Dynamic covalent polypeptides showing tunable secondary structures and Thermoresponsiveness

Article abstract of DOI:10.1002/pola.27433

Lysine-based polypeptides can be afforded with steerable secondary structures and tunable thermoresponsiveness through dynamic covalent OEGylation. These polypeptides were formed through dynamic imine linkage via reactions of amino moieties from poly(L-ly

Full text of DOI:10.1002/pola.27433

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Dynamic Covalent Polypeptides Showing Tunable Secondary Structures
and Thermoresponsiveness
Jiatao Yan, Kun Liu, Xiuqiang Zhang, Wen Li, Afang Zhang
Laboratory of Polymer Chemistry, Department of Polymer Materials, College of Materials Science and Engineering,
Shanghai University, Nanchen Road 333, Shanghai 200444, China
Correspondence to: A. Zhang (E-mail: azhang@shu.edu.cn)
Received 21 July 2014; accepted 29 September 2014; published online 20 October 2014
DOI: 10.1002/pola.27433
ABSTRACT: Lysine-based polypeptides can be afforded with
steerable secondary structures and tunable thermoresponsive-
ness through dynamic covalent OEGylation. These polypep-
tides were formed through dynamic imine linkage via reactions
of amino moieties from poly(L-lysine)s with aldehydes from
oligoethylene glycol (OEG)-based dendrons. In addition to
solution concentrations and pH values, macromolecular effect
was found to play an important role on the imine formation.
OEGylated polypeptides showed characteristic thermorespon-
sive properties, and their phase transition temperatures were
governed predominately by terminal groups and the coverage
of OEG dendrons. Notably, thermally induced aggregation
would enhance the imine formation even at elevated tempera-
ture. In contrast to the covalent polypeptide representatives,
the dynamic covalent polypeptides conveyed different thermor-
esponsiveness due to imine linkages, and their phase transi-
tion temperatures could be tuned simply by varying ratios
of OEG dendrons with different hydrophilicity. Furthermore,
helical conformation of these polypeptides was enhanced with
attachment of OEG dendrons, and could be reversibly
switched through thermally induced aggregation. V
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KEYWORDS: dendrimers; dynamic covalent chemistry; helical
conformation; peptides; secondary structures; stimuli-sensitive
polymers
INTRODUCTION Polypeptides have formed a class of impor-
tant bio-mimicking polymers, whose ordered secondary
structures, such as a-helix and b-sheet, play crucial roles in
mediating their biological functions and material applica-
peptides from glutamate and glucosylated allyl- or propargyl-
glycine exhibited characteristic pH-switchable conformations
1
0
and bioactivity, together with enhanced helicity. Through
the responsive thioether linkage, glycosylated poly(S-alkyl-L-
homocysteine)s underwent redox-triggered helix-to-coil tran-
1
tions. The stability of these secondary structures is domi-
1
1
nated by the peptide chain length, amino acid sequence, and
sition. Further studies on stimuli-responsive polypeptides
with tunable secondary structures will not only help to
understand and control some abnormal conformation trans-
2
also architectures. Charges along peptide chains provide
water solubility, but always decrease helicity due to coulomb
3
12
repulsion. Many strategies such as side-chain crosslinking,
formation related to amyloid-based diseases but also be
4
5
hydrogen-bond surrogate, metal coordination, salt bridge
potentially helpful in designing for drug delivery system, tis-
6
7
13
formation, and self-assembly have been used to modulate
the secondary structures of peptides with specific sequences.
Alternatively, by elongating the distance between charged
pendants and polypeptide backbone, ionic polypeptides were
found to show remarkable helical stability against changes in
sue engineering, and smart materials.
Thermoresponsive polypeptides are one of the most attrac-
tive stimuli-responsive polypeptides, and have found promis-
ing applications in various areas. One intriguing class of
thermoresponsive polypeptides are those based on elastin-
8
pH, temperature, and denaturing reagents. This structural
1
4
effect was proposed to be attributable to the decrease of
charge repulsion and increase of side-chain hydrophobicity.
Recently, much attention has been paid to synthesize stimuli-
responsive polypeptides, whose secondary structures or
macroscopic properties can be switched by external stimuli
like polypeptides (ELPs).
