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Reversible Thermal Transition of Polydiacetylene Based on KTTKS
Collagen Sequence
Pasquale Palladino,a Valeria Castelletto,a Ashkan Dehsorkhi,a Dmitry Stetsenkoa and Ian W. Hamleya*
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Here we explore the physico-chemical properties of a peptide
amphiphile obtained by chemical conjugation of the collagenstimulating peptide KTTKS with 10,12-pentacosadiynoic acid
which photopolymerizes as a stable and extended
polydiacetylene. We investigate the self-assembly of this new
polymer and rationalize its peculiar behavior in terms of a
thermal conformational transition. Surprisingly, this polymer
shows a thermal transition associated with a non-cooperative
increase in β-sheet content at high temperature.
conjugation length and planarity. 1
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Inter-monomer topochemical addition of diacetylenes
produces a colored polymer, 1-3 characterized by an extensive
π-conjugation, with applications ranging from sensing to drug
delivery. 4 This conjugation takes place in the solid state, 5 on
surface,6 or in solution. 7-11 In case of peptide amphiphiles
(PAs), self-assembly allows a close packing of diacetylene
moieties favoring the inter-monomer topochemical addition. 7-9
The supramolecular arrangement of PAs is driven by
attractive hydrophobic interactions between the tails and by
attractive polar and electrostatic interactions between the
headgroups. 12 In this regard, we have previously investigated
the properties of a PA derived from collagen-stimulating
peptide KTTKS which generates β-sheet structure upon
aggregation. The strand conformation assumed by this peptide
determines a bilayer arrangement stabilized by inter-peptide
backbone-backbone hydrogen bonds. 13,14 We explored here
the physico-chemical properties of synthetic peptide
amphiphile
10,12-pentacosadiynoyl-Lys-Thr-Thr-Lys-Ser
(C25-KTTKS) photopolymerized as reported in Figure 1. We
have chosen the 10,12-pentacosadiynoic acid because its
diacetylene position within the hydrophobic tail was recently
reported to improve the stability and extension of conjugation
of polydiacetylene (PDA). 9 We have observed that C25KTTKS self-assembles in bilayer, assuming a β-sheet
conformation and forming nanofiber structures. The selfassembly, with the help of the β-sheet structure, facilitates the
polydiacetylene formation via topochemical addition between
hydrophobic tails as reported in Figure 1 giving a purple
solution at room temperature. This polymer shows a thermal
transition associated with a non-cooperative increase in βsheet content at high temperature. In detail, it is well known
that, within this kind of polymers, the color of PDA depends
on the supramolecular arrangement and in particular on its πThis journal is © The Royal Society of Chemistry [year]
Figure 1. Polydiacetylene formation scheme between hydrophobic tails of
C25-KTTKS monomers.
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Figure 2. Electronic absorption spectra of C 25-KTTKS polymer
changing temperature from 5 °C to 45 °C.
A blue phase is obtained at low temperature and it is
associated to a flat ribbon assembly. A red phase is usually
associated to non-planar conformation reversibly obtained by
heating. However, high temperature could also produce an
irreversible nanotube-like arrangement in the red state. 1-4
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Figure 3. Electronic absorption spectra of C 25-KTTKS as red phase. The sample was heated (A) and cooled (B) between 5 ºC and 95 ºC.
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The purple color obtained in this work for C 25-KTTKS in solution is scarcely observed in PDAs and dentotes a configuration of
C25-KTTKS polydiacetylene backbone intermediate between those corresponding to the to blue and red phases. Figure 2 shows
the thermochromic transition properties of C 25-KTTKS after polymerization, heated from 5 ºC to 45 ºC, analyzed by UV-vis
spectroscopy. Relevant details of the backbone configuration in the pu rple phase are still missing. 1-4 Therefore we will analyze
spectroscopy results in the purple phase as a contribution of elements already identified for the red and the blue phases. Th e main
absorption peaks observed are around 541 nm and 500 nm, characteristic of red phase, and 641 nm, distinctive of blue phase. 1-3
Similar spectra were previously reported for diacetylene PAs. 8 Altogether the additive mixing of red and blue absorbance in
Figure 2 gives the purple appearance of polymer solution. Unfortunately, we were not able to isolate the transient blue phase for a
full characterization. However, the blue peak intensity decreases irreversibly with heating (Figure 2 and S1) and disappears
around 45 ºC, thereby suggesting that the blue phase was a kineticall y trapped conformation, whereas the red peaks increase at the
same time (Figure 2 and S1), and this sample in the stable red phase was further investigated.
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Figure 4. (A) Far UV CD spectra of C25-KTTKS polymer at different temperatures. (B) CD value (mdeg) for C25-KTTKS at 222 nm versus temperature
increase (open circle) and decrease (closed circle).
