Water-soluble Poly(γ-propargyl- l -glutamate) containing pendant sulfonate ions and terminal fluorophore: Aggregation-enhanced emission and secondary structure

Article abstract of DOI:10.1021/ma500886v

Tetraphenylthiophene (TP) with aggregation-enhanced emission (AEE) property was used as terminal fluorophore of the water-soluble poly(γ-propargyl-l- glutamate) (PPLG)-based polymers of TP-iPPLGs to probe the relationship between the secondary structure (

Full text of DOI:10.1021/ma500886v

Article
pubs.acs.org/Macromolecules
Water-Soluble Poly(γ-propargyl‑L‑glutamate) Containing Pendant
Sulfonate Ions and Terminal Fluorophore: Aggregation-Enhanced
Emission and Secondary Structure
Ke-Ying Shih, Tai-Shen Hsiao, Shiang-Lin Deng, and Jin-Long Hong*
Department of Materials and Optoelectronic Science, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
S
* Supporting Information
ABSTRACT: Tetraphenylthiophene (TP) with aggregation-enhanced emission
(AEE) property was used as terminal fluorophore of the water-soluble poly(γ-
propargyl-L-glutamate) (PPLG)-based polymers of TP-iPPLGs to probe the
relationship between the secondary structure (α-helix) of polypeptides and the
AEE-related emission behavior. Intermolecular aggregation of the terminal TP
unit in TP-iPPLG is sterically blocked by the large α-helical PPLG chains, leading
to the weak AEE-related fluorescence in water. In contrast, the intermolecular
approach between the terminal TP units of TP-iPPLG is accessible if TP was
connected by peptide chin in random coil structure. Therefore, the helix-to-coil
transition induced in the alkaline aqueous solution successfully enhances the
emission intensity of the TP-iPPLG solution. The pH-induced conformation
change in relationship to the AEE-related emission behavior is therefore evaluated
in this study.
variations resulted in different emission and FL decay
behaviors. Study on the fluorophore-labeled PBLG provided
routes for the evaluation of aggregation behavior of the peptide
chains.
INTRODUCTION
■
Synthetic polypeptides are mimics of natural peptides and able
to form stable secondary structures (e.g., α-helix, β-sheets, and
random coil) in solution and solid state.1−3 As the cornerstone
of the three-dimensional architecture of proteins, the α-helical
structure functioned to regulate numerous biological activities.
The unique folding/unfolding property and the rigid rodlike
morphology have made the α-helix the subject of intense study
and building block in the design of therapeutics and molecular
assemblies.4−13 However, the usefulness of these structures is
limited because of their poor aqueous solubility and
processability. Therefore, interest has been largely focused on
the design and synthesis of water-soluble α-helical polypeptide.
Therefore, charged amino groups14,15 and cyclodextrin16 have
been incorporated with polypeptides as the side chains to
render in the good water solubility. The ultrastable, water-
soluble α-helical polypeptides14 can be produced by elongating
charge-containing amino acid side chains to position the
charges distant from the polypeptide backbone.
As fluorescence (FL) spectroscopy constitutes one of the
most powerful tools to study the aggregation of polymers,
traditional fluorescent probes such as carbazolyl,17 dansyl,18 and
pyrene19 have been chemically incorporated with poly(γ-
benzyl-L-glutamate) (PBLG) to monitor the chain conforma-
tions of PBLG in solution. For example, the absence of excimer
emission in the pyrene-labeled poly(ethylene glycol)-b-PBLG19
indicated that the pyrene groups were separated from each
other in the self-assembled structure. A study on triblock
PBLG−polyfluorene−PBLG copolymers20 suggested that
formations of rod−rod−rod and coil−rod−coil conformations
depend on the solvent casting history and the morphological
Traditional planar fluorophores (e.g., pyrene and anthracene)
are generally weakly emissive in the solid state. In contrast,
noncoplanar silole molecule (1-methyl-1,2,3,4,5-pentaphenylsi-
lole)21,22 was found to have strong emission in the aggregated
solution and solid states despite that it is nonemissive in the
dilute solution state. Aggregation-induced emission (AIE) was
therefore designated for this peculiar phenomenon in view that
the originally nonluminescent solution of silole can be tuned to
emit strongly when aggregates formed by nonsolvent inclusion.
Since the first discovery of the silole system, several organic and
polymeric materials23−28 with AIE or aggregation-enhanced
emission (AEE) properties have been prepared and charac-
terized. With chemical structure similar to silole, tetraphenylth-
iophene (TP) and TP-derived organic and polymeric
materials29−31 were developed in our lab, and their AIE and
AEE characters were identified. Also, this TP fluorophore was
served as the terminal and central fluorophores of PBLG-based
polymers of TP1PBLG and TP2PBLG,32 respectively, to probe
the relationship between the secondary structure (α-helix) of
polypeptides and the AEE-related fluorescence. Mainly,
intermolecular aggregation of the central TP units in the
disubstituted TP2PBLGs is sterically blocked by the large α-
helical PBLG chains, leading to the reduced AEE-oriented
Received: April 29, 2014
Revised: June 3, 2014
Published: June 12, 2014
© 2014 American Chemical Society
4037
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
Scheme 1. Chemical Structure of TP-iPPLGs and the Feasibility of Intermolecular Aggregation among TP Terminals
Connected by Different Secondary Structures
emission. By adding trifluoroacetic acid (TFA), the rigid helical
chain of TP2PBLG can be transformed into flexible random
coil structure; therefore, adding TFA largely intensified the
emission intensity of TP2PBLG since intermolecular aggrega-
tion of the TP centers in random-coil chain can be readily
achieved. Comparatively, the terminal TP units in the
monosubstituted TP1PBLGs have no problem in approaching
each other, resulting in strong emission intensity. The
secondary structure of polypeptides is therefore correlated
with the AEE activity.
In contrast to the water-insoluble TP1PBLG and
TP2PBLG,32 a new water-soluble polypeptide of TP-iPPLG
(Scheme 1) containing an AEE-active TP terminal and ionic
sulfonate side groups were prepared and characterized in this
study. Instead of the commonly used organic charged
amino14,15 or β-cyclodextrin (CD)16 side groups, ionic sodium
sulfonate groups were introduced through the facile click
reaction and were used as the side chains of TP-iPPLGs to
deliver the desired water solubility of the peptide polymers.
These ionic TP-iPPLGs provide access to study the influence of
sulfonate side groups on the secondary structure of the peptide
chain, and the relation between AEE-related emission behavior
and secondary structure of peptide chain can be explored.
