Peptide functionalised discotic amphiphiles and their self-assembly into supramolecular nanofibres

Article abstract of DOI:10.1039/c1sm05896g

The multicomponent co-assembly of discotic amphiphiles provides a modular and versatile approach to prepare RGDS- and PHSRN-peptide functionalised supramolecular nanofibres, bearing pendant paramagnetic Gd(iii)-chelates.

Full text of DOI:10.1039/c1sm05896g

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Peptide functionalised discotic amphiphiles and their self-assembly into
supramolecular nanofibres†
ab
*
P. Besenius,‡
Y. Goedegebure,c M. Driesse,b M. Koay,c P. H. H. Bomans,d A. R. A. Palmans,ab
P. Y. W. Dankersac and E. W. Meijer
abc
*
Received 15th May 2011, Accepted 8th July 2011
DOI: 10.1039/c1sm05896g
and PHSRN), and fluorophore [carboxyfluorescein (CF)] pendant
discotics, with MRI (GdIII–DOTA) labelled amphiphiles (Fig. 1).
Nanorods, functionalised with synergistic peptide units as well as
MRI labels could be used as targeted contrast agents in molecular
imaging applications. These nanoscopic, multifunctional and multi-
valent constructs would thereby allow for high contrast agent effi-
cacies in combination with accumulation in an area of interest. We
show that stacking of peptide bearing discotic amphiphiles into one-
dimensional nanorods can lead to reinforced aggregation, bundling
and hence the formation of nanofibres. Furthermore, by mixing in
GdIII–DOTA labelled discotic amphiphiles that act as diluting co-
monomers, secondary interactions are avoided and well-defined
nanorods of 50 nm in length are obtained. In line with our previous
investigations, we will highlight the need to correlate the self-assembly
pathways of the ordered supramolecular polymers with morpho-
logical characterisations.1a,7,9
The multicomponent co-assembly of discotic amphiphiles provides
a modular and versatile approach to prepare RGDS- and PHSRN-
peptide functionalised supramolecular nanofibres, bearing pendant
paramagnetic Gd(III)-chelates.
Supramolecular engineering of soft-condensed matter has allowed
chemists to develop robust and versatile methodologies for the
preparation of solution based macromolecular architectures, gels and
bulk materials.1 Particularly for biomedical applications,2 non-cova-
lent architectures in aqueous environments are indispensable, relying
on self-assembled linear amphiphiles, such as phospholipids,3 block
copolymers4 or peptide amphiphiles.5 Stupp and coworkers have
shown that the latter are extremely appealing owing to their modular
synthetic approach: multifunctional and multivalent non-covalent
nanofibres can be prepared conveniently without the need for
repeated syntheses, obtaining scaffolds that are dynamic and adapt-
able, yet inherently degradable into their small molecular weight
constituent subunits.2b,5b,6
Recently, we1a,7 and others1h,8 have reported a new class of C3-
symmetrical discotic amphiphiles that are designed to polymerise into
columnar aggregates, via a combination of hydrogen bonding, p–p
interactions and solvophobic effects. In order to demonstrate the
versatility of the benzene-1,3,5-tricarboxamide (BTA) based supra-
molecular polymers, we hereby report the preparation of multifunc-
tional supramolecular nanorods, via the multicomponent co-
assembly of two different peptide functionalised discotics (RGDS
The synthetic route was designed around a common C3-symmet-
rical intermediate that was prepared in a convergent approach
(Scheme 1). Thereafter Gd(III)–DOTA and CF labels could be
attached via their activated ester moieties (Scheme 2).1a In compar-
ison, the preparation of peptide functionalised discotics was thought
to be more challenging. Since the purification of multivalent supra-
molecular monomers that are highly amphiphilic is not straightfor-
ward, our approach involved an orthogonal and highly efficient
ligation step that could be performed under mild conditions and at
the same time, avoid protecting group strategies. For this reason, the
aInstitute for Complex Molecular Systems, Eindhoven University of
Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
bLaboratory of Macromolecular and Organic Chemistry, Department of
Chemical Engineering and Chemistry, Eindhoven University of
Technology, The Netherlands. E-mail: e.w.meijer@tue.nl; Tel: +31 40
247 3101
cLaboratory of Chemical Biology, Department of Biomedical Engineering,
Eindhoven University of Technology, The Netherlands
dLaboratory of Materials and Interface Chemistry and Soft Matter
CryoTEM Research Unit, Department of Chemical Engineering and
Chemistry, Eindhoven University of Technology, The Netherlands
† Electronic supplementary information (ESI) available: Detailed
experimental procedures and materials characterisation, additional CD
and PL data. See DOI: 10.1039/c1sm05896g
Fig.