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Figure 5. Amide I′ FTIR spectrum for C25-KTTKS.
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In particular, the peak intensity around 541 nm decreases upon increasing the temperature (5 ºC to 95 º C) but it was recovered by
cooling, whilst the absorbance around 500 nm is not strongly affected by temperature changes (Figure 3 and Figure S2). This
result suggests that absorbance around 541 nm is more sensitive to polydiacetylene structural change than the absorbance around
500 nm. The CD spectra measured for the PA are reported in Figure 4. The spectra show a strong negative band close to 220 nm
typically associated with β-sheet conformation. 9 C25-KTTKS conformation was confirmed by Fourier transform infrared analysis
(FTIR) in the amide I′ region (Figure 5), as shown by the presence of a main peak at 1625 cm -1, obtained at room temperature,
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which is consistent with β-sheet-structure. 13 Interestingly, the absence of any significant visible CD signal in the polydiacetylene
region (350 nm - 680 nm) (Figure S3), in contrast to earlier reported data for diacetylene PA systems, is likely due to the lack of a
chiral enviroment of the polydiacetylene backbone. 8 Additional results on C 25-KTTKS self-assembly at room temperture, obtained
using transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS), respectively (Supplementary
Information), show the presence of very thin fibres with a bilayer structure.
Unexpected results were observed concerning the thermal response of C 25-KTTKS. In fact, C 25-KTTKS folding shows a linear
and reversible dependence with temperature, such that the β-sheet increases upon heating the sample from 10 ºC to 90 ºC (Figure
4A). This behavior is opposite to data in the literature. It was reported for other PAs with conjugated polydiacetylene backbone
that their content in β-sheet structure decreases upon heating the sample from room temperature, such that complete unfolding
occurs at 90 ºC. 9,10 Moreover, the same peptide sequence with shorter conjugated saturated hydrophobic tail showed a common
coil-to-β transition with temperature.15 Nevertheless, it was previously shown that a polyalanine-poloxamer-polyalanine
copolymer underwent a reversal sol-to-gel transition associated with coil-to-β-sheet conformation change as the temperature
increased.16 Accordingly, our results could be explained by backbone dehydration by heating which increases the β -sheet content
of C 25-KTTKS polymer because of increased intermolecular contacts and interactions. The main difference with respect to
previously reported peptidic PDAs resides in much more stable inter-peptide hydrogen bonds for the KTTKS sequence explored
here. These results confirm the fundamental role of the hydrophilic region in controlling the thermal transition in
polydiacetylenes. 11,13,17
Another remarkable aspect of C 25-KTTKS thermal behavior is the linear change of peptide conformation with temperature as
shown in Figure 4B. This kind of structural change without the typical sigmoidal trend of transition was previously interpreted as
indicative of very low folding energy barrier in β-sheet-forming peptides due to non-cooperative intramolecular hydrogen bonds
formation between the β-strands. 18 However, more mechanistic studies are necessary to understand the β-sheets increases with
heating here reported. Furthermore, in our case it is worth noting that the above mentioned linear isomerization is related to the
red phase thermochromic behavior, suggesting that the peptide β-sheet conformation adapts to different geometries of the
polydiacetylene backbone. Finally, an important feature of C 25-KTTKS thermal transition is represented by its reversibility.
Besides the electronic absorption data in Figure 3, the heating and coo ling CD data reported in Figure 4B show a very good
superposition. These results are in agreement with the recently reported investigation that describes the enhancement of
intermolecular hydrogen-bonds connecting the headgroups, and the increase of intramolecular flexibility of the region between
the diacetylenic moiety and first amide group, as essential molecular features to achieve reversible thermal transition of PD As. 16
We believe that our successful rational design of polydiacetylene polymer based on the β-sheet-forming KTTKS collagen
sequence,13,14 and on 10,12-pentacosadiynoic acid, which forms a stable and extended polydiacetylene framework, 9 should be
useful in the further development of smart materials responding to an external stimulus, such as a temperature change, with
applications in nanotechnology.
Notes and references
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School of Chemistry, Food and Pharmacy, University of Reading, Reading RG6 6AD, U.K. Fax: +44 1183788450; Tel: +44 1183786341. E-mail:
i.w.hamley@reading.ac.uk
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Footnotes
† Electronic Supplementary Information (ESI) available: Experimental details, including synthesis, polymerization, FTIR, TEM and SAXS data of C25KTTKS. See DOI: 10.1039/b000000x/
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This work was supported by EPSRC grants EP/F048114/1, EP/G026203/1 and EP/G067538/1 to IWH. We thank Dr Peter Harris for assistance with TEM.
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