Theoretically, the AEE-related emission of TP-iPPLG is
strongly correlated to the aggregation tendency of the TP
terminals, which in turn are affected by the steric shielding
imposed by the neighboring peptide chains. It is envisaged that
the two-dimensional α-helical rod, which has diameter larger
than the molecular length of the TP terminal, affords effective
steric shielding on the TP terminals and thus is effective in
preventing intermolecular aggregation of the TP units. In
contrast, no serious steric shielding effect is expected when TP
terminals are connected by peptide chains in flexible random
coil structure. Readiness of the fluorescent TP units to undergo
intermolecular aggregation will affect the AEE-related emission
behavior. By varying the secondary structure in alkaline
solution, the corresponding helix-to-coil transition occurs to
result in TP-iPPLG with random coil chains; with them mutual
approaches of the TP terminals are accessible, resulting in
enhanced emission with the prevailing AEE effect. The
fluorescence response can then be measured, and the
relationship between AEE-related emission behavior and
secondary structure of the water-soluble polypeptide can be
therefore identified.
EXPERIMENTAL SECTION
■
Materials. All reagents and chemicals were purchased from Aldrich
Chemical Co. and used as received. DMF was refluxed and distilled
over CaH2 under reduced pressure to a flask containing alumina and
was used directly after distillation. CuBr (98%, Aldrich) was stirred
overnight in acetic acid, filtered, washed with ethanol and diethyl ether,
and dried in vacuo before use. Compounds of TPNH2,32 SAES,31and
γ-propargyl-L-glutamate N-carboxyanhydride (PLG-NCA)33,34 were
prepared according to the reported procedures. Detailed procedures
for the ring-opening polymerization and click reaction (Scheme 2) are
given below.
Ring-Opening Polymerization of N-Carboxyanhydride To
Prepare TP-PPLGs. Preparation of TP-PPLG(L) was used as an
example to illustrate its preparation procedures as follows: a solution
of PLG-NCA (3 g, 14.2 mmol) in anhydrous DMF (20 mL) was
stirred and bubbled with argon gas for 20 min before a solution of
TPNH2 (0.03 mL, 0.48 mmol) in anhydrous DMF (5 mL) was added.
After stirring for 2 days at room temperature, the polymer was
precipitated from diethyl ether and dried in a vacuum oven. The
4038
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
Scheme 2. Ring-Opening Polymerization of PLG-NCA Monomer by a TPNH2 Initiator and the Following Click Reaction to
Obtain TP-iPPLG
syntheses of TP-PPLG(H) was operated by the same procedure but
with different formulation. TP-PPLG(L): 1H NMR (500 MHz,
CD2Cl2): δ 8.34 (broad, 1H, Hf), 7.5−7.03 (broad, 19H, Hg), 4.70 (s,
2H, Hb), 3.99 (broad, 1H, He), 2.62 (broad, 2H, Hc), 2.50 (s, 1H, Ha),
2.32−2.19 (broad, 2H, Hd) (Figure 2a). 13C NMR (125 MHz,
CD2Cl2): δ 177.0, 173.3, 132−128, 78.7, 75.9, 57.3, 52.8, 31.4, 25.8.
cycle of freeze−thaw−pump. The reaction was then performed at 50
°C under argon for 24 h. After cooling to room temperature, the
resultant mixtures were passed through a neutral alumina column to
remove the copper catalysts. The eluent was concentrated by rotary
evaporation before precipitation from diethyl ether. The precipitate
was filtered off and dried under vacuum at room temperature to obtain
the final product. The syntheses of TP-iPPLG(H) was operated by the
same procedure but with different formulation. TP-iPPLG(L): 1H
NMR (500 MHz, d6-DMSO): δ 8.18 (broad, 1H, Hf′), 8.06 (s, 1H,
Ha′), 7.48−6.98 (broad, 19H, Hg′), 5.08 (s, 2H, Hb′), 4.65 (broad, 2H,
Hh), 4.57 (broad, 2H, Hi), 3.02 (broad, 1H, He′), 2.31 (broad, 2H,
Hc′), 1.89−1.74 (broad, 2H, Hd′) (Figure 2b). 13C NMR (125 MHz,
d6-DMSO): δ 175.0, 173.3, 142.3, 131.1−125.7, 125.3, 67.4, 57.3, 51.6,
46.6, 29.7, 26.9. (Figure 1b). TP-iPPLG(H): 1H NMR (500 MHz, d6-
DMSO): δ 8.16 (broad, 1H, Hf′), 8.06 (s, 1H, Ha′), 7.25−6.85 (broad,
19H, Hg′), 5.08 (s, 2H, Hb′), 4.56 (broad, 2H, Hh), 4.49 (broad, 2H,
Hi), 3.02 (broad, 1H, He′), 2.39−2.23 (broad, 2H, Hc′), 2.03−1.82
(broad, 2H, Hd′) (Figure S1b). 13C NMR (125 MHz, d6-DMSO): δ
175.0, 173.3, 142.3, 131.1−125.7, 125.4, 67.4, 55.1, 51.7, 46.6, 30.3,
26.3 (Figure S2b).
1
(Figure 1a). TP-PPLG(H): H NMR (500 MHz, CD2Cl2): δ 8.21
(broad, 1H, Hf), 7.25−6.9 (broad, 19H, Hg), 4.61 (s, 2H, Hb), 3.94
(broad, 1H, He), 2.61 (broad, 2H, Hc), 2.48 (s, 1H, Ha), 2.23−2.11
(broad, 2H, Hd) (Figure S1a, Supporting Information). 13C NMR
(125 MHz, CD2Cl2): δ 177.0, 173.9, 132−128, 78.2, 75.9, 57.3, 52.8,
31.4, 25.8 (Figure S2a).
Preparations of TP-iPPLGs by Click Reaction. Preparation of
TP-iPPLG(L) was used as an example to illustrate its preparation
procedures as follows: SAES (0.57 g, 3.29 mmol), TP-PPLG(L) (0.5 g,
2.99 mmol), and CuBr (0.04 g, 0.3 mmol) were dissolved in DMF (20
mL) in a two-neck flask equipped with a magnetic stirrer and a
condenser. After three freeze−thaw−pump cycles, N,N,N′,N″,N″-
pentamethyldiethylenetriamine (PMDETA) (0.063 mL, 0.3 mmol)
was added, and the whole reaction mixture was subjected to another
4039
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
collected at right angle to the excitation beam was used in the spectral
measurement. Solution quantum yield in water was determined by
comparison with a quinine sulfate standard solution (10−4 M in 1 N
H2SO4). The integrating sphere was used to measure the quantum
yields of the solid samples. Circular dichroism spectra were recorded
using a JASCO J-810 spectrometer, with sample in deionized water at
a concentration of 10−4 M. Chain dimension of the α-helical structure
was calculated from the minimum-energy conformation simulated
from the Materials Studio software.
