Thermoresponsive oligoprolines

Article abstract of DOI:10.1039/c2sm25451d

Monodispersed oligoprolines decorated covalently with hydrophobic units show characteristic thermoresponsive behavior with fast and sharp phase transitions at certain concentrations. The phase transition temperatures are dependent on the shape and locatio

Full text of DOI:10.1039/c2sm25451d

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links <
C
Soft Matter
Cite this: Soft Matter, 2012, 8, 4869
www.rsc.org/softmatter
COMMUNICATION
Thermoresponsive oligoprolines†
*
Feng Chen, Xiuqiang Zhang, Wen Li, Kun Liu, Yifei Guo, Jiatao Yan and Afang Zhang
*
Received 26th February 2012, Accepted 16th March 2012
DOI: 10.1039/c2sm25451d
including the X units, the peptide chain length, solution pH value
and the salt concentration, most of the reported ELP-based
polymers exhibit broad phase transitions (temperature range
>3 ꢀC), and the hysteresis during the heating and cooling
processes is less addressed. Therefore, other peptide-based
polymers were developed to show improved thermoresponsive-
ness. Recently, poly(glutamate)s carrying oligo(ethylene glycol)
(OEG) side-units were reported by Li et al.11 Depending on the
length of OEG units, these polypeptides could adopt ordered
secondary structures which, in turn, afford the polypeptides with
thermoresponsiveness. By varying OEG length and the ratios of
the comonomers, the LCSTs of the (co)polypeptides can be
tuned. Though thermoresponsive polypeptides have been paid
some attention, only few examples were reported due to their
relatively harsh synthesis and structural complexities. Devel-
oping novel (poly)peptides with unique thermoresponsive
properties remains a challenge. To date, most thermoresponsive
macromolecules reported are polydispersed.
Monodispersed oligoprolines decorated covalently with hydrophobic
units show characteristic thermoresponsive behavior with fast and
sharp phase transitions at certain concentrations. The phase tran-
sition temperatures are dependent on the shape and location of the
hydrophobic units, and can be also tuned via supramolecular host–
guest interactions.
Thermoresponsive polymers are of great interest in sensors and
biomedical applications.1 These polymers show a lower critical
solution temperature (LCST) in aqueous solutions. Based on
entropy-driven processes, the polymer chains start to dehydrate right
below this temperature, followed by chain collapse and forming
mesoglobules or aggregates upon heating. The tunability of the
LCST is critical,2 and normally can be mediated by the overall
hydrophobicity–hydrophilicity balance of the polymers. Incorpora-
tion of hydrophilic or hydrophobic units,3 end-group modifications,4
or even controlling particle size5 have been employed to tune the
phase transition temperatures of polymers. Recently, polymer
architecture has also been proved to show significant influence on the
phase transitions. On changing polymer architecture from linear
shape into cyclic6 or dendritic ones,7 the thermoresponsive properties
vary. Furthermore, the phase transition temperature can also be
influenced by supramolecular interactions. With introduction of
hydrophilic host molecules such as b-cyclodextrin, the thermores-
ponsiveness of hydrophobic guest molecules can be tuned.8
Oligoprolines are an intriguing class of peptides which are capable
of adopting two different helical conformations: the compact, right-
handed polyproline I (PPI) in less polar solvents like aliphatic alco-
hols, and the stretched, left-handed polyproline II (PPII) in polar
solvents like water.12 PPII has been found to be a common secondary
structure in natural proteins, and plays important roles in many
biological processes. Oligo- or polyprolines themselves are fully
water-soluble and not thermoresponsive. To extend the idea to
combine advantages from oligoprolines and the thermoresponsive
behavior in one matter, and to explore the effects of structure and
architecture on the thermoresponsiveness of the monodispersed
substances, here we report on the synthesis and characterization of
novel thermoresponsive oligoprolines covalently decorated with
hydrophobic units (Fig. 1). Both ball-shaped adamantyl (Ada) and
linear n-decyl (Dec) groups were selected to investigate the possible
Oligo- or polypeptides showing thermoresponsive behavior
may form a promising class of thermoresponsive polymers due to
their improved biofunctions and bioactivities together with the
ordered secondary structures. The most intensively investigated
thermoresponsive polypeptides are elastine-like peptides (ELPs)
with VPGXG as the main amino acid sequence, where X can be
any amino acid except proline. It was found that either long or
short ELPs showed phase transition behavior and were followed
by the conformation changing from random coil to b-spiral.9
Inspired by these, different kinds of homopolymers and block
and random copolymers carrying VPGXG units were reported
and their LCST behavior investigated.10 Though the LCSTs of
ELP-based polymers could be finely tuned by several parameters
Department of Polymer Materials, Shanghai University, Chengzhong
Street 20, Shanghai, China. E-mail: wli@shu.edu.cn; azhang@shu.edu.
cn; Fax: +86 21 69982827; Tel: +86 21 69980375
Fig. 1 Cartoon presentation of the oligoprolines modified with different
† Electronic supplementary information (ESI) available: Experimental
procedures and spectroscopic data. See DOI: 10.1039/c2sm25451d
hydrophobic moieties at different positions.
This journal is ª The Royal Society of Chemistry 2012
Soft Matter, 2012, 8, 4869–4872 | 4869

