Tuning the pH-triggered self-assembly of dendritic peptide amphiphiles using fluorinated side chains

Article abstract of DOI:10.1039/c4ob02185a

We report the synthesis of a series of anionic dendritic peptide amphiphiles of increasing hydrophobic character. By establishing state diagrams we describe their pH and ionic strength triggered self-assembly into supramolecular nanorods in water and highlight the impact of hydrophobic shielding in the supramolecular polymerisation process. Via the incorporation of fluorinated peptide side chains the pH-triggered monomer to polymer transition at physiological ionic strength is shifted from pH 5.0 to pH 7.4. We thereby show that compensating attractive non-covalent interactions and hydrophobic effects with repulsive electrostatic forces, a concept we refer to as frustrated growth, is a sensitive tool in order to manipulate one-dimensional supramolecular polymerisation processes in water.

Full text of DOI:10.1039/c4ob02185a

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Tuning the pH-triggered self-assembly of dendritic
peptide amphiphiles using fluorinated side chains†
Cite this: DOI: 10.1039/c4ob02185a
Ralph Appel,a,b Sebastian Tacke,c Jürgen Klingaufc and Pol Besenius*a,b
We report the synthesis of a series of anionic dendritic peptide amphiphiles of increasing hydrophobic
character. By establishing state diagrams we describe their pH and ionic strength triggered self-assembly
into supramolecular nanorods in water and highlight the impact of hydrophobic shielding in the
supramolecular polymerisation process. Via the incorporation of fluorinated peptide side chains the
pH-triggered monomer to polymer transition at physiological ionic strength is shifted from pH 5.0 to pH 7.4.
We thereby show that compensating attractive non-covalent interactions and hydrophobic effects with
repulsive electrostatic forces, a concept we refer to as frustrated growth, is a sensitive tool in order to
manipulate one-dimensional supramolecular polymerisation processes in water.
Received 15th October 2014,
Accepted 12th November 2014
DOI: 10.1039/c4ob02185a
www.rsc.org/obc
well, as shown by the groups of Ulijn and Adams,28–32 yielding
enzyme or pH-responsive hydrogels.33,34
Introduction
Fabrication of nanoscale materials using supramolecular non-
We have recently focused on developing strategies to manip-
covalent interactions provides access to many exciting material ulate one-dimensional (1D) supramolecular polymerisation
properties. Compared to traditional covalent synthetic processes in water. For that purpose we used the concept of
approaches, the self-assembly of molecular building blocks to frustrated growth that relies on compensating attractive non-
supramolecular polymeric morphologies and nanostructures covalent interactions with repulsive steric and electrostatic
leads to dynamic materials,1–6 that are responsive towards forces. The supramolecular synthons we have used in an
optical,7–9 mechanical10,11 or biological12 stimuli. Some of the earlier study consisted of dendritic peptide amphiphiles,
attractive features for supramolecular materials that arise based on a small benzene-1,3,5-carboxamide branching agent.
thereof are self-healing properties,10,13 controlled release of C3-symmetrical hydrophobic cores are indeed known to direct
cargo,8,14–16 or emergent behaviour like self-replication.11,17,18 the supramolecular polymerisation into strictly 1D mor-
In the area of aqueous responsive supramolecular systems, phologies with negligible secondary aggregation.35–44 We have
peptide materials such as nanofibres,19–24 nanotubes11,25 and reported the synthesis of dendritic nonaphenylalanines
artificial β-barrels26 have attracted much interest. Particularly which were linked to peripheral carboxylic acid Newkome
aliphatic β-sheet encoded peptide amphiphiles, functionalised dendrons45,46 via a 3-(hydroxypropylthio)propionic acid type
with hydrophobic alkyl tails and charged hydrophilic head linker. Similarly to the examples of van Esch,35 Hartgerink,47
groups, as developed by Stupp and coworkers,19 are prominent Goldberger48 and others49–52 we were able to study the
building blocks for the fabrication of bioactive gels and sup- pH-dependent self-assembly.44 Very recently we have then
ports for tissue engineering and regenerative medicine.21,27 expanded the core of the branching agent, using 3,4-ethylene-
Aromatic peptide amphiphiles have shown growing interest as dioxythiophene (EDOT) extended 1,3,5-substituted benzene:
using dendritic hexaphenylalanine we realised that the loss of
non-covalent interactions due to the omission of one phenyl-
alanine per side arm in the supramolecular monomer, was
aOrganic Chemistry Institute, Westfälische Wilhelms-Universität Münster,
compensated by the larger π-system of the hydrophobic core.53
Corrensstrasse 40, 48149 Münster, Germany. E-mail: p.besenius@uni-muenster.de;
We hereby aim to systematically study the effect of increas-
Tel: +49 251 83 63928
ing the hydrophobic shielding of dendritic peptide amphi-
philes, and the impact on the pH and ionic strength triggered
self-assembly into supramolecular nanorods (Fig. 1). By estab-
lishing state diagrams for three different molecules of varying
hydrophobic character, we show that the supramolecular poly-
merisation is fully switched off if the apolar hexyl linker
between the dendritic nonaphenylalanines and peripheral
bCenter for Nanotechnology (CeNTech), Heisenbergstrasse 11, 48149 Münster,
Germany
cDepartment of Cellular Biophysics, Institute of Medical Physics and Biophysics,
Westfälische Wilhelms-Universität Münster, Robert-Koch-Strasse 31, 48149 Münster,
Germany
†Electronic supplementary information (ESI) available: Experimental proce-
dures, materials synthesis and characterisation, additional CD data, negative
stain TEM and STEM micrographs. See DOI: 10.1039/c4ob02185a
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Results and discussion
Synthetic routes
The synthesis of the dendritic peptide amphiphiles 6 and 12
followed a convergent approach (Scheme 1A; full details can be
found in the ESI†). First, the Cbz-protected tri-L-phenylalanine
2 was synthesized using conventional solid-phase peptide syn-
thesis (SPPS). Using standard peptide coupling reagents, the
tert-butyl protected Newkome dendron46 1, or 6-aminohexa-
noic acid extended dendron 8 were coupled to 2 in order to
obtain the Cbz-protected dendronised triphenylalanines 3 and 9.