RESULTS AND DISCUSSION
■
Synthesis of Water-Soluble, Ionic Polymer of TP-
iPPLG. The water-soluble TP-iPPLG polymers were synthe-
sized according to the procedures illustrated in Scheme 2. The
facile click reaction between azide-functionalized SAES and the
alkyne side groups of TP-PPLG is the key step to generate the
ionic sulfonate side groups of the final TP-iPPLG products.
Primarily, three key reactants including SAES, TP-PPLG, and
the AIE-active initiator of TP-NH2 are prerequisite for the
preparation of the desired TP-iPPLG. Two polymeric TP-
PPLGs with low and high MWs were prepared from the ring-
opening polymerization of N-carboxyanhydride monomer of
PLG-NCA by the AIE-active initiator of TP-NH2. The
propargyl side group of TP-PPLG was then “clicked” by the
azide group of SAES, resulting in two products of TP-
iPPLG(L) and TP-iPPLG(H) with relatively low and high
MWs, respectively. Here, the cyclic monomer of PLG-NCA
needed to be prepared from the facile cyclization of propargyl
glutamate (PLG) in the presence of triphosgene while another
key intermediate of TP-NH2 was obtained from a two-step
reaction procedure, starting from the initial nitration of TP
followed by reduction of the nitro group to obtain the desired
amino function in TPNH2. The azide-functionalized SAES was
synthesized by a simple substitution reaction between sodium
azide (NaN3) and SBES. All the intermediate reactant and final
products were carefully identified, and the detailed spectral data
are included in the Supporting Information.
Figure 1. 13C NMR spectra of (a) TP-PPLG(L) (in CD2Cl2 + 15 vol
% TFA) and (b) TP-iPPLG(L) (in d6-DMSO).
FTIR spectroscopic analysis can be primarily applied to
confirm the success of the click reaction, by which the neutral
TP-PPLG can be converted to ionic TP-iPPLG. FTIR spectra
revealed the complete disappearance of the characteristic
signals for the azide and acetylene groups (Figure 3). The
signals at 2130 cm−1, representing the alkyne group of TP-
PPLG(L), and 2105 cm−1, representing the azide group of
SAES, were absent in the spectrum of TP-iPPLG(L). A signal
Figure 2. 1H NMR spectra of (a) TP-PPLG(L) (in CD2Cl2 + 15 vol %
TFA) and (b) the protonated TP-iPPLG(L) (in d6-DMSO).
Measurements. 1H NMR spectra were recorded at room
temperature using a Bruker AM 500 (500 MHz) spectrometer with
tetramethylsilane (TMS) as the external standard. 13C CP-MAS NMR
spectra were acquired on a Bruker 14.1 T wide-bore Avance III
spectrometer equipped with a 4 mm double-resonance magic-angle-
spinning (MAS) probe head. The Larmor frequency used for 13C
NMR is 150.92 MHz. The samples were spun at 12 kHz. FTIR spectra
were recorded from a Bruker Tensor 27 FTIR spectrophotometer; 32
scans were collected at a spectral resolution of 1 cm−1. The solid
polymer powders were mixed with KBr before being pressed to make
pellets for measurement. Mass spectra were obtained from a Bruker
Daltonics Autoflex III MALDI-TOF mass spectrometer and operated
by the following voltage parameters: ion source 1, 19.06 kV; ion source
2, 16.61 kV; lens, 8.78 kV; reflector 1, 21.08 kV; reflector 2, 9.73 kV. A
wide-angle X-ray diffraction (WAXD) pattern was obtained from a
Siemens D5000 X-ray diffractometer with a source of Cu Kα (11/4
0.154 nm) radiation at 40 kV and 30 mA. Diffraction patterns were
collected with a scan rate of 3 s per 0.1° in the 2θ ranges of 2°−40°.
The UV−vis absorption spectra were recorded with an Ocean Optics
DT 1000 CE 376 spectrophotometer. The PL emission spectra were
obtained from a LabGuide X350 fluorescence spectrophotometer
using a 450 W Xe lamp as the continuous light source. A small quartz
cell with dimensions 0.2 × 1.0 × 4.5 cm3 was used to accommodate
the solution sample. A “right angle” geometry that fluorescence was
Figure 3. Solid FTIR spectra of SAES, TP-PPLG(L), and TP-
iPPLG(L).
4040
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
Table 1. Molecular Weights of TP-PPLG and TP-iPPLG Species Determined from MALDI-TOF and 1H NMR Spectroscopies
a
1
from MALDI-TOF
from H NMR
Mn
a
a
a
b
b
Mn
Mw
PDI
N
N
TPPPLG(L)
TPPPLG(H)
TP-iPPLG(L)
TP-iPPLG(H)
2900 ( 3%)
4490 ( 1%)
3050 ( 1%)
4840 ( 2%)
1.05
1.08
17
27
3480 ( 4%)
5170 ( 1%)
7020 ( 3%)
10370 ( 1%)
20
31
20
30
a
b
Mn = number-average molecular weight, Mw = weight-average molecular weight, PDI = polydispersity. N = number of repeat units.
for the amide I groups of the TP-PPLG(L) at 1660 cm−1 also
appeared in the spectrum of TP-iPPLG(L). The well-defined
structures of the neutral TP-PPLGs were confirmed in the
corresponding MALDI-TOF MS spectra (Figure S4), which
revealed only one apparent distribution for both TP-PPLG(L)
and TP-PPLG(H). Unfortunately, no MS peaks can be resolved
from the ionic TP-iPPLG polymers because the difficulty to
find appropriate matrix to overcome the strong electrostatic
interaction forces among the sulfonate pendant groups of the
ionic polymers. The MWs of the neutral TP-PPLG species
from the MALDI-TOF analysis are listed in Table 1, which
indicates that number (N) of the repeat unit in the low- and
high-MW TP-PPLG(L) and TP-PPLG(H) are 17 and 27,
respectively. To unambiguously determine the MWs of the
singlet at 2.50 ppm corresponded to the alkyne protons [Ha].
The resonance peaks of side-chain alkyl CH2 protons (Hc and
Hd) appeared upfield as multiplets between 2.0 and 2.7 ppm.