View Article Online
geometry effects of hydrophobic units on their thermoresponsive
behavior. Moreover, the linear hydrophobic unit Dec is located either
at chain-end or in the center of the oligopeptides to examine the
possible architecture effects on this property. Furthermore, their
thermoresponsive behavior was also examined through host–guest
interaction.
All modified oligoprolines P8-Ada, P8-Dec and P8(Dec) are water-
soluble at room temperature, but quickly collapse from water and
their aqueous solutions turn turbid when heated up to certain
temperatures. Their thermoresponsive behavior was thus investigated
and their cloud points (Tcs) were determined by turbidity measure-
ments using UV/Vis spectroscopy. Typical phase transition curves
from P8-Dec and P8(Dec) are shown in Fig. 2a. The Tcs for P8-Dec
and P8(Dec) are 25.6 ꢀC and 33.5 ꢀC, respectively, while the Tc from
P8-Ada is higher than 90 ꢀC. These results indicate: (1) although both
Ada and Dec units contain 10 carbons, the linear hydrophobic decyl
unit contributes more hydrophobicity than that from the ball-shaped
adamantyl group, resulting a much lower Tc (compare P8-Dec with
P8-Ada); (2) location of the linear hydrophobic unit in the centre of
the oligopeptide shows a relatively weak hydrophobicity than that
attached at the chain end, leading to a higher Tc [compare P8-Dec
with P8(Dec)]; and (3) all phase transitions from P8-Dec and P8(Dec)
are very sharp (<1 K), and the hystereses are small (<2 K), which
should be resulted from the absence of strong hydrogen bonding
from the secondary amide linkage within oligoprolines in contrast to
convenient peptides. The effects of peptide concentration on the
thermoresponsive behavior of P8-Dec and P8(Dec) were also inves-
tigated, and results are summarized in Fig. 2b (for turbidity curves,
see Fig. S1 in ESI†). Overall, the Tc of P8-Dec shows much high
concentration dependence than that for P8(Dec). From concentra-
tion of 0.25 wt% to 1.0 wt%, the Tc for P8-Dec decreases 12 K, while
the Tc for P8(Dec) decreases only 5 K. This result indicates that alkyl
The monodispersed oligoprolines were synthesized by a similar
procedure according to the previous report via solution peptide
synthesis strategy as shown in Scheme 1. Starting from the proline
octamer ester 1a,13 saponification with LiOH afforded the acid 1b,
which was then reacted with pentafluorophenol (Pfp-OH) to form the
active ester 1c. Amidation of this active ester with either ada-
mantylamine or n-decylamine furnished, respectively, the peptides
P8-Ada and P8-Dec with hydrophobic units at the chain end. The
proline octamer with alkyl chain located in the middle of peptide
chain [P8(Dec)] was synthesized through multiple steps. Williamson
etherification between hydroxyproline 2 and 1-bromo-decane affor-
ded the ester 3a. After hydrolysis and reacting with Pfp-OH, the
active ester 3c was obtained. Amide coupling between compound 4
and 5 afforded the tripeptide 6a, which was deprotected with TFA to
furnish the ammonium 6b. Amidation of 3c with 6b afforded the
tetrapeptide 7a, which was deprotected with TFA to form the
ammonium 7b. Reaction with the tetrapeptide active ester 813 fur-
nished the octamer P8(Dec). All key compounds were characterized
1
via H NMR and high resolution mass spectroscopy to prove their
high purity (for spectra, see ESI†).
Scheme 1 Synthesis of modified oligoprolines P8-Ada, P8-Dec and P8(Dec). Reagents and conditions: (a) LiOH$H2O, MeOH, H2O, ꢁ5 ꢀC–r.t., 4 h
(92–98%); (b) pentafluorophenol, EDC$HCl, DCM, ꢁ15 ꢀC–r.t., overnight (60–74%); (c) Ada-NH2, DIEA, DCM, 0 ꢀC–r.t., overnight (64%); (d)
n-decylamine, DCM, 0 ꢀC–r.t., overnight (60%); (e) 1-bromo-decane, KI, 15-crown-5, NaH, THF, r.t., 20 h (40%); (f) DiPEA, DCM, ꢁ15 ꢀC–r.t.,
overnight (91%, 64% and 65%, respectively); and (g) TFA, DCM, 0 ꢀC–r.t., 4 h (100%).
4870 | Soft Matter, 2012, 8, 4869–4872
This journal is ª The Royal Society of Chemistry 2012

