Kinetically Controlled Stepwise Self-Assembly of AuI-Metallopeptides in Water

Article abstract of DOI:10.1021/jacs.7b08189

The combination of attractive supramolecular interactions of a hydrophobic Au^I-metallopeptide with the shielding effect of flexible oligoethylene glycol chains provides access to a stepwise self-assembly of a Au^I-metalloamphiphile in water. Kinetic control of the supramolecular polymer morphology is achieved using a temperature-dependent assembly protocol, which yields low dispersity supramolecular polymers (metastable state I) or helical bundled nanorods (state II).

Full text of DOI:10.1021/jacs.7b08189

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Communication
Kinetically Controlled Stepwise Self-Assembly of AuI-Metallopeptides in Water
Benedict Kemper, Lydia Zengerling, Daniel Spitzer, Ronja Otter, Tobias Bauer, and Pol Besenius
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08189 • Publication Date (Web): 22 Dec 2017
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Kinetically Controlled Stepwise Self-Assembly of AuI-
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Benedict Kemper, Lydia Zengerling, Daniel Spitzer, Ronja Otter, Tobias Bauer, Pol Besenius*
Institute of Organic Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 10–14, 55128 Mainz, Germany
Supporting Information Placeholder
interactions,15 a van der Waals-like interaction between closed-
ABSTRACT: The combination of attractive supramolecular
interactions of a hydrophobic AuI-metallopeptide with the shield-
shell gold(I) centers. We here report the kinetically controlled
supramolecular polymerization of a AuI-metallopeptide into meta-
ing effect of flexible oligoethylene glycol chains provides access
to a stepwise self-assembly of a AuI-metalloamphiphile in water.
stable nanorods at room temperature. Upon heating these nano-
rods undergo thermal fibrillation to form dimeric bundles.
The discotic AuI-metalloamphiphile 1 was synthesized in a
convergent approach. First, the hydrophobic C3-symmetrical
Kinetic control of the supramolecular polymer morphology is
achieved using a temperature-dependent assembly protocol, which
yields low dispersity supramolecular polymers (metastable state I)
or helical bundled nanorods (state II).
diphenylalanine
core
bearing
the
chlo-
rido(diphenylphosphane)gold(I) complex was prepared based on
peptide coupling chemistry, using a carboxylic acid functionalized
diphenylphosphane gold(I) complex (see SI). Secondly, the anion-
ic ligand exchange of the chlorido ligand in the AuI-complex with
the hydrophilic thiol carrying a tetraethylene glycol dendron, was
followed by NMR spectroscopy in degassed DMF-d7 and DIPEA
The supramolecular polymerization of molecular building
blocks is a promising approach for the construction of functional
nanostructures.1 Such complex architectures are formed by chem-
ically encoded self-assembly based on multiple non-covalent and
reversible interactions.2 Traditionally, synthetic supramolecular
polymerizations have been considered as equilibrium structures
and investigations into the kinetic pathways of product formation
have been largely neglected.3 This is surprising since the field of
DNA-directed supramolecular assemblies have exploited multi-
step kinetic pathways in order to guide structure formation of
colloids and nanoparticles.4 Recently, a number of pioneering
studies have identified pathway selection and the power of kinetic
control in steering the structure and function of supramolecular
polymers and materials,5 for applications from organic electron-
ics6 to cellular adhesion.7 Meijer and coworkers studied the path-
way complexity of chiral oligophenylvinylenes in organic sol-
vents, where temperature protocols could determine the self-
assembly into kinetically or thermodynamically controlled aggre-
gates, with opposite handedness.5d The groups of Sugiyasu and
Takeuchi reported on a living supramolecular polymerization
using functionalized porphyrins that assemble into either kinet-
ically stable J-type or thermodynamically stable H-type aggre-
gates.8 De Cola and coworkers presented an example of a PtII-
metalloamphiphile, which upon dissolution in water/dioxane
mixtures spontaneously self-assembles into spherical, red-
emitting nanoparticles.9 This kinetically trapped state slowly
interconverts into anisotropic green-emitting assemblies before
the thermodynamic stable state consisting of blue-emitting micro-
crystalline structures is obtained. In water, the key to success in
the preparation of supramolecular nanostructures is to combine
hydrogen bonding, Coulombic forces, π–π stacking and dispersive
interactions, with solvophobic shielding.10 Based on our previous
strategy of frustrated growth using dendritic peptide amphiphiles11
and the surface confined assembly of supramolecular copoly-
mers,12 we designed amphiphilic AuI-metallopeptides. AuI-
complexes have prompted renewed interest in the field of lumi-
nescent materials,13 homogeneous catalysis,14 where many of the
unique chemical and physical properties arise due to the soft
nature of the gold(I) metal ion and its ability to undergo aurophilic
1
as base. Disappearance of the thiol triplet in the H NMR as well
as a shift from ꢀ=31.0 ppm (P-AuI-Cl) to ꢀ=34.8 ppm (P-AuI-S)
in the 31P{1H} NMR, provided evidence for the successful ligand
exchange. Using the same procedure, we synthesized further
derivatives of AuI-metalloamphiphile 1, containing one or three
phenylalanines per side arm (molecules 2 and 3, respectively).
