Charge-Determined LCST/UCST Behavior in Ionic Polypeptoids

Article abstract of DOI:10.1021/acs.biomac.8b00240

Stimuli-responsive polymers have received increasing interest for a variety of applications. Here, we report a series of unique charge-determined thermoresponsive polypeptoids synthesized by a combination of ring-opening polymerization and click chemistry. The LCST-type and UCST-type behavior is mainly dominated by the charge state on the side chain. Further, the phase transition temperature highly depends on the degree of polymerization, the side-chain architecture, the pH value, and so on. The obtained polypeptoid solutions exhibit good stability against temperature and salt concentration. To our knowledge, this report presents the first charge-determined LCST/UCST-type polymer from identical homopolymer backbone that displays a wide range of tunable cloudy points in aqueous media. We propose the hydrogen-bonding interaction plays a critical part on the solution behavior. These features make polypeptoids ideal candidates for highly designable stimuli-responsive polymeric materials.

Full text of DOI:10.1021/acs.biomac.8b00240

Article
Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
pubs.acs.org/Biomac
Charge-Determined LCST/UCST Behavior in Ionic Polypeptoids
Chao Xing, Zhekun Shi, Jiliang Tian, Jing Sun,* and Zhibo Li*
Key Laboratory of Biobased Polymer Materials, Shandong Provincial Education Department; School of Polymer Science and
Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China
*
S Supporting Information
ABSTRACT: Stimuli-responsive polymers have received increasing interest for a variety of applications. Here, we report a series
of unique charge-determined thermoresponsive polypeptoids synthesized by a combination of ring-opening polymerization and
click chemistry. The LCST-type and UCST-type behavior is mainly dominated by the charge state on the side chain. Further, the
phase transition temperature highly depends on the degree of polymerization, the side-chain architecture, the pH value, and so
on. The obtained polypeptoid solutions exhibit good stability against temperature and salt concentration. To our knowledge, this
report presents the first charge-determined LCST/UCST-type polymer from identical homopolymer backbone that displays a
wide range of tunable cloudy points in aqueous media. We propose the hydrogen-bonding interaction plays a critical part on the
solution behavior. These features make polypeptoids ideal candidates for highly designable stimuli-responsive polymeric
materials.
INTRODUCTION
polymers were reported to display UCST behavior in aqueous
11,16
■
solution, particularly under physiologic conditions.
Poly-
Thermoresponsive polymers, a class of promising smart
materials, have received extensive interest for both fundamental
research and applications in the past two decades.
particular, water-soluble thermoresponsive polymers are
capable of exhibiting a reversible phase transition to temper-
ature in aqueous media. They offer great potential for
mers that can exhibit both LCST and UCST behavior under
17,18
1
,2
mild and physiologic conditions have been barely reported.
In
It has been reported that copolymerization is one of the
strategies by tuning the types and compositions of mono-
13
mers. A recent study shows that copolymers composed of N-
3
acryloylglycinamide and diacetone acrylamine have either
LCST- or UCST-type transitions, depending on the
compositions, degree of polymerization (DP), polymer
biomedical applications such as protein delivery, smart
hydrogels, and surface science. A majority of thermoresponsive
4
polymers show typical lower critical solution temperature
19
concentration, and so on. A new generation of smart
5
−9
(
(
LCST) behavior,
such as poly(N-isopropylacrylamide)
polymers with finely tunable LCST/UCST properties is
PNIPAM), OEG-grafted (meth)acrylates polymers, and
20
desired.
polypeptides. The LCST-type polymer undergoes a phase
transition from a hydrated state to a dehydrated state upon
heating. In contrast, the reported polymers exhibiting upper
critical solution temperature (UCST) behavior are relatively
rare, such as poly(sulfobetaine) and poly(N-acryloylglycina-
Polypeptoids are a promising class of peptidomimetic
21
polymers based on an N-substituted glycine backbone.
Special Issue: Biomacromolecules Asian Special Issue
1
0−15
mide).
The UCST-type polymer shows a miscibility gap at
Received: February 10, 2018
Revised: March 29, 2018
low temperature. In most cases, the UCST behavior is observed
in organic solvents or organic/water mixtures. Only a few
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.biomac.8b00240
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
They offer great advantageous properties for both fundamental
research and applications in biotechnology. Due to the
similarity in structure to polypeptides, the polypeptoids exhibit
Nano ZS. Zeta-potentials (ζ) were calculated with the Smoluchowski’s
formula: ζ = μEη/ϵ, where η denotes the viscosity and ϵ the
permittivity of the solution. Solutions were prepared by dissolving the
polymer in water at desired concentrations, followed by titration of the
initial solution to the desired pH using 1 M HCl or 1 M NaOH,
respectively.
22−24
excellent biocompatibility and potential bioactivities.
The
lack of inter- and intrachain hydrogen bonding and chirality in
the main chain results in a potentially flexible backbone. The
properties of polypeptoids are mainly dominated by the side-
chain identity, which offers a unique and facile way to tailor the
properties of the polymers by structural design of the side
chains. Further, the polypeptoids are highly designable in terms
of synthetic approaches. In addition to solid-phase method, the
ring-opening polymerization (ROP) has emerged as an effective
method to produce the polypeptoids with high molecular
Synthesis of 2-(Allylamino)acetic Acid Hydrochloride (a).