These polypeptides exhibit
reversible thermally induced phase transition behavior in
aqueous solutions, accompanying the secondary structure
change from random coil to b-spiral. The excellent biocom-
patibility makes ELPs useful for a wide variety of biomedical
applications. However, linear ELPs always show broad-phase
9
such as temperature, pH, and redox. For example, copoly-
Additional Supporting Information may be found in the online version of this article.
2014 Wiley Periodicals, Inc.
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FIGURE 1 Molecular structures of dynamic OEGylated polypeptides (Et-P, Me-P), monomeric imines (Et-M, Me-M), and nondy-
namic OEGylated polypeptide (Et-NP) discussed in this work.
2
3
transitions and large hysteresis owing to the strong hydrogen
bonding from amide moieties. To avoid these, various ther-
moresponsive polypeptides with different chemical structures
or architectures have been developed to mimic ELPs. Oligo-
peptides or polypeptides can be afforded with thermorespon-
sive properties through covalent modification to elegantly
them with thermoresponsiveness and, at the same time, to
2
4
switch their secondary structures. To combine thermores-
ponsive polypeptides with dynamic characteristics, we here
present the synthesis of OEGylated polypeptides through DCC
(Fig. 1). Dendritic OEG moieties were selected on one side to
efficiently shield hydrogen bonding of amide units in polypep-
tide backbones, and on the other side to provide thermores-
ponsiveness. These dynamic covalent polypeptides inherit
superior thermoresponsive properties from their parent OEG-
1
5
tune the hydrophilicity–hydrophobicity balance.
Oligo
(
ethylene glycol)s (OEGs) were utilized as motifs to mediate
polypeptides with superior thermoresponsiveness. The poly-
peptides such as poly(L-lysine)s, poly(L-glutamate)s, and
poly(L-cysteine)s carrying linear OEG units in their side chains
were found to be thermoresponsive, and their phase transi-
tion temperatures can be modulated by varying OEG length,
25
based dendronized polymers; simultaneously, their thermor-
esponsiveness and secondary structures can be finely tuned
and controlled through the dynamic covalent linkages.
1
6
ratios of comonomers, and even chirality of polypeptides.
EXPERIMENTAL
Alternatively, polyprolines grafted with bulky OEG dendrons
show appealing thermoresponsive behavior, whose thermores-
ponsive properties and secondary structures are dependent
on both dendron generation and arrangement of dendrons
along the polypeptide backbones. Despite the great progress
in synthetic strategies, most thermoresponsive polypeptides
reported to date are constructed through covalent linkage.
Their chemical structures or architectures are fixed once the
polypeptides were prepared. It is hard to tune their thermor-
esponsive properties at will without breaking them, and
therefore, it remains a challenge to develop novel strategies
for preparing thermoresponsive polypeptides with truly tuna-
ble phase transition temperatures.
Materials
Ethoxyl- and methoxyl-terminated first-generation OEG-
based dendritic alcohols were synthesized according to our
2
5(b)
1
7
previous reports.
molecular weight by viscosity 5 48,600) was purchased
from Sigma-Aldrich. Buffer solutions were prepared by add-
Poly-L-lysine hydrochloride (PLL)
(
ing appropriate amounts of NaOH to 30 mM NaHCO aque-
ous solutions, and pH values (11, 10, 9) were calibrated with
a Metler Toledo seven compact S220-B pH meter. Dichloro-
3
methane (DCM) was dried over CaH . Other reagents and
2
solvents were purchased at reagent grade and used without
further purification. All reactions were run under a nitrogen
atmosphere. Macherey-Nagel precoated TLC plates (silica gel
60 G/UV254, 0.25 mm) were used for thin-layer chromatog-
raphy (TLC) analysis. Silica gel 60 M (Macherey-Nagel, 0.04–
0.063 mm, 200–300 mesh) was used as the stationary phase
for column chromatography.
