Supramolecular gel formation and self-correction induced by aggregation-driven conformational changes

Article abstract of DOI:10.1039/b816234d

The formation of self-assembled fibrillar networks by low molecular weight peptidomimetics containing a Pro-Val moiety is reported; insight into the aggregation mechanism is provided revealing that it is associated to an unfolding process and that a fibri

Full text of DOI:10.1039/b816234d

View Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Supramolecular gel formation and self-correction induced
by aggregation-driven conformational changesw
Francisco Rodrıguez-Llansola, Juan F. Miravet* and Beatriu Escuder*
´
Received (in Cambridge, UK) 16th September 2008, Accepted 3rd November 2008
First published as an Advance Article on the web 13th November 2008
DOI: 10.1039/b816234d
The formation of self-assembled fibrillar networks by low
molecular weight peptidomimetics containing a Pro-Val moiety
is reported; insight into the aggregation mechanism is provided
revealing that it is associated to an unfolding process and that a
fibrillar network formed under kinetic control can self-correct
into a thermodynamically stable one.
peptidomimetic compounds 1a–c are involved in an intriguing
folding vs. aggregation behavior reminiscent of proteins.
Compounds 1a–c (Scheme 1) were prepared in good yields
by conventional solution peptide synthesis and fully charac-
terized. These compounds showed a high tendency to aggre-
gate in several organic solvents. The aspect of the aggregates
was observed either macroscopically (gels or crystalline
precipitates) as well as at the microscopic level by SEM
(see Fig. 1 and ESIw). All the xerogels revealed the presence
of an entangled fibrillar network characteristic of organogels.7
The aggregation of compounds 1a–c in CH3CN was
followed by CD spectroscopy. Samples were prepared by
gentle heating of acetonitrile and the solid gelator until
complete dissolution. Then spontaneous cooling at 25 1C took
place in a few minutes. In the case of compound 1a upon
increasing the concentration of the gelator from 0.6 mM to
30 mM important changes in shape, position and intensity of
the bands were found (Fig. 2A). These changes can be
associated to the aggregation that results in the formation of
a supramolecular gel at concentrations near to 30 mM
(minimum concentration for gel formation is 34 mM).
Strikingly, very significant differences were observed in the
CD spectrum of 1a if a slow, controlled cooling protocol was
used for samples at concentrations close or above 30 mM. In
this case, above 40 1C the CD spectrum was basically identical
to that obtained by spontaneous cooling. However, at 35 1C a
new band appeared at 210 nm that increased in intensity with
time (Fig. 2B). Indeed, a strong increase of De for this band
was observed after ca. 1.5 h together with the formation of a
gel. These findings suggested that slow conformational
changes were taking place in these samples. It can be reasoned
that under spontaneous cooling conditions metastable aggre-
gates would be kinetically trapped in the gels whereas slow,
controlled cooling allowed the evolution into aggregates with
increased thermodynamic stability. This difference, depending
on the cooling protocol, is also evident in the size and aspect of
the materials at the microscopic level (Fig. 1). Xerogels
obtained from kinetically trapped acetonitrile gels show larger
fibres, whereas those derived from thermocontrolled gels
Conformational preferences of the amino acids determine
peptide secondary and tertiary structure and play an impor-
tant role in protein function. For example, it is well reported
that misfolding of the peptidic chain may produce unwanted
processes that provoke fatal diseases. One of these problematic
processes could be peptide assembly or aggregation. For
instance, amyloid proteins may suffer conformational changes
during folding processes leading to intermediate misfolded
structures that can evolve into amyloid assemblies. These
assemblies may precipitate as plaques over important parts
of the body such as neural tissues and produce the so-called
amyloidogenic diseases.1,2 In general, aggregation of proteins
may have a dramatic effect on their function (structural,
catalytic or as a carrier). This has lead to an increasing interest
in the study of low molecular weight peptidomimetics in order
to understand those processes and design active inhibitors for
them.3
In the last years, our research has been focused in the study
of the self-assembly of small peptidomimetic compounds. In
this sense, we have studied bolaamphiphilic compounds
capable of assemble into fibrillar networks and gels in different
solvents.4 Here we report on the study of peptidomimetics
1a–c containing L-Pro residues. A bolaamphiphilic L-Val
scaffold has been used because we and others had shown
before that this is an effective fragment for the construction
of fibrillar aggregates.5 L-Pro residues were introduced for
their future application as functional materials. It is well
known that L-Pro introduces specific conformational demands
due to the presence of the heterocyclic moiety in alpha position
to the peptidic bond. For instance, it appears involved in many
exotic secondary structure fragments such as polyproline I and
II helices or triple helix collagen structures where it is respon-
sible of the formation of turns in the backbone crucial
for the final structure.6 The current study revealed that
Universitat Jaume I, Dpt. Quı´mica Inorga`nica i Orga`nica, 12071
´
Castello, Spain. E-mail: escuder@qio.uji.es.
E-mail: miravet@qio.uji.es; Fax: +34-964729155
w Electronic supplementary information (ESI) available: Synthesis
and characterization of compounds, NMR spectra and additional
graphs, SEM pictures and WAXD diffractograms. See DOI:
10.1039/b816234d
Scheme 1
ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 209–211 | 209

