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Cite this: DOI: 10.1039/c0xx00000x

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]

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1

Reversible Thermal Transition of Polydiacetylene Based on KTTKS
Collagen Sequence

Pasquale Palladino,
a

Valeria Castelletto,
a

Ashkan Dehsorkhi,
a

Dmitry Stetsenko
a

and Ian W. Hamley
a
*

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX

DOI
: 10.1039/b000000x
5

Here we explore the physico
-
chemical properties of a peptide
amphiphile obtained by chemical conjugation of the collagen
-
stimulating peptide KTTKS with 10,12
-
pentacosadiynoic acid
which photopolymerizes as a stable and extended
polydiace
tylene. We investigate the self
-
assembly of this new
10

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.


15

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 soli
d state,
5

on
surface,
6

or in solution.
7
-
11

In case of peptide amphiphiles
20

(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
25

headgroups.
12

In this regard, we have previously investigated
the properties of a PA derived from collagen
-
stimulating
peptide KTTKS which genera
tes β
-
sheet structure upon
aggregation. The strand conformation assumed by this peptide
determines a bilayer arrangement stabilized by inter
-
peptide
30

backbone
-
backbone hydrogen bonds.
13,14

We explored here
the physico
-
chemical properties of synthetic peptid
e
amphiphile 10,12
-
pentacosadiynoyl
-
Lys
-
Thr
-
Thr
-
Lys
-
Ser
(C
25
-
KTTKS) photopolymerized as reported in Figure 1. We
have chosen the 10,12
-
pentacosadiynoic acid because its
35

diacetylene position within the hydrophobic tail was recently
reported to improve the s
tability and extension of conjugation
of polydiacetylene (PDA).
9

We have observed that C25
-
KTTKS self
-
assembles
in bilayer
, assuming a

β
-
sheet
conformation
and forming nanofiber structures
.
The self
-
40

assembly, with the help of the β
-
sheet structure, facilit
ates 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 β
-
45

sheet conte
nt 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 π
-
conjugation length and planarity.
1



50


Figure 1.
Polydiacetylene formation
scheme

between

hydrophobic tails of

C
25
-
KTTKS monomers.


Figure 2.
Electronic absorption spectra of C
25
-
KTTKS polymer
55

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 phas
e is usually
associated to non
-
planar conformation reversibly obtained by
heating. However, high temperature could also produce an
60

irreversible nanotube
-
like arrangement in the red state.
1
-
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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
.

The purple color obtained in this work for C
25
-
KTTKS in solution is scarcely observed in PDAs and dentotes a configuration of
C
25
-
KTTKS polydiacetylene backbone intermediate between t
hose corresponding to the to blue and red phases. Figure 2 shows
5

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, character
istic 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
10

Figure 2 gives the purple appearance of polymer solution.
Unfortunatel
y, 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 C
25
-
KTTKS polymer at different temperatures. (B) CD value (
mdeg) for C
25
-
KTTKS at 222 nm
versus

temperature
increase (open circle) and decrease (closed circle).


5

Figure 5. Amide I′ FTIR spectrum for C
25
-
KTTKS.

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
10

typically associated with β
-
sheet conformation.
9
C
25
-
KTTKS conformation was confirmed by Fourier transform i
nfrared 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 r
oom 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.

5


Unexpected results were observed con
cerning 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 t
he 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 pept
ide sequence with shorter conjugated

saturated hydrophobic tail

showed a common
10

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 as
sociated 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 co
ntacts 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
15

here. These results confirm the fundamental role of the hydrophilic regi
on 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 ty
pical 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
20

formation between the β
-
strands.
18

However, more mechanistic st
udies 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
25

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 intram
olecular 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
30

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

a
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


35


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Footnotes



Electronic Supplementary Information (ESI) available: Experimental details, including synthesis, polymerization, FTIR, TEM an
d SAXS data of C
25
-
KTTKS. See DOI:

10.1039/b000000x/


This work was supported by EPSRC grants EP/F0
48114/1, EP/G026203/1 and EP/G067538/1 to IWH. We thank Dr Peter Harris for assistance with TEM.

60