Self-assembly and fibrillization of a Fmoc-functionalized polyphenolic amino acid

Article abstract of DOI:10.1039/c3sm52222a

Self-assembly of Fmoc-l-DOPA leads to the formation of twisted fibers at pH 2. Temperature dependent circular dichroism (CD) reveals the formation of a linear superstructure via π-π stacking. SAXS experiments together with TEM imaging confirm the existence of semi-flexible twisted-multistranded fibers. The self-assembly is found to be pH-dependent, with no aggregation at pH 5 and 7.

Full text of DOI:10.1039/c3sm52222a

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ARTICLE TYPE
Self-assembly and Fibrillization of a Fmoc-functionalized polyphenolic
amino acid
Abhijit Saha, Sreenath Bolisetty, Stephan Handschin and Raffaele Mezzenga*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Self-assembly of Fmoc-
twisted fibers at pH 2. Temperature dependent Circular
Dichroism (CD) reveals the formation of linear
superstructure via π-π stacking. SAXS experiments together
L-DOPA leads to the formation of
a
10 with TEM imaging confirm the existence of semi flexible
twisted- multistranded fibers. The self-assembly is found to
be pH-dependent, with no aggregation at pH 5 and 7.
Selfꢀassembly plays a pivotal role in the formation of complex
biological building blocks. The simplest and bestꢀknown example
15 of selfꢀassembly in living system is naturally occurring DNA,
which assembles from single strands into a double helical form.1
Taking inspiration from nature, selfꢀassembly has been widely
implemented on naturally occurring molecules and their
derivatives to create novel nanomaterials. Notable examples of
Fig. 1 Synthesis of Fmocꢀ
L
ꢀDOPA.
50
mammals, including men. ꢀDOPA is a psychoactive drug and it
L
is used in the clinical treatment of Parkinson's disease and
dopamineꢀresponsive dystonia23,24 as it inhibits amyloid fibril
formation.24,25 It can cross the bloodꢀbrain barrier and acts as the
20 use of naturally occurring small molecules or their derivatives 55 precursor to the neurotransmitters dopamine, norepinephrine
and complexes as scaffolds for molecular selfꢀassembly include
bile acids,2 phytosterol,3 saccharides,4 vitamins,5 nucleic acid,6
sophorolipids,7 terpenic acid8, etc. Peptides, in particular, have
been widely used in the last two decades as building blocks for
(noradrenaline), and epinephrine (adrenaline). Besides it can
function as an antioxidant due to the presence of two phenolic
OH groups.
LꢀDOPA has multiple interacting sites (phenolic ring
for πꢀπ stacking, ꢀNH2 and ꢀCO2H for Hꢀbonding), which may
25 molecular selfꢀassembly due to the possibility of templating, 60 facilitate the formation of supramolecular nanostructures in
biocompatible, biodegradable, tuneable and being stimuli
responsive materials.9ꢀ16 They can associate nonꢀcovalently in
solution through multiple supramolecular interactions such as Hꢀ
bonding, hydrophobic and πꢀπ stacking, to form fibrils, tubes,
30 ribbons etc.
Fluorenylꢀ9ꢀmethoxycarbonyl (Fmoc)ꢀfunctionalized amino acids
and peptides are a unique class of molecules to form fibrillar
structures in water.16ꢀ21 Fmoc groups attached to the amino acids
promote hydrophobic and πꢀπ interactions between π electrons in
35 the aromatic fluorenyl rings and hence facilitate the formation of
oneꢀdimensional (1D) supramolecular polymers in solutions, and
eventually, nanostructured fibrils with an internal structure.
To the best of our knowledge, Selfꢀassembly of chiral
polyphenols has been not deeply investigated, despite the
solution. The Fmoc group on the Nꢀterminal further allows
hydrophobic and πꢀπ interactions.
Samples of FmocꢀLꢀDOPA were prepared at three different pH
conditions (pH 2, 5 and 7) by adjusting the pH of the solutions
65 with diluted HCl or NaOH solutions and by heating. At pH 2,
°
upon incubating the samples at 25 C, turbidity appeared first, to
vanish gradually with time. The experiments were performed
°
after incubating all the solutions overnight at 25 C, and the
samples were investigated by microscopic, spectroscopic and
°
°
70 scattering techniques at 25 C and 70 C, respectively. At pH 2,
the 0.5 wt% sample formed a weak gel, which broke upon
mechanical agitation. Even at lower conc. (∼0.25 wt%) a very
weak gel like behavior could be observed although this could
flow upon slight agitation. At pH 5 and pH 7 no gel or viscous
40 additional functionalities that polyphenols can bring, such as for 75 fluid formation was observed and the solutions turned brown in
colour with time.
