Probing the self-assembly and stability of oligohistidine based rod-like micelles by aggregation induced luminescence

Article abstract of DOI:10.1039/c6ob00292g

The synthesis and self-assembly of a new C₂-symmetric oligohistidine amphiphile equipped with an aggregation induced emission luminophore is reported. We observe the formation of highly stable and ordered rod-like micelles in phosphate buffered

Full text of DOI:10.1039/c6ob00292g

Organic &
Biomolecular Chemistry
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COMMUNICATION
Probing the self-assembly and stability of
oligohistidine based rod-like micelles by
Cite this: DOI: 10.1039/c6ob00292g
aggregation induced luminescence†
Received 4th February 2016,
Accepted 7th March 2016
a
a
b
b
c
DOI: 10.1039/c6ob00292g
Hendrik Frisch, Daniel Spitzer, Mathias Haase, Thomas Basché, Jens Voskuhl*
a
and Pol Besenius*
www.rsc.org/obc
The synthesis and self-assembly of a new C
2
-symmetric oligohisti- oligohistidine amphiphile equipped with an aggregation
4
4–49
dine amphiphile equipped with an aggregation induced emission induced emission luminophore.
The development of
luminophore is reported. We observe the formation of highly intrinsically luminescent supramolecular assemblies is of high
stable and ordered rod-like micelles in phosphate buffered saline, interest, since it avoids the need for additional labelling of
with a critical aggregation concentration below 200 nM. Aggrega- monomers. In addition self-assembling luminophores that
tion induced emission of the luminophore confirms the high stabi- operate in luminescence “turn-on” mode further allow one to
lity of the anisotropic assemblies in serum.
probe and track self-assembled states in more complex
5
0,51
media,
for example serum where highly abundant proteins
Self-assembly of small molecules into macromolecular and
polymeric architectures in water has been an increasingly
popular synthetic strategy in organic materials chemistry since
the pioneering work on supramolecular polymers 25 years
make the use of electron microscopy and conventional lumine-
scence “turn-off” probes very challenging.
2
The self-assembly unit is based on a C -symmetric 4,5-bis-
(
phenylthio)phthalonitrile (BPTP) as the central hydrophobic
1
,2
ago. The use of directional non-covalent interactions in the
molecular subunits can yield highly ordered one-dimensional
moiety extended with a β-sheet encoded alternating phenyl
alanine–histidine peptide sequence (FHFHF) which bears a
C-terminal hydrophilic polycationic dendron (Fig. 1A). The
3
–9
supramolecular assemblies.
These display dynamic behav-
iour and possess biological function that are not accessible
52
synthetic route is based on a convergent approach. We have
1
0
through conventional covalent polymers. Particularly power-
ful design motifs to produce nanoscopic and ordered polymers
in water are synthetic oligopeptides, using for example cyclic
incorporated hydrophobic FH alternating amino acid
sequences in the side arm of the self-assembly unit, including
an apolar hexyl-spacer separating the hydrophobic block from
the water solubilising dendritic amine block (Fig. 1). The BPTP
luminescent core was prepared by reacting 4,5-dichlorophthalo-
nitrile first with 4-hydroxythiophenol, then with bromo-tert-
butyl acetic acid in two high yielding steps. After deprotection
of the C-terminal carboxylic acid, PyBOP mediated amidation
of 4 with the protected dendronised peptide 8, and subsequent
deprotection of the histidine as well as the primary amine
functional groups yielded the target molecule 1. The detailed
synthesis, purification and full characterisation of all inter-
mediates can be found in the ESI.†
1
1,12
13–15
16,17
designs,
aliphatic
or aromatic amphiphiles,
Promising biological function
and
1
8,19
20
dendritic oligopeptides.
2
1–28
arises through applications as drug delivery vehicles,
2
9–32
33,34
vaccines,
imaging agents
or supports for tissue
1
5,35–38
engineering.