Figure 1. C3-symmetrical AuI-metalloamphiphile 1 (A) and its
self-assembly in water into 1D supramolecular nanorods and
intertwined fibrils (B).
We first decided to study the temperature-dependent self-
assembly of the discotic AuI-monomer 1 using circular dichroism
(CD) spectroscopy (Figures 2-3). Due to changes in the secondary
structure during the supramolecular polymerization process, CD is
a powerful tool to investigate the self-assembly of peptidic am-
phiphiles and chiral supramolecular monomers into ordered su-
pramolecular polymers.11 The amphiphile 1 quickly dissolves in
the cold phosphate buffer. Note, that 1 was isolated form a solu-
tion in DMF, where it is molecularly dissolved as clearly shown
in the NMR measurements during the characterization (Figures
S42-S44). In 5 °C buffer, the CD spectrum shows a weak negative
CD band at λ=213 nm (state 0, Figure 2), which is very similar to
the CD spectrum of a molecularly dissolved species obtained in a
CH3CN/H2O (1:1) solvent mixture (Figure S3). Upon increasing
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the temperature from 5 °C to 25 °C using 5 °C intervals, the CD
full conversion to state I is already achieved after 2 min (Figure
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spectra change significantly. A strong (λ=219 nm) and a weak
(λ=250 nm) negative CD band is observed (state I, Figure 2). This
is indicative of the formation of ordered supramolecular polymers.
In temperature-dependent transmission electron microscopy
(TEM) experiments we were able to assign this first assembly
state I to anisotropic nanorod-like structures. Surprisingly, when
we increased the temperature further to above room temperature,
we observed a transition to a second state, with a different CD
signature. In the CD spectrum at 60 °C (state II, Figure 2), a weak
negative band at λ=219 nm and a positive band at λ=237 nm is
observed. These results are indicative of a change in the second-
ary structure of the peptidic core due to a rearrangement of the
supramolecular assemblies, in agreement with reports on red-
shifted β-sheet signals due to a twisting of fibrillar structure for-
mation.16 TEM micrographs of state II reveal the presence of
dimer helical bundles. Note, that at temperatures T>70 °C, the
solutions become turbid most likely due to dehydration of the
tetraethylene glycol chains. This behavior has been ascribed to a
lower critical solution effect, often observed for PEGylated mac-
romolecular structures in water.17 The clouding of the solution is
fully reversible after cooling the solution to temperatures
T<70 °C. We point out that in our supramolecular engineering
approach, the removal of one phenylalanine unit per side-arm in
the AuI-metallopeptide 2 significantly reduced the hydrophobicity
of the amphiphile. In this case, CD and TEM did not provide any
indications for self-assembly. If, on the other hand, one additional
phenylalanine per side-arm is introduced in amphiphile 3, the
driving force for self-assembly is so strong that highly aggregated
fibrils were obtained (Figures S11-S12).
3C). Further incubation at 35 °C for 2 h induces the formation of
II. From these experiments, we conclude that at T<20 °C, a meta-
stable state I is produced, with a lifetime of two hours (15 °C) to
several days (5 °C) and at T>20 °C a thermodynamically pre-
I
ferred state II is slowly formed. The energetic barrier Ea for the
self-assembly of monomers into supramolecular polymers must be
significantly smaller than EaII, that is assigned to the bundling
process. Once state II is reached, cooling the solutions at 5 °C for
a week does not lead to the re-formation of state I or state 0.
