fibrilli-
zation of amyloidogenic peptides, to the best of our knowl-
edge, this is the first time that the intermolecular interaction of
a simple molecular hydrogelator, as opposed to larger oligo-
peptides, and a b-amyloid fragment has been studied. It has
been shown that a low-molecular-weight compound can have
a crucial effect on the molecular conformation of a larger pep-
tide.
Currently we are investigating in detail the interaction of
compound 1 and amyloid compounds Ab1-40 and pentapep-
tide KLVFF at the molecular level. We believe that the fact that
this compound forms fibrillar aggregates under physiological
conditions may also be an advantage for its potential use as
an amyloid scavenger because aggregation or hydrogelation
would retard the degrading activity of proteases.[24] Moreover,
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Turner, A. Saiani, R. V. Ulijn, Adv. Mater. 2008, 20, 37; c) G. Cheng, V. Cas-
telletto, C. M. Moulton, G. E. Newby, I. W. Hamley, Langmuir 2010, 26,
4990; d) L. Chen, K. Morris, A. Laybourn, D. Elias, M. R. Hicks, A. Rodger,
L. Serpell, D. J. Adams, Langmuir 2010, 26, 5232; e) I. W. Hamley, Soft
Matter 2011, 7, 4122; f) R. J. Swanekamp, J. T. M. DiMaio, C. J. Bower-
man, B. L. Nilsson, J. Am. Chem. Soc. 2012, 134, 5556.
it is known that molecular hydrogels are reversible dynamic
systems in which free molecules are constantly exchanging
from solution to the aggregate phase and this feature has
been recently used in drug delivery.[25] In this sense, hydrogels
of compounds 1 and 5 could allow a smooth release of the
active free tetrapeptide into, for example, the blood stream in
which it could capture misfolded amyloids.
[9] a) H. A. Lashuel, S. R. LaBrenz, L. Woo, L. C. Serpell, J. W. Kelly, J. Am.
Chem. Soc. 2000, 122, 5262; b) S.-Y. Qin, Y. Pei, X.-J. Liu, R.-X. Zhuo, X.-Z.
Zhang, J. Mater. Chem. B 2013, 1, 668.
[10] M. Ma, Y. Kuang, Y. Gao, Y. Zhang, P. Gao, B. Xu, J. Am. Chem. Soc. 2010,
132, 2719.
[11] D. J. Adams, M. F. Butler, W. J. Frith, M. Kirkland, L. Mullen, P. Sanderson,
Soft Matter 2009, 5, 1856.
Acknowledgements
[12] a) C. Tang, A. M. Smith, R. F. Collins, R. V. Ulijn, A. Saiani, Langmuir 2009,
25, 9447; b) L. Chen, S. Revel, K. Morris, L. C. Serpell, D. J. Adams, Lang-
muir 2010, 26, 13466.
[13] A. Barth, C. Zscherp, Q. Rev. Biophys. 2002, 35, 369.
[14] a) D. A. S. Grahame, C. Olauson, R. S. H. Lam, T. Pedersen, F. Borondics, S.
Abraham, R. G. Weiss, M. A. Rogers, Soft Matter 2011, 7, 7359; b) S.
Abraham, Y. Lan, R. S. H. Lam, D. A. S. Grahame, J. J. H. Kim, R. G. Weiss,
M. A. Rogers, Langmuir 2012, 28, 4955.
This work was supported by the Ministry of Economy and
Competitiveness of Spain (Grants CTQ2009-13961 and
CTQ2012-37735) and Universitat Jaume I (Grant P1-1B2009-42).
M.T.-S. thanks the Ministry of Education, Culture and Sport of
Spain for an FPU fellowship.
[15] M. Suzuki, M. Yumoto, H. Shirai, K. Hanabusa, Chem. Eur. J. 2008, 14,
2133.
Keywords: aggregation · fibers · gels · peptides · self-
assembly
[16] a) N. Sreerama, R. W. Woody in Circular Dichroism: Principles and Appli-
cations, (Eds.: N. Berova, K. Nakanishi and R. W. Woody) Wiley, Hoboken,
2000; b) S. M. Kelly, N. C. Price, Curr. Prot. Pept. Sci. 2000, 1, 349.
[17] N. Amdursky, M. Molotskii, D. Aronov, L. Adler-Abramovich, E. Gazit, G.
Rosenman, Nano Lett. 2009, 9, 3111.
[18] U. Anand, M. Mukherjee, Langmuir 2013, 29, 2713.
[19] K. J. Robbins, G. Liu, V. Selmani, N. D. Lazo, Langmuir 2012, 28, 16490
and references cited therein.
[1] a) R. V. Ulijn, A. M. Smith, Chem. Soc. Rev. 2008, 37, 664; b) H. Cui, M. J.
Webber, S. I. Stupp, Biopolymers 2010, 94, 1; c) J. P. Jung, J. Z. Gasiorow-
ski, J. H. Collier, Biopolymers 2010, 94, 49; d) C. A. E. Hauser, S. Zhang,
Chem. Soc. Rev. 2010, 39, 2780.