Glycoxylic acid solution (60 g) was mixed with 300 mL of CH Cl in
2
2
the reaction flask. A total of 15 mL of allylamine was added slowly, and
the reaction was stirred for 6 h at room temperature. The CH Cl was
2
2
then removed by rotavapor. An aqueous solution of HCl (300 mL, 1.0
M) was added, followed by refluxing in 110 °C and stirring overnight.
Brown solid was further purified by recrystallization in methanol/THF
(
30 mL/300 mL) at −10 °C overnight after water was removed by
25−27
rotavapor. The resulting compound was obtained by filtered and dried
under vacuum (14.1 g, 46.5% yield). H NMR (500 MHz, D O) δ
weights in high yields.
The postmodification of the well-
1
2
defined polypeptoid precursors offer a convenient method to
5
.81 (m, 1H), 5.43 (t, 2H), 3.79 (s, 2H), 3.64 (d, 2H).
Synthesis of 2-(Allyl(tert-butoxycarbonyl)amino)acetic Acid
28,29
prepare polypeptoids with versatile functionalities.
We have previously prepared a family of pegylated nonionic
polypeptoids that exhibits lower critical solution temperature
(b). A total of 12 g a was dissolved in 200 mL of water. A total of 21.4
g di-tert-butyl dicarbonate and 27 mL of triethylamine was then added
and stirred overnight at room temperature. The mixture was washed
by hexane (3 × 100 mL) to remove unreacted di-tert-butyl dicarbonate
followed by adjusting pH to 3 with 4 M aqueous HCl; an aqueous
phase was then extracted with ethyl acetate (3 × 100 mL). The
supernatant was separated and washed with brine and dried with
anhydrous Na SO . The white solid was collected after the solvent was
3
0
(
LCST) behavior. Unlike our previous study, we synthesized
a family of ionic polypeptoids with tunable thermal-responsive
property in this study. The synthetic method combines ring-
opening polymerization (ROP) technique with thiol−ene/yne
click chemistry. The obtained polypeptoids show either LCST-
type or UCST-type behavior, depending on the side-chain
charges. Moreover, the phase transition of the polypeptoids in
both cases is pH-sensitive. The influence of the side-chain
architecture, the degree of polymerization (DP), and the
polymer concentration on the solution properties of the
polypeptoids has been systematically investigated. We propose
the phase transition temperature is highly dependent on the
hydrogen bonding of the system. To our best knowledge, this is
the first report of a charge-determined thermoresponsive
polymer from an identical homopolymer backbone.
2
4
1
removed by rotavapor (6.1 g, 71.7%). H NMR (500 MHz, CDCl ) δ
3
5.77 (m, 1H), 5.16 (t, 2H), 3.98 (s, 2H), 3.88 (d, 2H), 1.43 (s, 9H).
Synthesis of N-Allyl N-Carboxyanhydride (Allyl-NCA, c). A
total of 6 g b was dissolved in 250 mL of anhydrous CH Cl under
2 2
nitrogen in a 500 mL flask. A 2.5 mL aliquot of PCl was then added
3
dropwise to the reaction solution and the system was stirred for 3 h in
an ice bath. After the solvent was removed under vacuum, the oil was
extracted with 15 mL of CH Cl and filtered. In a glovebox, further
2
2
purification was performed from anhydrous THF/hexane. After 4−5
times dissolving/precipitating cycles, clear oil was obtained (2.2 g,
1
5
3.4%). H NMR (500 MHz, CDCl ) δ 5.78 (m, 1H), 5.34 (d, 2H),
3
4
.07 (s, 2H), 4.01 (d, 2H).
Synthesis of Poly(N-allyl glycine) (PNAG). In a typical
EXPERIMENTAL SECTION
■
procedure, the allyl-NCA (300 mg, 2.12 mmol) dissolved in 3 mL
of anhydrous THF, followed by adding a stock solution of
benzylamine (3.61 wt % in THF). The solution was stirred at 50 °C
for 24 h with nitrogen protection, and the polymerization progress was
monitored by FTIR. The solution was precipitated into an excess of
hexane. The product was dried under reduced pressure to yield a white
solid (74.2% yield). All the other polymers were prepared in a similar
way according to the designed NCA to initiator ratio.
Materials and Methods. Glyoxylic acid (50 wt % in H O) and
2
allylamine (98%) were purchased from Shandong Xiya Chemical Co.,
Ltd. Progargylamine was purchased from Nantong Camry Chemical
Co., Ltd. Benzylamine (99%), cysteamine hydrochloride (98%),
mercaptoacetic acid (99%), di-tert-butyl dicarbonate, and 2,2-
dimethoxy-2-phenylacetophenone (DMPA) were purchased from
Aladdin reagent. Phosphorus trichloride and trimethylamine were
purchased from Sinopharm Chemical Reagent Co., Ltd. 3-
Mercaptopropionic acid was purchased from Adamas Reagent Co.,
Ltd. Dichloromethane (DCM), hexane, and tetrahydrofuran (THF)
were purified by passing through activated alumina columns prior to
use. The rest of chemicals were purchased from commercial suppliers,
which were used without further purification unless otherwise stated.
