Altering peptide fibrillization by polymer conjugation

Article abstract of DOI:10.1021/bm3007117

A strategy is presented that exploits the ability of synthetic polymers of different nature to disturb the strong self-assembly capabilities of amyloid based β-sheet forming peptides. Following a convergent approach, the peptides of interest were synthesized via solid-phase peptide synthesis (SPPS) and the polymers via reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by a copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) to generate the desired peptide-polymer conjugates. This study focuses on a modified version of the core sequence of the β-amyloid peptide (Aβ), Aβ(16-20) (KLVFF). The influence of attaching short poly(N-isopropylacrylamide) and poly(hydroxyethylacrylate) to the peptide sequences on the self-assembly properties of the hybrid materials were studied via infrared spectroscopy, TEM, circular dichroism and SAXS. The findings indicate that attaching these polymers disturbs the strong self-assembly properties of the biomolecules to a certain degree and permits to influence the aggregation of the peptides based on their β-sheets forming abilities. This study presents an innovative route toward targeted and controlled assembly of amyloid-like fibers to drive the formation of polymeric nanomaterials.

Full text of DOI:10.1021/bm3007117

Article
pubs.acs.org/Biomac
Altering Peptide Fibrillization by Polymer Conjugation
Sabrina Dehn,† Valeria Castelletto,‡ Ian W. Hamley,‡ and Sebastien Perrier*,†
́
†Key Centre for Polymers and Colloids, School of Chemistry, The University of Sydney, NSW, 2006, Australia
‡Department of Chemistry, University of Reading, Whiteknights, Reading, RG6 6AD, United Kingdom
S
* Supporting Information
ABSTRACT: A strategy is presented that exploits the ability of
synthetic polymers of different nature to disturb the strong self-
assembly capabilities of amyloid based β-sheet forming peptides.
Following a convergent approach, the peptides of interest were
synthesized via solid-phase peptide synthesis (SPPS) and the
polymers via reversible addition−fragmentation chain transfer
(RAFT) polymerization, followed by a copper(I) catalyzed azide−
alkyne cycloaddition (CuAAC) to generate the desired peptide−
polymer conjugates. This study focuses on a modified version of
the core sequence of the β-amyloid peptide (Aβ), Aβ(16−20) (KLVFF). The influence of attaching short poly(N-
isopropylacrylamide) and poly(hydroxyethylacrylate) to the peptide sequences on the self-assembly properties of the hybrid
materials were studied via infrared spectroscopy, TEM, circular dichroism and SAXS. The findings indicate that attaching these
polymers disturbs the strong self-assembly properties of the biomolecules to a certain degree and permits to influence the
aggregation of the peptides based on their β-sheets forming abilities. This study presents an innovative route toward targeted and
controlled assembly of amyloid-like fibers to drive the formation of polymeric nanomaterials.
the context of protein misfolding diseases,13 such as
INTRODUCTION
■
Alzheimer’s (AD)14 and Parkinson’s (PD) diseases.15 In the
case of AD, the β-amyloid peptide (Aβ) is believed to aggregate
into fibrils and form amyloids,16,17 and several studies suggest
that a critical sequence for fibrillization is Aβ(16−20),
KLVFF.5,18 It has also recently been shown that addition of
two more phenylalanine residues to that core sequence
(FFKLVFF) permits even stronger aggregation via hydro-
phobic and aromatic interactions.19 Recent work has focused
on attaching synthetic polymers to these β-sheets forming
peptide sequences to investigate the influence on the β-sheet
forming properties.4,20 Synthetic strategies for peptide/
protein−polymer conjugates are well described,3,11,21,22 and
the properties of the peptide-polymer conjugates have been
examined. For instance Pochan and co-workers used ethylene
glycol modified amino acids to synthesize polypeptides that
retain the α-helical structure of the amino acids.23 In some
cases, the self-assembly abilities of the conjugates and
corresponding properties such as gel formation can be
controlled by the degree of polymerization and the balance
between α-helical and β-sheet structures.24 Studies on the
influence of conjugated synthetic polymeric chains to β-sheets
forming peptides and their β-sheets forming properties in
particular have also been reported.4,25−28 For instance, Adams
et al. have used polyethylene oxide (PEO) with side chain
conjugated peptides to form vesicles,29 and Tzokova et al. have
demonstrated the importance of the nature of the peptide and
Inspiration from nature and improved synthetic strategies have
triggered dramatic advances in the development of multifunc-
tional materials over the past two decades. The combination of
biological materials such as proteins and peptides with synthetic
polymers is of particular interest because the resultant
conjugates benefit from the properties of both components.1
These hybrid materials have found many diverse applications,
such as tissue engineering,2 drug delivery,3 and structured
nanomaterials.4 Nanomaterials, in particular, benefit greatly
from the self-organization properties of peptides, because they
lead to hierarchical nanostructures of much higher complexity
than those achievable with synthetic polymers.5 For instance,
nanostructures such as nanofibers6 or nanotubes7 based on self-
assembled polypeptides can be applied in regenerative
medicine8 or nanoelectronics.7 Various approaches to design
peptide-based nanomaterials and how to control and trigger
their self-assembly, have been described, including their use to
guide the formation of supramolecular structures and related
functionalities.9,10 The challenge has now shifted to the
incorporation of additional functional molecules such as
polymers for the generation of highly functional nanostructured
materials.3,11 The self-organization properties of peptides or
proteins can be used to encode structural information in the
polymeric nanomaterials at the molecular level. In addition, the
synthetic polymer section of these nanomaterials can also affect
the self-organization properties of the peptides or proteins, thus
changing their reactivity. Peptides and proteins are well-known
to strongly self-assemble into β-sheets, which can further
aggregate into ribbons and fibrils.12 Indeed, β-sheet forming
peptides have been intensively discussed in the past decade in
Received: May 8, 2012
Revised: July 8, 2012
Published: July 9, 2012
© 2012 American Chemical Society
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Scheme 1. (a) Solid Phase Supported Peptide Synthesis of N3C4−FVLKFF−OH (1) and N3C4−FFVLKFF−OH (2); (b) RAFT
Polymerization To Yield PNIPAAM20 (5) and PHEA20 (6); (c) Microwave-Assisted CuAAC Reaction To Generate the
FFVLKFF Conjugates 8 (PNIPAAM20) and 10 (PHEA20) and the FVLKFF Conjugates 7 (PNIPAAM20) and 9 (PHEA20)
the relative balance between polymer and peptide for the self-
assembly process.30 However, there is limited precise
investigation on the self-assembly properties of the resultant
conjugates4,27,31−36 and there is still a high demand on
understanding this process to exploit the self-assembly
properties with full capacity. Poly(ethylene glycol) (PEG) is
the most common polymer used in research on peptide−
polymer conjugates aggregation because it improves bio-
compatibility and decreases immunoresponse.37 To date, PEG
is the only polymer considered in studies involving the core
sequence of the β-amyloid peptide (Aβ16−20),20,25,31,38,39 and
it has been reported that hydrophobically modified Aβ(16−20)
sequences (FFKLVFF) form very strong self-assembled
structures19 that are retained even after PEGylation.25 These
initial results suggest that the nature of the peptide may dictate
the suitability of a given conjugate polymer to alter the strong
self-assembly properties of the β-sheet forming peptides.