After hydrogenation using Pd/C as catalyst the amine deriva-
tives 4 and 10 were coupled to 1,3,5-benzenetricarbonyl tri-
chloride to obtain the branched self-assembly units 5 and 11.
Finally after acidic deprotection of 5 and 11, the dendritic
nona-phenylalanine peptide amphiphile 6 was obtained after
four steps in a moderate non-optimised overall yield of 13%,
and 12 after four steps in a non-optimised overall yield of 22%
(Scheme 1A). In 6 the hydrophobic core and hydrophilic
Fig. 1 (A) Chemical structures of the three dendritic peptide amphi-
philes 6, 12 and 18. (B) The schematic representation of their pH and
ionic strength triggered self-assembly into one-dimensional (1D)
nanorods.
carboxylic acid Newkome dendrons is omitted. If the hydro-
phobic character of the peptide core is increased further by
introducing trifluoromethyl groups via thiol–ene click chem-
istry in O-allyl derivatised tyrosine groups, the thermodynamic
driving force for polymerisation is increased and the pH-trig-
gered monomer to polymer transition at physiological ionic
strength is shifted from pH 5.0 to pH 7.4. Note that the fluori-
nated analogues of hydrophobic aliphatic amino acids have
been introduced in peptide structures in the past:54–62 it was
for example shown that the thermodynamic stability of coiled-
coil motifs was increased due to an increased hydrophobic
core;63,64 very recently it has been shown that an increased flu-
orine content increases the hydrophobic character of peptides,
decreases their α-helix propensity and increases the formation
rates of β-sheet-rich amyloids.65
We are particularly interested in well-defined and stimuli-
responsive nanomaterials66–74 that are designed to undergo
morphological transitions triggered by specific changes in a
physiological environment. These hold much promise for
applications in biomedical imaging and therapy.75 We are
planning to develop methodologies whereby the acidity in the
microenvironment of a tumour interior or inflamed tissue
induces the triggered self-assembly of small molecules into
larger nanorods. The additional benefit of fluorinated building
blocks is their potential use in 19F-MRI,76–78 which would
allow to quantify the selective tumour retention and accumu-
lation in cancer diagnosis and therapy.79
Scheme 1 Synthetic schemes for the preparation of the dendritic peptide
amphiphiles 6, 12 and 18: (A) (i) PyBOP, DIPEA (64%); (ii) H2, Pd/C (91%); (iii)
benzene-1,3,5-tricarbonyl trichloride, DIPEA (50%); (iv) TFA–DCM, 1 : 1
(44%); (v) PyBOP, DIPEA (81%); (vi) H2, Pd/C (89%); (vii) benzene-1,3,5-tri-
carbonyl trichloride, DIPEA (63%); (viii) TFA–DCM, 1 : 1 (48%); (B) (ix) PyBOP,
DIPEA (71%); (x) piperidine, CH3CN (44%); (xi) benzene-1,3,5-tricarbonyl tri-
chloride, DIPEA (50%); (xii) TFA–DCM, 1 : 1 (93%), (xiii) trifluoroethanethiol,
DMPA, THF/H2O, hν (38%).
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dendron are directly connected, while 12 contains an apolar prevent supramolecular polymerisation into large macro-
hexyl linker between the same dendritic nonaphenylalanine molecular structures. Surprisingly we were not able to screen these
core and the peripheral carboxylic acid Newkome dendron.
charge-charge repulsive interactions by increasing the ionic
In order to create a dendritic core that is more hydrophobic strength up to 1 M NaCl at pH 9.0 (Fig. 2A) or by acidifying the
than the one in 12, we incorporated a L-phenylalanine-di- buffer to pH 6 (Fig. 2B). Since the CD spectra are identical in
(O-allyl-L-tyrosine) into the same convergent synthesis as pH 9.0, 7.4 and 6.0, both at low and high ionic strength
described before (Scheme 1B) including an apolar hexyl linker (Fig. S1†) it thereby becomes apparent that an apolar spacer
between the dendritic nonapeptide core and the peripheral between the hydrogen bonding hydrophobic core and hydro-
carboxylic acid Newkome dendron. The allyl ether functional philic shell is critical for self-assembly.