Poly(N-propargylglycine) (PNPG) was synthesized by following a
2
8
published procedure.
General Procedure for Modification with Cysteamine
Hydrochloride/3-Mercaptopropionic Acid. Typically, cysteamine
hydrochloride (1.19 g, 10.5 mmol) or mercaptoacetic acid (1.12 g,
10.5 mmol), PNAG (100 mg, [SH] = 1.05 mmol, [SH]/[CC] =
1
13
H and C NMR spectra were recorded on Bruker AV500 FT-NMR
1
0), and DMPA (13 mg, 0.053 mmol) were dissolved in 1 mL of
spectrometer. Tandem gel permeation chromatography (GPC) was
performed at 50 °C using an SSI pump connected to Wyatt Optilab
DSP with 0.02 M LiBr in DMF as eluent at flow rate of 1.0 mL/min.
The concentrations of GPC samples were ∼5 mg/mL. The molecular
weights were calibrated against polystyrene (PS) standards. Fourier
transform infrared (FTIR) spectra were recorded from 2000 to 1400
cm on a Horiba−Jobin Yvon FluoroMax-4 spectrofluorometer. All
the sample solutions were cast on KBr plates at different temperatures
before measurement. We defined the cloud points (CPs) as the
temperature at the transmittance of 50% during the heating process.
All the CPs were measured by monitoring the transmittance of a 500
nm light beam through a quartz sample cell on a Shimadzu UV-2910
spectrometer. The heating/cooling rate is 1 °C/min. Circular
dichroism spectra were recorded on an applied photophysics chirascan
CD spectrometer. The solution was placed into a quartz cell with a
path length of 0.1 cm. The electrophoretic mobility (μE) of the
aggregates in dependence of pH was measured on a Malvern Zetasizer
DMF. The system was degassed and then irradiated with UV light at
room temperature for 3 h. After dialyzed for 3 days and lyophilized,
white solid was achieved (75.4% yield). All the other PNAG and
PNPG were modified in a similar way.
−1
RESULTS AND DISCUSSION
The N-propargyl N-carboxyanhydride (NPG-NCA) and N-allyl
N-carboxyanhydride (Allyl-NCA) monomers were synthesized
following reported methods (Scheme S1). The chemical
structure of the monomers was confirmed by H NMR
■
31
1
spectroscopy (Figures S1 and S2). The homopolymers were
then synthesized by ring-opening polymerization (ROP) of
both NCA monomers with a nucleophilic initiator benzylamine
(Scheme 1). The polymerization was monitored by FTIR. The
disappearance of two characteristic ν
peaks of the
CO
B
DOI: 10.1021/acs.biomac.8b00240
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Scheme 1. Synthetic Pathways of Polymerization of NPG-
NCA and Allyl-NCA Monomers and Subsequent Radical
Thiol Addition
Table 1. Molecular Parameters of Homopolypeptoids
a
b
c
c
a
feed ratio
M
n
M
n
dispersity
b
samples
(NCA/initiator)
n
(kg/mol) (kg/mol)
(Đ)
PNPG41
PNPG83
PNPG107
PNAG21
PNAG46
PNAG55
PNAG79
40
80
110
20
40
60
80
41
83
107
21
46
55
79
3.8
7.8
10.7
1.8
4.4
5.8
7.6
2.8
4.1
4.9
4.8
4.5
5.2
6.2
1.21
1.27
1.16
1.31
1.13
1.10
1.09
a
Feed molar ratio of NCA/initiator. Calculated from 1H NMR
b
c
spectra. Determined from GPC; n represents the average DP of
PNPG or PNAG.
dispersity (Đ) ≤ 1.31, indicating the well-controlled polymer-
ization (Figure S3).
The polypeptoids were subsequently modified with thiol-
terminated regents to yield functionalized polypeptoids
(
Scheme 1). PNAG was conjugated with cysteamine hydro-
chloride and mercaptoacetic acid to yield PNAG-NH and
PNAG-COOH, respectively. Figure 1b shows a typical H
2
1
NMR spectrum of PNAG-NH , where the protons of newly
2
a
Reaction conditions are described in the text.
formed CH SCH linkage at δ ∼ 2.53−2.88 ppm are visible.
2
2
Further the protons of alkenyl group at 5.01−5.94 ppm entirely
monomer at 1790 and 1860 cm− was considered as complete
conversion of NNCA monomers to polypeptoids. All peaks of
the obtained polypeptoids are well assigned in the H NMR
1
disappear, which indicates the virtually quantitative conversion
32
of alkenyl group. The chemical structure of PNAG-NH was
2
1
further confirmed by FTIR, shown in Figure 2. A characteristic
1
spectra, which confirm the chemical structures (Figures 1 and
S2). A series of homopolypeptides with different degrees of
polymerization (DPs) were then synthesized by varying the
ratio of NCA to the initiator. The average DPs of PNPG and
PNAG were in the range of 41−107 and 21−79, respectively.