The resonance of the main-chain alkyl CH protons [He]
appeared as a singlet at 3.99 ppm, which became the resonance
peak of TP-iPPLG(L) at 3.02 ppm (He′ in Figure 2b) in d6-
DMSO. For TP-iPPLG(L), the resonance at 8.06 ppm was due
to the protons [Ha′] in the triazole structure resulting from the
click reaction, which confirms the successful synthesis of TP-
iPPLG(L). For TP-PPLG(L), the signal of the CH2 [Hb]
neighboring to acetylene group was located at 4.7 ppm (Figure
2a), and it shifted downfield significantly to 5.08 ppm (Hb′ in
Figure 2b) for TP-iPPLG(L). The peak area ratio between
proton Hb′ and the aromatic protons Hg′ can be used to
calculate the number-average MW (Mn) of TP-iPPLG(L). The
same calculation can be made on TP-iPPLG(H) (Figure S1b),
too, and the results listed in Table 1 indicate good correlation
between the number (N) of repeat unit of TP-PPLGs and TP-
iPPLGs.
1
ionic TP-iPPLG(L) and TP-iPPLG(H) polymers, 13C and H
NMR spectroscopies were further applied.
Figure 1 presents the 13C NMR spectra of TP-PPLG(L) and
TP-iPPLG(L). The 13C NMR spectrum of TP-PPLG(L) in
CD2Cl2/TFA (Figure 1a) revealed signals for the ester CO
[Cd] and amide CO carbon [Ch] atoms at 177 and 173.3
ppm, respectively, whereas those of the alkyne carbon [Ca and
Cb] atoms appeared at the respective positions of 75.9 and 78.7
ppm. The signals for the amino acid α-carbon atoms (Cg)
appeared at 52.8 ppm. For TP-iPPLG(L) (Figure 1b), the ester
CO carbon [Cd′], the amide CO carbon [Ch′] and the
amino acid α-carbon (Cg′) atoms resonated at positions in close
vicinities of those in the spectrum of TP-iPPLG(L). However,
the resonances of the two carbon atoms [Ca′ and Cb′] of the
triazole ring appeared at 142.3 and 125.3 ppm. No signal of the
alkyne carbons (Ca and Cb in Figure 1a) can be detected in
Figure 1b, indicating the completeness of the click reaction. In
addition, before click reaction the signal for the carbon (Cc,
Figure 1a) neighboring to alkyne appeared at 57.3 ppm; after
click reaction this signal shifted considerably to 67.4 ppm (Hc′
in Figure 1b). Results from the 13C NMR spectra indicated that
click reaction carried out successfully to result in quantitative
transformation from alkyne to triazole ring with sulfonate
function.
Conformational Transformation from the Solid TP-
PPLG to the Solid TP-iPPLG. The conformations of the TP-
PPLG and TP-iPPLG solids can be evaluated by analyzing the
amide absorptions in their FTIR spectra (Figure 4). Analysis of
Initially, MWs of TP-iPPLGs cannot be determined from the
1H NMR spectra because no phenyl proton signal of TP
terminal is present in the spectra of TP-iPPLGs in D2O. The
TP terminals buried in the ionic aggregates of TP-iPPLG
polymers are supposed to be inert to the stimuli of external
magnetic field; thereby, acidic HCl was used to dissociate the
ionic interaction, and as expected, the resulting protonated TP-
iPPLG showed the resolvable aromatic resonances of the TP
Figure 4. FTIR spectra of (a) TP-PPLG(L), (b) TP-iPPLG(L), (c)
TP-PPLG(H), and (d) TP-iPPLG(H).
1
units. Figure 2 presents the H NMR spectra of TP-PPLG(L)
these spectra using the second-derivative technique revealed
that the amide I band at 1655 cm−1 was characteristic of α-
helical secondary structure while for polypeptides possessing a
β-sheet conformation, the amide I band was located at 1627
cm−1. Random coil or turn populations were characterized by a
signal at 1693 cm−1, and the free CO unit of the side chain
group of PPLG provided a signal at 1740 cm−1. The spectra in
and the protonated TP-iPPLG(L) dissolved in CD2Cl2/
trifluoroacetic acid (TFA, 15 vol %) and d6-DMSO,
respectively. For TP-PPLG(L), the aromatic protons of the
TP initiator resonated as multiplets (Hg in Figure 2a) in the
range from 7.03 to 7.5 ppm. The signal for the amide protons
[Hf] of TP-PPLG(L) appeared as a singlet at 8.34 ppm; the
4041
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
Table 2. Secondary Structures of TP-PPLG and TP-iPPLG Species Determined from Infrared Spectroscopy
random coil
α-helix
β-sheet
sample
ν (cm−1
)
Af (%)
ν (cm−1
)
Af (%)
ν (cm−1
)
Af (%)
TP-PPLG(L)
TP-PPLG(H)
TP-iPPLG(L)
TP-iPPLG(H)
1691
1691
1691
1691
10.6 ( 0.2)
8.7 ( 0.1)
8.8 ( 0.1)
5.6 ( 0.2)
1656
1655
1655
1655
62.7 ( 0.2)
80.9 ( 0.1)
80 ( 0.1)
1623
1623
1625
1625
26.7 ( 0.1)
10.4 ( 0.2)
11.2 ( 0.1)
6.2 ( 0.1)
88.2 ( 0.1)
the range from 1500 to 1800 cm−1 were then deconvoluted into
a series of Gaussian curves to quantify the fraction of each of
the peaks. Table 2 summarizes our results obtained from curve
fitting of the amide I group for the random coil, α-helical, and
β-sheet structures. The result suggested that increasing MW of
the peptide chains tends to promote the rigid α-helical
secondary structure. Therefore, high-MW TP-PPLG(H) and
TP-iPPLG(H) contain more fractions of α-helical structures
than the low-MW analogues of TP-PPLG(L) and TP-
iPPLG(L), respectively. This result is similar to the finding
reported by Papadopoulos et al. for PBLG.35 Besides the MW
effect, the ionic pendant groups of TP-iPPLGs are also
beneficial for the formation of α-helical chain. Therefore, the
ionic TP-iPPLGs always have higher fraction of α-helical
secondary structure than their neutral precursors of TP-PPLGs.
It may be envisaged that the α-helical conformation possesses
long intrachain distance between the neighboring ionic side
groups; thus, electrostatic repulsion forces can be reduced in
the α-helical conformation of TP-iPPLG(L) and TP-iPPLG-
(H).
relative fraction of α-helical and β-sheet structures; clearly, the
ionic TP-PPLGs polymers contain higher fraction of α-helical
secondary structure than the neutral TP-iPPLGs. The result
coincides with the finding from the FTIR spectra.