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Figure 3. Time-dependent CD analysis (λ=219 nm) of a freshly
prepared solution of 1 (50 µM) in cold phosphate buffer (10 mM)
at T=5 °C (A), 25 °C (B) and 35 °C (C).
In order to correlate the states 0, I and II detected by CD spec-
troscopy with the morphology of theses assemblies, we performed
temperature- and time-dependent TEM experiments (Figure 4).
For this purpose, a freshly prepared solution of the AuI-
metalloamphiphile 1 in ice-cold Tris buffer was quickly deposited
on a carbon film coated copper grid cooled to 6 °C (Figure S17)
and stained with a cold solution of uranyl acetate. Negative stain-
ing is generally employed to artificially enhance the contrast of
soft materials and biological samples where contrast is usually too
low. It furthermore fixates the sample on the carbon surface by
acting like a mold and prevents structural changes of the material
during the drying process.18 In agreement with the CD data, where
no secondary structure was observed in state 0, only small spheri-
cal objects were obtained (Figure 4A). Assuming a diameter of
8 nm for the stretched-out conformation of the hydrophobic core
and 11.5 nm including the hydrophilic shell of the AuI-
metalloamphiphile 1, these isotropic structures of 12 nm (σ=4.9,
n=182, Figure S5) are assigned to be monomeric or small oligo-
meric species. In contrast, after dissolving the amphiphile at
20 °C, immediate deposition and staining on TEM grids, we
obtained elongated micelles (Ln=28 nm, Lw=41 nm, Đ=1.5, σ=19,
n=666, Figures 4B and S6). These worm-like or necklace micelles
appear very flexible. After allowing the solution to age for 5 min
at 20 °C, the cylindrical micelles become considerably longer and
more rigid (Ln=103 nm, Lw=140 nm, Đ=1.4, σ=61, n=210, Fig-
ures 4C and S7), and have a homogeneous thickness of d=8 nm,
which agrees well with the stretched-out conformation of the
hydrophobic core. The more rigid nature of the 1D nanorod-like
morphologies are in agreement with the more pronounced nega-
tive CD band (λ=219 nm), which is characteristic for assembly
state I. This is most likely due to more efficient supramolecular
packing. A second contribution to the increase of the CD band
likely originates from their increased length and thus higher de-
gree of polymerization. In agreement with temperature-dependent
Figure 2. CD spectra of 1 with a stepwise temperature increase
from 5 °C to 60 °C starting from a cold solution of 1 (50 µM) in
phosphate buffer (10 mM).
Furthermore, we performed temperature-dependent CD meas-
urements and applied a temperature ramp of 0.5 °C/min, while
monitoring the intensity of the CD band at λ=219 nm (Figure S2).
Starting from a cold solution of monomers or disordered oligo-
mers (state 0), very sharp transitions to state I are observed at
21.5 °C. Upon heating the solution further, a gradual temperature
induced transition to state II is observed. These observations
corroborate the initial steady state CD and TEM experiments.
Given our great interest in elucidating the kinetics of supramo-
lecular polymer formation, we performed time-dependent CD
experiments. When monitoring the CD band at λ=219 nm at 5 °C,
a very slow self-assembly process is observed that takes 48 h to
reach the plateau value for state I (Figure 3A). Importantly, the
intensity of the CD band remains constant for more than 5 days.
When we performed the same experiment at 25 °C, the first tran-
sition is complete after 10 min (Figures 3B and S4D), and thereaf-
ter a steady increase of the CD intensity is observed, indicating a
fast transition from 0→I and slow transition I→II. Note that at 15
°C the first transition takes 60 min (Figure S4B), while at 35 °C,
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Figure 4. Negative stain TEM micrographs and corresponding histograms of a solution of 1 (50 µM) in Tris buffer (10 mM). Sample
preparation was carried out at 6 °C (t=0 min) (A), at 20 °C t=0 min (B), t=5 min (C) and t=25 min (D), and after heating to 60 °C (E).
CD experiments, the transition from small and disordered mor-
phologies to long, stiff and ordered nanorods is fast at 20 °C.