groups are very useful since they can easily be modified via
This was verified by investigating the supramolecular poly-
thiol–ene type click chemistry,44,80,81 using 2,2,2-trifluoroethane- merisation of the dendritic peptide amphiphile 12 in full
thiol to incorporate fluorinated side chains into the hydro- detail. At neutral pH 7.4, in 10 mM phosphate buffer and 0 M
phobic peptide block. Note that we had to install a NaCl, a CD spectrum with at weak negative band at 210 nm is
phenylalanine amino acid on the N-terminus rather than a observed, that is very similar to the one obtained for com-
protected tyrosine, since we encountered very low yields when pound 6 (Fig. 3 and S2†). At pH 7.4, the addition of NaCl leads
coupling tri-(O-allyl-tyrosine) derivatives to the benzenetri- the appearance of a positive band at λ = 203 nm, to a shift of
carbonyl trichloride core, most likely due to steric issues. Using the negative band from λ = 210 nm to 220 nm and a significant
conventional Fmoc-SPPS chemistry we prepared Fmoc-phenyl- increase in the intensity. This is a good indication for the self-
alanine-di-(O-allyl-L-tyrosine). In four similar steps as assembly into ordered chiral supramolecular polymers. By
described for compound 12 the amphiphiles 17 was success- electrostatic screening of the negative charges in the periphery
fully obtained. After UV-initiated thiol–ene click chemistry of the supramolecular nanorods, the polymerisation becomes
using 2,2-dimethoxy-2-phenylacetophenone (DMPA) in a THF– more favourable.44,53 Similarly after lowering the pH from 7.4
water mixture 1 : 1 we isolated the target fluorinated dendritic to pH 5.0 the same transitions in the CD spectra are observed,
peptide amphiphile 18, after five steps in a non-optimised which indicates the formation of supramolecular polymers.
overall yield of 6% (Scheme 1B).
These findings suggest that at physiological pH the self-assem-
bly of the polyanionic dendritic peptide 12 is dictated by a
mechanism of frustrated growth: attractive supramolecular
interactions, hydrogen-bonding and hydrophobic effects, that
drive the monomers to polymerise are opposed by repulsive
Coulomb interactions which disfavour self-assembly.35,37,39,44,53
The multistimuli-responsive self-assembly of these peptidic
supramolecular polymers can be most conveniently visualised
in an ionic strength and a pH dependent contour plot, which
we refer to as state diagram‡ (Fig. 3D). It can clearly be
observed that at 100 mM NaCl the transition from a dis-
assembled state at pH 7.4 to a fully polymerised state at pH 5.0
is much sharper than the transition at pH 7.4, from 0 to 1 M
NaCl. Screening of the repulsive interactions by protonation of
the dendritic carboxylic acids occurs at a pH value between
Spectroscopic investigations into the pH and ionic strength
triggered self-assembly
The dendritic peptide amphiphiles 6, 12 and 18 equipped with
nine carboxylic acids groups turned out to be highly soluble in
aqueous buffers.
First, we investigated the self-assembly of dendritic peptide
amphiphile 6 in phosphate buffer using circular dichroism
(CD) spectroscopy. At pH 9.0 in 10 mM phosphate buffer, the
CD spectra suggest that no aggregation takes place (Fig. 2A
and S1†). This agrees with observations we have made
before:44 in slightly basic pH and at low ionic strength
repulsive Coulombic interactions from the charged dendritic
carboxylate groups in the hydrophilic shell of the peptides
Fig. 2 Ionic strength dependent CD spectra for dendritic peptide amphiphile 6 (62.5 × 10−3 mM, 293 K) in 10 mM phosphate buffer at pH 9.0 (A)
and pH 6.0 (B).
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Fig. 3 Ionic strength dependent CD spectra for dendritic peptide amphiphile 12 (62.5 × 10−3 mM, 293 K) in 10 mM phosphate buffer at pH 7.4 (A),
pH 6.0 (B) and pH 5.0 (C); (D) state diagram depicting the dimensionless degree of aggregation (0 referring to the molecularly dissolved state and 1
to a fully polymerised system, monitored at the CD band of λ = 220 nm) of 12 (62.5 × 10−3 mM), in 10 mM phosphate buffer at 293 K, as a function of
the pH and the concentration of added NaCl.‡1
pH 5.0 and pH 6.0, which is at least one unit higher than expected shows that the supramolecular polymerisation for the fluori-
for a common organic carboxylic acid in water. The pKa for a nated monomer 18 is clearly shifted towards higher pH values
Newkome-type dendritic carboxylic acid was reported to be and lower ionic strength compared to dendritic peptide
about 4.82 We have previously observed a similar shift in the amphiphile 12 (Fig. 3D and 4D). The pH-triggered monomer
apparent pKa value for amphiphilic dendritic carboxylic acids to polymer transition at physiological ionic strength is shifted
which we attributed due to be driven by the aggregation from pH 5.0 (for monomer 12) to pH 7.4 (for monomer 18).