To date, we have focused our attention on preparing
dynamic supramolecular nanorods obtained via the self-
1
9,39–42
assembly of C -symmetric amphiphilic peptides,
and
3
luminescent spherical micelles or 2D-sheets based on linear
4
3
Au(I)-metallopeptides. Here we report a new C -symmetric
2
In order to investigate the morphology of self-assembled
C2-symmetrical peptide amphiphile 1, we carried out negative
stain transmission electron microscopy (TEM) experiments.
a
Institute of Organic Chemistry, Johannes Gutenberg-Universität Mainz, Duesbergweg
40
1
0-14, D-55128 Mainz, Germany. E-mail: besenius@uni-mainz.de;
Solutions of 1 at 100 µM concentrations in Tris buffer (10 mM)
at pH 7.4 showed one-dimensional semiflexible objects with a
length that could not be resolved (>500 nm) and an average
thickness of 7.0 ± 1.4 nm (Fig. 1B). The diameter of the rods is
twice as large as expected compared to the approximated
chain extended length of a single molecule of about 3.7 nm.
The semi-flexible rods are therefore best described as
Tel: +49-6131-39-22355
b
Institute of Physical Chemistry, Johannes Gutenberg-Universität Mainz,
Duesbergweg 10-14, D-55128 Mainz, Germany
c
Institute of Organic Chemistry, University of Duisburg-Essen, Universitätsstrasse 7,
D-45117 Essen, Germany. E-mail: jens.voskuhl@uni-due.de; Tel: +49-201-183-2404
†
Electronic supplementary information (ESI) available: Additional Fig. S1–S6,
detailed Experimental section: materials, synthesis, characterisation, analysis,
methods and instrumentation. See DOI: 10.1039/c6ob00292g
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5
3
content of β-sheet secondary structure (Fig. 2). This indicates
that the high level of molecular order leading to the formation
of anisotropic nanostructures arises from the formation of
hydrogen-bonded β-sheet domains along the fibre axis,
which is well known for the self-assembly of peptide
1
3,54,55
amphiphiles.
In order to confirm that the self-assembly is driven by the
hydrophobicity of the BPTP moiety and the peptide sequence,
assisted by hydrogen bonding we performed disassembly
experiments using a chemical denaturant. The addition of an
UV-transparent organic solvent like methanol leads to the dis-
ruption of the hydrophobic shielding of the hydrogen bonding
3
9,41,42
sequences and therefore disassembly of 1.
CD spectra
show the disappearance of the β-sheet characteristic negative
CD band at 216 nm and a new positive CD band at 220 nm
appears, which we have previously assigned to molecularly dis-
solved building blocks based on alternating hydrophobic and
hydrophilic sequence pairs of phenyl alanine and glutamic
4
0,52
acid (FEFEF) or phenyl alanine and lysine (FKFKF).
It is
interesting to point out that by lowering the pH value, we
could not observe the same changes in the CD spectra
compared to the spectra recorded in methanol. We initially
expected the protonation of the histidine to increase the hydro-
philicity of the peptide side arms and thereby induce dis-
assembly, similar to our previous work on the C
FXFXF sequences. Note that a reduction in the β-sheet band at
16 nm by 31% is indicative of a decrease in the degree
3
-symmetric
2
of order, but not full disassembly, likely due to protonation
and swelling in the peptide core. These pH-titrations
are indicative of highly stable assemblies. In summary, the
TEM and CD experiments in buffered aqueous media demon-
strate the self-assembly into ordered and stable rod-like
micelles.
2
Fig. 1 (A) Schematic representation of the self-assembly of C -sym-
metric cationic peptide amphiphiles
1 into luminescent rod-like
micelles; (B) negative stain TEM image of the rod-like micelles (de-
posited from a 100 μM solution of 1 in Tris buffer (10 mM, pH = 7.4)) and
the distribution of the rod diameter.
micellar-like arrangement of amphiphiles with the hydro-
phobic block pointing into the core of one-dimensional assem-
blies, solvated via the cationic dendrons in the shell.