After 25 min, the supramolecular polymers grew slightly longer
(Ln=128 nm, Lw=181 nm, Đ=1.4, σ=82, n=381). (Figures 4D and
S9). Note, that even if the TEM micrographs suggest lateral ag-
gregation of the supramolecular polymers, they remain colloidally
stable. The formation of precipitates or hydrogels was not ob-
served. Intriguingly, close inspection of the TEM micrographs
indicated the presence of a few bundled nanorods (Figure S9).
When heating the sample to 60 °C, followed by cooling down to
room temperature, TEM images reveal the full conversion into
fibrils, state II of the kinetically controlled step-wise assembly
process (Figure 4E and S10). The upper TEM micrograph in
Figure 4E shows a rare example of a single nanorod located next
to a helical nanofibril. The thickness of the thermally induced
fibrils (10–14 nm) suggests that they consist of two intertwined
nanorods. Using AFM experiments we have confirmed the pres-
ence of nanofibrils (Figure S13). We hypothesize that bundling
reduces the solvent exposure of hydrophobic domains of the
1D supramolecular polymer morphology using temperature de-
pendent assembly conditions and avoids the need for selective
solvent techniques. At low temperatures, CD and TEM experi-
ments describe the supramolecular polymerization of monomeric
or small oligomeric disordered species (state 0) into nanorod-like
morphologies of high molecular weight. At 5 °C the lifetime of
the disordered state 0 is very short, suggesting that the energetic
barrier for the self-assembly of monomers into 1D supramolecular
polymers is very low. At temperatures T<20 °C the 1D nanorod-
like species (state I) are stable on the time-scale of several hours
to days. Upon increasing the temperature T>20 °C the metastable
state I is converted into the thermodynamically stable helical
fibrils, consisting of two intertwined nanorods (state II). The large
activation energy required for this bundling most likely originates
from the shielding effect of the flexible oligoethylene glycol
chains that form a hydrated shell around the nanorods. Our ap-
proach demonstrates the unique opportunities provided by the
combination of strong supramolecular interactions in water with
repulsive forces, in order to direct the assembly process into non-
equilibrium systems. This feature is crucial for the development
of multicomponent supramolecular materials with optimized
functional properties.
diphenylalanine
coupled
to
the
chlo-
rido(diphenylphosphane)gold(I) complexes in the self-assembled
nanorods of 1. If the peptide sequence is extended with one phe-
nylalanine moiety per side arm (3), the self-assembly is dominat-
ed by secondary aggregation and clustered nanorods as shown in
TEM images obtained from H2O/HFIP solvent mixtures (Figures
S11-S12). In order to test our hypothesis, we plan to tune the
hydrophobic character of the oligopeptide aromatic side chains to
either favor bundling by including more hydrophobic 19F-labeled
pentafluorophenylalanine,19 or disfavor bundling using tyrosine as
a hydrogen bonding partner for water molecules.20 In a comple-
mentary approach, the Stupp laboratory has disclosed the β-sheet
directed self-assembly of anionic peptide amphiphiles, where a
thermal treatment at 80 °C led to the dehydration of the supramo-
lecular nanofibers and to the formation of infinitely long bundled
filaments.21 Note, that the contour length dispersities of our 1D
supramolecular polymers (Đ≤1.5) are unusually low. In theory,
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website: Additional experimental CD, TEM
and AFM data as well as synthetic details including full character-
ization using MALDI and HR-MS, 1H, 13C{1H} and 31P{1H}
NMR spectroscopy (PDF).
AUTHOR INFORMATION
Corresponding Author
*besenius@uni-mainz.de
we would have expected dispersities of 2 at such high degrees of
ORCID
3c, 22
polymerization.1c,
We tentatively assign the significantly
Pol Besenius: 0000-0001-7478-4459
lower values to a fast nucleation coupled to a slower elongation
step, which define the chain-growth polymerization process. The
presented supramolecular polymerization can therefore be likened
to a living polymerization.5a, 5b, 5e, 5h In comparison, it has been
shown for anionic polymerizations that a slow initiation process,
or the existence of chain ends that exchange slowly through mul-
tiple states, lead to broadening of the molecular weight distribu-
tion and dispersity values that exceed 1.3.23
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank Prof. Angelika Kühnle for providing access to the AFM
facilities. This work was supported by the DFG (SFB 858).
In conclusion, we present the stepwise self-assembly of AuI-
metallopeptides in water. This approach provides control over the
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