process;44 this is a well-known phenomenon for polymeric The state diagram thereby highlights the importance of hydro-
weak acids and self-assembled fatty acids.83–85
phobic effects in the self-assembly of amphiphilic dendritic
Finally we investigated the self-assembly of the most hydro- carboxylic acids. The previously discussed apparent pKa shifts
phobic compound out of the small series of dendritic peptide (the intrinsic pKa of the dendritic carboxylic acid is about 4)82
amphiphiles 6, 12 and 18. CD bands for compound 18 become are increased by another two units in the case of monomer 18
red-shifted compared to 6 and 12, due to the presence of the compared to 12: due to the higher thermodynamic driving
functionalised O-allyl-tyrosine side chains (Fig. 4 and S3†). force for supramolecular polymerisation the carboxylic acids
At pH 9.0 and 0 M NaCl we expect the carboxylic acids to fully in the hydrophilic shell become weaker.
deprotonated and the dendritic amphiphiles to be molecularly
These findings are in good agreement with pKa sifts
dissolved. The CD spectra reveal a negative band at λ = 210 nm observed in the self-assembly of linear peptide amphiphiles:
and a positive band at λ = 225 nm. Upon lowering the pH from the Goldberger lab has reported amphiphiles based on a
pH 9.0 to pH 7.4, pH 6.0 and pH 5.0, or by increasing the ionic palmic acid tail coupled to a XAAAEEEE oligopeptide.48 Due to
strength, the observed bands shift significantly: a strong the electrostatic repulsion of the deprotonated glutamic acids
positive band at λ = 205 nm appears, the negative band shifts (E) at neutral pH, these molecules are present as isolated
from λ = 210 nm to λ = 220 nm, and the positive one from monomers at low concentration. By acidifying the solution
λ = 225 nm to λ = 230 nm. Both CD bands increase in intensity, self-assembly into nanofibers occurs, driven by the formation
while another additional weakly negative band at λ = 245 nm of intermolecular β-sheets. The balance of repulsive to
appears. The pH and ionic strength dependent contour plot attractive interactions can be manipulated by varying the
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Fig. 4 Ionic strength dependent CD spectra for dendritic peptide amphiphile 18 (62.5 × 10−3 mM, 293 K) in 10 mM phosphate buffer at pH 7.4 (A),
pH 6.0 (B) and pH 5.0 (C); (D) state diagram depicting the dimensionless degree of aggregation (0 referring to the molecularly dissolved state and
1 to a fully polymerised system, monitored at the CD band of λ = 230 nm) of 18 (62.5 × 10−3 mM), in 10 mM phosphate buffer at 293 K, as a function
of the pH and the concentration of added NaCl.‡
hydrophobic amino acid (X) embedded in the β-sheet forming intrinsic pKa of the base/acid is shifted, an observation which
region: if the driving force for β-sheet formation in the oligo- was summarized in the intermediate internal charge repulsion
alanine block XAAA is enhanced via the incorporation of a (ICR) model.86,87 Indeed, the delicate balance of attractive and
more hydrophobic amino acid ((tyrosine (Y), valine (V), phenyl- repulsive features in these barrel-stave supramolecules is not
alanine (F), or isoleucine (I), the propensity for β-sheet too dissimilar to the frustrated assembly term we have used to
formation follows the trend I > F > V > Y), the triggered self- describe the supramolecular 1D nanorod formation: lack of
assembly is shifted from pH 6.0 to pH 6.6; the apparent pKa of ICR has been thought to account for ‘implosion’, low ICR for
the oligoglutamic acid EEEE block is therefore shifted from contraction, high ICR for expansion, and excess ICR for
their intrinsic pKa of 4.7–4.9 by up to two units.
‘explosion’ of the β-barrels. Furthermore contributions of the
Similarly, weakening of the basic character of protonated surrounding bilayer have to be taken into account: the external
lysine and histidine side chains (apparent pKa of 7 and 4.5 membrane pressure (EMP) model is supposed to protect the
respectively), as well as a weakening of the acidic character of overcharged barrels from exploding and to promote the implo-
aspartic acid side chains (apparent pKa of 6) has been observed sion of undercharged barrels.86,87 This effect has important
by Matile and coworkers in the β-sheet directed self-assembly consequences for the catalytic activity of the synthetic pores.87
of p-octiphenyl β-barrel pores embedded in lipid bilayers.86
In summary, we have been able to build state diagrams,
Due to the spacial proximity and electrostatic repulsion of the which are well known for self-assembled natural spherical or
multiply charged bases or acids in these synthetic pores, the filamentous viruses,88–92 in order to investigate pH and ionic
strength dependent self-assembly of dendritic peptide amphi-
philes into supramolecular polymers in water. We have high-
lighted the impact of hydrophobic shielding in the
supramolecular polymerisation process: via the incorporation
of fluorinated peptide side chains the pH-triggered monomer
to polymer transition at physiological ionic strength is shifted
from pH 5.0 to pH 7.4.
‡The state diagrams were plotted from experimental pH and ionic strength
dependent data points. The contour plot was generated using the 2D Interp-
olation/Extrapolation function in OriginPro 9.1. The boundaries are approxi-
mately correct for a monomer concentration of 62.5 × 10−6 M at 293 K. They are
likely to shift, favouring aggregation, when the monomer concentration is
increased.