Figure 6 displays the WAXD patterns of TP-PPLGs and TP-
iPPLGs recorded at room temperature; these diffraction
Figure 5 displays the 13C CP/MAS spectra of TP-PPLGs and
TP-iPPLGs. The different 13C chemical shifts of the Cα and
Figure 6. WAXD patterns (recorded at room temperature) of (a) TP-
PPLG(L), (b) TP-iPPLG(L), (c) TP-PPLG(H), and (d) TP-
iPPLG(H).
patterns exhibit certain differences relative to the content of
the secondary structure. For TP-PPLG(L) containing certain
fractions of β-sheet chain (cf. Table 2), the sharp peak at q of
1.34 (d = 0.468 nm, the intermolecular distance between
adjacent peptide chains within one lamella) was clearly visible
(Figure 6a). This result is different from that obtained for TP-
PPLG(H) (Figure 6c). The corresponding diffraction at q =
1.34 became inseparate from the amorphous background due
to the low content of β-sheet structure. For TP-PPLG(L) and
TP-PPLG(H), the three reflections at higher angles, with
positions 1:31/2:41/2 relative to the primary peak (q*), are due
to the (10), (11), and (20) reflections of a 2D hexagonal
cylindrical packing composed of 18/5 α-helices with a cylinder
distance of 1.18 nm.35 The structure of PPLG has been
described as a nematic-like paracrystal with a periodic packing
of α-helices in the direction lateral to the chain axis. The broad
amorphous region at q = 1.54 is mainly due to the long
amorphous side chain group in TP-PPLG. After the click
reactions, the ionic TP-iPPLGs contain mainly the α-helical
secondary structure; therefore, the WAXD spectra of TP-
iPPLG(L) and TP-iPPLG(H) (Figure 6b,d) indicate the
presence of three reflections, at positions 1:31/2:41/2 relative
to the primary peak diffracted at q = 0.25, which correspond to
a 2D hexagonal cylindrical packing with a cylinder distance of
2.51 nm.
Figure 5. Solid state 13C NMR spectra of (a) TP-PPLG(L), (b) TP-
iPPLG(L), (c) TP-PPLG(H), and (d) TP-iPPLG(H).
amide CO carbon atom nuclei relate to the local
conformations of the individual amino acid residues, charac-
terized by their dihedral angles and types of intermolecular and
intramolecular H-bonds.36,37 In the case of TP-PPLGs, the α-
helical secondary structure was characterized by the signals of
the Cα and amide CO resonances appeared at 57.5 and 176
ppm, respectively. In the β-sheet conformation, these signals
were shifted upfield, by approximately 4−5 ppm, to 52.7 and
172 ppm, respectively.38 The overlapped CO absorptions in
the range from 167 to 181 ppm can be used to evaluate the
Figures 7a and 7b provide a schematic depiction of the α-
helical conformation and cylindrical organization of the TP-
4042
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
Figure 7. α-Helical rods (upper) and the simulated chain arrangements (lower) of TP-PPLG and TP-iPPLG.
PPLG and TP-iPPLG, respectively. The arrangement of the α-
helical conformation for TP-PPLG can be represented by the
2D hexagonal packing (Figure 7a) with a cylinder distance of
1.18 nm, as calculated from the WAXD data, with a
characteristic spacing close to the reported value of PPLG.35
Similar hexagonal packing of cylinders was expected for TP-
iPPLG (Figure 7b); however, the cylinder distance for TP-
iPPLG is 2.51 nm, as evaluated from a q value of 0.25 according
to the WAXD diffraction peak. The observed spacing was close
to the thickness (2.5 nm) expected for the repeated packing of
a TP-iPPLG bilayer structure, as estimated from the sum of
twice the ionic group length (2 × 0.66 nm = 1.32 nm)16 and
the cylinder distance of (1.18 nm) of TP-PPLG (1.32 + 1.18 =
2.5 nm).
Emission Behavior of the TP-iPPLG Polymers. As the
main focus of this study, the AEE-related emission behavior was
characterized by the solution emission responses toward
concentration and aggregation. Effect of concentration was
primarily emphasized by the unnormalized solution emission
spectra of TP-iPPLGs in water (Figure 8), which exhibited the
continuous accession of the emission intensity with increasing
concentration. In contrast to the emission quenching caused by
solution thickening, solutions of TP-iPPLGs become more
emissive with increasing concentration. When concentrations of
TP-iPPLG(L) and TP-iPPLG(H) in water were increased from
10−5 to 10−3 M, more than 10-fold emission enhancements
were observed. More drastic emission enhancements were
observed in the spectra of the solid samples, which are
correlated with the fact that molecular motions are seriously
retarded in the solid state. In the solution and solid sates,
Figure 8. (a) Concentration effect on the PL emission spectra of (a)
TP-iPPLG(L) and (b) TP-iPPLG(H) solutions and solids (λex = 325
nm).
emissions of the low-MW TP-iPPLG(L) are all higher in
intensity than those of the high-MW TP-iPPLG(H) under the
same experimental conditions. As suggested above, the rigid α-
helical chain is effective in preventing the intermolecular
aggregation of the TP terminals; with higher fraction of α-
helical chain, the TP-iPPLG(H) polymer therefore emitted
with lower intensity than the TP-iPPLG(L) polymer with lower
fraction of α-helical chain.
4043
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
All emission spectra in Figure 8 consist of two overlapping
bands, with the short-wavelength monomer emission at 420 nm
and the long-wavelength aggregate emission shown as the right
shoulder of the monomer emission. The aggregate emission
became significant for the solid polymers, which is conceivable
in considering the feasible intermolecular aggregations of the
TP terminals in the condensed solid states. Comparatively, the
solid TP-iPPLG(L) emits with higher intensity than the high-
MW TP-iPPLG(H), which can be further certified from the
respective ΦF values of 0.29 and 0.23 measured from
integrating sphere. With higher content of fluorescent TP
unit, TP-iPPLG(L) therefore emits more efficiently than TP-
iPPLG(H) with lower TP content. The relative intensity
between the aggregate and the monomer emissions is also an
index for the intermolecular aggregation of the TP units in TP-
iPPLG(L) and TP-iPPLG(H). The solid emission spectrum of
TP-iPPLG(H) (Figure 8b) reveals a higher aggregate/
monomer emission ratio than the spectrum of TP-iPPLG(L)
(Figure 8a). As concluded above, intermolecular aggregation of
TP terminals is more effectively blocked when they are linked
to the large α-helical rod; therefore, with less fraction of α-
helical chain TP-iPPLG(L) therefore emits with lower
aggregate/monomer ratio than TP-iPPLG(H) with higher
fraction of α-helix.