View Article Online
Fig. 3 Plots of Tc with the different ratio a-CD/oligopeptides for both
P8-Dec and P8(Dec). Heating and cooling rate ¼ 0.2 ꢀC minꢁ1
.
Concentration of the peptides ¼ 0.5 wt%.
Fig. 4 1H NMR spectra of complex from P8-Dec and a-CD with the
ratio of 1 : 0.5 below (23 ꢀC), (a) and above (50 ꢀC), (b) the Tc. The
spectrum of pure P8-Dec in D2O at 23 ꢀC (c) is also included for
comparison. The dotted lines are a guide for the eyes.
Fig. 2 a) Plots of transmittance vs. temperature for 0.5 wt% aqueous
solutions of P8-Dec and P8(Dec) and (b) dependence of Tcs on concen-
trations of P8-Dec and P8(Dec). Heating and cooling rate ¼ 0.2 ꢀC minꢁ1
.
native a-CD into the inclusion complexes, (2) the inclusion complexes
are dynamic with fast exchange between CD host and peptide guests,
and (3) alkyl moieties at the end of peptide chains facilitate the
complexation with a-CD because of less steric hindrance. In addition,
temperature-varied 1H NMR spectroscopy was utilized to follow the
thermally induced dehydration of the supramolecular complexes
(Fig. 4), and found: (1) most proton signals from oligoprolines
became broad at elevated temperatures due to the dehydration and
(2) proton signals from the terminal methyl group split into two
groups at elevated temperature, indicating partial decomposition of
the supramolecular complex due to the steric hindrance formed from
the chain collapse.16
moieties at the end of peptide chains enhance the inter-molecular
aggregation more pronouncedly. Besides the Tc, the broadness of
the phase transitions for both peptides is also dependent on the
concentration. With the decrease of solution concentrations, the
transitions became significantly broad (Fig. S1 in ESI†). Above
results demonstrate that the geometry and location of the hydro-
phobic units not only show significant influence on the phase tran-
sition temperature of the oligopeptides, but also afford them different
aggregation behavior. The large concentration dependence of the
thermoresponsive behavior of these oligopeptides is characteristic of
thermoresponsive scaffolds with low molar masses.14
Thermoresponsive behavior of the oligoprolines was also investi-
gated through host–guest interaction. a-Cyclodextrin (a-CD) can
form complexes via host–guest interaction with alkyl chains with
constants in the range of several hundreds,15 thus, different amount of
a-CD was introduced into the aqueous solutions of the oligoprolines
to form supramolecular complexes. The influence of the supramo-
lecular interaction on the thermoresponsiveness of both P8-Dec and
P8(Dec) was examined, and the results are summarized in Fig. 3 (for
the turbidity curves, see Fig. S2 in ESI†). Both oligopeptides show
increased Tcs with increase of the a-CD/oligopeptide ratio. When
equivalent a-CD was added, Tcs for P8-Dec and P8(Dec) increased to
ꢀ
68 and 55 C, respectively. Even when excess amount of CD was
added, Tcs increased further (data are not shown). The Tc change for
P8-Dec is more pronounced than that for P8(Dec). These results
suggest: (1) complexation introduces strong hydrophilicity from
Fig. 5 Circular dichroism spectra of P8-Dec and P8(Dec) in aqueous
solutions at 25 ꢀC and 60 ꢀC.
This journal is ª The Royal Society of Chemistry 2012
Soft Matter, 2012, 8, 4869–4872 | 4871