[2] a) C. M. Dobson, Phil. Trans. R. Soc. Lond. B 2001, 356, 133; b) I. W.
Hamley, Angew. Chem. 2007, 119, 8274; Angew. Chem. Int. Ed. 2007, 46,
8128; c) I. W. Hamley, Chem. Rev. 2012, 112, 5147.
[20] R. Sabate, L. Rodriguez-Santiago, M. Sodupe, S. J. Saupe, S. Ventura,
Chem. Commun. 2013, 49, 5745.
[3] A. Rauk, Chem. Soc. Rev. 2009, 38, 2698.
[4] a) A. K. Mehta, K. Lu, W. S. Childers, Y. Liang, S. N. Dublin, J. Dong, J. P.
Snyder, S. V. Pingali, P. Thiyagarajan, D. G. Lynn, J. Am. Chem. Soc. 2008,
130, 9829; b) M. J. Krysmann, V. Castelletto, A. Kelarakis, I. W. Hamley,
R. A. Hule, D. J. Pochan, Biochemistry 2008, 47, 4597; c) F. T. Senguen,
T. M. Doran, E. A. Anderson, B. L. Nilsson, Mol. BioSyst. 2011, 7, 497.
[5] a) L. O. Tjernberg, J. Nꢂslund, F. Lindqvist, J. Johansson, A. R. Karlstrçm,
J. Thyberg, L. Terenius, C. Nordstedt, J. Biol. Chem. 1996, 271, 8545; b) T.
Takahashi, H. Mihara, Acc. Chem. Res. 2008, 41, 1309; c) C. Nerelius, A.
Sandegren, H. Sargsyan, R. Raunak, H. Leijonmarck, U. Chatterjee, A.
Fisahn, S. Imarisio, D. A. Lomas, D. C. Crowther, R. Strçmberg, J. Johans-
son, Proc. Natl. Acad. Sci. USA 2009, 106, 9191.
[6] a) V. J. Bradford, B. L. Iverson, J. Am. Chem. Soc. 2008, 130, 1517; b) A.
Wahlstrçm, L. Hugonin, A. Perꢃlvarez-Marꢄn, J. Jarvet, A. Grꢂslund, FEBS
J. 2008, 275, 5117; c) W. Yang, Y. Wong, O. T. W. Ng, L.-P. Bai, D. W. J.
Kwong, Y. Ke, Z.-H. Jiang, H.-W. Li, K. K. L. Yun, M. S. Wong, Angew.
Chem. 2012, 124, 1840; Angew. Chem. Int. Ed. 2012, 51, 1804; d) H. Yu,
M. Li, G. Liu, J. Geng, J. Wang, J. Ren, C. Zhao, X. Qu, Chem. Sci. 2012, 3,
3145.
[21] a) E. Gazit, FASEB J. 2002, 16, 77; b) W. Kim, M. H. Hecht, Proc. Natl.
Acad. Sci. USA 2006, 103, 15824; c) P. Marek, A. Abedini, B. Song, M. Ka-
nungo, M. E. Johnson, R. Gupta, W. Zaman, S. S. Wong, D. P. Raleigh, Bio-
chemistry 2007, 46, 3255; d) C. J. Bowerman, D. M. Ryan, D. A. Nissan,
B. L. Nilsson, Mol. BioSyst. 2009, 5, 1058; e) F. T. Senguen, N. R. Lee, X.
Gu, D. M. Ryan, T. M. Doran, E. A. Anderson, B. L. Nilsson, Mol. BioSyst.
2011, 7, 486; f) A. Lakshmanan, D. W. Cheong, A. Accardo, E. Di Fabrizio,
C. Riekel, C. A. E. Hauser, Proc. Natl. Acad. Sci. USA 2013, 110, 519.
[22] For example, see: A. T. Petkova, R. D. Leapman, Z. Guo, W.-M. Yau, M. P.
Mattson, R. Tycko, Science 2005, 307, 262.
[23] J. Sato, T. Takahashi, H. Oshima, S. Matsumura, H. Mihara, Chem. Eur. J.
2007, 13, 7745.
[24] K. J. C. Van Bommel, M. C. A. Stuart, B. L. Feringa, J. van Esch, Org.
Biomol. Chem. 2005, 3, 2917.
[25] a) F. Zhao, M. L. Ma, B. Xu, Chem. Soc. Rev. 2009, 38, 883; b) M. D. Segar-
ra-Maset, V. J. Nebot, J. F. Miravet, B. Escuder, Chem. Soc. Rev. 2013, 42,
7086.
[7] a) A. M. Jonker, D. W. P. M. Lçwik, J. C. M. van Hest, Chem. Mater. 2012,
24, 759; b) C. J. Bowerman, B. L. Nilsson, Biopolymers 2012, 98, 169.
[8] a) S. Ray, A. K. Das, M. G. B. Drew, A. Banerjee, Chem. Commun. 2006,
4230; b) A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R. F. Collins, M. L.
Received: July 8, 2013
Published online on December 11, 2013
Chem. Eur. J. 2014, 20, 1023 – 1031
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