Circular dichroism (CD) spectroscopy experiments were
carried out in order to gain insight into the molecular order of
these nanostructures. The CD spectra of solutions of 1 ana-
lysed via TEM showed a minimum at around 216 nm, which is
Fig. 2 CD experiments of self-assembled 1 in buffered water (Tris
buffer, 10 mM) at pH values of 7.4 (green curve, 100 µM) and 3.2 (red
curve, 100 µM) and the disassembled state recorded in methanol (black
characteristic of supramolecular assemblies with a high curve, 25 µM).
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To investigate whether the luminescence properties of
Using the aggregation induced luminescence properties of
BPTP can be utilised to probe the self-assembly of 1 in various 1, we further investigated the stability of the rod-like micelles
media, we first of all performed a series of photoluminescence towards dilution and aimed to determine the critical aggre-
spectroscopy measurements of 1 in DMSO/H
Fig. 3A). Similarly to MeOH,‡ DMSO is an efficient denaturant buffer (10 mM, pH 7.4) from 12.5 µM to 200 nM leads to a
since it is known to disrupt hydrogen bonded assemblies in linear decrease of the aggregate specific emission intensity
2
O mixtures gation concentration (CAC). The dilution of 1 in phosphate
(
5
6,57
water.
–40 vol% we observe an increase in the emission band at mine the CAC since it is below the detection limit of our
em = 460 nm (Fig. 3B). At 50 vol% H O the emission intensity luminescence spectrometer, but we can estimate an upper
Upon increasing the water content in DMSO from (Fig. S1†) at λem = 460 nm. It was thus not possible to deter-
0
λ
2
reaches a plateau value, which is indicative of a fully aggre- limit of 200 nM. To further investigate the high stability of
gated state. The observed aggregation induced emission (AIE) the self-assembled dendronised cationic amphiphile 1, we
of 1 most likely arises from a combination of the restriction of performed pH dependent photoluminescence experiments,
4
9
intramolecular motions (RIM) and twisted intramolecular complementary to CD spectroscopic investigations (Fig. 4A).
5
8
charge transfer (TICT). In the aggregated state the intra- By lowering the pH from 7.4 to pH 3.2, the aggregate
molecular rotations and vibrations of 1 are restricted, blocking specific emission band λem = 460 nm decreases, but only by
radiationless relaxation channels and promoting the radiative 10% in intensity. The TEM images of 1 (100 µM) at pH 3.2
decay pathway. Furthermore, solvatochromic effects like TICT
could influence the luminescence, since in DMSO the molecu-
larly dissolved 1 is exposed to a highly polar environment,
while upon aggregation in H O the luminophore is embedded
2
in the apolar inner core of the micelles.
Fig. 3 (A) Photoluminescence emission of 1 (25 µM) in DMSO/H
25 mM phosphate buffer, pH 7.4) mixtures. (B) Intensity of the photo-
luminescence emission at λem = 460 nm (λexc = 285 nm) as a function of
2
O
(
Fig. 4 (A) pH dependent photoluminescence measurements in 25 mM
phosphate buffer. (B) Negative stain TEM image of the rod-like micelles
deposited from a 100 μM solution of 1 at pH 3.2 (10 mM Tris buffer) and
the distribution of the rod diameter.
2
water content in vol%. (C) Photograph of compound 1 in buffered H O
and in DMSO under UV light irradiation.
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reveal the presence of semi-flexible nanorods with a uniform
thickness of 6.8 ± 0.8 nm, thereby confirming that the
assemblies are very stable and stay intact even after protona-
tion of the imidazole groups. The addition of a base to the
pH 3.2 solution of 1 restores the aggregate specific band to
its original intensity at a neutral pH of 7.2 (Fig. 4A). This
observation is in agreement with our tentative conclusion
from the pH dependent CD data. Reversible protonation of
the histidine side chain residues leads to an increased
hydrophilicity and lateral swelling of the ordered core in the
rod-like micelles.