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Morphological characterisation of the supramolecular
nanorods via electron microscopy
We performed conventional negative stain transmission elec-
tron microscopy (TEM) and scanning transmission electron
microscopy (STEM) experiments in order to investigate the
morphology of the supramolecular polymers based on mono-
mers 12 and 18. Negative stain TEM images were recorded
after depositing a 1 mg mL−1 concentrated clear solution of
dendritic peptide 12 in 10 mM phosphate buffer at pH 7.4 and
500 mM NaCl on carbon film coated EM grids (Fig. 5A). Note
that after deposition, the EM grids were washed with an
aqueous 2% w/v uranyl acetate solution for 15 s to remove the
inorganic salts and in order to fixate the organic peptidic
material,93 before blotting excess liquid with filter paper. The
TEM micrographs clearly show the presence of nanorods with
a diameter of approximately 5 nm in diameter and a contour
Fig. 6 STEM image of the dendritic peptide amphiphile 18, deposited
length distribution ranging from about 150 to 200 nm on an EM grid covered with an amorphous carbon film from
a
1 mg mL−1 clear solution of 18 in 10 mM phosphate buffer, pH 5.0 and
(Fig. 5A). The morphology thereby justifies the terminology of
1D nanorod-like supramolecular polymers. STEM experiments
were furthermore performed using the same solution of 12 to
0 mM NaCl; the scale bar represents 500 nm.
investigate the influence of the staining agent and washing
steps on the morphology of the nanorods. After omitting the
washing and staining procedures, STEM micrographs reveal
the presence of nanorods, albeit with a much higher density
on the carbon film coated TEM grid (Fig. S4†).
In contrast, conventional negative stain TEM experiments
of the monomer solution of 12 at pH 7.4 and 0 M NaCl only
shows small nanostructures of around 10 nm in size, which
are likely monomers or very small oligomers (Fig. 5B). Screen-
ing of the repulsive interactions in the anionic amphiphilic
building blocks leads to a monomer–polymer transition at
higher ionic strength, as expected based on our previous find-
ings.44 Note that ionic strength dependent lengths profiles
have also been reported in worm-like micellar solutions of
charged surfactants.94–98 These results are therefore in full
agreement with the detailed pH and ionic strength titrations
probed via CD spectroscopy.
Finally we were also able to visualise the pH-triggered for-
mation of supramolecular polymers from fluorinated amphi-
philic monomer 18. At pH 5.0 and 0 mM NaCl STEM images
show the presence of nanorods (Fig. 6). At this pH, the proto-
nation of the dendritic carboxylic acid side chains is likely to
favour bundling of the nanorods into thin fibres. By increasing
the ionic strength at pH 5.0 from 0 mM NaCl to 500 mM NaCl,
the tendency for bundling becomes more pronounced and
larger structures were observed in these samples (Fig. S5†). In
summary, negative stain TEM and STEM experiments corrobo-
rate the CD investigations and the pH and ionic strength trig-
gered formation of one-dimensional supramolecular polymers,
with a nanorod-like morphology.
Fig. 5 TEM images of the negative stained dendritic peptide amphiphile
12, deposited on a carbon coated EM grid from a 1 mg mL−1 clear solu-
tion of 12 in 10 mM phosphate buffer, pH 7.4 and 500 mM NaCl (A) and
0 mM NaCl (B) (negative staining was performed using 2% w/v uranyl
acetate), the scale bars represent 200 nm.
Experimental
Unless stated otherwise, all reagents and chemicals were
obtained from commercial sources at the highest purity available
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and used without further purification. Water was demineral-
(5) 4 (159 mg; 0.169 mmol; 3.0 eq.) and 1,3,5-benzenetricar-
ised prior to use. Some solvents were dried using the follow- bonyl trichloride (15 mg; 0.056 mmol; 1.0 eq.) were dissolved
ing drying agents: dichloromethane over sodium hydride, under argon in dry DMF (5 mL) and DIPEA (109 mg;
tetrahydrofurane over sodium and benzophenone. Purification 0.845 mmol; 15.0 eq.) were then added. The reaction mixture
via preparative flash column chromatography was carried out was stirred at room temperature for 20 h, after 2 h PyBOP
using silica gel with an average grain size of 15–40 µm (179 mg; 0.344 mmol; 7.0 eq.) was added to react hydrolysed
(MERCK). Technical grade solvents that were used as a mobile acid chloride with remaining amine 10. The organic solvent
phase were distilled before use. Analysis of the collected frac- was removed under reduced pressure. The residue was purified
tions was performed via TLC on silica coated aluminum sheets via precipitation in water and isolated via centrifugation. The
(60 F254, MERCK). Size exclusion chromatography was carried residue was purified again via SEC in methanol. Rf (DCM–
out using Sephadex™ LH-20 beads (GE Healthcare Bio- EtOAc 1 : 1 + 20 vol% MeOH, SiO2) = 0.90. Yield: 83 mg
Sciences, Uppsala) as stationary phase and distilled methanol (0.028 mmol, 50%) C165H222N12O39, light brownish solid.
as mobile phase or Sephadex™ G-25 beads (GE Healthcare 1H NMR (300 MHz, DMSO-d6): δ [ppm] = 8.70–8.54 (m, 3H,
Bio-Sciences, Uppsala) as stationary phase and demineralised NH), 8.43–7.97 (m, 9H, NH and CHaroBTA), 7.60–6.82 (m, 48H,
CHaro and NH), 4.80–4.49 (m, 9H, CHα), 3.67–3.44 (m, 36H,
Phe
water as mobile phase.