Table 1 summarized the molecular characteristics of both
polymers PNPG and PNAG , where the subscript n represents
the average DP of both polymers. H NMR spectroscopy was
used to determine the molar ratio and molecular weight of the
blocks. The DPs were obtained from the proton integral ratios
of allyl group or propargyl group to the phenyl group. The
GPC trace shows a narrow molecular weight distribution with
stretching band of at 3081 cm− (ν C−H) is completely absent

after the modification, confirming the quantitative conversion.
The successful modification of PNAG with mercaptoacetic acid
1
was also confirmed by H NMR and FTIR (Figures 1a and 2).
PNPG was modified in a similar way to yield PNPG-(NH2)2
and PNPG-(COOH) with branched side chains, respectively.
2
Similarly, the results suggest the successful modification
n
n
1
13
(Figures S4 and S5). In a typical C NMR spectrum, the
absence of protons at 73.8−79.7 ppm suggests the complete
conversion of the alkynyl groups (Figure S4).
All of the polypeptoids modified with mercaptoacetic acid
can readily dissolve in alkaline solution. Due to the presence of
1
Figure 1. Representative H NMR spectra of (a) PNAG -COOH; (b) PNAG -NH ; (c) PNAG in DMSO (* indicates DMSO).
46
46
2
46
C
DOI: 10.1021/acs.biomac.8b00240
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
interactions via inter- and intrachain hydrogen bonding
between amide groups on the backbone and −COOH groups
36
on the side chain. Upon heating, the phase transition
occurred driven by thermally controlled reversible hydrogen
bonding. This is not unexpected as the thermal-responsive
property is dependent on many effects. It was previously
reported that the zwitterionic groups can lead to the cluster
formation that largely influences the thermal-responsive
37
properties of the polypeptoid system. Furthermore, the
transition of cis backbone conformation to a mixture of cis
and trans conformation is also related to the phase transition
38
behavior. FTIR was further used to explore the influence of
hydrogen bonding on phase transition behavior (Figure S6).
−1
The characteristic band of −COOH at ∼3400 cm (νOH) is
significantly reduced at high temperature, indicating the
hydrogen bonding formation between the polymer with water
molecule. Further decreasing pH to 4.0 results in a turbid
solution in the absence of the UCST behavior, due to the
increased protonation degree of the −COOH groups.
Interestingly, the UCST behavior is pH-responsive with
extremely narrow transition of ΔpH = ∼0.2.
Figure 2. FTIR spectra of PNAG , PNAG -NH , and PNAG -
COOH.
46
46
2
46
carboxylic acid groups on the side chains, the polymers are
expected to show pH-responsive behavior in aqueous solution.
The desired pH value was adjusted by HCl or NaOH solutions.
At pH ≥ 4.5, a clear solution of PNPG -(COOH) is obtained
To quantify the percent protonation/deprotonation of the
4
1
2
−
COOH groups, we performed zeta-potential (ζ) measure-
at a concentration of 2 mg/mL over the entire experimental
temperature window (Figure 3a). As pH is decreased to 4.2, the
solution appears cloudy at ambient temperature. This is due to
the increasing content of protonated −COOH moieties that
ment. The ζ percent based on the ζ (= −23.1 mV) of the
solution at pH ≥ 7 is plotted as a function of pH (Figure S7).
An abrupt increase is observed as pH ranges from 4 to 5. The
percentages of ζ are observed to 6% and 97%, suggesting nearly
complete protonation and deprotonation at pH 4.0 and 4.5,
respectively. At pH 4.2, the ζ percent of 72% shows that the
same amount of −COOH group is deprotonated. This
indicates the slight variation in ζ percent can lead to the
different thermal-responsive property. It is noteworthy that the
previously reported poly(N-(2-carboxyethyl)glycine) with
33
decrease the solubility of the polypeptoid. Note that the
poly(L-glutamic acid) analog was reported to show a pK of
a
3
4
∼
4.3. Upon heating, the polymer solution shows phase
transitions from cloudy to clear. Figure 3a shows the
transmittance increases from 0% to 100% as the temperature
increases from 20 to 80 °C. We defined the cloud point (CP)
as the temperature at the transmittance of 50% during the
heating process, which was determined to be 35 °C in this case.
During the cooling process, reversible phase transition is
observed. The transmittance recovers completely to 0%,
resulting in the CPcooling of 33 °C. This indicates a typical
UCST behavior. The CP determined in the cooling ramp is
slightly lower (2 °C) than that in the heating ramp. The
hysteresis is possibly due to the overcooling during
39
pendant COOH groups exhibit excellent water solubility.
Distinct from the reported one, PNPG -(COOH) contains
n
2
additional thioether groups on side chains with branched
architecture, which are generated by thiol−yne click chemistry.
The difference in structure varies the hydrophilic−hydrophobic
balance of the system. All of these features result in the pH-
dependent UCST-type thermoresponsive behavior in the
polymer. Note that the polypeptoids barely show CD signals.
This is due to the lack of chiral center, suggesting the absence
of the secondary structure (data not shown).