Dynamic covalent chemistry (DCC) relies on the reversible for-
mation and breaking of covalent bonding under thermody-
namic control, which has been proven to be a powerful
strategy to mediate polymers with responsive and adaptive
1
8
properties. The dynamic covalent linkages allow the compo-
nent exchanges and structure reshuffles under certain condi-
Instrumentation and Measurements
19
1
13
tions, which can be kinetically fixed after removal of catalyst.
H and C NMR spectra were recorded on a Bruker AV 500
1
13
Various reversible covalent linkages, including disulfide, imine,
and acylhydrazone, have been consciously selected to construct
dynamic covalent (poly)peptides, which show promising appli-
( H: 500 MHz, C: 125 MHz) spectrometer. High-resolution
MALDI-TOF-MS analyses were performed on IonSpec Ultra
instruments. UV/vis turbidity measurements were carried
out on a PE UV/vis spectrophotometer Lambda 35 equipped
with a thermostatically regulated bath. Sample solutions
2
0
21
cations in synthetic receptors, self-replication, and respon-
22
sive assembly. However, less attention was paid to afford
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were placed in the spectrophotometer (path length 1 cm),
ꢀ
21
and heated or cooled at a rate of 0.2 C min . The absorp-
tions of the solution at k 5 500 nm were recorded every 5 s.
The cloud point temperature (T ) was determined as the
cp
one at which the transmittance at k 5 500 nm had reached
50% of the value difference between the initial and final
stages. Circular dichroism (CD) measurements were per-
formed on a JASCO J-815 spectropolarimeter with a thermo-
controlled 0.2 mm quartz cell (five accumulations; “continues
21
scanning” mode; scanning speed: 100 nm min ; data pitch:
.5 nm; response: 1 s; bandwidth: 2.0 nm).
0
Synthesis of Et-OEG
Pyridinium chlorochromate (0.51 g, 2.37 mmol) was added
to a solution of ethoxyl-terminated first-generation OEG-
based dendritic alcohols (1.00 g, 1.57 mmol) in DCM
ꢀ
(
20 mL) at 0 C, and then the reaction temperature was ele-
1
FIGURE 2 H NMR spectra of Et-OEG (a), an equivalent mixture
of Et-OEG and PLL (b), and an equivalent mixture of Et-OEG
and pentylamine (c) in pH 5 11 buffer at room temperature. [Et-
OEG] 5 3 mM.
vated to room temperature. After stirring for 3 h, the solvent
was evaporated under vacuum, and the residue was purified
twice by column chromatography (DCM/MeOH, 40:1 v/v) to
afford Et-OEG (0.80 g, 80%) as a colorless oil.
1
H NMR (CDCl ): d 5 1.19–1.22 (t, 9H, CH ), 3.51–3.89 (m,
1
3
3
solutions, which was verified with H NMR spectroscopy
[
3
1
6
6H, CH ), 4.21–4.28 (m, 6H, CH ), 7.14 (s, 2H, CH), 9.82 (s,
2
2
Fig. 2(a,b)]. Upon addition of 1 eq of PLL (based on the
1
H, CHO). HR-MS: m/z calcd for C H O Na [M 1 Na]
3
1 54 13
amino moieties) to the buffer solution of Et-OEG at pH 5 11,
the resolved proton signals (peaks 1 and 2) from monomeric
Et-OEG at d 5 9.70 and 7.26 ppm remained but their inten-
sities significantly reduced. At the same time, new broad
proton signals appeared at d 5 7.93 and 6.84 ppm, which
correspond to the imine and benzoic moieties from the poly-
peptide, respectively. The intensity reduction of the proton
signals from Et-OEG and appearance of new broad signals
prove that dendritic OEG units were attached onto PLL
through the amine/aldehyde condensation to form the OEGy-
lated polypeptide Et-P. The coexistence of Et-P and Et-OEG
suggests thermodynamic equilibrium for the imine forma-
tion. By comparing the benzoic signal intensity from Et-P at
d 5 6.84 ppm with that from Et-OEG at d 5 7.26 ppm, the
imine yield was calculated to be about 59.0%. Similarly, the
more hydrophilic polymeric imine Me-P was formed by mix-
ing equivalent PLL and Me-OEG with a yield around 50.9%
57.3462; found 657.3454.