The NMR-spectra were recorded on the spectrometers CH2OCH2), 2.90–2.58 (m, 18H, CH2Phe), 2.47–2.33 (m, 18H,
AV 300, ARX 300 and AV 400 (BRUKER). All measurements were OCH2CH2), 1.37 (s, 81H, CH3tBu). MALDI-MS (positive mode)
carried out in deuterated solvents. The chemical shift (δ) is (DHB [EtOAc]): m/z calcd for [M + Na]+ 3020.57; found: 3020.71
recorded in parts per million (ppm) and relative to the residual [M + Na]+.
solvent protons.99 Mass spectra were recorded on the electro-
(6) 5 (80 mg; 0.027 mol) was stirred three times in 3.0 mL of
spray ionisation spectrometers (ESI) Micro Tof (BRUKER) and a mixture of TFA and DCM (1 : 1) for 1 h. The organic solvent
Orbi-Trap LTQ-XL (THERMO SCIENTIFIC). Molecules of a high was removed in each step under reduced pressure. Finally the
molecular weight were analysed using matrix assisted laser residue was taken up in water and freeze-dried over night.
desorption ionization time of flight (MALDI-TOF) spectrometry Yield: 29 mg (0.012 mmol, 44%) C129H150N12O39, white solid.
using an Autoflex Speed (BRUKER). CD spectra were recorded 1H NMR (300 MHz, DMSO-d6): δ [ppm] = 12.18 (brs, 9H,
on a JASCO J-815.
COOH), 8.69–8.52 (m, 3H, NH), 8.39–7.97 (m, 9H, NH and
Phe
For transmission electron microscopy (TEM) investigations, CHaroBTA), 7.59–6.82 (m, 48H, CHaro
and NH), 4.78–4.51
standard 200 mesh EM grids, covered with a carbon coated (m, 9H, CHα), 3.75–3.41 (m, 36H, CH2OCH2), 3.05–2.64
plastic foil (Pioloform), were utilized. To render the support (m, 18H, CH2Phe), 2.47–2.33 (m, 18H, OCH2CH2). MALDI-MS
film more hydrophilic, the EM grids were plasma cleaned for (positive mode) (DHB [H2O/ACN]): m/z calcd for [M + Na]+
30–45 seconds. For sedimentation, 2–3 μL sample material was 2515.00; found: 2515.00 [M + Na]+.
left 1 min and afterwards the residual material was blotted.
(11) 10 (171.5 mg; 0.162 mmol; 3.3 eq.) and 1,3,5-benzene-
Thereafter, the grids were washed once with 2–3 μL 2% w/v tricarbonyl trichloride (13.0 mg; 0.049 mmol; 1.0 eq.) were dis-
uranyl acetate (15 s). Followed by a second blotting step, the solved under argon in dry DMF (5 mL) and DIPEA (82 mg;
sample material was then stained by uranyl acetate for 45 s, 0.64 mmol; 4.0 eq.) were then added. The reaction mixture was
blotted and left for air drying. The samples were examined on stirred at room temperature for 18 h. The organic solvent was
a Philips CM10 (FEI, Eindhoven, the Netherlands) operated at removed under reduced pressure. The residue was purified via
80 kV. Electron micrographs were recorded by a side-mounted precipitation from THF into a mixture of n-pentan and diethyl-
CCD camera (IDS, Obersulm, Germany) utilizing acquisition ether (1 : 1) and then isolated via centrifugation. The residue
software written in LabView.
was purified via flash chromatography over SiO2 (DCM–EtOAc–
For scanning transmission electron microscopy (STEM) MeOH 1 : 1 : 1), Rf (DCM–EtOAc–MeOH 1 : 1 : 1, SiO2) = 0.50.
investigations, EM high-resolution grids were utilised. Here, a Yield: 105 mg (0.031 mmol, 63%) C183H255N15O42, light brown-
1
holey carbon film is covered with a 2–4 nm thick amorphous ish solid. H NMR (300 MHz, DMSO-d6): δ [ppm] = 8.72–8.61
carbon film, which acts as a support for the sample material. (m, 3H, NH), 8.41 (d, J = 8.5 Hz, 3H, NH), 8.25–7.99 (m, 9H,
BTA
As in the case for TEM investigations, the grids were plasma CHaro
and NH), 7.80 (t, J = 5.6 Hz, 3H, NH), 7.33–6.87 (m,
cleaned prior to the sample preparation. Since no staining was 48H, CHaro and NH), 4.82–4.38 (m, 9H, CHα), 3.59 (m, 36H,
Phe
performed for STEM investigations the preparation protocol CH2OCH2), 3.08–2.67 (m, 24H, CH2 and NHCH2Ahx), 2.36 (t,
Phe
was reduced to the following steps: 2–3 μL sample material J = 6.3 Hz, 18H, OCH2CH2), 2.01 (t, J = 7.0 Hz, 6H, CH2CONAhx),
tBu
was left for sedimentation (1 min). Hereafter, grids were either 1.50–1.28 (m, 99H, CH3
and CH2Ahx). MALDI-MS (positive
washed briefly with buffer or blotted directly and left for air mode) (DHB [EtOAc]): m/z calcd for [M + Na]+ 3357.82; found:
drying. The samples were examined in a high-resolution in- 3357.86 [M + Na]+.