1 Schematic representation of the self-assembly of discotic
amphiphiles: the hydrophobic BTA core directs the self-assembly into
a helical architecture whilst the peripheral hydrophilic functional groups
introduce an amphiphilic character.
‡ Current address: Organic Chemistry Institute and CeNTech,
€
Westfalische Wilhelms-Universitat Munster, Corrensstrasse 40, 48149
Munster, Germany; E-mail: p.besenius@uni-muenster.de
€
€
€
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Scheme 1 Synthesis of the nucleophilic, hydrophobic cores of the dis-
cotic synthetic precursors 12 and 15: (i) HATU, DiPEA, DMF, 25 h,
90%; (ii) TFA–DCM (1 : 1), 5.5 h, 98%; (iii) PyBOP, DiPEA, THF, 12 h,
80%; (iv) piperidine, CH3CN, 1.5 h, 53%; (v) THF–DMF (1 : 9), DiPEA,
36 h, 72%; (vi) TFA–DCM (1 : 1), 2.5 h, 72%; (vii) 14, DiPEA, DMF, 2 h,
49%; (viii) TFA–DCM (1 : 1), 0 ꢂC, 1 h, quantitative.x
use of oxime ligation seemed to meet all these requirements (Scheme
2).10 The target peptides were the well-known integrin binding ligands
Gly-Arg-Gly-Asp-Ser (GRGDS) and Pro-His-Ser-Arg-Asn
(PHSRN).11 A short, flexible glycine spacer (GGG) and a serine (S)
were introduced to the N-terminus of the peptides and synthesized by
solid phase peptide synthesis, using standard Fmoc-based protocols.
Without purification of the peptides, the N-terminal serine of both
peptides was selectively oxidized using periodate (NaIO4) at neutral
pH.10b
Aggregation and therefore steric hindrance are the most likely
explanations for the unexpectedly long reaction times to achieve full
conversion for the oxime ligations. However, the conversions and
isolated yields for all the intermediate synthetic steps were very high
and highly pure products were obtained (Fig. 2). Only in the final
purification step via preparative liquid chromatography, material was
lost due to the tendency for the structures to aggregate during
chromatography.
Scheme 2 Synthesis of the peptide functionalised discotic amphiphiles 1
and 2, carboxyfluorescein (CF, mixture of isomers) and Gd(III)–DOTA
pendant discotics 3 and 4: (i) anilinium acetate buffer (0.1 M, pH 4.5), 5
days, 10%; (ii) CF–NHS, DiPEA, DMF, 25 h, 76%; (iii) DOTA–NHS,
DiPEA, DMF, 12 h, quantitative; (iv) Gd(OAc)3$H2O, ultra pure water,
pyridine, 50%.x
curves started to level off or even increase. These additional transi-
tions in the cooling curves strongly indicate a secondary process, most
likely arising from interactions of the peptide side arms within one
stack. This would also explain the extraordinary high stability of the
rod-like structures compared to the amphiphiles reported pre-
viously.1a To find further evidence for this hypothesis we performed
cryo-TEM, in the same buffered conditions. Very long rods in the
micrometre range were observed which exhibited a high tendency to
In the simplest case of a single peptide discotic aggregate, solutions
of 1 or 2 in dilute buffered conditions give rise to strong Cotton effects
indicating the formation of helical supramolecular polymers
(Fig. 3A). However, when monitoring the negative CD band at
280 nm—which is characteristic for ordered aromatic chromophores
of the hydrophobic wedge—at different temperatures, we observed
extraordinarily high stability of these aggregates (Fig. 3B). At
1 ꢀ 10ꢁ5 M of 1, the CD effect only dropped by about 10% after
heating the solutions to 368 K. Only upon addition of 5 vol%
DMSO, a strong hydrogen bond acceptor, and further diluting the
system to 2.5 ꢀ 10ꢁ6 M, were the non-covalent polymers fully dis-
assembled. Peptide discotic 2 showed very similar aggregation
behaviour. This is a rare case in which we have observed such stable
ordered supramolecular polymers in aqueous environments (Fig. 4).
By monitoring the temperature dependence of the CD band at
280 nm from a molecularly dissolved (i.e. 368 K) to fully aggregated
state (i.e. 283 K), we studied the assembly process in more detail. The
cooling curves or growth profiles observed were reminiscent of
a nucleation–elongation type mechanism at high temperatures.
However at about 320 K for 1 or already at 340 K in the case of 2, the
Fig. 2 LC-MS analysis of purified peptide discotic 1: (A) TIC trace, (B)
PDA trace, and (C) ESI-MS.