Synthesis of Me-OEG
Pyridinium chlorochromate (1.10 g, 5.10 mmol) was added
to a solution of methoxyl-terminated first-generation OEG-
based dendritic alcohols (2.03 g, 3.41 mmol) in DCM
ꢀ
(
60 mL) at 0 C, and then the reaction temperature was ele-
vated to room temperature. After stirring for 3 h, the solvent
was evaporated under vacuum, and the residue was purified
twice by column chromatography (DCM/MeOH, 40:1 v/v) to
afford Me-OEG (1.60 g, 79%) as a colorless oil.
1
H NMR (CDCl ): d 5 3.37 (m, 9H, CH ), 3.51–3.89 (m, 30H,
3
3
CH ), 4.21–4.28 (m, 6H, CH ), 7.14 (s, 2H, CH), 9.82 (s, 1H,
CHO). HR-MS: m/z calcd for C H O Na [M 1 Na]
6
2
2
1
2
8 48 13
15.2993; found 615.2988.
(
Supporting Information Fig. S2). For comparison, mono-
Synthesis of Et-NP
Excess of NaBH (200 mM) was added to the solution of Et-P
meric imines Et-M and Me-M were prepared by reactions of
pentylamine with Et-OEG and Me-OEG, respectively, to verify
possible polymeric effects on the imine formation. H NMR
4
(3 mM) in pH5 11 buffer at room temperature. After stirring
1
overnight, the Et-NP with around 56% OEG coverage was
spectrum of the reaction mixture for Et-OEG is shown in Fig-
ure 2(c). The well-resolved new-born proton signals at
d 5 8.15 and 7.06 ppm correspond to imine and benzoic
moieties from the monomeric imine Et-M, respectively. By
counting the intensity ratio of benzoic signal at 7.06 ppm
from Et-M with that at 7.26 ppm from Et-OEG, the imine
yield was calculated to be 22.0%, which is much lower than
that in polymeric imines Et-P and Me-P. The enhancement of
imine formation with PLL could be mainly ascribed to local
concentration enrichment of amino moieties along PLL
chains. Certainly, pKa decrease of amino moieties in PLL
when compared with pentylamine should be another reason
yielded, which was purified with dialysis against deionized
water. This reduction reaction was verified by H NMR spectra
1
(
Supporting Information Fig. S1). After the addition of NaBH4,
the proton signals from Et-OEG and Et-P disappeared, while
some new proton signals corresponding to dendritic alcohols
Et-OEG-OH and Et-NP emerged. This demonstrates that Et-P
was totally reduced to form nondynamic Et-NP.
RESULTS AND DISCUSSION
Imine Formation at Room Temperature
The formation of dynamic covalent polypeptides was con-
ducted through mixing PLL with dendritic OEGs in aqueous
7
(a)
to contribute partially to the enhancement.
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through imine linkage will change with external conditions,
such as concentration and pH, which may lead to different
expression of thermoresponsiveness for the OEGylated PLLs.
Herein, these two external conditions were modulated inde-
pendently to examine the effects of OEG coverage on the
thermoresponsive behavior, and Et-P was selected as a rep-
2
7
resentative. The plots of its Tcp and OEG coverage (x) ver-
sus concentration or pH are shown in Figure 4 (for
corresponding turbidity curves, see Supporting Information
Fig. S5). When the concentration of Et-P decreased from 3 to
0
6
.5 mM (at pH 5 11), x reduced significantly from 59.0 to
.8%. At the same time, T increased dramatically from 37.5
cp
ꢀ
to 53.3 C (D 5 15.8 K), and the phase transition became
broad simultaneously together with the appearance of
obvious hysteresis. On the other hand, a decrease of pH from
1
1 to 9 also led to a large decrease of x, which causes a
FIGURE 3 Plots of transmittance versus temperature for Et-P,
Me-P, and Et-M in pH 5 11 buffer. [c] 5 3 mM.
