Separated coil and chain aggregation behaviors on the miscibility and helical peptide secondary structure of poly(tyrosine) with poly(4-vinylpyridine)

Article abstract of DOI:10.1021/ma301179t

In this study, we synthesized a low-molecular-weight polytyrosine (PTyr) through living ring-opening polymerization of the α-amino acid-N-carboxyanhydride and then blended it with poly(4-vinylpyridine) (P4VP) homopolymer in N,N-dimethylformamide (DMF) and MeOH solutions, thereby controlling the miscibility behavior and secondary structures of the PTyr. Infrared spectroscopy revealed that the PTyr/P4VP mixture featured strong hydrogen bonds between the OH groups of PTyr and the pyridyl groups of P4VP. Differential scanning calorimetry revealed that the glass transition temperatures of the PTyr/P4VP complexes formed from MeOH solutions were higher than those of the corresponding PTyr/P4VP miscible blends obtained from DMF solutions. The behavior of the PTyr/P4VP blends obtained after evaporation of the DMF solutions was consistent with separated random coils of the PTyr chains. The increased degree of hydrogen bonding within the PTyr/P4VP complexes formed from MeOH solutions resulted in interpolymer complex aggregates; the corresponding enhanced intermolecular hydrogen bonding of PTyr with P4VP resulted in β-sheet conformations for PTyr, as evidenced from Fourier transform infrared spectroscopy, solid state nuclear magnetic resonance spectroscopy, and wide-angle X-ray diffraction analyses. This model, which takes advantage of the well-defined secondary structures (α-helices, β-sheets) of PTyr, can, therefore, be used to identity the behavior of separated coils and aggregated chains in polymer blend and complex systems.

Full text of DOI:10.1021/ma301179t

Article
pubs.acs.org/Macromolecules
Separated Coil and Chain Aggregation Behaviors on the Miscibility
and Helical Peptide Secondary Structure of Poly(tyrosine) with
Poly(4-vinylpyridine)
Yi-Syuan Lu, Yung-Chih Lin, and Shiao-Wei Kuo*
Department of Materials and Optoelectronic Science, Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University,
Kaohsiung, Taiwan
ABSTRACT: In this study, we synthesized a low-molecular-
weight polytyrosine (PTyr) through living ring-opening
polymerization of the α-amino acid-N-carboxyanhydride and
then blended it with poly(4-vinylpyridine) (P4VP) homopol-
ymer in N,N-dimethylformamide (DMF) and MeOH
solutions, thereby controlling the miscibility behavior and
secondary structures of the PTyr. Infrared spectroscopy
revealed that the PTyr/P4VP mixture featured strong
hydrogen bonds between the OH groups of PTyr and the
pyridyl groups of P4VP. Differential scanning calorimetry revealed that the glass transition temperatures of the PTyr/P4VP
complexes formed from MeOH solutions were higher than those of the corresponding PTyr/P4VP miscible blends obtained
from DMF solutions. The behavior of the PTyr/P4VP blends obtained after evaporation of the DMF solutions was consistent
with separated random coils of the PTyr chains. The increased degree of hydrogen bonding within the PTyr/P4VP complexes
formed from MeOH solutions resulted in interpolymer complex aggregates; the corresponding enhanced intermolecular
hydrogen bonding of PTyr with P4VP resulted in β-sheet conformations for PTyr, as evidenced from Fourier transform infrared
spectroscopy, solid state nuclear magnetic resonance spectroscopy, and wide-angle X-ray diffraction analyses. This model, which
takes advantage of the well-defined secondary structures (α-helices, β-sheets) of PTyr, can, therefore, be used to identity the
behavior of separated coils and aggregated chains in polymer blend and complex systems.