Herein, we describe a systematic investigation on the capability
of a polar polymer (poly(hydroxylethyl acrylate), PHEA) and a
less polar polymer (poly(N-isopropyl acrylamide), PNIPAAM)
to influence the self-assembly properties of a β-sheet forming
peptide. A modified Aβ(16−20) sequence with two additional
phenylalanine units (FFVLKFF) was used as a model system.
In addition, the Aβ(16−19) sequence, similarly modified with
one phenylalanine units, (FVLKFF) was also investigated to
assess the importance of phenylalanine in the peptide sequence.
We opted for a convergent approach, in which the peptides and
polymers were first synthesized via solid-phase peptide
synthesis (SPPS) and reversible addition−fragmentation chain
transfer (RAFT) polymerization respectively, followed by
copper(I) catalyzed azide−alkyne cycloaddition (CuAAC) to
generate the desired peptide-polymer conjugates (Scheme 1).
MATERIALS AND METHODS
■
Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium (HBTU) were
obtained from GL Biochem and used as supplied. Butyl-trithiocar-
bonate propanoicacid (BTCPA) was obtained from Dulux and used as
supplied, and azoisobutyronitrile (AIBN) was obtained from Aldrich
and precipitated from methanol prior to use. Hunig’s base N,N-
̈
diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), thioansiol,
triisopropylsilane, sulfurylchloride, and all solvents were ordered
through Sigma-Aldrich and used as received. N-Isopropylacrylamide
(NIPAAM) was purchased from Sigma-Aldrich and recrystallized
before usage. Hydroxyethyl acrylate (HEA) was purchased from
Sigma-Aldrich and was deinhibited before polymerization. All other
chemicals were ordered through Sigma-Aldrich and used without any
further purification.
Nuclear Magnetic Resonance (NMR). NMR analyses were
carried out on Bruker Ultra Shield Avance Spectrometers (200 or 300
MHz). For all NMR analyses deuterated solvents as stated were used.
Size Exclusion Chromatography (SEC). SEC analyses were
performed at 50 °C using a Agilent SEC system (PL-GPC50 Plus),
equipped with a guard column and two Polar Gel-M columns (300 ×
7.5 mm) and a PL-RI detector. The system was set at 32 °C with DMF
using 0.1 wt % LiBr, 0.05 g/L hydroquinone with a flow rate of 0.7
mL/min. For sample analysis, a poly-styrene calibration has been used.
Transmission Electron Microscopy (TEM). Samples were
prepared by placing a drop of sample solution on a carbon coated
copper grid covered with thin film of pioloform. Samples were air-
dried for at least 24 h before the analysis. TEM images were obtained
using a JEOL1400 electron microscope.
Fourier Transform Infrared (FT-IR). FT-IR spectra were obtained
using a Bruker ISF66v spectrometer FT-IR from dried film of sample
solution. The numbers of scans were set at 32.
Electrospray Ionization Mass Spectroscopy. ESI analysis was
performed using a Finnigan LCQ mass spectrometer.
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Circular Dichroism Spectroscopy (CD). CD spectra were
recorded using a Chirascan spectropolarimeter (Applied Photophysics,
U.K.). Solutions of the peptides and conjugates in methanol were
loaded in parallel plaque cells (Hellma quartz Suprasil), with a 0.1 or 1
mm path length. The CD data were measured using 1 s acquisition
time per point and 0.5 nm step. The postacquisition smoothing tool
from Chirascan software was used to remove random noise elements
from the averaged spectra. A residual plot was generated for each curve
in order to verify whether or not the spectrum has been distorted
during the smoothing process. The CD signal from the methanol was
subtracted from the CD data of the peptide solutions.
Small-Angle X-ray Scattering (SAXS). Experiments were
performed on beamline ID02 at the ESRF, Grenoble, France. Samples,
dissolved in methanol, were placed in a glass capillary mounted in a
brass block for temperature control. Micropumping was used to
minimize beam damage, by displacing a drop of the sample by 0.01−
0.1 mm for each exposure. The sample-to-detector distance was 1.2 m,
and the X-ray energy was 12.46 keV. The q = 4π sin θ/λ (2θ is the
scattering angle and λ is the wavelength) range was calibrated using
silver behenate. Data processing (background subtraction, radial
averaging) was performed using the software SAXSUtilities.
CO, amide I) cm−1. HighRes MS found, 1072.59785 (M + H+);
calcd, 1071.59.
1
Analysis of 2. H NMR (d-TFA, 300 MHz) δ = 7.143−7.280 (m,
20H, Har), 6.775 (s, br, NH, NH2), 4.933−4.977 (d, J = 13.2 Hz, 4H,
CHNH backbone), 4.574−4.666 (d, J = 27.6 Hz, 2H, CHNH
backbone), 4.322 (s, br, 1H, CHNH backbone), 3.077−3.359 (m,
10H, CH2N3, 14H, CH2Phe), 2.490 (s, br, 2H, CH2NH2), 1.441−
1.837 (m, 16H, CH, CH2), 0.862−1.031 (m, 12H, CH3 peptide side
chains) ppm. 13C NMR (d-TFA, 300 MHz) δ = 181.05 (COOH),
178.54 (NHCO−), 176.12 (NHCO−), 175.43 (NHCO−), 175.12
(NHCO−), 136.57 (Car-CH2), 131.20 (Car, Car-CH2), 129.92 (Car-
CH2), 120.15 (Car), 118.62 (Car), 115.07 (Car), 112.36 (Car), 18−
62.27 (CH3, CH2, CH, 26C) ppm. MS (ESI+) m/z = 1072.80 (M +
H+, 100). IR (ATR FT-IR) ν = 3420 (m, NH-H), 2113 (w, N3), 1681
(s, CO, amide I), 1629 (m, CO, amide I) cm−1. HighRes MS
found, 925.52944 (M + H+); calcd, 924.52.
Synthesis of 5-Azido Pentanoic Acid 3. The synthesis of the
C4N3 linker followed a procedure from Srinivasan40 and Kakwere.33
Under inert conditions, bromovaleric acid (40 mmol, 7.24 g) was
dissolved in 8 mL of MeOH and cooled down to 0 °C. Thionyl
chloride (120 mmol, 14.27 g) was added dropwise under a N2
atmosphere within 45 min and stirred for 30 min at 0 °C. The
reaction mixture was allowed to warm up to room temperature and
was stirred for 19 h. After solvent evaporation, the residue was
suspended in 50 mL of ethyl acetate and extracted with NaHCO3 (3 ×
30 mL), H2O (3 × 30 mL), and brine (1 × 30 mL). Drying over
NaSO4 and removing the solvent under reduced pressure lead to a
brown liquid.