example, a great antioxidant power. Here we report the selfꢀ
assembly of Fmocꢀfunctionalized polyphenolꢀderivative, “Nꢀ(9ꢀ
fluorenylmethyloxycarbonyl)ꢀ3,4ꢀdihydroxyꢀLꢀphenylalanine
The 0.25 wt% sample at pH 2 was investigated by polarized
optical microscopy (POM). Interestingly, the sample showed
birefringence under crossꢀpolarised (Fig. 2a) condition. The
80 observed birefringence of the sample might be indicative of
construction of nematic phase by selfꢀassembled the FmocꢀLꢀ
DOPA molecules. When the sample was heated at 70 C, the
birefringence disappeared (Fig. 2b) which might be due to the
(FmocꢀLꢀDOPA),” in water at various pH conditions. Fmocꢀ
45 DOPA was synthesized by reaction of Fmoc chloride with
DOPA in a 10% aqueous NaHCO3ꢀDioxane mixture (Fig. 1).22
Lꢀ
Lꢀ
Lꢀ
°
DOPA is a chiral amino acid which is generated via biosynthesis
from the amino acid Lꢀtyrosine available from plants and
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nematic to isotopic phase transition with the loss of the
supramolecular structure. No characteristic birefringence was
assembled aggregated state. The change of molar ellipticity,[θ],
with temperature at 307 nm is shown in the inset of Fig. 3. At 25
°C the value was ꢀ1618 at 307 nm and when increasing
temperature it gradually reduced and finally reached the baseline
40 by 50 °C. These two CD experiments infer supramolecular
chirality during the dynamic assembly of small molecules at pH 2
and amplification of chirality due to the higher order helical
structure formation in the selfꢀassembled fibrillar state.
Spontaneous disassembly of the supramolecular polymer into
observed for 0.25 wt% Fmocꢀ
LꢀDOPA at pH 5 and pH 7.
45 individual synthons (FmocꢀLꢀDOPA) occurred at higher
temperatures. In contrast, CD spectra of 0.1 wt% sample at pH 5
and pH 7 showed no Cotton coupling in the region from 270 nm
to 370 nm (Supp. Fig. 1). This is indicative that there was no
assembly of small molecules under these pH conditions and
50 hence, no helical superstructure was formed.
°
°
5
Fig. 2 POM images (a) at room temperature (25 C) and (b) at 70 C for
0.25 wt% FmocꢀLꢀDOPA in water at pH 2 (Scale bar is 100 ꢁm).
To further understand the molecular organization of Fmocꢀ
DOPA in water, circular dichroism (CD) spectroscopy was
10 carried out. Circular dichroism is powerful tool for
understanding the arrangement of chiral molecules in the
associated state. In the selfꢀassembled state intermolecular
interactions between chromophores may generate pronounced
chiroptical responses and hence may produce signals often much
15 stronger than their isolated or molecularly solvated state. We
Lꢀ
Dynamic light scattering (DLS) measurements for 0.25 wt%
FmocꢀLꢀDOPA at pH 2 showed a hydrodynamic radius (Rh) of
820 nm at 25 °C due to the supramolecular association of
molecules (Fig. 4a). The hydrodynamic radius was reduced to 2
a
°
55 nm by increasing the temperature of the solution to 70 C where
the supramolecular aggregates dissociated into the molecularly
solvated state. The time correlation function and decrease in
scattering intensity count rate is shown in the Supp. Fig. 2. The
DLS results were also in accordance with the optical microscopy
60 and circular dichroism measurements, confirming the
disassembly of molecules at higher temperature.
prepared a dilute solution of 0.1 wt% FmocꢀLꢀDOPA at pH 2 for
the CD spectroscopy. The spectra (Fig. 3) showed a large
negative Cotton effect at 307 nm, a small negative Cotton effect
at 297 nm and small positive Cotton effect at 302 nm. All the
20 Cotton effects in the region of 270–310 nm have been attributed
to linear πꢀstacking of the Fmoc moieties (π–π* transitions).26,27
The observed Cotton effects imply superhelical arrangement
formed by the Fmoc moieties in the selfꢀassembled state. A first
large induced negative CD signal at higher wavelength (at 307
25 nm) and then a positive Cotton effect (at 301 nm) are indicative
for the formation of left handed helical structures due to πꢀπ
stacking of Fmoc moieties during the fibril formation.27 To
confirm this, temperature dependent CD studies were performed
with the same sample at 0.1 wt % concentration. With the
30 increase in temperature, all the signals gradually decreased. At 50
We then performed smallꢀangle Xꢀray scattering (SAXS)
measurements on the 0.5 wt% sample of FmocꢀLꢀDOPA at pH 2
to understand the structure of the aggregates. The SAXS
65 spectrum measured at room temperature is shown in Fig. 4b. The
scattering intensities follow the asymptotic slope of ꢀ1.7 in the q
range 0.05 nmꢀ1ꢀ 1 nmꢀ1. This scattering pattern indicates the
aggregates having a selfꢀavoiding semi flexible (nominal fractal
exponent 5/3) fibrillar structure within the length scale of 125 nm
70 to 6 nm. The same sample was measured at higher solution
°
temperature of 70 C. At this condition, the scattering intensity
counts dropped down and became equal to the background
solvent scattering intensity (see Supp. Fig. 3). This conclusively
indicates the dissolution of fibrillar aggregates at higher
75 temperature.