View Online
Fig. 1 SEM images of xerogels of compound 1a: (A) CH3CN,
spontaneous cooling, (B) CH3CN, slow cooling; Bars 1 mm.
Fig. 3 Models of compound 1a in solution (left) and in the gel (right)
obtained with MACROMODEL 8.0, AMBER* force field.
d and e supporting the folded structure of compounds 1a–c in
diluted solution. Additionally, CD spectra of a 0.6 mM
solution of 1a showed the typical shape of an helix with two
negative bands at 217 and 205 nm and a positive lobe at ca.
187 nm. A possible model obtained by molecular mechanics
that agrees with these evidences is shown in Fig. 3 (see ESIw).
Concentration dependent 1H NMR of compounds 1a–c
before gel formation under spontaneous conditions showed a
downfield shift of amide NH a evidencing its participation in
aggregation by intermolecular H-bonding. On the other hand
the chemical shift of the amide signal b did not change in that
range of concentrations. These results indicate that the initial
stages of spontaneous aggregation are associated to the rupture
of some the intramolecular H-bonds of the folded conforma-
tions (Scheme 2). This unfolding mechanism was confirmed by
transfer-NOE experiments with the formed gels. This experi-
ment, widely used in protein science and only recently applied
by us in the field of supramolecular gels, gave information
about the structure of 1a–c in the aggregated state.9 The
comparison of the NOE correlations obtained for gel and
diluted samples confirmed the partial unfolding of the mole-
cule upon the aggregation that takes place upon spontaneous
cooling of concentrated samples of gelator (see ESIw). Indeed,
the unfolding process was also evidenced by CD where the
Fig. 2 (A) CD spectra of compound 1a at different concentrations in
CH3CN under spontaneous cooling. (B) Evolution of CD spectrum of
1a (30 mM) in CH3CN at 35 1C after slow cooling (step 5 min).
reveal a network of thinner fibrils that form a cloth-like high
surface material.
1H NMR was used to investigate the structure of these
compounds in solution and in the gels. It was envisaged that,
as previously reported for other analogues, the presence of a
flexible alkyl spacer and several H-bonding groups may allow
intramolecular folding in solution.5a Analogue 2 was prepared
for comparison and amide NH signals (a, b in Scheme 1) were
taken as probes. A relevant finding was that amide signal b,
appearing at 8.1 ppm was quite insensitive to concentration,
temperature and solvent polarity for all the compounds
suggesting that even at low concentrations this proton is
involved in a strong intramolecular H-bond with Pro Na lone
pair.8 On the other hand, amide signal a that appeared at
6.6 ppm for compound 2 was shifted downfield in diluted
samples of bola analogues (1a, 7 ppm, 1b, 6.7 ppm and 1c,
6.7 ppm). These results indicate that amide protons a are most
likely involved in intramolecular H-bonding and that this
effect particularly strong for compound 1a which presents
the shortest alkyl spacer. NOE experiments revealed
the spatial proximity of proton a to c and f, and proton b to
Scheme 2
ꢀc
This journal is The Royal Society of Chemistry 2009
210 | Chem. Commun., 2009, 209–211