Aggregates formed in solvent/nonsolvent can be used to
identify the AEE effect; here, DMF was used as nonsolvent to
induce aggregation of TP-iPPLGs in water. Upon excitation at
325 nm, dilute aqueous solutions (10−4 M) of TP-iPPLGs
already emitted with discernible intensity (Figure 9), and
increasing DMF in the solution (while keeping concentration
of TP-iPPLGs at 10−4 M) caused little but progressive gain on
the emission intensity. Upon increasing concentration of TP-
iPPLGs, the monomer emission peak progressively shifted to
higher wavelengths. Suggestively, the fluorescent TP unit may
adopt a more planar geometry with increasing steric constraint
imposed by aggregates formed in the solutions. The emission
spectra in Figure 9 indicated that both TP-iPPLG(L) and TP-
iPPLG(H) are AEE-active materials.
Figure 9. Emission spectra of (a) TP-iPPLG(L) and (b) TP-
iPPLG(H) in water/DMF mixtures of different compositions (λex
=
325 nm).
The α-helix of poly(L-glutamic acid) species were reported to
undergo a helix-to-coil transition at pH value of 6.18.39,40
A
similar transition was observed in β-CD-grafted PPLG,16
however, at a higher pH range in between 11 and 11.5 due
to the steric hindrance or the stable helical structure inherited
by the β-CD grafts. For TP-iPPLGs, similar conformational
transformation induced in the highly alkaline solution was
expected to vary the AIE-related emission behavior due to
different aggregation states accompanied by conformational
change. Indeed, the aqueous TP-iPPLG(L) showed a
progressive transformation on the emission peaks upon
increasing pHs from 7 to 13.3 (Figure 10). Emission spectra
of the aqueous TP-iPPLG(L) at pH 2 and 7 are essentially the
same; however, when NaOH was gradually added to the
aqueous TP-iPPLG(L) solution, the initial monomer emission
was continuously transformed into a major aggregate emission.
Among all, the major transformation occurred at a pH value
somewhere between pH 11 and pH 13.3. At pH 13.3, the TP-
iPPLG(L) solution emitted with a large aggregate emission,
whose intensity is almost 3-fold to the intensity obtained at pH
7. In highly alkaline solution, the main chain of TP-iPPLG(L)
underwent a helix-to-coil transformation, and once again, the
easy intermolecular aggregation of TP terminals in the random
coil chains was responsible for the highly fluorescent
Figure 10. PL emission spectra of the aqueous TP-iPPLG(L)
solutions with different pH values (λex = 325 nm).
aggregation emission observed in the spectra. The correspond-
ing conformational transformation was characterized next.
Conformational Transformation of the TP-iPPLG(L)
Polymer in Water. We recorded circular dichroism spectra to
characterize the conformational transformation of the water-
soluble TP-iPPLG(L) polymer in alkaline aqueous solution.
The α-helical structure was characterized by a triply inflected
spectrum, corresponding to two negative bands at 208 and 222
nm and a strong positive band at 192 nm. The β-sheet structure
was characterized by a negative minimum band near 218 nm
and a positive maximum near 198 nm. The random coil
4044
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
structure was characterized by a small positive band at 218 nm
and a negative band near 200 nm.39 The fourth principal
structure of the C10-helix (i.e., a repetitive H-bonded ring
containing 10 atoms)41 possesses a tighter, more elongated, and
higher potential energy than the α-helix structure (C13-helix).
Nevertheless, the C10-helix was characterized by a small positive
band at 195 nm, with a smaller intensity and a much lower
ellipticity ratio ([θ]222/[θ]208) than that of the α-helix.42
Next, we tested the effect of pH on the conformation of TP-
iPPLG(L). The circular dichroism spectra in Figure 11 display
solutions. Effect of chain conformation on the AEE-related
emission behavior is thus verified.
With the ionic pendant group, the present TP-iPPLG system
exhibits the helix-to-random coil transformation in alkaline
solution. However, the previous system of TP-2PBLG32 was
reported to proceed this conformational transformation at
acidic environment (pH = 2 in THF−H2O (v/v = 1/9)). For
the neutral TP-2PBLG, acidic species added in the solution
tend to penetrate into the peptide chain, dissociating the
intramolecular H bonds dominant in the α-helical structure of
TP-2PBLG, to result in random-coil chains. But for the present
TP-iPPLG, the ionic sulfonate pendant groups greatly enhance
the stability of the α-helical chains in the acid media. The ionic
sulfonate groups are considered to serve as efficient electro-
static shields, protecting the interior peptide chains from the
attacks of the acidic species. In strong acidic media, the
sulfonate pendant groups may just be protonated to result in
sulfonic acid groups with sustained stability in the acid media.
The quantum efficiencies (ΦFs) of the aqueous solutions
(10−4 M) (Table 3) at different pH values are in the ranges
Table 3. Quantum Efficiency (ΦF) Measured from the
a
Solution at Different pHs
TP-iPPLG(L)
solution (10−4 M)
pH
7−11
7
12
13
13.1
12.7
13.2
14.2
13.3
18.7
ΦF (%)
6.8
8.7
Figure 11. Effect of pH on the circular dichroism spectrum of TP-
iPPLG(L) in NaOH solution.
a
Quantum yield, obtained by using quinine sulfate as refer ence
standard (10−4 M in 1 N H2SO4).
the pH-induced conformational changes of the TP-iPPLG(L)
solutions. By increasing the basicity of the solution from pH 7
from 0.07 to 0.187. As common dye for biomaterials,
fluorescein and rodamine 101 have extremely high ΦFs of
0.95 (in aqueous 0.1 M NaOH solution, pH = 13) and 1.0 (in
ethanol),46 respectively. The weak solution emission efficiency
of TP-iPPLG(L) is typical of AIE-active materials since free
molecular rotation in the mobile solution state activates the
nonradiative decay pathways, resulting in the weak solution
emission.
TP-iPPLG(L) as Sensor for Bovine Serum Albumin.