Fig. 4 a) DLS measurements of 0.25 wt% Fmocꢀ
2 at the indicated temperatures and b) SAXS at 25 C of 0.5 wt % Fmocꢀ
ꢀDOPA at pH 2.
LꢀDOPA in water at pH
°
L
Fig. 3 Temperature dependent CD spectra of 0.1 wt% FmocꢀLꢀDOPA in
80 Transmission electron microscopy (TEM) and cryogenicꢀTEM
(cryoꢀTEM) were carried out to understand the morphology of
the selfꢀassembled FmocꢀLꢀDOPA at pH 2 (Fig. 5 and Supp. Fig.
water at pH 2.
°C, the signals approached the baseline implying the molecules
35 were in the molecularly dissolved state and not in the selfꢀ
4ꢀ6). TEM and cryoꢀTEM images of FmocꢀLꢀDOPA at 0.1 wt%
2
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Fig. 5 TEM (a,c; scale bar is 200 nm) and cryoꢀTEM (b,d,e; scale bar 100 nm) of 0.1 wt% (a,b,e) and 0.25 wt% (c,d) of FmocꢀLꢀDOPA at pH 2.
and 0.25 wt% are presented in Fig. 5. Both single and
multistranded fibers were observed in all the images. The width
of the single fibers varied approximately from 6 to 13 nm. The
width of the twisted fibers varied from 7 to 39 nm. Left handed
twisted nature of the multistranded fibers could be clearly
observed, confirming CD analysis. Interestingly, branching and
entanglement could also be observed for the fibers, although at
pH 5 and 7, even at a concentration as high as 0.5 wt% only a
very few fibers could be found (Supp. Fig. 7 and 8). This
10 indicates that supramolecular assembly in the form of fibers is not
occurring at these higher pH conditions, in agreement with the
other observations at the same pH.
The pKa of FmocꢀLꢀDOPA was determined to be 3.98. At higher
pH conditions (pH 5 and 7), most of the molecules are ionised
15 and therefore they do not assemble to form superstructures rather
5
Fig. 6 Schematic for the proposed model for selfꢀassembly of FmocꢀLꢀ
35 DOPA at pH 2.
possibility of intermolecular Hꢀbonding between the carbonyl and
NꢀH of the adjacent carbamate groups, which may participate in
the selfꢀassembly scheme. A twisting is generated along the
40 length of the fibril during fibrillation as a consequence of the
amplification of chirality of amino acids. With increasing
concentration, multistranded fibrils are generated first, and
staying in
a molecularly solvated state. Zeta potential
measurements were carried out for 0.1 wt% samples at the three
aboveꢀmentioned pHs. Zeta potentials at pH 2, 5 and 7 were ꢀ
17.2, ꢀ64.2 and ꢀ64.7 mV respectively (Supp. Fig. 9).
20 Corresponding electrophoretic mobilities were ꢀ0.571, ꢀ5.072 and
ꢀ5.033 ꢁmcm/Vs at pH 2, 5 and 7. This supported the fact that at
pH 2, the majority of the monomers were nonꢀionised and could
selfꢀassemble to form fibers, whereas at higher pH conditions (pH
5 and 7), the majority of the molecules are ionised and the selfꢀ
25 assembly into linear fibers is disfavoured. Also at higher pHs
conditions, there is a possibility of degradation of polyphenols.28
Based on the above observations, a schematic model for the selfꢀ
successively, networks of fibrils by branching and entanglement.
In summary, the selfꢀassembly of FmocꢀLꢀDOPA has been
45 investigated in water at different pH conditions. Temperature
dependent CD studies revealed the molecules aggregate linearly
via πꢀπ stacking to form fibers at pH 2. SAXS results indicated
the occurrence of semiꢀflexible linear objects. TEM and cryoꢀ
TEM studies confirmed the formation of multistranded left
50 handed twisted fibers. At higher pH conditions (pH 5 and 7) the
majority of FmocꢀLꢀDOPA molecules were ionized due to
deprotonation of the carboxylic group, and resulted in inhibition
of fibril formation.
assembly of FmocꢀLꢀDOPA at pH 2 is proposed (Fig. 6). The
monomers stack linearly along the axis of the fibril. The
30 molecules inside the fibril get stabilized via πꢀπ interactions
among the Fmoc group. Wideꢀangle Xꢀray scattering (WAXS)
The authors gratefully acknowledge the Swiss National Science
55 Foundation (SNSF, project no. 200021_135146) for financial
support.
patterns further confirmed the πꢀπ stacking of the FmocꢀLꢀDOPA
(0.4 nm) inside the aggregates (Supp. Fig. 10). There is a
Notes and references
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ETH Zurich, Food & Soft Materials Science, Institute of Food, Nutrition
& Health, Schmelzbergstrasse 9, LFO , 8092 Zürich, Switzerland.
E-mail: raffaele.mezzenga@hest.ethz.ch
† Electronic Supplementary Information (ESI) available: [details of
experimental procedures, sample preparation, CD spectra at different pHs,
.DLS, SAXS, WAXS, TEM and CryoꢀTEM images]. See
DOI: 10.1039/b000000x/
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