View Online
helical band found in diluted solution suffered a red shift
until 235 nm associated with the conformational change and
aggregation.
H-bonds and in consequence to lower stability of their folded
conformations. Nevertheless, transfer-NOE experiments on
those gels supported the partial unfolding of the molecule as
a general mechanism.
The observed evolution of the CD of concentrated mixtures
upon slow cooling suggested that an additional slow confor-
mational reorganization was taking place yielding a thermo-
stable gel (Scheme 2).z The higher degree of organization in
this gel is clearly supported by several facts. First, the WAXD
pattern of the xerogels obtained by slow cooling showed sharp
reflections denoting a high degree of crystallinity in the fibers
whereas that of the xerogel obtained after spontaneous
cooling gelation only showed a broad signal at 4.5 A and a
background of an amorphous material, in accordance with a
loosely defined aggregation as a result of the reduced number
on intermolecular interactions. This peak is typical for inter-
molecular distance in the H-bonding direction (see Fig. 3 and
ESIw) as described for analogues.4a Secondly, a kinetic study
of the aggregation under controlled cooling conditions at
35 1C revealed an activation free energy for the reorganization
process of 28 kJ molꢁ1 in agreement with the rupture of
several hydrogen bonds. Finally, the solubility of the gel
formed by 1a under kinetic conditions determined by
1H NMR was 12 mM whereas for the thermodynamic gel was
8 mM reflecting stronger intermolecular interactions and
increased gelation ability in the latter case.10 These properties
are related to the higher thermal stability measured for the gels
obtained by slow cooling (Tgel = 75 1C) as compared to the
kinetic gels (Tgel = 55 1C).
In summary, we have presented an example of small
peptidomimetics that evidence the paramount importance of
folding and aggregation even for a priori simple molecules. We
have shown that very subtle structural and environmental
changes may have a dramatic effect on supramolecular aggre-
gation. We have also proved that, upon overcoming the
required energy barrier these reversible supramolecular
systems are capable of self-correction to produce a more stable
and organised material. Current work is being done with
the aim of controlling the switching between soluble and
aggregated states.
We thank MEC (Grant CTQ2006-14984) and Universitat
Jaume I-Bancaixa (Grant P1ꢂ1A2006-1) for financial support.
F. R. Ll. thanks Generalitat Valenciana for a FPI fellowship.
Notes and references
z CD experiments of gels formed by fast cooling at different tempera-
tures (10, 0, ꢁ20 1C) confirmed the strong influence of cooling rate and
allowed the observation of kinetically trapped intermediate partially
unfolded conformations.
1 (a) Mechanisms of Protein Folding, ed. R. H. Pain, Oxford
University Press, 2000; (b) Self-assembling Peptide Systems in
Biology, Medicine and Engineering, eds. A. Aggeli, N. Boden and
S. Zhang, Kluwer, Dordrecht, 2001.
Very interestingly, a kinetically trapped gel was able to self-
correct when it was kept for 48 h at 30 1C being converted into
a gel with properties similar to those of the material obtained
by slow cooling (solubility and WAXD pattern of the xerogel).
Some differences were observed in the aggregation of com-
pounds 1b and 1c as compared to 1a. Firstly, spontaneous
cooling had to be employed in order to obtain gels whereas
slow cooling produced crystalline precipitates. On the other
hand, CD investigation of the aggregation process did not
reveal a strong influence of the cooling methodology as
described for compound 1a. Finally, WAXD of xerogels and
precipitates were identical (see ESI). Altogether these results
suggested that in these two compounds the energetic barrier
for conformational changes is much lower than for compound
1a probably related to the higher flexibility of the alkyl spacer
that, as already mentioned, led to weaker intramolecular
2 I. W. Hamley, Angew. Chem., Int. Ed., 2007, 46, 8128.
3 L. D. Estrada and C. Soto, Curr. Top. Med. Chem., 2007, 7, 115.
4 (a) B. Escuder, S. Martı and J. F. Miravet, Langmuir, 2005, 21,
´
6776; (b) J. F. Miravet and B. Escuder, Chem. Commun., 2005,
5796; (c) J. F. Miravet and B. Escuder, Org. Lett., 2005, 7, 4791.
5 (a) K. Hanabusa, R. Tanaka, M. Suzuki, M. Kimura and
H. Shirai, Adv. Mater., 1997, 9, 1095; (b) M. Doi, A. Asano,
H. Yoshida, M. Inouguchi, K. Iwanaga, M. Sasaki, Y. Katsuya,
T. Taniguchi and D. Yamamoto, J. Pept. Res., 2005, 66, 181.
6 V. Ottani, D. Martini, M. Franchi, A. Ruggeri and M. Raspanti,
Micron, 2002, 33, 587.
7 (a) Molecular Gels: Materials with Self-assembled Fibrillar Networks,
eds. P. Terech and R. G. Weiss, Springer, Dordrecht, The
Netherlands, 2006; (b) F. Fages, Top. Curr. Chem., 2005, 256, 1.
8 H. C. Ahn and K. Choi, Org. Lett., 2007, 9, 3853.
9 B. Escuder, M. Llusar and J. F. Miravet, J. Org. Chem., 2006, 71,
7747.
10 A. R. Hirst, I. A. Coates, T. R. Boucheteau, J. F. Miravet,
B. Escuder, V. Castelletto, I. W. Hamley and D. K. Smith,
J. Am. Chem. Soc., 2008, 130, 9113.
ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 209–211 | 211