General Procedure for the Synthesis of Peptides 1 and 2.
The azide modified peptides N3C4−FVLKFF (1) and N3C4−
FFVLKFF (2) have been synthesized via standard solid-phase 9-
fluorenylmethoxycarbonyl (Fmoc) peptide synthesis on a 2-chloro-
trityl resin. A total of 2 g (1.5 mmol g−1) 2-chlorotrityl resin was
suspended in 5 mL DCM for 30 min in a fritted syringe (10 mL). After
the solvent has been filtered off, a solution of Fmoc-L-phenylalanine−
OH (6.0 mmol, 2.322 g) and N,N-diisopropylethylamine (DIPEA,
Upon addition of 30 mL of DMSO, NaN3 (77 mmol, 5 g) was
added under rapid stirring. This solution has been stirred at 50 °C for
24 h and the resultant white suspension has been taken up with 20 mL
H2O and was extracted with Et2O (4 × 40 mL). Washing with brine,
drying over NaSO4 and removing the solvent under reduced pressure
yielded in a brown oil. After dissolving in 30 mL of THF/H2O = 3:1
(v/v), 20 mL of aqueous LiOH (73 mmol) was added and the mixture
was stirred for 4 h at room temperature. THF was removed under
reduced pressure and the aqueous phase was combined with 50 mL of
ethyl acetate. Washing with 1 N HCl (3 × 50 mL), H2O (3 × 50 mL),
and brine (2 × 50 mL) and drying the combined organic phases over
NaSO4 and removing the solvent under reduced pressure yielded in 5
as brown oil (70.1%, 4.01 g).
Hunig’s Base; 12 mmol, 3.08 g) in DMF (5 mL) was added and
̈
shaken for 2 h. The solvent was filtered off and the resin was washed
with DCM/MeOH/DIPEA 17:2:1 (3 × 10 mL) to cap any unreacted
peptide chains. After washing the resin with DCM (3 × 10 mL), DMF
(3 × 10 mL), and DCM (3 × 10 mL), it was dried in vacuum to be
used for further SPPS. UV−vis was used to determine a loading of 1.06
mmol Fmoc-L-phenylalanin−OH per g of resin. A total of 0.5 g resin
was used for further SPPS. For coupling of each Fmoc-protected L-
amino acid, the resin was swollen in 5 mL of DCM for 30 min and
deprotected with 20% solution piperidin in DMF (2 × 5 mL) for 3
min. After washing the resin with DMF (3 × 10 mL), DCM (3 × 10
mL), and DMF (3 × 10 mL), a solution of Fmoc-L-amino acid−OH
(1.5 equiv), HBTU (2 equiv) to activate the N-terminus, and DIPEA
(5 equiv) in DMF (5 mL) was added and shaken overnight (16 h).
The solvent was filtered off and the resin washed with DMF (5 × 10
mL) and unreacted chains capped with DCM/MeOH/DIPEA 17:2:1
(3 × 10 mL) for 2 × 3 min. After washing with DMF (5 × 10 mL), the
next coupling step was performed. Upon addition of the last amino
acid, the azide linker C4N3 3, was coupled under the same reaction
conditions. A mixture of TFA/thioanisol/triisopropylsilane: H2O =
88:5:2:5 (10 mL for 3 h) was used to isolate the desired peptide
sequences 1 and 2 from the solid phase. The obtained solution was
concentrated to near dryness, dissolved in a small amount of MeOH
and precipitated from ice-cold Et2O. If necessary, preparative HPLC
(ACN, H2O, TFA) was performed for purification. Drying in vacuum
yielded the opaque solids 1 and 2 (0.127 g, 41%).
1H NMR (CDCl3, 300 MHz) δ = 11.442 (s, 1H, OH), 3.233−3.276
(t, J = 12.9 MHz, 6.6 MHz, 2H, CH2N3), 2.328−2.375 (t, J = 14.1
MHz, 6.9 MHz, 2H, CH2(CH2)3N3), 1.547−1.726 (m, 4H,
CH2CH2N3) ppm. 1H NMR data are in agreement with literature
results.33
Synthesis of (Prop-2-ynyl propanoate)yl Butyltrithiocarbon-
ate (PPBTC, 4). The alkyne-modified RAFT agent PPBTC 4 was
synthesized as described by Konkolewicz et al.41 Butyltrithiocarbonate
propanoic acid (BTCPA; 2.06 g, 8.60 mmol) was dissolved in 50 mL
DCM and cooled down to 0 °C. Propargyl alcohol (2.42 g, 23.02
mmol), EDCI (3.02 g, 12.62 mmol), and DMAP (0.13 g, 1.1 mmol)
was added and stirred at 0 °C for 2 h. The reaction mixture was
allowed to warm up to room temperature and was stirred for
additional 16 h. Washing with H2O (5 × 20 mL), drying over MgSO4,
and removing the solvents yielded a yellow oil. Purification was
achieved via passing over a silica pad using toluene/ethyl acetate 9:1.
Removing solvents and drying in vacuum gave the desired product 4 as
1
Analysis of 1. H NMR (d-TFA, 300 MHz) δ = 7.223−7.393 (m,
15H, Har), 6.860 (s, br, NH, NH2), 4.994−5.060 (d, 3H, 19.8 Hz,
CHNH backbone), 4.668−4.745 (d, J = 23.1 Hz, 2H, CHNH
backbone), 4.467 (s, br, 1H, CHNH backbone), 3.442 (s, br, 2H,
CH2N3), 3.176−3.324 (d, br, J = 44.4 Hz, 6H, CH2Phe), 2.597 (s, br,
2H, CH2NH2), 2.144−2.212 (m, 3H, COCH2-, CH(CH3)2CH),
1.379−2.024 (m, br, 13H, CH2CH2N3, CH2CH2CH2N3,
CH2CH2CH2NH2, CH2CH2NH2, CH2(CH2)3NH2, CH2(CH(CH3)2),
CH(CH3)2CH2), 1.056−1.119 (m, br, 12H, CH3 peptide side chains)
ppm.
1
yellow oil (91.7%, 2.16 g). H NMR (CDCl3, 300 MHz) δ = 4.802−
4.876 (q, J = 7.5 Hz, 1H, SCHCH3CO), 4.726 (t, J = 1.2 Hz, 2H,
CH2CCH), 3.30−3.379 (t, J = 7.2 Hz, 2H, CH3(CH2)2CH2S-), 2.482−
2.498 (t, J = 2.4 Hz, 1H, CH2CCH), 1.595−1.725 (m, 5H,
CH3CH2CH2CH2S-, SCHCH3CO), 1.385−1.484 (m, 2H,
CH3CH2(CH2)2S-), 0.901−0.949 (t, J = 7.2 Hz, 3H, CH3(CH2)3S-).