biological point of view, β-sheet secondary structures have
emerged as important features in the development of several
neurodegenerative disorders (e.g., prion diseases).29 As a result,
conformational studies of model polypeptides are important
steps toward mimicking the biological activity of more-complex
proteins.30 In previous studies,31,32 we found that the secondary
structures of the polypeptides poly(r-methyl L-glutamate)
(PMLG), poly(r-ethyl L-glutamate) (PELG), and poly(r-benzyl
L-glutamate) (PBLG) could be altered through blending with
other random-coil nonpeptide oligomers [namely, phenolic
resin or poly(vinylphenol) (PVPh)], mediated by hydrogen
bonding interactions. The content of α-helical conformations in
these blend systems correlated strongly with the strengths of
intermolecular hydrogen bonding to the hydrogen-bond-donor
polymers. Nevertheless, these poly(L-glutamate)s formed only
relatively weak intermolecular hydrogen bonds between their
side chain CO groups and the OH groups of phenolic resin
or PVPh. The strongest interassociation equilibrium constant
(KA), a relatively low value of only 50, was found in the
phenolic/PELG blend system, consistent with CO groups in
polymers being relatively weak hydrogen bond acceptor groups
INTRODUCTION
■
Amino acid-based polymers, including polypeptides, have been
studied widely because of their potential applications as
biocompatible materials with useful chemical properties and
functions that derive from the amino acid moieties.1,2
Modification of a polypeptide with a functional polymer, such
as poly(ethylene glycol) (PEG), is used typically for the
development of new materials.3−5 In addition, multiblock
copolymers have also been prepared to model silk-based
materials, which formed nanostructures through β-sheet self-
assembly.6 Most poly(peptide-b-nonpeptide) (rod/coil) block
copolymers have been studied for their potential applications in
tissue engineering and drug delivery.7−21 The synthesis of
diblock copolymers is, however, a difficult and time-consuming
means of modifying polypeptides. From both practical and
economic points of view, polymer blending of existing polymers
is a more effective and convenient route toward creating new
and useful materials exhibiting greater versatility and flexibility
than is the development of new polypeptides.22−27
Polypeptides can form hierarchically ordered structures
containing fundamental secondary motifs: α-helices, which
can be regarded as rigid rods stabilized through intramolecular
hydrogen bonding interactions, and β-sheets, stabilized by
intermolecular interactions.28 From a synthetic point of view,
the α-helical structures of polypeptides cause them to behave as
rigid rod−like polymers in solution and in solid states. From a
Received: June 10, 2012
Revised: July 22, 2012
Published: August 10, 2012
© 2012 American Chemical Society
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product recrystallized three times from THF/hexane. Yield: 5.6 g, 27.1
mmol, 61.3%. 1H NMR (DMSO): δ (ppm) = 2.90 (d) 2H, CH2), 4.69
(t, 1H), 6.69 (d, 2H, Ar), 6.95 (d, 2H, Ar), 9.02 (s, 1H), 9.33 (s, 1H,
NH). FTIR (KBr, cm−1): 3313 (NH), 1846 (CO), 1762 (CO),
1601 (OH), 1590, 753, 698 (Ar).
Propargyl-Terminated Poly-L-Tyrosine (PTyr). Tyr−NCA
(4.00 g, 19.3 mmol) was weighed in a drybox under N2, placed in a
three-neck bottle, and dissolved in anhydrous DMF (20 mL). The
solution was stirred for 10 min and then propargylamine (0.25 mL,
96.5 mmol) was added using a N2-purged syringe. After stirring for 2
days at 0 °C, the polymer was recovered through precipitation in Et2O
and drying under high vacuum. Table 1 lists the degree of
[KA < KB (self-association equilibrium constant) in most
cases].31
Random coil nonpeptide polymers blended with OH-
containing polymers [e.g., PVPh] with poly(4-vinylpyridine)
(P4VP) are typically strongly hydrogen-bonding blend systems
(in this case, KA = 1200) and have been investigated
widely.33−38 In this study, we choose polytyrosine (PTyr) as
our hydrogen-bond-donor polypeptide and blended it with
P4VP to investigate the influence of their strong hydrogen
bonds on the secondary structure of the peptide and their
miscibility behavior. In PVPh/P4VP systems, the solvent has a
dramatic effect on complex formation.39,40 For example, a
mixture of PVPh/P4VP yields a precipitated complex in low-
polarity solvents (e.g., MeOH, EtOH) that exhibits a single
glass transition temperature (Tg) of 210 °C that is significantly
higher than that of each component of the blend (ca. 150
°C).39,40 In contrast, no precipitation occurs in N,N-
dimethylformamide (DMF) because this polar solvent also
participates in hydrogen bonding and competes with P4VP for
the OH groups of PVPh. Because DMF is a much stronger
hydrogen bond acceptor than MeOH, it does not allow the
formation of the interpolymer complex.41 The polymer coils in
the PVPh/P4VP mixture are well separated in DMF solutions;
therefore, the polymer chains are rather extended prior to
solvent evaporation because the interassociation hydrogen
bonding between PVPh and DMF is stronger than that
between PVPh and P4VP.38,42 As a result, the stronger
hydrogen bonding between PVPh and P4VP in MeOH tends
to induce polymer complex aggregation. In previous stud-
ies,43−46 we observed, from transmission electron microscopy
(TEM) analyses, distinct chain behaviorseparated coils and
chain aggregatesalso from mixtures of poly(styrene-b-vinyl-
phenol) (PS-b-PVPh) and poly(4-vinylpyridine-b-methyl meth-
acrylate) (P4VP-b-PMMA) in different polar solvents as a
result of their ability to self-assemble into well-defined
nanostructures.