steep increase of Tcp (D 5 23.9 K) and broadening of phase
transitions. Certainly, reduction of pH values also leads to
enhanced protonation of amino groups as well as helicity
decrease of polypeptide, which should also contribute to the
increase of Tcp and broadness of phase transitions. These
results demonstrate that thermoresponsiveness of the
dynamic OEGylated polypeptides is closely related to cover-
age of the dendritic OEG along PLL chains. Coverage
Thermoresponsive Properties
2
8
The dendritic OEGs or PLL alone are not thermoresponsive
ꢀ
even up to 80 C. In contrast, the formed OEGylated polypep-
tides (Et-P and Me-P) are water soluble at room tempera-
ture, but exhibit typical thermoresponsive behavior at
elevated temperature. Therefore, their thermally induced
phase transition processes were followed by using UV/vis
spectroscopy, and the typical turbidity curves are shown in
Figure 3. Both OEGylated polypeptides show quite fast and
sharp phase transitions during heating (D < 2 K). For hydro-
phobic Et-P a very small hysteresis (D < 1 K) was observed,
but hydrophilic Me-P showed an abnormal reversible behav-
ior during the cooling process, which is caused by thermally
decrease leads to the higher T , broader phase transition, as
well as larger hysteresis. To verify effects of imine dynamic
cp
induced precipitation. The cloud point temperatures (T ) for
cp
ꢀ
Et-P and Me-P were determined to be 37.5 and 65.5 C,
respectively. This large difference of Tcps indicates their high
dependence on terminal groups of OEG moieties. Although
OEG dendron coverage on PLL chains is relatively low at the
given conditions (50–59%), these polypeptides adopt similar
thermoresponsive behavior as the covalent OEGylated
2
5
dendronized polymethacrylates (OEG coverage 5 100%) in
both phase transition behavior and transition temperatures.
These suggest that polypeptides could be endowed with
superior thermoresponsiveness when reasonable amount of
OEG units are attached onto their side chains. For compari-
son, thermoresponsiveness of monomeric imines (Me-M and
Et-M) was also examined. Ethoxyl-terminated Et-M is ther-
ꢀ
moresponsive but its Tcp is higher than 70 C. Furthermore,
its phase transition is quite broad (>20 K). Methoxyl-
terminated Me-M is too hydrophilic to show any thermores-
ꢀ
ponsiveness even at very high temperature (90 C). The sig-
nificant difference in thermoresponsive properties between
polymeric and monomeric imines can be ascribed to the
macromolecular effect, which facilitates both formation of
the imines and the thermally induced collapse and aggrega-
tion kinetics of neighboring dendrons.
As imine bonds in aqueous solutions are dynamic in
FIGURE 4 Plots of Tcp and x versus concentration (fixed pH5 11)
(a) and pH (fixed [c] 5 3 mM) (b) for both Et-P and Et-NP.
2
6
nature,
the coverage of dendritic OEG on PLL chains
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hysteresis [Fig. 5(a)]. Their T s are completely dependent
cp
on the mole ratios of two homopolypeptides [Fig. 5(b)], and
ꢀ
were determined to be 41.8, 47.2, and 54.1 C for the mole
ratios of Me-P/Et-P at 1:3, 1:1, and 3:1, respectively. These
results indicate that the phase transition temperatures of the
dynamic OEGylated polypeptides are fully tunable in the
ꢀ
range of 37–66 C simply through adjusting mole ratios of
two dendritic OEGs with different hydrophilicities.
Effects of Thermally Induced Phase Transitions
on Imine Linkage
1
Temperature-varied H NMR spectroscopy was used to
examine the effect of thermally induced phase transitions on
the dynamic imine linkage. The monomeric imine Et-M was
selected as the representative because its proton signals are
well resolved when compared to its polymeric counterpart.