Data are in agreement with results previously reported by Konkolewicz
et al.41
13C NMR (d-TFA, 400 MHz) δ = 178 (COOH), 177 (NHCO−),
175 (NHCO−), 136 (Car-CH2), 131 (Car, Car-CH2), 129 (Car-CH2),
120 (Car), 118 (Car), 116 (Car), 113 (Car), 19.5−62.5 (CH3, CH2, CH,
24C) ppm. MS (ESI+) m/z = 925.25 (M+H+, 100). IR (ATR FT-IR) ν
= 3282 (m, NH-H), 2153 (w, N3), 1679 (s, CO, amide I), 1631 (m,
General Procedure for the Synthesis of Polymers. The RAFT
polymers were prepared according to a procedure previously reported
by our group.34,41,42 A mixture of monomer, removed from inhibitors,
AIBN (AIBN/RAFT = 0.1:1 equiv) and solvents were added. The
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Table 1. Synthetic Details and Characterization for Polymers
a
a
b
b
polymer
[M]/ [RAFT]
33:1
time (h)
conversion (%)
DP
Mn
Đ
5
6
PNIPAAM20
PHEA20
4
3
81
88
20
23
2540
2950
5400
4600
33:1
a
b
1
Determined via H NMR spectroscopy. Determined via SEC. SEC results uncorrected relative to PS standards.
Table 2. Microwave-Assisted Click Reaction between Azide Peptide and Alkyne Polymers
molar equivalents
mL
polymer
azide peptide
CuSO4·5H2O
sodium ascorbate
DMF
7
PNIPAAM20
PNIPAAM20
PHEA20
1.2
1.2
1.2
1.2
N3C4-FVLKFF
N3C4-FFVLKFF
N3C4-FVLKFF
N3C4-FFVLKFF
1
1
1
1
2
2
2
2
10
10
10
10
5
5
5
5
8
9
10
PHEA20
Figure 1. (a) 1H NMR spectrum of PNIPAAM20-FVLKFF conjugate 7 confirming the successful click reaction. (b) FT-IR spectrum from dried films
of 1 wt % methanol solutions of PHEA20-FFVLKFF conjugate 10, showing the disappearance of the azide peak at 2153 cm−1 (black box) as a result
of the successful click reaction.
reaction vessel was purged with N2 for 15 min and left under a
nitrogen atmosphere of 1 atm for the polymerization. N-
isopropylacrylamide polymerization was performed in dioxane at 60
°C, hydroxyethyl acrylate in tert-butanol at 70 °C, and purified via
precipitation from ice-cold hexane/Et2O (4:1) or Et2O. The molecular
weight of the polymers were characterized by NMR and SEC.
Analysis of 5 (Table 1). 1H NMR (d-TFA, 300 MHz) δ = 4.805 (s,
2H, CH2, -O-CH2-CCH), 4.142 (s, 22H, CH, -NH-CH(CH3)2), 3.460
(s, 2H, CH2, CH3-(CH2)2-CH2-S-), 2.533, 2.378, 1.934, 1.769, 1.290−
1.406 (polymer backbone, CH3-CH2-CH2-CH2-S-, -O-CH2-CCH),
0.856−0.987 (m, 5H, CH2, CH3-CH2-(CH2)2-S-, CH3, CH3-(CH2)3-S-
) ppm.
copper was removed via filtration through neutral alumina. The
solvent was evaporated under reduced pressure; the resultant brown
residue was washed with H2O to remove any remains of sodium
ascorbate. After centrifugation and drying in vacuum a brown solid
could be isolated (64 mg, 43%).
The conjugates were characterized by using 1H NMR. The
disappearance of the peak for the alkyne proton and the methyl
group next to the triple bond indicated a complete reaction of the
alkyne polymer. In addition, ESI-MS has been used but none of the
starting materials were detected, meaning peptide and polymer have
been completely consumed by the click reaction. Furthermore the FT-
IR spectra do not show any peaks correspondent to the azide,
concluding that all azide has been completely reacted in the click
reaction.
Analysis of 6 (Table 1). 1H NMR (DMSO-d6, 300 MHz) δ = 4.769
(s, 23H, OH), 4.675 (s, 1H, −O−CH2-CCH), 4.008 (s, 41H, -O-CH2-
CH2-OH), 3.553 (s, 46H, -O-CH2-CH2-OH), 3.402 (s, 2H, CH3-
(CH2)2-CH2-S-), 1.066−2.335 (m, polymer backbone, CH3-CH2-CH2-
CH2-S-, CH3-CH2-CH2-CH2-S-), 0.863−0.901 (t, 3H, CH3-(CH2)3-S-
) ppm.
1
Analysis of 7. H NMR (d-TFA, 300 MHz) δ = 8.587 (s, 1H,
CCHtriazole), 7.257−7.395 (d, br, 15H, Har), 5.599 (s, 2H,
OCH2Ctriazole), 4.758−5.095 (m, 10H, OH polymer, CHNH back-
bone, NH), 0.974−4.502 (PNIPAAM backbone, RAFT side chain,
peptide side chains) ppm.
General Procedure for the Synthesis of Peptide−Polymer
Conjugates 7−10. The azide peptide sequences 1 and 2 were
combined with the correspondent alkyne polymers 5 and 6,
CuSO4·5H2O, sodium ascorbate and 5 mL DMF. After vortex mixing,
the reaction was carried out in a microwave reactor at 100 °C, 200 W,
for 15 min under continuous stirring and N2 cooling.
All four peptide polymer conjugates were purified by a second
microwave-assisted click reaction at 80 °C using an azide resin to
remove any unreacted polymers. The azide resin was synthesized
according to procedure previously used in this group.34 Excess of
1
Analysis of 8. H NMR (d-TFA, 300 MHz) δ = 8.385 (s, 1H,
CCHtriazole), 7.347 (s, br, 20H, Har), 5.234 (s, 2H, OCH2Ctriazole),
1.045−4.993 (CHNH backbone, PNIPAAM backbone, RAFT side
chain, peptide side chains) ppm.
1
Analysis of 9. H NMR (d-TFA, 300 MHz) δ = 8.902 (s, 1H,
CCHtriazole), 7.646−7.778 (d, br, 15H, Har), 5.976 (s, 2H,
OCH2Ctriazole), 1.046−5.203 (CHNH backbone, PNIPAAM backbone,
RAFT side chain, peptide side chains) ppm.
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Figure 2. (a) FT-IR spectrum of amino peptides and correspondent azide modified peptides 1 and 2 displaying the characteristic bands for β-sheets
around 1679 cm−1 and 1631 cm−1. (b) Circular dichroism spectrum of azide peptides 1 and 2 showing the characteristic negative and positive bands
for β-sheet structures.
Figure 3. Typical TEM images of aggregates obtained from self-assembly of the conjugates (dried films of 1 wt % methanol solutions): (a) TEM
image of N3C4−FVLKFF−OH 1; (b) TEM image of N3C4−FFVLKFF−OH 2; (c) SEM image of N3C4−FFVLKFF−OH 2.