lens scanning electron microscope (S-5000, HITACHI, Japan),
equipped with a homemade annular dark-field (ADF) detector. 2.5 mL of a mixture of TFA and DCM (1 : 1) for 1 h. The
ADF images were taken at 30 kV acceleration voltage. organic solvent was removed after each step under reduced
Full experimental procedures, materials synthesis and pressure. Finally the residue was taken up in water and
(12) 11 (105 mg; 0.031 mol) was stirred three times in
characterisation for compounds 1–18 can be found in the ESI.†
freeze-dried over night. Yield: 42 mg (0.015 mmol, 48%)
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C147H183N15O42, white solid. 1H NMR (300 MHz, DMSO-d6): with water as a mobile phase. Yield: 13.5 mg (0.0035 mmol,
δ [ppm] = 12.14 (brs, 9H, COOH), 8.73–8.58 (m, 3H, NH), 38%), C177H225F18N15O48S6, white solid. 1H NMR (300 MHz,
8.49–8.32 (m, 3H, NH), 8.15 (s, 3H, CHaroBTA), 8.10– DMSO-d6): δ [ppm] = 8.71–8.61 (m, 3H, NH), 8.41–8.06 (m, 9H,
7.96 (m, 3H, NH), 7.84–7.62 (m, 3H, NH), 7.33–6.87 (m, 48H, NH and CHaroBTA), 7.89–7.76 (m, 3H, NH), 7.27 (d, J = 7.4 Hz,
Phe
Haro
and NH), 4.83–4.37 (m, 9H, CHα), 3.53 (m, 36H, 6H, CHaroTyr), 7.21–7.06 (m, 21H, CHaroPhe and Tyr), 6.93 (s, 3H,
Phe
CH2OCH2), 3.08–2.67 (m, 24H, CH2
and NHCH2Ahx), NH), 6.84–6.64 (m, 12H, CHaroTyr), 4.80–4.66 (m, 3H, CHα),
2.40 (t, J = 6.3 Hz, 18H, OCH2CH2), 2.01 (t, J = 7.0 Hz, 6H, 4.56–4.32 (m, 6H, CHα), 3.95–3.80 (m, 12H, ArOCH2), 3.60–3.50
CH2CONAhx), 1.46–1.11 (m, 18H, CH2Ahx). ESI-MS (positive (m, 36H, CH2OCH2), 3.0–2.7 (m, 36H, CH2Tyr and Phe, NHCH2
Ahx
mode): m/z calcd for [M + 2Na]2+ 1438.6231; found: 1438.6249 and CH2CF3), 2.40 (t, J = 6.3 Hz, 18H, OCH2CH2), 2.02 (t, J =
[M + 2Na]2+.
6.7 Hz, 6H, CH2CONAhx), 1.48–1.34 (m, 6H, CH2Ahx), 1.30–1.23
(16) 15 (89 mg; 0.07 mmol; 3.3 eq.) and 1,3,5-benzenetri- (m, 6H, CH2Ahx), 1.18–1.09 (m, 6H, CH2Ahx). 19F NMR
carbonyl trichloride (6 mg; 0.02 mmol; 1.0 eq.) were dissolved (282 MHz, DMSO-d6): δ [ppm] = −65.01 (t, J = 11.0 Hz, 9F),
in 3 mL DMF (SPPS grade) and treated with DIPEA (89 mg; −65.20 (t, J = 10.8 Hz, 9F).