3
5
dehydration. We have previously reported that pegylated
polypeptoids show LCST behavior due to the presence of
3
0
oligo(ethylene glycol) (OEG) groups. Unlike previous
results, herein we incorporated the carboxylic acid groups on
the side chains, which largely enhance the polymer−polymer
The influence of the concentration on the solution properties
of PNPG -(COOH) samples was investigated. Generally,
41
2
Figure 3. (a) Plots of transmittance as a function of temperature for aqueous solutions (2 mg/mL) of PNPG -(COOH) at different pH. Filled
41
2
symbol: cooling ramp; open symbol: heating ramp. (b) Plots of transmittance as a function of temperature for aqueous solutions of PNPG -
4
1
(
COOH) at pH = 4.2 at different concentrations.
2
D
DOI: 10.1021/acs.biomac.8b00240
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Figure 4. (a) Plots of CP as a function of NaCl concentration for PNPG107-(COOH) solution at a concentration of 0.5 mg/mL at pH 4.2. (b)
2
Transmittance of PNPG -(COOH) aqueous solution at a concentration of 0.5 mg/mL at pH 4.2 vs 8 heating and cooling cycles between 20 and
41
2
8
0 °C.
increasing the concentration results in lower CP (Figure 3b),
16,19
which is different from the most reported UCST system.
The deviation is likely due to the charge state of the polymers.
Considering the pH-sensitive property, slight variation of
protonation degree may change the solubility of the polymer.
We further investigated the addition of salt on the solution
behavior of the polypeptoids. A typical plot of CP of PNPG107
-
(
COOH) as a function of NaCl concentration show that the
2
CP remains consistent as the NaCl concentration is increased
from 0 to 100 mM (Figure 4a). This suggests that the solution
is relatively stable to the salt, despite the presence of ionic
groups. We also incorporated CaCl at a concentration up of 50
2
mM into the system. Interestingly, the complex solution lacks
UCST behavior over the entire experimental window. We
attribute this to the coordination of −COOH groups in the
Figure 5. Plots of CPs as a function of DP for Bn-PNPG -(COOH)
n
2
2+
polymer with Ca ions, which disrupt the hydrogen bonding of
and Bn-PNAG -COOH at a concentration of 0.5 mg/mL at pH 4.2.
n
40
the system (Figure S8). This further confirms that the
hydrogen bonding plays a vital part in the thermal-responsive
property of the system. Moreover, the phase transition is
recovered after 8 times heating and cooling run between 20 and
modified with cysteamine to obtain PNPG -(NH ) and
n
2 2
PNAG -NH . The obtained polymers are expected to show
n
2
8
0 °C, suggesting good stability of the polypeptoid solution
pH-responsive behavior due to the presence of amine groups
on the side chains. In this case of PNAG -NH , the polymers
(Figure 4b).
n
2
We further systematically studied the temperature effect on
are well soluble at pH ≤ 12.9 at a concentration of 10 mg/mL
over the entire measurable temperature (Figure 6). Interest-
ingly, unlike the reported poly(N-(2-aminoethyl)glycine) with
the solution properties of PNPG -(COOH) samples. A family
n
2
of PNPG -(COOH) , with n ranging from 41 to 107 were
n
2
39
prepared. We plotted the CP of PNPG -(COOH) solutions
pendant NH groups with good water solubility, the samples
n
2
2
versus DP of PNPG. It is observed that increasing the DP
results in decreased CP (Figure 5). It was reported the longer
polymer chain enables less hydrophobic residues exposed in
aqueous media. More of the deionized −COOH groups
embedded in the longer polymer chain can reduce the
aggregation of the polymers and increase the solubility of the
polymer in aqueous solution. To probe the influence of the side
chain architecture, we investigated the temperature effect on
exhibit reversible phase transition from clear to cloudy upon
heating with increasing pH to 13.2. This suggests a typical
LCST behavior, consistent with the positively charged
polypeptoids. It is conceivable that the pH effect on the
solution behavior is extremely important. The incorporated
amine groups on the side chains behave as the hydrogen bond
donor and form strong interaction with water, which results in
the LCST-type behavior. It is known that the UCST behavior is
ascribed to the strong polymer−polymer and solvent−solvent
interactions. Instead, the strong polymer−solvent interaction
3
0
the solution properties of PNAG -COOH. Interestingly, the
n
PNAG -COOH samples exhibit UCST-type behavior as well.
n
11,17
Plotting the CP of PNAG -COOH solutions versus DP of
generally leads to the LCST behavior.
In this case, the
n
hydrogen bonding between the amine groups and amide
groups in PNAG -NH is relatively weak as compared to
n
2
PNAG -COOH, which results in the LCST behavior rather
n
than UCST behavior. FTIR was used to determine the
influence of hydrogen bonding on the phase transition behavior
(Figure S6b). Similarly to positively charged polypeptoid, the
−
1
broader characteristic band of amine groups at ∼3460 cm
NH2
(ν ) at low temperature, indicative of the hydrogen bonding
E
DOI: 10.1021/acs.biomac.8b00240
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
Figure 6. (a) Plots of transmittance as a function of temperature for aqueous solutions (10 mg/mL) of PNAG -NH at different pH. Filled symbol:
79
2
heating ramp; open symbol: cooling ramp. (b) Plots of CP as a function of pH for aqueous solutions (10 mg/mL) of PNAG -NH .