Figure 4. FT-IR spectra of conjugates 7 (a), 9 (b), 8 (c), and 10 (d) of dried films of 1 wt % methanol solutions. Arrows indicate the relevant bands
or changes in band shapes.
1
Analysis of 10. H NMR (d-TFA, 300 MHz) δ = 8.614−8.690 (d,
br, 1H, CCHtriazole), 7.341−7.486 (d, br, 20H, Har), 5.683 (s, 2H,
OCH2Ctriazole), 0.501−5.131 (CHNH backbone, PNIPAAM backbone,
RAFT side chain, peptide side chains) ppm.
FVLKFF (1) and FFVLKFF (2) were synthesized via solid-
phase peptide synthesis (SPPS), and the azide group was
introduced at the N terminal. The polymers were synthesized
via reversible addition−fragmentation chain transfer (RAFT)
polymerization. The synthesis of the azide modified peptides 1
and 2 via SPPS leads to white solids in 41% yield (Scheme 1a),
and were characterized by NMR spectroscopy, ESI-MS
spectroscopy, high resolution mass spectrometry (High Res
RESULTS AND DISCUSSION
■
The azide and alkyne functionalities were introduced in the
peptide and polymer segment, respectively, following proce-
dures previously published by our group.43 Azide-modified
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Table 3. Amount (in Percentage) of Remaining β-Sheet
Features in Conjugates 7−10
β-sheet presence in samples
parallel
antiparallel
a
b
a
b
1621−1639 cm−1
1614−1648 cm−1
26
1660−1697 cm−1
1656−1708 cm−1
10
PNIPAAM20-FVLKFF
PNIPAAM20-FFVLKFF
PHEA20-FVLKFF
c
17
8.5
13
24
12
Figure 7. (a) Circular dichroism spectrum of FVLKFF 1 and
correspondent PNIPAAM20 and PHEA20 conjugates 7 and 9 from 1 wt
% methanol solution; (b) circular dichroism spectrum of FFVLKFF 2
and correspondent PNIPAAM20 and PHEA20 conjugates 8 and 10
from 1 wt % methanol solution.
PHEA20-FFVLKFF
a
Characteristic bands for parallel and antiparallel β-sheets found for
b
FVLKFF-C4N3. Characteristic bands for parallel and antiparallel β-
c
sheets found for FFVLKFF-C4N3. Conjugate 9 only displayed one
broad band from overlapping bands of antiparallel and parallel β-sheet
features.
(SI, Figure 6). The desired peptide−polymer conjugates 7−10
(Table 2) were synthesized via a convergent approach42,47
using microwave-assisted copper-catalyzed azide−alkyne cyclo-
addition (CuAAC; Scheme 1c). Successful cycloaddition
1
reaction was confirmed via H NMR (Figure 1a; SI, Figures
7 and 8), FTIR (Figure 1b), and SEC (SI, Figure 9).
The 1H NMR spectrum (Figure 1a) of PNIPAAM20-
FVLKFF conjugate 7 reveals the characteristic peak for the
proton (H13′) at the newly triazole ring formed during the
CuAAC at 8.587 ppm. The proton next to the ester
functionality (H12) has been shifted downfield from 4.805 to
5.590 ppm (H12′), which confirms the successful coupling
reaction. The disappearance of the azide band at 2153 cm−1 in
the FT-IR spectrum (Figure 1b) of PHEA20-FFVLKFF
conjugate 10 verifies a successful conjugate formation. A shift
of the SEC traces for PNIPAAM20 and PHEA20 indicates the
successful coupling between the azide and alkyne. The SEC
traces of the PNIPAAM conjugates 7 and 8 (SI, Figure 9) are
shifted toward a longer retention time which indicates a higher
hydrodynamic volume compared to the pure PNIPAAM20. For
both PHEA20 conjugates 9 and 10, the SEC traces are shifted to
shorter retention times due to a lower hydrodynamic volume of
the conjugates compared to the pure PHEA20 (SI, Figure 9b).
Self-Assembly Studies. The self-assembly properties of
peptides 1 and 2 and of the correspondent PNIPAAM20 and
PHEA20 conjugates 7−10 have been studied in 1 wt %
methanol solutions or as the related dried films of 1 or 5 wt %
methanol solutions. FT-IR spectroscopy of dried films (1 wt %
solutions in methanol) of 1 and 2 all show the characteristic
bands for β-sheet assemblies at around 1631 cm−1 (parallel β-
sheets) and 1679 cm−1 (antiparallel β-sheets),48 indicating that
the additional azide C4-linker does not disturb the ability to
form β-sheet assemblies (Figure 2a). Circular dichroism (CD)
spectra of peptides 1 and 2 also reveal characteristic β-sheet
features.49 A negative band around 230 nm and a positive band
at 218 nm is observed for 1 and a negative band at 235 nm and
a positive band at 225 nm for 2 (Figure 2b).
Figure 5. SAXS data for conjugates 7−10 (1 wt % in methanol) The
solid line through the data for conjugate 7−10 is the model form
factor fit for the Gaussian coil structure and the sheet-like structure,
respectively, described in the text. (RG is the radius of gyration, eta is
the contrast, BG is background.)
Figure 6. (a) Circular dichroism spectrum of PNIPAAM20 conjugates
7 and 8 (1 wt % methanol); (b) circular dichroism spectrum of
PHEA20 conjugates 9 and 10 (1 wt % methanol).
The classical β-sheet circular dichroism spectrum has a
minimum at 216−220 nm.5,50 The red-shift for this minimum
to 230 and 235 nm, respectively, is due to the formation of J-
aggregates from a head-to-tail stacking of the aromatic units51
and is a known feature for fiber formation.49 These findings
imply the presence of β-sheet structures in both azide peptides
1 and 2. A strong band at 225 nm observed for 2, which results
from the fourth phenylalanine residue, shows the influence of
these additional π−π stacking interaction on the secondary
structure of the peptide. Measurements using different
concentrations display a concentration dependency of the
MS), and Fourier transform infrared spectroscopy (FT-IR; SI).
Polymerization of hydroxyethyl acrylate and N-isopropyl-
acrylamide via reversible addition−fragmentation chain transfer
(RAFT) polymerization44−46 using chain transfer agent (CTA)
4 gave the functional polymers PHEA20 5 and PNIPAAM20
6
(Scheme 1b) with low polydispersities of 1.1 and 1.2,
1
respectively (SI, Figure 5) and were verified via H NMR
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Figure 8. Typical TEM images of aggregates obtained from self-assembly of the conjugates (dried films of 1 wt % methanol solutions): (a)
PNIPAAM20-FVLKFF, (b) PNIPAAM20-FFVLKFF, (c) PHEA20-FVLKFF, and (d) PHEA20-FFVLKFF conjugates. Magnification is displayed on the
top right corner of each image.