For comparison, the nonthermoresponsive Me-M was also
1
checked. Typical H NMR spectra of Et-M formed from
equivalent Et-OEG and pentylamine in the range of d 5 7.9–
ꢀ
1
0.9 ppm from 30 to 70 C are shown in Figure 6(a) (for
full spectra, see Supporting Information Fig. S7). Below the
phase transition temperature, the proton signals from Et-M
and Et-OEG coexist and remain in single group. As the tem-
ꢀ
perature increases to 46 C which is right below the aggre-
ꢀ
gation point (46.7 C), proton signals from Et-M split into
0
0
two groups (peaks 3, 3 , 4, and 4 ), while those from Et-OEG
remain one group (peaks 1 and 2). Proton splitting for Et-M
suggests that OEG collapse from water renders Et-M into
two physicochemical states, which possesses
a
slow
exchange process on the NMR timescale. The downfield sig-
FIGURE 5 (a) Plots of transmittance versus temperature for the
mixture of Me-P and Et-P at different mole ratios (0:1, 1:3, 1:1,
nals (3, 4) correspond to Et-M in hydrated state, and the
0
0
upfield (3 , 4 ) to Et-M in dehydrated state. With further
increase of solution temperature, the content of dehydrated
imine increases gradually, while the content of hydrated
imine decreases. By comparing these signal intensities, the
yield of Et-M (both in hydrated and dehydrated states) can
be calculated, and the results are plotted in Figure 6(b). As
expected, the yield of Et-M decreases linearly with the
3:1, and 1:0) in pH 5 11 buffer. (b) Plot of Tcp against the mole
ratio of Me-OEG. [c] 5 3 mM.
nature on the thermoresponsive behavior, Et-P was reduced
with NaBH to form the corresponding nondynamic polypep-
4
tide Et-NP. This nondynamic polypeptide possesses an OEG
coverage around 56%, and its thermoresponsiveness was
investigated (for turbidity curves, see Supporting Informa-
increase of temperature below T . However, as the tempera-
cp
ture increases above T , the yield increases abruptly. This
tion Fig. S6). The T of Et-NP increased slightly from 41.1
cp
cp
ꢀ
tendency is enhanced with the further increase of solution
to 43.6
C (D 5 2.5 K) when the concentration was
ꢀ
ꢀ
temperature, and the imine yield reaches 35.6% at 70 C,
decreased from 3 to 0.5 mM, while it reduced moderately
ꢀ
which is even slightly higher than that (28.1%) at 23 C,
from 45.2 to 41.1 C (D 5 4.1 K) when solution pH was
indicating that thermally induced aggregation facilitates the
imine formation in the case of Et-M. For comparison, the
yield of hydrophilic Me-M was also followed, which is found
to be decreased continuously during the whole heating pro-
cess [Fig. 6(b)]. Above results demonstrate that the ther-
mally induced phase transition can effectively promote the
formation of imine in aqueous solutions even at high tem-
peratures, providing that the collapsed phase is hydrophobic
enough. Enhancement of imine formation in thermally
induced aggregation of Et-M was further verified by mixing
Et-M with a thermoresponsive covalent dendronized polymer
Et-PG1 (for its molecular structure, see Supporting Informa-
changed from 9.0 to 11.0. These small variations make sharp
contrast to the case of dynamic Et-P, indicating that dynamic
imine linkage plays a key role in mediating thermorespon-
siveness of the polypeptides.
Based on reversibility of the dynamic imine linkage in OEGy-
lated polypeptides, it should be possible to tune the overall
hydrophilicity–hydrophobicity
balance
through
imine
exchange, and this can be done by using dendritic OEGs of
different hydrophilicity. Through mixing homopolypeptides
Et-P and Me-P at different mole ratios, the random copoly-
peptides would be formed via the imine exchange based on
similar reactivities of Me-OEG and Et-OEG toward PLL. In
the same way as the parent homopolypeptides, these
copolypeptides show quite fast phase transitions and small
2
5(b)
tion Fig. S8)
at different temperatures. The addition of
Et-PG1 into the mixture of Et-OEG and pentylamine shows
negligible influence on the yield of Et-M at room
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of dynamic OEGylation on the secondary structures, PLLs
with different OEG coverage were prepared by changing the
mole ratio of Et-OEG to amino moieties from PLL ([Et-
OEG]/[NH ]), and their CD spectra are shown in Figure 7(a).