79
2
forming between the polymer and water molecule. As pH is
increased from 13.2 to 13.6, the CP of PNAG -NH decreases
n
2
from 54 to 22 °C (Figure 6b). Further increasing pH to 14.0
results in the turbid solution without exhibiting the LCST
behavior. We presume that this is due to the increased
deprotonation degree of the polymer that reduces the solubility
of the polymer. To confirm this, we performed zeta-potential
(ζ) measurement. The ζ percent based on the ζ (= 20.4 mV) of
the solution at pH ≥ 7 is plotted versus pH (Figure S7). An
abrupt increase is observed in the pH range of 13−14. As pH
increases from 13.2 to 13.6, the ζ percent decreases from 88%
to 49%, suggesting increased deprotonation degree of the
polymer. Further decreasing pH to 14.0 results in the ζ percent
of 10%, consistent with the previous conclusion. Note that the
PNPG -(NH ) with branched side chains lacks both LCST
n
2 2
and UCST behavior in the experimental window, confirming
the importance of the molecular architecture.
Figure 7. Plots of CP as a function of DP for aqueous solutions (10
The solution property of PNAG -NH was further system-
n
2
mg/mL) of PNAG -NH at pH 13.6.
n
2
atically investigated. Decreasing the concentration results in the
lower CP and incomplete recovery of transmittance of the
solution, suggesting high concentration is essential for the
LCST behavior (Figure S9). We also investigated the stability
of the polypeptoid solution at a concentration of 10 mg/mL.
The heating and cooling cycles were performed for 8 times
between 20 and 80 °C. The phase transition is entirely
recovered, suggesting good stability to temperature (Figure
S10). Similar to the polymer with pedant carboxylic acid
groups, the influence of salts on the CP is barely visible (Figure
S11). Further, we synthesized a series of PNAG -NH with
side-chain charges. Further, the phase transition of the
polypeptoids in both cases is pH-sensitive due to the presence
of charge. A systematic study of the influence of the
temperature and pH on the solution properties of the
polypeptoids was performed. We propose the hydrogen
bonding interaction plays an extremely critical part on the
solution behavior.
ASSOCIATED CONTENT
Supporting Information
■
n
2
*
S
different DP. All the samples show LCST-type behavior. A plot
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.8b00240.
of the CP versus DP for the PNAG -NH shows that the CP
n
2
decreases with increasing DP, which is likely due to less
solubility of longer molecular chains, irrespective of the
41
1
13
Additional H/ C NMR data, FTIR spectra, GPC data,
details of CPs, and turbidity measurements (PDF).
presence of ionized groups (Figure 7). Note that this differs
from the results of polypeptoids with −COOH groups on the
side chain. We attribute it to the different molecular
interactions of the systems.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: jingsun@qust.edu.cn.
E-mail: zbli@qust.edu.cn.
■
CONCLUSION
■
*
In conclusion, a family of ionic polypeptoids were synthesized
by a combination of ROP and thiol−ene/yne click chemistry.
The polypeptoids prepared in this work show reversible LCST-
type or UCST-type behavior in aqueous media, which is fairly
distinct from the previously reported positively and negatively
charged polypeptoids and pegylated nonionic polypeptoids.
The thermoresponsive behavior is highly dependent on the
ORCID
Jing Sun: 0000-0003-1267-0215
Zhibo Li: 0000-0001-9512-1507
Funding
This work was supported by National Natural Science
Foundation of China (51503115, 21674054, 51722302, and
F
DOI: 10.1021/acs.biomac.8b00240
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
1434008), Qingdao Innovation Leader Talent Program
Article
2
(17) Zhu, Y.; Batchelor, R.; Lowe, A. B.; Roth, P. J. Design of
Thermoresponsive Polymers with Aqueous LCST, UCST, or Both:
Modification of a Reactive Poly(2-vinyl-4,4-dimethylazlactone) Scaf-
fold. Macromolecules 2016, 49, 672−680.
(third), and Taishan Scholars Program.
Notes
The authors declare no competing financial interest.
(
18) Wu, G.; Chen, S. C.; Zhan, Q.; Wang, Y. Z. Well-Defined
Amphiphilic Biodegradable Comb-Like Graft Copolymers: Their
Unique Architecture-Determined LCST and UCST Thermorespon-
sivity. Macromolecules 2011, 44, 999−1008.
ACKNOWLEDGMENTS
■
This work was supported by National Natural Science
Foundation of China (51503115, 21674054, 51722302, and
1434008), Qingdao Innovation Leader Talent Program
(19) Sun, W.; An, Z.; Wu, P. UCST or LCST? Composition-
Dependent Thermoresponsive Behavior of Poly(N-acryloylglycina-
mide-co-diacetone acrylamide). Macromolecules 2017, 50, 2175−2182.
(20) Boustta, M.; Colombo, P. E.; Lenglet, S.; Poujol, S.; Vert, M.
Versatile UCST-based thermoresponsive hydrogels for loco-regional
sustained drug delivery. J. Controlled Release 2014, 174, 1−6.
(21) Sun, J.; Zuckermann, R. N. Peptoid polymers: a highly
designable bioinspired material. ACS Nano 2013, 7, 4715−4732.
̈
̂
(22) Klinker, K.; Schafer, O.; Huesmann, D.; Bauer, T.; Capeloa, L.;
2
(
third), and Taishan Scholars Program.