Figure 9. TEM images of self-assembled aggregates (dried films of 1 wt % methanol solution): (a) freshly prepared PHEA20-FVLKFF-OH 9 (1 wt
%); (b) incubated PHEA20-FVLKFF-OH 9 (1 wt %); (c) incubated PHEA20-FVLKFF-OH 9 (5 wt %); incubated PHEA20-FFVLKFF-OH 10 (5 wt
%).
formation of β-sheet structures. At low concentration (0.16 wt
%), only a weak band around 230 nm could be observed, but
upon increasing the concentration to 0.63 and 0.9 wt %, clear
features for β-sheet structures could be obtained (SI, Figure
10). This observation confirms that the addition of the azide
linker does not destroy the β-sheet forming abilities.
TEM and SEM images were obtained from dried films of 1
wt % methanol solutions on carbon coated copper TEM grids.
One of the advantages of applying CuAAC to generate the
desired conjugates is that no additional staining of the TEM
samples is required.42 The azide peptides 1 and 2 clearly show
strong entangled networks (Figure 3), which points out the
strong ability of 1 and 2 to not only form β-sheet assemblies as
supported by FT-IR and CD data, but also to aggregate in
larger structures.
The aggregation properties of the conjugates were
investigated by FT-IR, CD, TEM, and SAXS. FT-IR spectra
show that FFVLKFF conjugates 8 (PNIPAAM20) and 10
(PHEA20) and FVLKFF conjugates 7 (PNIPAAM20) and 9
(PHEA20) are still able to form β-sheet assemblies (Figure 4).
The FT-IR spectrum of PNIPAAM20-FVLKFF conjugates 7
displays a slight shoulder at 1691 cm−1 (Figure 4a, black
arrow), which implies the presence of antiparallel β-sheets. The
correspondent band around 1630 cm−1 from the parallel β-
sheets is covered under the broad band from the PNIPAAM
residue at 1652 cm−1. We conclude that the broadness of the
band for conjugate 7 around 1652 cm−1 and the appearance of
the shoulder at 1691 cm−1 is evidence for the presence of β-
sheet structures that overlap with the CO stretching band of
the PNIPAAM. The FT-IR spectrum of the PNIPAAM20-
FFVLKFF conjugate 9 (Figure 4b) shows a strong band around
1733 cm−1 resulting from the CO stretching band from the
ester group of the PHEA side chain. At 1637 and 1685 cm−1
appear two bands that are assigned to β-sheet structures (β-
sheet and antiparallel β-sheet, respectively). From these results
we conclude that the attachment of PHEA20 does prevent the
formation of strong fiber like networks but β-sheets are still
formed. For PNIPAAM20-FVLKFF conjugate 8, two bands at
1550 and 1652 cm−1 (Figure 4c) were observed resulting from
the PNIPAAM residues and no shoulder around 1691 cm−1was
observed. However, since the broadness of the band at 1652
cm−1 for 8 is much more pronounced than the band for the
pure PNIPAAM (Figure 4c, indicated with arrows). We
conclude that the CO stretching band from PNIPAAM
overlay the bands from parallel and antiparallel β-sheets.
Further proof for the existence of β-sheet structures is discussed
below. The FT-IR spectrum of the PNIPAAM20-FFVLKFF
conjugate 10 shows also a strong band around 1733 cm−1 from
the ester group of the PHEA side chain. In addition, bands at
1687 and 1644 cm−1 are observed (Figure 4d, marked with
arrows), which are assigned to antiparallel and parallel β-sheets,
respectively.
FTIR second derivatives (sav-Golay) was used to determine
the remaining amount of antiparallel and parallel β-sheets in the
four conjugates. The desired bands of the second derivatives of
the FTIR spectrum were integrated and compared to the
correspondent integrals of the peptides N3C4−FVLKFF−OH 1
(1660−1697 cm−1, parallel) and N3C4−FFVLKFF−OH 2
(1621−1639 cm−1, antiparallel; Table 3). We find the amount
of β-sheet features has been reduced from 100% in the pure
peptides 1 and 2 to values between 10 and 25%. Interestingly,
the nature of the conjugate seems to affect the type of β-sheet
assembly observed for the FVLKFF-based conjugate, varying
from a majority of parallel when conjugated to PNIPAAM20 to
antiparallel when conjugated to PHEA20. In the case of
FFKLVFF, the mixture of both types is kept, although peak
overlap did not allow for differentiation between parallel and
antiparallel β-sheet in the case of PNIPAAM20-FVLKFF.
To investigate whether the FT-IR features are due to the
conjugates 7−10, FT-IR mixing experiments were performed
using peptides 1 and 2 and PNIPAAM20 and PHEA20 in a 1:1
mixture (SI, Figures 11 and 12). For all four conjugates, a clear
red-shift of the amide bands is obtained, which is indicative of
the formation of sheet-like aggregates. Moreover, the mixture of
FVLKFF 1 and PNIPAAM20 5 displays a distinct shoulder in
the related amide band around 1650 cm−1, indicating the
presence of two different species in the solution with
overlapping FT-IR bands. The FT-IR spectrum of the mixing
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experiment with FFVLKFF 2 and PNIPAAM20 5 in a 1:1
mixture (SI, Figure 11b) shows a splitting of the band at 1652
cm−1 that is associated with the presence of two discrete
compounds.
conjugate. PNIPAAM chains interact with the peptide segment,
possibly as it is less favored by the solvent (methanol) and via
H-bond formation between the amide groups found in the
peptide and the acrylamide repeating units. Such interactions
do not completely prevent β-sheet assemblies, but strongly
disturb aggregation in larger structures. The introduction of a
fourth phenylalanine residue in the peptide segment
strengthens the peptide assemblies and allows for the formation
of larger aggregates, despite the presence of the PNIPAAM
chain. The more polar PHEA chains have greater interactions
with the solvent and do not affect to the same extent β-sheet
formation. The PHEA conjugate only partially prevents
aggregation and favors dispersion in the solvent, thus, leading
to the formation of smaller structures. We have previously
observed that conjugating PHEA to the β-sheet forming
peptide P11-2 prevents the assembly of the peptidic segment.33
This highlights the much stronger tendency of peptides 1 and 2
compared to peptide P11-2 to aggregate into β-sheet-like
structures and the importance of the functionality of the
polymer to interfere with the peptide fibrillization.
SAXS measurements on the FFVLKFF conjugates 8
(PNIPAAM20) and 10 (PHEA20) and on the FVLKFF
conjugates 7 (PNIPAAM20) and 9 (PHEA20) in methanol (1
wt %) do not show any aggregation for conjugates 8−10 under
the conditions modeled by SAXS (Figure 5). In fact, the SAXS
data for conjugates 8 (PNIPAAM20) and 10 (PHEA20) can be
modeled as Gaussian coils with a radius of gyration 2−2.5 nm,
which is reasonable considering the polymer chain dimensions.