2
At pH 5 11, the native PLL adopts a typical a-helical confor-
mation at room temperature as indicated by Cotton effects
with a maximum at k 5 191 nm and two minima at k 5 208
and 222 nm. Upon successive addition of Et-OEG, OEG cov-
erage on polypeptide chains increased gradually, and the
mean residual molar ellipticity at k 5 222 nm ([h]222
increased accordingly [Fig. 7(b)]. When [Et-OEG]/[NH2]
reached 0.5, [h] tends to reach a maximum value of about
)
2
22
ꢀ
2
21
3
5,000 cm dmol , corresponding to nearly 100% helic-
ity. Similar helical conformation enhancement was also
3
0
observed at pH 5 10 where a relatively low OEG coverage
(
21.4%) was reached (Supporting Information Fig. S10). This
demonstrates that attachment of OEG units onto PLL chains
could effectively enhance the polypeptide helicity, which
should result from shielding of static or charge repulsion
among residual amino pendants through OEG dendrons.
Based on their unique thermoresponsiveness, influence of
temperature on the secondary structures of these OEGylated
1
FIGURE 6 (a) Temperature-varied H NMR spectra of Et-M in
buffer. (b) Plots of transmittance and imine yield versus tem-
perature for Et-M in buffer. The imine yield for Me-M on the
temperature is also included. pH 5 11, [c] 5 5 mM.
temperature (Supporting Information Fig. S9). However, as
ꢀ
the temperature increased to 32 C, which is right around
the phase transition temperature of Et-PG1 but far below
that of Et-M, the proton signals from Et-M split into two
groups, suggesting that Et-M was partially encapsulated in
the hydrophobic domain formed by the dehydration and
aggregation of Et-PG1. As a result, the yield of Et-M
increases obviously from 26.7 to 39.1% when temperature
ꢀ
increases from 30 to 49 C. Hence, the dehydration and col-
lapse of OEG units from the water could provide a unique
hydrophobic microenvironment, which effectively facilitates
imine formation and inhibits to some extent imine hydrolysis
in aqueous solutions. Similar enhancement of imine forma-
tion in aqueous conditions was also observed in other
2
9
amphiphilic self-assembly systems.
FIGURE 7 (a) CD spectra of PLL in pH 5 11 buffer
Secondary Structures of the OEGylated Polypeptides
Secondary structures of these OEGylated polypeptides were
investigated by CD spectroscopy. To examine possible effect
([NH ] 5 3 mM) with different amounts of Et-OEG. [Et-OEG]/
2
[NH ] represents the mole ratio of Et-OEG to lysine units in
2
2
PLL. (b) Plot of 2[h]222 and x against [Et-OEG]/[NH ].
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above the crucial turn point for Et-P. To further illustrate the
effects of thermally induced collapse and aggregation on the
secondary structure transitions of polypeptides, PLLs with
different OEG coverage Et-P(0.5) and Et-P(0.2) were pre-
pared by changing the ratios of [Et-OEG]/[NH ] to be 0.5
2
and 0.2, respectively. Their [h]222 versus temperature is plot-
ted in Figure 8(b) (for CD spectra, see Supporting Informa-
tion Fig. S12). For the native PLL, [h]222 decreased slowly
ꢀ
with increase of temperature from 20 to 60 C, and tended
ꢀ
to be unchanged at 60 C as a result of the a-helix-to-b-sheet
transition. However, the OEGylated polypeptide Et-P with a
high OEG coverage (59.0%) underwent great drop of [h]2
at the onset of the phase transition (38 C), and decreased
22
ꢀ
sharply with increase of temperature. When OEG coverage
was reduced to around 29.4% in the case of Et-P(0.5), this
OEGylated polypeptide showed similar thermoresponsive
ꢀ
behavior as Et-P, except an increased Tcp (41.8 C) together
with a slightly broader phase transition and larger hysteresis
(
for turbidity curves, see Supporting Information Fig. S13).