REFERENCES
■
(
̈
1) Stuart, M. A.; Huck, W. T.; Genzer, J.; Muller, M.; Ober, C.;
Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.
Emerging applications of stimuli-responsive polymer materials. Nat.
Mater. 2010, 9, 101−113.
Braun, L.; Stergiou, N.; Schinnerer, M.; Dirisala, A.; Miyata, K.
Secondary Structure-Driven Self-Assembly of Reactive Polypept(o)-
ides: Controlling Size, Shape and Function of Core Cross-Linked
Nanostructures. Angew. Chem., Int. Ed. 2017, 56, 9608−9613.
(
2) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-responsive nanocarriers
for drug delivery. Nat. Mater. 2013, 12, 991−1003.
3) Roy, D.; Brooks, W. L.; Sumerlin, B. S. New directions in
thermoresponsive polymers. Chem. Soc. Rev. 2013, 42, 7214−7243.
4) Dimitrov, I.; Trzebicka, B.; Muller, A. H. E.; Dworak, A.;
Tsvetanov, C. B. Thermosensitive water-soluble copolymers with
doubly responsive reversibly interacting entities. Prog. Polym. Sci. 2007,
(
(23) Gangloff, N.; Ulbricht, J.; Lorson, T.; Schlaad, H.; Luxenhofer,
R. Peptoids and Polypeptoids at the Frontier of Supra- and
Macromolecular Engineering. Chem. Rev. 2016, 116, 1753.
(
̈
(24) Knight, A. S.; Zhou, E. Y.; Francis, M. B.; Zuckermann, R. N.
Sequence Programmable Peptoid Polymers for Diverse Materials
Applications. Adv. Mater. 2015, 27, 5665.
3
(
2, 1275−1343.
5) Huang, J.; Heise, A. Stimuli responsive synthetic polypeptides
(25) Zhang, D.; Lahasky, S. H.; Guo, L.; Lee, C. U.; Lavan, M.
derived from N-carboxyanhydride (NCA) polymerisation. Chem. Soc.
Rev. 2013, 42, 7373.
6) Lahasky, S. H.; Hu, X.; Zhang, D. Thermoresponsive Poly(α-
Polypeptoid Materials: Current Status and Future Perspectives.
Macromolecules 2012, 45, 5833−5841.
(
(26) Fetsch, C.; Grossmann, A.; Holz, L.; Nawroth, J. F.; Luxenhofer,
peptoid)s: Tuning the Cloud Point Temperatures by Composition
R. Polypeptoids from N-Substituted Glycine N-Carboxyanhydrides:
Hydrophilic, Hydrophobic, and Amphiphilic Polymers with Poisson
Distribution. Macromolecules 2011, 44, 6746−6758.
and Architecture. ACS Macro Lett. 2012, 1, 580−584.
(
̈
7) Xia, Y.; Yin, X.; Burke, N. A. D.; Stover, H. D. H. Thermal
Response of Narrow-Disperse Poly(N-isopropylacrylamide) Prepared
(27) Tao, X.; Du, J.; Wang, Y.; Ling, J. Polypeptoids with Tunable
by Atom Transfer Radical Polymerization. Macromolecules 2005, 38,
Cloud Point Temperatures Synthesized from N-Substituted Glycine
2
(
275−2283.
8) Vancoillie, G.; Frank, D.; Hoogenboom, R. Thermoresponsive
poly(oligo ethylene glycol acrylates). Prog. Polym. Sci. 2014, 39, 1074−
095.
9) Lutz, J. F.; Akdemir, O.; Hoth, A. Point by point comparison of
N-Thiocarboxyanhydrides. Polym. Chem. 2015, 6, 3164−3174.
(28) Lahasky, S. H.; Serem, W. K.; Guo, L.; Garno, J. C.; Zhang, D.
Synthesis and Characterization of Cyclic Brush-Like Polymers by N-
Heterocyclic Carbene-Mediated Zwitterionic Polymerization of N-
Propargyl N-Carboxyanhydride and the Grafting-to Approach. Macro-
molecules 2011, 44, 9063−9074.
1
(
̈
two thermosensitive polymers exhibiting a similar LCST: is the age of
poly(NIPAM) over? J. Am. Chem. Soc. 2006, 128, 13046−13047.
10) Woodfield, P. A.; Zhu, Y.; Pei, Y.; Roth, P. J. Hydrophobically
Modified Sulfobetaine Copolymers with Tunable Aqueous UCST
through Postpolymerization Modification of Poly(pentafluorophenyl
acrylate). Macromolecules 2014, 47, 750−762.
11) Glatzel, S.; Laschewsky, A.; Lutz, J. F. Well-Defined Uncharged
Polymers with a Sharp UCST in Water and in Physiological Milieu.
Macromolecules 2011, 44, 413−415.
12) Seuring, J.; Bayer, F. M.; Huber, K.; Agarwal, S. Upper Critical
Solution Temperature of Poly(N-acryloyl glycinamide) in Water: A
Concealed Property. Macromolecules 2013, 45, 374−384.