View Article Online
The helical conformations of the thermoresponsive oligoprolines
were investigated with circular dichroism spectroscopy, and the
results are plotted in Fig. 5. Typical circular dichroism spectra for
PPII helices (a weak positive band around 228 nm and a strong
negative band around 204 nm) were obtained for both P8-Dec and
P8(Dec). This helical conformation was retained without significant
change at different temperatures, which suggests that the hydro-
phobic environment formed from the collapsed oligoprolines above
their Tcs cannot lead to the conformation change from the more
hydrophilic PPII to the more hydrophobic PPI. This PPII confor-
mation remained even after keeping at elevated temperatures for
several days. The reason for this high conformation stability may be
due to the insufficient driving force from the hydrophobic aggregates
or the cap effect from the terminal group.17
Notes and references
1 (a) H. G. Schild, Prog. Polym. Sci., 1992, 17, 163; (b) C. H. Alarcon,
S. Pennadam and C. Alexander, Chem. Soc. Rev., 2005, 34, 276; (c)
C. Pietsch, U. S. Schubert and R. Hoogenboom, Chem. Commun.,
2011, 47, 8750.
€
2 (a) W. Li, A. Zhang and A. D. Schluter, Chem. Commun., 2008, 5523;
(b) Y. Zhou and D. Yan, Chem. Commun., 2009, 1172; (c) X.-Y. Liu,
F. Cheng, Y. Liu, W.-G. Li, Yu. Chen, H. Pan and H.-J. Liu,
J. Mater. Chem., 2010, 20, 278; (d) D. J. Philips and M. I. Gibson,
Chem. Commun., 2012, 48, 1054; (e) M. A. Ward and
T. K. Georgiou, Soft Matter, 2012, 8, 2737.
3 (a) M. A. Ward and T. K. Georgiou, J. Polym. Sci., Part A: Polym.
Chem., 2010, 48, 775; (b) B. S. Murray, A. W. Jackson,
C. S. Mahon and D. A. Fulton, Chem. Commun., 2010, 46, 8651.
4 S. Jana, S. P. Rannard and A. I. Cooper, Chem. Commun., 2007, 2962.
5 N. S. Ieong, K. Brebis, L. E. Daniel, R. K. O’Reilly and M. I. Gibson,
Chem. Commun., 2011, 47, 11627.
6 J. Xu, J. Ye and S. Liu, Macromolecules, 2007, 40, 9103.
7 (a) Z. Jia, H. Chen, X. Zhu and D. Yan, J. Am. Chem. Soc., 2006, 128,
€
8144; (b) M. J. N. Junk, W. Li, A. D. Schluter, G. Wegner,
Conclusion
H. W. Spiess, A. Zhang and D. Hinderberger, Angew. Chem., Int.
Ed., 2010, 49, 5683.
This work has demonstrated a powerful novel route to synthesize
thermoresponsive peptides, which can be extended to other func-
tional (poly)peptides. Both the shape and location of the hydro-
phobic units have been proved to have significant effects on the phase
transition temperatures of the modified peptides. Due to the absence
of strong hydrogen bonding of the secondary amide linkages, these
peptides show unique thermoresponsive behavior with sharp transi-
tions and small hysteresis. Additionally, the thermoresponsiveness of
the oligoprolines can also be tuned easily through the host–guest
interaction between the alkyl chain and a-CD. This work provides an
alternative strategy for the design of promising thermoresponsive
chiral scaffolds from polymers for bio-applications.
8 (a) H. Ritter, O. Sadowski and E. Tepper, Angew. Chem., Int. Ed.,
2003, 42, 3171; (b) H. Ohashi, Y. Hiraoka and T. Yamaguchi,
Macromolecules, 2006, 39, 2614.
9 (a) D. W. Urry, J. Phys. Chem. B, 1997, 101, 11007; (b)
ꢀ
J. C. Rodrıguez-Cabello, J. Reguera, A. Girotti, F. J. Arias and
M. Alonso, Adv. Polym. Sci., 2006, 200, 119; (c) H. Nuhn and
H.-A. Klok, Biomacromolecules, 2008, 9, 2755.
10 (a) L. Ayres, M. R. J. Vos, P. J. H. M. Adams, I. O. Shklyarevskiy and
J. C. M. van Hest, Macromolecules, 2003, 36, 5967; (b) R. M. Conrad
and R. H. Grubbs, Angew. Chem., Int. Ed., 2009, 48, 8328; (c) Y. Chen
and Z. Guan, J. Am. Chem. Soc., 2010, 132, 4577.
11 C. Chen, Z. Wang and Z. Li, Biomacromolecules, 2011, 12, 2859.
12 (a) P. M. Cowan and S. Mcgavin, Nature, 1955, 176, 501; (b)
W. Traub and U. Shmueli, Nature, 1963, 198, 1165.
13 A. Zhang and Y. Guo, Chem.–Eur. J., 2008, 14, 8939.
14 W. Li, A. Zhang, Y. Chen, K. Feldman, H. Wu and A. D. Schluter,
Chem. Commun., 2008, 5948.
We thank Dr Hongmei Deng (Instrumental Analysis and
Research Center of Shanghai University) for her assistance in NMR
measurements. This work is financially supported by National
Natural Science Foundation of China (nos. 21034004, 21104043, and
20974020) and the Science and Technology Commission of Shanghai
(no. 10520500300 and 11pj1404100).
€
15 A. Hashidzumea and A. Harada, Polym. Chem., 2011, 2, 2146.
16 J. Yan, W. Li, K. Liu, D. Wu, F. Chen, P. Wu and A. Zhang, Chem.–
Asian J., 2011, 6, 3260.
17 M. Kuemin, S. Schweizer, C. Ochsenfeld and H. Wennemers, J. Am.
Chem. Soc., 2009, 131, 15474.
4872 | Soft Matter, 2012, 8, 4869–4872
This journal is ª The Royal Society of Chemistry 2012