Among varieties of serum albumin, bovine serum albumin
(BSA)47 is the most studied one in the biochemistry or
biomedical science due to its structural similarity with human
serum albumin (HSA).48 As a large globular protein (66 kDa)
consisting of 583 amino acid residues in a single polypeptide
chain, BSA was often used as a binding material with certain
dyes and probes, which provides fluorescence for the study of
binding mechanisms.49
With the inferior quantum efficiency, TP-iPPLG(L) can still
be used as probe for detecting BSA. In this case, when BSA was
added to the alkaline (pH = 13.3) solution of TP-iPPLG(L),
emission of the solution mixtures became weaker (Figure 12)
with increasing BSA content from 0 to 5 mg/mL.
Comparatively, the long-wavelength aggregate emission exhibits
more drastic intensity reduction compared to the monomer
emission at short-wavelength region. Suggestively, intermolec-
ular aggregation of the TP terminals is largely hampered,
rendering in the reduced aggregate emission, when the TP-
iPPLG(L) chains bind to the large protein chains of BSA. The
random coil chain, as the main structure of TP-iPPLG(L) in
the alkaline solution, preferably binds to BSA, resulting in stable
homogeneous solution for measurement. In contrast, no such
measurement is possible for the acidic and neutral TP-iPPLG
solutions because in this case adding BSA caused immediate
to 11, we observed a slight variation on the ratio [θ]222/[θ]208
,
indicating that there are only minor conformational change
involved in this pH range. The polymer chains should remain
as a helical mixture of α-helix and C10-helix structures.39
Nevertheless, the major conformational transformation oc-
curred in highly alkaline solutions with pH > 12; at this stage,
the triply inflected spectrum reduced to a doubly inflected
dichroic spectrum. The α-helix of the TP-iPPLG(L) polymer
was converted to a random coil conformation by the alkaline
NaOH. The intramolecular H-bonds involved in the α-helical
chains were dissociated in the highly alkaline environment.
The conformational change in the alkaline solution is
therefore correlated with the AEE-related emission behavior,
which is closely associated with the extent of molecular
approaches (aggregation) of the TP fluorophores in TP-iPPLG.
The geometrical factor affecting the extent of intermolecular
aggregation was conceptually depicted in Scheme 1. In the
solution state, the TP fluorophores with a molecular width of 9
Å can be more effectively isolated from other TP units if they
are connected to a large α-helical chain with a diameter of 25 Å
(the diameters of the α-helix are in the ranges of 15−26
Å43−45) than those in the flexible random coil or in the β-sheet
chains. In acidic and neutral solutions (from pH 2 to pH 7), the
TP-iPPLG chains mainly existed in the large α-helical
conformation. The TP terminal in the large α-helical chains
are more isolate from other TP terminals, resulting in the
monomer emission with low emission intensity. In contrast, the
TP-iPPLG(L) chains transformed into the flexible random coil
structure in the alkaline solution. The TP terminals in the
random coil conformation underwent easy intermolecular
association, resulting in the major aggregate emission with
the intensity much higher than those in the acidic and neutral
4045
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
AUTHOR INFORMATION
■
Corresponding Author
*E-mail jlhong@mail.nsysu.edu.tw; Tel +886-7-5252000 ext
4065 (J.-L.H.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported financially by the National Science
Council, Taiwan, Republic of China, under Contracts NSC
102-2221-E-110-084-MY3 and NSC 102-2221-E-110-080-.
REFERENCES
■
Figure 12. Emission spectra of the alkaline TP-iPPLG(L) solution
(10−4 M, pH = 13.3) at different amounts of BSA from 0 to 5 mg/mL
(λex = 325 nm).
(1) Kricheldorf, H. R. Angew. Chem., Int. Ed. 2006, 45, 5752−5784.
(2) Deming, T. J. Prog. Polym. Sci. 2007, 32, 858−875.
(3) Deming, T. J. In Peptide Hybrid Polymers; Polypeptide and
Polypeptide Hybrid Copolymer Synthesis via NCA Polymerization; Klok,
H. A., Schlaad, H., Eds.; Springer: Berlin, 2006; pp 1−18.
(4) Munoz, V.; Serrano, L. Nat. Struct. Biol. 1994, 1, 399−409.
(5) Fiori, W. R.; Lundberg, K. M.; Millhauser, G. L. Nat. Struct. Biol.
1994, 1, 374−377.
precipitation of the reaction products. Water-insoluble products
were thus produced when BSA reacted to TP-iPPLG(L) chains
in the rigid α-helix or β-sheet structures.
(6) Goodman, C. M.; Choi, S.; Shandler, S.; DeGrado, W. F. Nat.
Chem. Biol. 2007, 3, 252−262.
CONCLUSIONS
(7) Fezoui1, Y.; Hartley, D. M.; Walsh1, D. M.; Selkoe1, D. J.;
Osterhout, J. J.; Teplow, D. B. Nat. Struct. Biol. 2000, 7, 1095−1099.
(8) Drin, G.; Casellal, J.-F.; Gautier, R.; Boehmer, T.; Schwartz, T.
U.; Drin, B. A. Nat. Struct. Mol. Biol. 2007, 14, 138−146.
(9) Bryson, J. W.; Betz, S. F.; Lu, H. S.; Suich, D. J.; Zhou, H. X.;
O’Neil, K. T.; DeGrado, W. F. Science 1995, 270, 935−941.
(10) Bang, D.; Gribenko, A. V.; Tereshko, V.; Kossiakoff, A. A.; Kent,
S. B.; Makhatadze, G. I. Nat. Chem. Biol. 2006, 2, 139−143.
(11) Zhang, S. G. Nat. Biotechnol. 2003, 21, 1171−1178.
(12) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Science 2001, 294,
1684−1688.
■
A new series of water-soluble TP-iPPLG polymers containing
an AEE-active fluorescent terminal and ionic side groups can be
successfully prepared from a two-step reaction scheme,
including the ring-opening polymerization of PLG-NCA
monomer, initiated by an AIE-active initiator of TP-NH2, to
generate the neutral TP-PPLG and the following click reaction
on TP-PPLG to introduce the desired ionic pendant groups.
The TP-iPPLG polymers are AEE-active materials according to
the emission behavior in H2O/DMF solution mixtures.
Ionic pendant group and MW of the polypeptides are found
to affect the secondary structure of the resulting peptide chain.
The ionic sulfonate pendant group in TP-iPPLGs enhances the
content of the α-helical structure when compared to the neutral
precursors of TP-PPLGs. With higher MW, solid TP-
iPPLG(H) also possesses higher fraction of α-helical chains
as compared to low-MW TP-iPPLG(L). Since α-helical chains
are efficient in blocking the intermolecular aggregation of the
TP units, the emission spectrum of solid TP-iPPLG(H)
therefore contains less aggregate to monomer emission ratio
as compared to TP-iPPLG(L) with lower α-helix content.