13) Seuring, J.; Agarwal, S. First Example of a Universal and Cost-
Effective Approach: Polymers with Tunable Upper Critical Solution
Temperature in Water and Electrolyte Solution. Macromolecules 2012,
(
29) Secker, C.; Robinson, J. W.; Schlaad, H. Alkyne-X modification
of polypeptoids. Eur. Polym. J. 2015, 62, 394−399.
30) Tian, J. L.; Sun, J.; Li, Z. Biomimetic Pegylated polypeptoids
with Thermoresponsive Properties. Polymer 2018, 138, 132−138.
31) Robinson, J. W.; Secker, C.; Weidner, S.; Schlaad, H.
Thermoresponsive Poly(N-C3 glycine)s. Macromolecules 2013, 46,
80−587.
32) Guo, L.; Zhang, D. Cyclic poly(alpha-peptoid)s and their block
(
(
(
(
5
(
(
copolymers from N-heterocyclic carbene-mediated ring-opening
polymerizations of N-substituted N-carboxylanhydrides. J. Am. Chem.
Soc. 2009, 131, 18072−18074.
(
(33) Murnen, H. K.; Rosales, A. M.; Jaworski, J. N.; Segalman, R. A.;
Zuckermann, R. N. Hierarchical self-assembly of a biomimetic diblock
copolypeptoid into homochiral superhelices. J. Am. Chem. Soc. 2010,
4
(
5, 3910−3918.
1
(
32, 16112−16119.
14) Seuring, J.; Agarwal, S. Polymers with Upper Critical Solution
34) Kukula, H.; Schlaad, H.; Antonietti, M.; Forster, S. The
̈
Temperature in Aqueous Solution: Unexpected Properties from
formation of polymer vesicles or ″peptosomes″ by polybutadiene-
block-poly(L-glutamate)s in dilute aqueous solution. J. Am. Chem. Soc.
2002, 124, 1658−1663.
Known Building Blocks. ACS Macro Lett. 2013, 2, 597−600.
(
15) Fu, W.; Luo, C.; Morin, E. A.; He, W.; Li, Z.; Zhao, B. UCST-
Type Thermosensitive Hairy Nanogels Synthesized by RAFT
Polymerization-Induced Self-Assembly. ACS Macro Lett. 2017, 6,
̈
(35) Lutz, J. F.; Weichenhan, K.; Akdemir, O.; Hoth, A. About the
1
(
27−133.
16) Shimada, N.; Ino, H.; Maie, K.; Nakayama, M.; Kano, A.;
Maruyama, A. Ureido-derivatized polymers based on both poly-
allylurea) and poly(L-citrulline) exhibit UCST-type phase transition
behavior under physiologically relevant conditions. Biomacromolecules
011, 12, 3418−3422.
Phase Transitions in Aqueous Solutions of Thermoresponsive
Copolymers and Hydrogels Based on 2-(2-methoxyethoxy)ethyl
Methacrylate and Oligo(ethylene glycol) Methacrylate. Macromolecules
2007, 40, 2503−2508.
(36) Takeshi, M.; Masashi, N.; Yasuhisa, F.; Keiji, M.; Masami, T.;
Maeda, Y. Soluble−Insoluble−Soluble Transitions of Aqueous Poly-
(
2
G
DOI: 10.1021/acs.biomac.8b00240
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
(
4
N-vinylacetamide-co-acrylic acid) Solutions. Langmuir 2006, 22,
336−4342.
37) Du, P.; Li, A.; Li, X.; Zhang, Y.; Do, C.; He, L.; Rick, S. W.;
(
John, V. T.; Kumar, R.; Zhang, D. Aggregation of cyclic polypeptoids
bearing zwitterionic end-groups with attractive dipole-dipole and
solvophobic interactions: a study by small-angle neutron scattering and
molecular dynamics simulation. Phys. Chem. Chem. Phys. 2017, 19,
1
(
4388−14400.
38) Ma, J.; Xuan, S.; Guerin, A. C.; Yu, T.; Zhang, D.; Kuroda, D. G.
Unusual molecular mechanism behind the thermal response of
polypeptoids in aqueous solutions. Phys. Chem. Chem. Phys. 2017,
1
(
9, 10878−10888.
39) Nam, K. T.; Shelby, S. A.; Choi, P. H.; Marciel, A. B.; Chen, R.;
Tan, L.; Chu, T. K.; Mesch, R. A.; Lee, B. C.; Connolly, M. D. Free-
floating ultrathin two-dimensional crystals from sequence-specific
peptoid polymers. Nat. Mater. 2010, 9, 454−460.
(
40) Wang, Q.; Mynar, J. L.; Yoshida, M.; Lee, E.; Lee, M.; Okuro,
K.; Kinbara, K.; Aida, T. High-water-content mouldable hydrogels by
mixing clay and a dendritic molecular binder. Nature 2010, 463, 339−
3
43.
(
41) Tao, X.; Deng, Y.; Shen, Z.; Ling, J. Controlled Polymerization
of N-Substituted Glycine N-Thiocarboxyanhydrides Initiated by Rare
Earth Borohydrides toward Hydrophilic and Hydrophobic Poly-
peptoids. Macromolecules 2014, 47, 6173.
H
DOI: 10.1021/acs.biomac.8b00240
Biomacromolecules XXXX, XXX, XXX−XXX