For the FVLKFF conjugate 9, it was also possible to fit the data
to a Gaussian coil model. However, the obtained radius of
gyration (4.4 nm) is larger than for the FFVLKFF conjugates.
Gaussian coil form factor fitting was less successful for the
SAXS data for conjugate 7 because the SAXS intensity exhibits
an extended range (including low q) with a q−2 dependence.
This indicates aggregation, consistent with the TEM images,
and this intensity scaling is in fact expected for a sheet-like
structure. Therefore, the SAXS data for conjugate 7 was fitted
to a model of a homogeneous sheet (i.e., a planar object with
extended lateral dimensions but finite thickness and a uniform
electron density) of thickness 1 nm (30% thickness
polydispersity). This demonstrates that the polarity of the
conjugate, mostly dictated by the polymer block, and the
polarity of the solvent used for the aggregation studies direct
the “nature” of the conjugates in solution. These somewhat
surprising results from SAXS analysis could be attributed to a
concentration effect and the state of the equilibrium of the self-
assembly process.
Circular dichroism was used as an additional technique to
verify the FTIR and SAXS data. Spectra were obtained from 1
wt % solutions of the conjugates in methanol. The CD spectra
of PNIPAAM20 conjugates 7 and 8 and PHEA20 conjugates 9
and 10 (Figure 6a and 6b) in methanol display bands for β-
sheet assemblies and confirm the FT-IR results. PNIPAAM20
conjugates 7 and 8 display a negative maximum at 235 nm and
a positive maximum at 220 nm (Figure 2a). The corresponding
PHEA20 conjugate 9 displays a minimum at 235 nm and a
dominating maximum at 225 nm, confirming the presence of β-
sheets and aromatic stacking (Figure 6b). For conjugate 10, a
broad minimum around 215−230 nm is observed as a result of
a strong β-sheet component and a small band at 225 nm for the
ππ-stacking from the aromatic residues (Figure 6b). The
observed red-shift of the minimum around 218 to 230 nm in all
four spectra are induced by strong maximum from the n−π*
aromatic interactions.52
TEM images of all four conjugates (Figure 8) reveal a small
number of large aggregates and confirm that attaching the
polymers to peptides 1 and 2, although not extensively
disturbing β-sheet formation, prevents the formation of
strongly entangled networks.
TEM also confirmed that the self-assembly process of the
conjugates into sheet-like aggregates is time- and concen-
tration-dependent, an observation also made by CD spectros-
copy. Indeed, TEM images from freshly prepared conjugate
solutions (1 wt % in methanol) show only small and thin
aggregates (Figure 9a; SI, Figures 13 and 14, left). However,
allowing the aggregates to self-assemble for 66 h lead to an
increased length (∼50−200 nm) and diameter (∼10−20 nm)
in the range expected for amyloidal structures52 (Figure 9b; SI,
Figures 13 and 14, right), indicating that the peptide part of the
conjugate still has an impact on the overall properties of the
conjugate and that the peptide properties become more
pronounced after a period of time. However, even after 66 h
of incubation of the self-assembly process the entanglement of
the aggregates are much less compared to the peptides 1 and 2
themselves. Increasing the concentration from 1 to 5 wt % in
PHEA20 conjugates 9 (Figure 9c) and 10 (Figure 9d) led to the
formation of larger and more structured aggregates.
CONCLUSION
■
In conclusion, we have described a synthetic strategy and self-
assembly studies for novel peptide−polymer conjugates,
utilizing the manipulation of the self-assembly process via
modified sequences from the β-amyloid peptide. Attaching
short polymers of different functionality, PNIPAAM20 and
PHEA20, to these peptide sequences disturbs their strong
fibrillization properties and offers some degree of control over
their aggregation. FTIR and circular dichroism measurements
display some β-sheet features in the peptide−polymer
conjugates 7−10. However, second derivative calculations of
the FTIR data confirm that conjugating PNIPAAM20 or
PHEA20 to the peptides 1 and 2 reduces the amount of β-
sheet features in the conjugate up to 80%. Models from SAXS
experiments displayed sheet like structures for FVLKFF
conjugates 7 (PNIPAAM20) and coil-like structures for the
other three conjugates, most likely due to concentration and
polarity effects of the conjugate and the solvent. Overall our
findings point out that a sensible choice in the nature of the
The CD spectra also support the conclusion by SAXS
experiments that the peptide aggregation is disturbed by
PNIPAAM20 and PHEA20. Indeed, FVLKFF conjugate 7
(PNIPAAM20) shows a red shift of the negative band from
230 to 235 nm and PHEA-FFVLKFF 10 displays a blue shift
and an intensive broadening of the band.
The CD spectrum of FFVLKFF conjugate 8 shows similar
bands to the unconjugated peptide, indicating that the
additional fourth phenylalanine residue strengthens the self-
assembled structure, despite the presence of PNIPAAM. CD
spectra of the PHEA conjugates 9 and 10 (Figure 7a,b) show
typical β-sheet bands with a maximum at 195 nm and a
minimum at 218 nm. We rationalize these observations by the
ability of the polymer chain to alter the aggregation of the
peptide segment depending on the nature of the polymer
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(21) Dehn, S.; Chapman, R.; Jolliffe, K.; Perrier, S. Polym. Rev. 2011,
51, 214−234.
polymer conjugate in relation to the solvent may allow control
over the self-assembly process. This approach is a promising
way to address the challenging task to control the strong
aggregation of β-sheet forming peptides and their use for
controlled synthesis of functionalized supramolecular polymeric
structures.
(22) Canalle, L. A.; Loewik, D. W. P. M.; Van Hest, J. C. M. Chem.
Soc. Rev. 2010, 39, 329−353.
(23) Yu, M.; Nowak, A. P.; Deming, T. J.; Pochan, D. J. J. Am. Chem.
Soc. 1999, 121, 12210−12211.
(24) Kotharangannagari, V. K.; Sanchez-Ferrer, A.; Ruokolainen, J.;
́
Mezzenga, R. Macromolecules 2012, 45, 1982−1990.
(25) Hamley, I. W.; Krysmann, M. J. Langmuir 2008, 24, 8210−8214.
(26) Johnson, J. C.; Wanasekara, N. D.; Korley, L. T. J.
Biomacromolecules 2012, 13, 1279−1286.
ASSOCIATED CONTENT
* Supporting Information
NMR, ESI, high resolution mass spectrometry (High Res MS),
Fourier transform infrared spectroscopy (FTIR), size exclusion
chromatography (SEC), transmission electron microscopy
(TEM), and circular dichroism (CD). This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
(27) Radu, L. C.; Yang, J.; Kopecek, J. Macromol. Biosci. 2009, 9, 36−
44.
(28) Shaytan, A. K.; Schillinger, E.-K.; Khalatur, P. G.; Mena-Osteritz,
E.; Hentschel, J.; Borner, H. G.; Bauerle, P.; Khokhlov, A. R. ACS
̈
̈
Nano 2011, 5 (9), 6894−6909.