The ionic sulfonate pendant groups of TP-iPPLG protect the
peptide main chain from attacking of the acidic species.
Therefore, TP-iPPLGs are rather stable in acidic media;
however, the stable α-helical structures are transformed into
random coil chains in the highly alkaline solution. Emission of
the alkaline TP-iPPLG solution contains a large aggregate
emission with the intensity much higher than the emission in
the acidic and neutral media. Effect of secondary structure on
the AEE-activity was therefore demonstrated. The alkaline
solution of TP-iPPLG(L) can be a probe for the detection of
BSA, and the corresponding emission spectra exhibit the large
reduction of the aggregate emission with increasing dosage of
BSA in the solution.
(13) Nowak1, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.;
Pochan, D.; Deming, T. J. Nature 2002, 417, 424−428.
(14) Lu, H.; Wang, J.; Bai, Y.; Lang, J. W.; Liu, S.; Lin, Y.; Cheng, J.
Nat. Commun. 2011, 2, 1−9.
(15) Zhang, Y.; Lu, H.; Lin, Y.; Cheng, J. Macromolecules 2011, 44,
6641−6644.
(16) Lin, Y. C.; Wang, P. I.; Kuo, S. W. Soft Matter 2012, 8, 9676−
9684.
(17) Doty, P.; Bradbury, J. H.; Holtzer, A. M. J. Am. Chem. Soc. 1956,
78, 947−954.
(18) Pokorna,
1, 185−190.
́
́ ́
V.; Vyprachticky, D.; Pecka, J. Macromol. Biosci. 2001,
(19) Kim, K. T.; Park, C.; Vandermeulen, G. W. M.; Rider, D. A.;
Kim, C.; Winnik, M. A.; Manners, I. Angew. Chem., Int. Ed. 2005, 117,
8178−8182.
(20) Rubatat, L.; Kong, X.; Jenekhe, S. A.; Ruokolainen, J.; Hojeij,
M.; Mezzenga, R. Macromolecules 2008, 41, 1846−1852.
(21) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.;
Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Z. Chem. Commun.
2001, 1740−1741.
(22) Tang, B. Z.; Zhan, X.; Yu, G.; Lee, P. P. S.; Liu, Y.; Zhu, D. J.
Mater. Chem. 2001, 11, 2974−2981.
(23) Aggregation-Induced Emission: Fundamentals; Qin, A., Tang, B.
Z., Eds.; John Wiley & Sons, Ltd.: New York, 2013.
(24) Liu, J.; Lam, J. W. Y.; Tang, B. Z. J. Inorg. Organomet. Polym.
2009, 19, 249−285.
(25) Hong, y.; Lam, J. W. Y.; Tang, B. Z. Chem. Commun. 2009,
4332−4353.
ASSOCIATED CONTENT
■
(26) Wu, J.; Liu, W.; Ge, J.; Zhang, H.; Wang, P. Chem. Soc. Rev.
2011, 40, 3483−3495.
S
* Supporting Information
Figures S1−S7. This material is available free of charge via the
(27) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Chem. Soc. Rev. 2011, 40,
5361−5388.
Internet at http://pubs.acs.org.
4046
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047

Macromolecules
Article
(28) Qin, A.; Lam, J. W. Y.; Tang, B. Z. Prog. Polym. Sci. 2012, 37,
182−209.
(29) Chien, R. H.; Lai, C. T.; Hong, J. L. J. Phys. Chem. C 2011, 115,
5958−5965.
(30) Lai, C. T.; Hong, J. L. J. Phys. Chem. B 2010, 114, 10302−10310.
(31) Lai, C. T.; Chien, R. H.; Kuo, S. W.; Hong, J. L. Macromolecules
2011, 44, 6546−6556.
(32) Li, S. T.; Lin, Y. C.; Kuo, S. W.; Chuang, W. T.; Hong, J. L.
Polym. Chem. 2012, 3, 2393−2402.
(33) Lin, Y. C.; Kuo, S. W. Polym. Chem. 2012, 3, 162−171.
(34) Lin, Y. C.; Kuo, S. W. Polym. Chem. 2012, 3, 882−891.
(35) Papadopoulos, P.; Floudas, G.; Klok, H. A.; Schnell, I.; Pakula,
T. Biomacromolecules 2004, 5, 81−91.
(36) Murata, K.; Katoh, E.; Kuroki, S.; Ando, I. J. Mol. Struct. 2004,
689, 223.
(37) Kricheldorf, H. R.; Muller, D. Macromolecules 1983, 16, 615.
(38) Sela, M.; Katchalski, E. Adv. Protein Chem. 1959, 14, 391−478.
(39) Hwang, J.; Deming, T. J. Biomacromolecules 2001, 2, 17−21.
(40) Zimmermann, R.; Kratzmu, T.; Erickson, D.; Li, D.; Braun, H.
G.; Werner, C. Langmuir 2004, 20, 2369−2374.
(41) Ananda, K.; Vasudev, P. G.; Sengupta, A.; Poopathi, K. M.;
Shamala, R. N.; Balaram, P. J. Am. Chem. Soc. 2005, 127, 16668−
16674.
(42) Toniolo, C.; Polese, A.; Formaggio, F.; Crisma, M.; Kamphuis, J.
J. Am. Chem. Soc. 1996, 118, 2744−2745.
(43) Torii, T.; Yamashita, T.; Horie, K. Eur. Polym. J. 1993, 29,
1265−1270.
(44) Doty, P.; Bradbury, J. H.; Holtzer, A. M. J. Am. Chem. Soc. 1956,
78, 947−954.
(45) Bovey, F. A.; Tiers, G. V. D. Adv. Polym. Sci. 1963, 3, 139−195.
(46) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed;
Kluwer Academic/Plenum Publishers: New York, 1999; p 53.
(47) Moriyama, Y.; Ohta, D.; Hadiya, K.; Mitsui, Y.; Takeda, K. J.
Protein Chem. 1996, 15, 265−272.
(48) He, X. M.; Carter, D. C. Nature 1992, 358, 209−215.
(49) Zhang, Y. Z.; Zhou, B.; Zhang, X. P.; Huang, P.; Li, C. H.; Liu,
Y. J. Hazard. Mater. 2009, 163, 1345−1352.
4047
dx.doi.org/10.1021/ma500886v | Macromolecules 2014, 47, 4037−4047