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Fig. 3 CD spectra of 1 at different concentrations in citrate buffer (100
mM, pH 6), and increasing amounts of DMSO: spectra at (A) 283 K and
(B) 368 K.
Fig. 6 Cryo-TEM of aggregates based on peptide discotics 1, 2 and Gd
(III) labelled discotic 4 (1 mg mlꢁ1, molar ratio of 1 : 1 : 12) in citrate
buffer (100 mM, pH 6); scale bars represent 50 nm.
Cryo-TEM images clearly show that single nanorods were produced
of at least 50 nm in length (Fig. 6), which corresponds to a degree of
polymerisation of about 150, assuming a disc–disc spacing of
0.35 nm,12 and molecular weights in the order of 500 kDa. This
implies that these rods are functionalised with around 66 peptide
units of RGDS and PHSRN (1 : 1) and provide a very promising
platform for potential multivalent and synergistic binding studies.
For further evidence of the successful co-assembly of peptide
functionalised and diluting discotics, a series of CD experiments were
performed. In contrast to the single monomer 1 and 2 based aggre-
gates, the supramolecular copolymerisation yielded CD effects
similar to the one-dimensional rods based on discotic 4 alone.1a
Importantly the cooling curves show a self-assembly process lacking
any secondary interactions. The shape is consistent with a nucle-
ation–elongation type mechanism, typical for a cooperative poly-
merisation and follows the growth profile reported previously for the
homopolymerisation of 4. Similar growth profiles were observed for
different ratios of peptide to Gd(III) labelled discotics, ranging from
1 : 6 to 1 : 3 (Fig. 7).
Fig. 4 Temperature dependent CD spectra of (A) 1 and (B) 2 (2.5 ꢀ 10ꢁ6
M), in citrate buffer (100 mM, pH 6, 5 vol% DMSO), monitored at l ¼
280 nm.
To follow the self-assembly process by temperature dependent
photoluminescence (PL), CF bearing discotic 3 was introduced to the
heterologous supramolecular aggregates. Critical temperatures in PL
emission intensities overlap well with those observed in CD cooling
curves (Fig. 7). The aggregation induced increase in fluorescence
intensity is significantly higher than the temperature dependent
emission quantum yield of CF in the same buffered conditions (see
ESI†). A series of control experiments at different ratios of CF
labelled discotics to diluting discotics confirm that at least a ratio of
1 : 10 is necessary to observe profiles as depicted in Fig. 7D. A lower
ratio results in quenching between laterally stacked CF units that are
in close proximity. The incorporation of the labelled discotics into
macromolecular stacks was confirmed by fluorescence anisotropy
experiments (see ESI†).
Fig. 5 Cryo-TEM of aggregates based on peptide discotic 1 at 1 mg mlꢁ1
in citrate buffer (100 mM, pH 6); scale bars represent 0.5 mm (left) and 50
nm (right).
bundle up into fibres (Fig. 5). This further confirms secondary
interactions within 1D aggregates.
In the past we have shown the cooperative self-assembly of Gd(III)–
DOTA discotics 4 into rods of 50–75 nm at millimolar concen-
trations.1a By combining peptide discotics 1 and 2 with 4 we were
aiming at targeted MRI labels. However, before using the co-
aggregates for this application insights into the processes of co-
assembly are required, since small changes in their composition can
alter the behaviour drastically. At an overall concentration of 0.5 mM
(1 mg mlꢁ1), a mixture of 4 were co-assembled with RGDS- and
PHSRN-labelled discotics 1 and 2 in a ratio of 12 : 1 : 1, respectively.
After addition of a buffer to the solid compounds and sonication
yielded clear solutions, which were previously not observed for the
peptide discotics themselves due to scattering of the clustered fibres.
In conclusion, we have shown that multicomponent self-assembly
is a versatile methodology to prepare nanosized soft materials with
peripheral functional groups, in this case, pendant peptides, Gd(III)
chelates and fluorescein groups. Stacking of the peptide discotic
amphiphiles into one-dimensional arrays leads to reinforced aggre-
gation induced by weak secondary interactions between the periph-
eral groups. Modular self-assembly, using diluting discotic
amphiphiles, prevents these secondary interactions due to the spatial
separation of peptide bearing discotics. Temperature dependent CD
and PL were sensitive techniques to investigate the self-assembly
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We thank IBOS (Project 053.63.310) and Marie Curie Actions FP7
(SahnMat, PIEF-GA-2009-235914) [PB] for funding, the Council for
Chemical Sciences of the Netherlands Organization for Scientific
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Hoeben (both SyMO-Chem) for providing DOTA–NHS and
compound 14.
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