To corroborate our hypothesis from the CD and lumine-
scence spectroscopic data, we further performed encapsulation
experiments with Nile Red (NR), a hydrophobic solvatochromic
dye, in order to probe the polarity of the core of the rod-like
micelles (Fig. S2†). When adding Nile Red (5 µM) to a solution
of 1 (25 µM) in phosphate buffer at pH 7.4, we observed a fluo-
rescence emission at λem = 621 nm (λexc = 550 nm). This value
compares very well with the published studies using encapsu-
lated NR in lipid bilayers, peptide aggregates and artificial
5
9–65
supramolecular systems,
and suggests that the dye is
incorporated in an apolar microenvironment. When reducing
the pH of a solution of self-assembled 1 and encapsulated NR
to pH 2.8, we observed a weak bathochromic shift of 4 nm to
λ
em = 625 nm, and a decrease in the fluorescence intensity of
%. Both indicate that the microenvironment becomes slightly
4
more hydrophilic but that the NR dye remains encapsulated
within the assemblies. In summary, we have used a host of
optical spectroscopic techniques to probe the molecular order
and stability of the rod-like micelles. Hydrophobic desolvation
and the β-sheet secondary structure lead to one-dimensional
self-assembly, which is not switched off by protonation of the
basic side chain moieties in the peptide core. In contrast to
the linear oligohistidine peptide amphiphiles reported by the
6
6
Fig. 5 (A) Negative stain TEM image of the rod-like micelles deposited
from a 100 μM solution of 1 in pH 7.4 phosphate buffered saline (PBS)
and the distribution of the rod diameter. (B) Photoluminescence
Stupp lab, a decrease in the pH therefore does not lead to a
disassembly of the nanostructures, but rather swelling of the
hydrophobic core.
−1
measurements of BSA (520 µM, 33 mg ml ) in isotonic PBS with 1
The high stability of the FHFHF based supramolecular (25 µM, 100 µg ml− , red curve) and without 1 (black curve).
1
nanostructures was investigated in physiologically relevant
media, under isotonic conditions and finally also in the pres-
ence of serum albumin. A solution of 1 (100 µM) in isotonic
7
0,71
phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, molecules.
The addition of bovine serum albumin (BSA,
pH 7.4) was characterised using TEM. Note that we usually use 520 µM, 33 mg ml− ) to a solution of 1 (25 µM, 100 µg ml ) in
1
−1
Tris buffer to perform negative stain TEM in order to avoid PBS buffer does not lead to disassembly of the aggregates,
4
0,67
artefacts due to phosphate salts.
Nevertheless, after depo- based on photoluminescence spectroscopy. The characteristic
sition of a solution of 1 in PBS buffer on the TEM grids, rod- emission band of 1 monitored at λem = 460 nm was detected
like nanostructures can clearly be observed in the electron over the autofluorescence of the serum albumin, which was
microscopy images, with an average diameter of 7.1 ± 1.1 nm present in a 330 fold excess by weight. These findings finally
(
Fig. 5A). Finally we used the aggregation induced lumine- confirm the excellent stability of the rod-like assemblies
scence of self-assembled 1 in order to probe the stability of the based on C -symmetric peptide amphiphiles 1, even in dilute
2
assemblies in artificial serum, which is critical for applications solutions and in the presence of a huge excess of BSA (Fig. 5B).
in supramolecular drug delivery vehicles and in synthetic vac- We have thereby designed a very robust peptide based self-
cines. Albumin accounts for approximately 60% of the proteins assembly building block using a 4,5-bis-(phenylthio)phthalo-
6
8
in blood, and is known to strongly effect the self-assembly of nitrile central core that enables us to probe aggregation pro-
3
4,69
amphiphiles due to molecular crowding effects
and to cesses in a complex and physiologically relevant serum-like
disrupt the formation of micelles by the absorption of single medium.
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1
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