ꢀ
As a result, its [h]222 reduced evidently at around 42 C, and
the decrease was less sharper than that of Et-P with
increase of temperature. When OEG coverage was further
decreased to around 7.8% for the case of Et-P(0.2), its
phase transition became very broad, and [h]222 showed
much slower decrease during the heating process, which
resembles very much as the native PLL. Above results dem-
onstrate that helicity of OEGylated polypeptides was
destroyed significantly once the solution temperature
increased above their cloud point temperatures. The tend-
ency for their helicity decrease during phase transitions was
dominated by OEG coverage: the larger the OEG coverage,
the more significant the decrease of helicity. This suggests
that the collapse and aggregation of dendritic OEG side
chains could probably weaken the intramolecular hydrogen
bonding of polypeptide backbone, which is the main driving
force for the formation of stable a-helical conformation. The
higher OEG coverage around the polypeptide chains could
lead to the formation of larger crowdedness and solubility to
prohibit intramolecular hydrogen bonding, thus forming
more disorder conformation. Certainly, steric hindrance from
collapsed OEG units may also contribute to diminishing the
ordered structures. Therefore, through finely tuning OEG
coverage, secondary structures of these OEGylated polypep-
tides can be reversibly switched to be ordered or disordered
based on their thermoresponsiveness.
FIGURE 8 (a) Temperature-varied CD spectra of Et-P in pH 5 11
buffer. (b) Plots of [h]222 versus temperature for Et-P, Me-P, Et-
P(0.5), and Et-P(0.2) in pH 5 11 buffer. Et-P(0.5) and Et-P(0.2)
2
represent these formed with [Et-OEG]/[NH ] ratios to be 0.5
and 0.2, respectively. [NH ] 5 3 mM.
2
polypeptides was examined, and resulting CD spectra from
Et-P are plotted in Figure 8(a). Below the phase transition
temperature, [h]
remains nearly the same at about
2
22
21
ꢀ
2
35,000 cm dmol with increase of temperature. However,
ꢀ
as temperature reached 38 C which is around the phase
ꢀ
ꢀ
transition point (37.5 C), [h]
reduced greatly to 27,000
2
22
2
21
cm dmol , indicating that the ordered conformation of
polypeptide was disrupted partially upon chain collapse
from water. With further elevation of solution temperature,
[
h]222 decreased continuously, and nearly down to zero at 50
C. Once the solution was cooled down to room temperature,
CONCLUSIONS
ꢀ
the ordered secondary structures of Et-P recovered com-
pletely [compare curves a with a in Fig. 8(a)]. Notably, the
presence of an isosbestic point at around k 5 200 nm
In summary, we have presented a novel strategy for endow-
ing polypeptides simultaneously with tunable thermorespon-
sive properties and secondary structures through DCC. These
polypeptides were formed through the reversible imine con-
densation between poly(L-lysine)s and dendritic OEG motifs
in aqueous solutions. Besides the contributions from solution
concentrations, pH values, component ratios, as well as
hydrophobicity of the components, the imine formation can
be enhanced significantly when reactions are performed on
macromolecules. Thermoresponsiveness of these OEGylated
0
(
marked with an arrow) suggests that polypeptide conforma-
tion was exclusively transformed between ordered a-helix
and disordered random conformation during the thermally
induced aggregation process. Similar phenomena were also
observed for the more hydrophilic Me-P [Fig. 8(b), for CD
spectra, see Supporting Information Fig. S11], but its [h]2
decreased evidently at around 66 C (its T ) which is far
cp
22
ꢀ
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polypeptides is dominated by terminal groups and coverage
of OEG dendrons. Thanks to the dynamic feature from
imines, their phase transition temperatures can be easily
tuned through imine exchange between dendritic OEGs of
different hydrophilicity. Secondary structures of these OEGy-
lated polypeptides are dependent on OEG coverage, and can
be tuned through thermally induced aggregation. Attached
OEG dendrons along polypeptide backbone provide shielding
effects to obviously enhance a-helical conformation. Ther-
mally induced aggregation can promote the imine formation
in aqueous environment when the reactants are hydrophobic
enough and, at the same time, distort the ordered secondary
structures probably due to the diminished intramolecular
hydrogen bonding. Based on the dynamic characteristics of
imine linkage, it is convenient to tune the thermoresponsive
properties and secondary structures of these OEGylated
polypeptides. Considering the excellent biocompatibility of
OEGylated polymers, these novel stimuli-responsive polypep-
tides would find promising applications in bio-related func-
tional materials. We thus expect that the novel strategy
developed in this work could inspire the exploration of novel
stimuli-responsive (poly)peptides to extend their applica-
tions in biological and biomedical systems.
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thank the National Natural Science Foundation of China (Nos.
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