0.69 mmol; 30.0 eq.). The solution was stirred at room temp-
erature for 45 h. The solvent was removed under reduced
pressure. The residue was purification via precipitation from
water and washing subsequently with methanol to remove
Conclusions
excess 15. Yield: 38 mg (0.01 mmol, 50%), C201H279N15O48, In conclusion, we disclose the synthesis of a series of three
white solid. 1H NMR (300 MHz, DMSO-d6): δ [ppm] = anionic dendritic peptide amphiphiles. We systematically
8.77–8.60 (m, 3H, NH), 8.26–8.08 (m, 9H, NH and CHaroBTA), study the effect of increasing the hydrophobic shielding of
7.84–7.69 (m, 3H, NH), 7.26 (d, J = 7.4 Hz, 6H, CHaroTyr), dendritic peptide amphiphiles, and the impact on the pH and
Phe and
7.18–7.05 (m, 21H, CHaro
Tyr), 6.88 (s, 1H, s, 3H, NH), ionic strength triggered self-assembly. By establishing state
6.79 (d, J = 8.5 Hz, 3H, CHaroTyr), 6.68 (d, J = 8.4 Hz, 3H, diagrams we show that the supramolecular polymerisation is
CHaroTyr), 5.92 (ddt, J = 17.1, 10.4, 5.1 Hz, 6H, OCH2CHvCH2), switched off if the apolar hexyl spacer between the dendritic
5.39–5.04 (m, 12H, OCH2CHvCH2), 4.81–4.65 (m, 3H, CHα), nonaphenylalanines and peripheral carboxylic acid Newkome
4.53–4.28 (m, 18H, CHα and OCH2CHvCH2), 3.57–3.45 (m, dendrons is omitted in compound 6, compared to molecule
Phe and Tyr
36H, CH2OCH2), 3.08–2.58 (m, 24H, CH2
and 12. If the hydrophobic character of the peptide core is
NHCH2Ahx), 2.36 (t, J = 6.1 Hz, 18H, OCH2CH2), 2.00 (t, J = increased further by introducing trifluoromethyl groups via
7.5 Hz, 6H, CH2CONAhx), 1.37 (m, 81H, CH3tBu), 1.30–1.04 (m, straightforward thiol–ene click chemistry in the O-allyl deriva-
18H, CH2Ahx). MALDI-MS (positive mode) (DHB [H2O/ACN]): tised tyrosine groups of 18, the thermodynamic driving force
m/z calcd for [M + Na]+ 3695.98; found: 3696.16 [M + Na]+.
for self-assembly is increased and the pH-triggered monomer
(17) 16 (38 mg; 0.01 mmol) was stirred twice in 2.0 mL of a to polymer transition at physiological ionic strength is shifted
mixture of TFA and DCM (1 : 1) for 45 min. The organic solvent from pH 5.0 to pH 7.4. Using TEM experiments we have
was removed after each step under reduced pressure. Yield: characterised the 1D supramolecular polymers as well-defined
30 mg (0.009 mmol, 93%), C165H207N15O48, white solid. nanorods with lengths distributions ranging from around
1H NMR (300 MHz, DMSO-d6): δ [ppm] = 8.68–8.58 (m, 3H, 100 nm to 500 nm, depending on the exact pH, ionic strength
NH), 8.27–8.08 (m, 9H, NH and CHaroBTA), 7.80–7.71 (m, 3H, and hydrophobicity of the peptide backbone. We thereby show
NH), 7.27 (d, J = 6.9 Hz, 6H, CHaroTyr), 7.21–7.06 (m, 21H, that by compensating attractive non-covalent interactions and
Tyr and
CHaro
Phe), 6.92 (s, 3H, NH), 6.81 (d, J = 8.7 Hz, 6H, solvophobic effects with repulsive electrostatic forces,
a
CHaroTyr), 6.70 (d, J = 8.6 Hz, 6H, CHaroTyr), 5.93 (ddt, J = 17.2, 10.4, concept we refer to as frustrated growth, is a sensitive tool in
5.2 Hz, 6H, CH2CHvCH2), 5.37–5.06 (m, 12H, OCH2CHvCH2), order to manipulate one-dimensional supramolecular poly-
4.81–4.71 (m, 3H, CHα), 4.55–4.27 (m, 21H, CHα and merisation processes in water.
CH2CHvCH2), 3.59–3.49 (m, 36H, CH2OCH2), 3.09–2.68
Tyr and Phe
(m, 24H, CH2
and NHCH2Ahx), 2.41 (t, J = 6.3 Hz,
18H, OCH2CH2), 2.02 (t,
1.46–1.10 (m, 18H, CH2Ahx). MALDI-MS (positive mode) (DHB
[EtOAc]): m/z calcd for [M + Na]+ 3191.5172; found: 3191.534 We thank U. Malkus, A. Ricker and H. Nüsse (Institute of
[M + Na]+.
Medical Physics and Biophysics, Münster) for performing TEM
J =
7.4 Hz, 6H, CH2CONAhx),
Acknowledgements
(18) 17 (29.5 mg; 0.0093 mmol; 1.0 eq.) was dissolved in a experiments, H. Frisch and B. Kemper for critical reading of
mixture of 6 mL THF and water (1 : 1). 1 mg 2,2-dimethoxy- the manuscript, and B. J. Ravoo for his support. P.B. acknowl-
2-phenylacetophenone (DMPA) and trifluoroethanethiol edges the ‘Fonds der Chemischen Industrie’ for a Liebig
(0.093 mmol, 10.8 mg 10.0 eq.) were added and the mixture Fellowship, the ‘Nordrhein-Westfälische Akademie der
was stirred for 18 h under irradiation with UV light (λ = Wissenschaften und der Künste’ for a Fellowship via the
365 nm and 405 nm). The solvent was removed under reduced ‘Junges Kolleg’, Marie-Curie Actions FP7 (Career Integration
pressure and the residue was washed with diethylether. After Grant, SupraBioMat, PCIG10-GA-2011-303872) and COST
dissolving the residue in basic water it was purified via SEC Action CM1005 (Supramolecular Chemistry in Water).
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