(29) Adams, D. J.; Atkins, D.; Cooper, A. I.; Furzeland, S.; Trewin,
A.; Young, I. Biomacromolecules 2008, 9, 2997−3003.
(30) Tzokova, N.; Fernyhough, C. M.; Butler, M. F.; Armes, S. P.;
Ryan, A. J.; Topham, P. D.; Adams, D. J. Langmuir 2009, 25 (18),
11082−11089.
(31) Burkoth, T. S.; Benzinger, T. L. S.; Jones, D. N. M.; Hallenga,
K.; Meredith, S. C.; Lynn, D. G. J. Am. Chem. Soc. 1998, 120, 7655−
7656.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +61 (2) 9351 3366. Fax: +61 (2) 9351 3329. E-mail:
sebastien.perrier@sydney.edu.au.
■
Notes
The authors declare no competing financial interest.
(32) Rathore, O.; Winningham, M. J.; Sogah, D. Y. J. Polym. Sci., Part
A 2000, 38, 352−366.
ACKNOWLEDGMENTS
■
(33) Kakwere, H.; Payne, R. J.; Jolliffe, K. A.; Perrier, S. Soft Matter
2011, 7, 3754−3757.
The ARC (Discovery Program DP1096651) and the EPSRC
(EP/F048114/1, EP/G026203/1, and EP/G067538/1) is
gratefully acknowledged for financial support. The authors
thank Ashkan Dehsorkhi for assistance with the CD measure-
ments and Dr. Elizabeth Carter for support with the FTIR data
fitting.
(34) Chapman, R.; Jolliffe, K.; Perrier, S. Polym. Chem. 2011, 2,
7977−7979.
(35) Hentschel, J.; Borner, H. G. Macromol. Biosci. 2009, 9, 187−194.
̈
(36) Borner, H. G. Prog. Polym. Sci. 2009, 34, 811−851.
̈
(37) Harris, J. M.; Chess, R. B. Nat. Rev. Drug Discovery 2003, 2,
214−221.
(38) Castelletto, V.; Newby, G. E.; Zhu, Z.; Hamley, I. W. Langmuir
2010, 26 (12), 9986−9996.
REFERENCES
■
(1) Klok, H.-A. J. Polym. Sci., Part A 2005, 43, 1−17.
(2) Drotleff, S.; Lungwitz, U.; Breunig, M.; Dennis, A.; Blunk, T.;
(39) Burkoth, T. S.; Benzinger, T. L. S.; Urban, V.; Lynn, D. G.;
Meredith, S. C.; Thiyagarjan, P. J. Am. Chem. Soc. 1999, 121, 7429−
7430.
Tessmar, J.; Gopferich, A. Eur. J. Pharm. Biopharm. 2004, 58, 385−407.
̈
(3) Klok, H.-A. Macromolecules 2009, 42, 7990−8000.
(40) Srinivasan, R.; Tan, L. P.; Wu, H.; Yang, P. Y.; Kalesh, K. A.;
Yao, S. Q. Org. Biomol. Chem. 2009, 7 (9), 1821−1828.
(41) Konkolewicz, D.; Gray-Weale, A.; Perrier, S. J. Am. Chem. Soc.
2009, 131, 19075−19077.
(4) Rosler, A.; Klok, H. -A.; Hamley, I. W.; Castelletto, V.;
̈
Mykhaylyk, O. O. Biomacromolecules 2003, 4, 859−863.
(5) Hamley, I. W. Angew. Chem., Int. Ed. 2007, 46, 8128−8147.
(6) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Science 2001, 294,
1684−1688.
(42) Zhao, Y.; Perrier, S. Chem. Commun. 2007, 41, 4294−4296.
(43) Poon, C. K.; Chapman, R.; Jolliffe, K. A.; Perrier, S. Polym.
Chem. 2012, 3, 1820−1826.
(7) Reches, M.; Gazit, E. Science 2003, 300, 625−627.
(8) Yang, Y. L.; Khoe, U.; Wang, X. M.; Horii, A.; Yokoi, H.; Zhang,
S. G. Nano Today 2009, 4, 193−210.
(9) Ulijn, R. V.; Smith, A. M. Chem. Soc. Rev. 2007, 37, 664−675.
(10) Zelzer, M.; Ulijn, R. V. Chem. Soc. Rev. 2010, 39, 3351−3357.
(11) Gauthier, M. A.; Klok, H. -A. Chem. Commun. 2008, 2591−
2611.
(44) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2009, 62,
1402−1472.
(45) Perrier, S.; Takolpuckdee, P. J. Polym. Sci., Part A 2005, 43,
5347−5493.
(46) Semsarilar, M.; Perrier, S. Nat. Chem. 2010, 2, 811−820.
(47) Kakwere, H.; Chun, C. K. Y.; Jolliffe, K. A.; Payne, R. J.; Perrier,
S. Chem. Commun. 2010, 46, 2188−2190.
(12) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.;
McLeish, T. C. B.; Pitkeathly, M.; Radford, S. E. Nature 1997, 386,
259−262.
(48) Haris, P.; Chapman, D. Biopolymers 1995, 37, 251−263.
(49) Hamley, I. W.; Nutt, D. R.; Brown, G. D.; Miravet, J. F.;
Escuder, B.; Rodriguez-Liansola, F. J. Phys. Chem. B 2010, 114 (2),
940−951.
(13) Dobson, C. M. Nature 2003, 426, 884.
(14) Goedert, M.; Spillantini, M. G. Science 2006, 314, 777.
(15) McLean, P. J.; Kawamata, H.; Hyman, B. T. Neuroscience 2001,
104, 901−912.
(16) Kahn, S. E.; Andrikopoulos, S.; Verchere, C. B. Diabetes 1999,
48, 241−253.
(17) Chimon, S.; Shaibat, M. A.; Jones, C. R.; Calero, D. C.; Aizezi,
B.; Ishii, Y. Nat. Struct. Mol. Biol. 2007, 14 (12), 1157−1164.
(18) Tjernberg, L. O.; Lilliehook, C.; Callaway, D. J. E.; Naslund, J.;
Hahne, S.; J., T.; Terenius, L.; Nordtsedt, C. J. Biol. Chem. 1997, 272,
12601.
(50) Kelly, S. M.; Jess, T. J.; Price, N. C. Biochim. Biophys. Acta 2005,
1751, 119−139.
(51) Whitten, D. G. Acc. Chem. Res. 1993, 26, 502−509.
(52) Castelletto, V.; Hamley, I. W.; Harris, P. J. F. Biophys. Chem.
2008, 138, 29−35.
(19) Krysmann, M. J.; Castelletto, V.; Hamley, I. W. Soft Matter
2007, 3, 1401−1406.
(20) Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Hamley, I. W.;
Hule, R. A.; Pochan, D. J. Biochemistry 2008, 47, 4597−4605.
2747
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