In this present study, we suspected that PTyr, with its OH
groups, would be another model polymer that might exhibit
separated coil/aggregated chain behavior in polymer blend
systems because this polypeptide can form well-defined
secondary structures (α-helices, β-sheets). In addition, we
expected these well-defined secondary structures to be different
in the separated coils and the aggregated chains when blended
with P4VP in different common solvents. Here, we used
differential scanning calorimetry (DSC), Fourier transform
infrared (FTIR) spectroscopy, solid state nuclear magnetic
resonance (NMR) spectroscopy, and wide-angle X-ray
diffraction (WAXD) to investigate the miscibility behavior,
hydrogen bonding interactions, and secondary structures of
PTyr/P4VP blends and their corresponding complexes.
Table 1. Characterization of the PTyr Prepared in This
Study
a
b
b
c
c
polymer
PTyr8
Mn
Mn
PDI
Mn
PDI
1030
3300
1.05
1350
1.05
a
b
Determined from 1H NMR spectrum. Determined from GPC
c
analysis. Determined from MADLI−TOF mass spectrum.
polymerization (DP) of PTyr used in this study, as determined
1
using H NMR spectroscopy and MALDI−TOF mass spectrometry,
and the synthesis is depiced in Scheme 1. Yield: 2.5 g, 2.85 mmol,
14.8%. 1H NMR (DMSO): δ (ppm) = 4.40 (t, 1H), 6.69 (d, 2H, Ar),
6.95 (d, 2H, Ar), 7.94 (s, 1H, NH), 9.14 (s, 1H). FTIR (KBr, cm−1):
3288 (NH), 1601 (benzene), 753, 698 (Ar).
Blend Preparation. Mixtures of PTyr/P4VP at various blend
compositions were prepared through solution-casting. A DMF
solution containing the polymer mixture (5 wt %) was stirred for
6−8 h and then the solvent was evaporated slowly at 50 °C for 1 day.
The film of the blend was then dried at 80 °C for 2 days to ensure total
removal of residual solvent.
Complex Preparation. PTyr/P4VP complexes were prepared by
dissolving pure PTyr and pure P4VP separately in MeOH. Each
solution was stirred overnight to ensure complete dissolution. The two
solutions were then mixed together to give a white precipitate, which
was filtered off and washed with MeOH. The samples were then dried
under vacuum to remove any residual solvent. The ratio of the content
of the complex to the total amounts of the two polymers in the initial
solutions was considered as the yield of the complex. The bulk
compositions of the complexes were determined through elemental
analyses; the results are summarized in Table 2.
Characterization. 1H NMR spectra were recorded at room
temperature using a Bruker AM 500 (500 MHz) spectrometer, with
the residual proton resonance of the deuterated solvent used as the
internal standard. High-resolution solid state NMR spectra were
recorded at room temperature using a Bruker DSX-400 spectrometer
1
operated at resonance frequencies of 399.53 and 100.47 MHz for H
and 13C nuclei, respectively. 13C cross-polarization (CP)/magic angle
sample spinning (MAS) spectra were measured using a 3.9-μs 90°
pulse, a 3-s pulse delay time, a 30-ms acquisition time, and 2048 scans.
All NMR spectra were recorded at 300 K using broad band proton
decoupling and a normal CP pulse sequence. An MAS rate of 5.4 kHz
was used to avoid absorption overlapping. Mass spectra were obtained
using a Bruker Daltonics Autoflex MALDI−TOF mass spectrometer.
The following voltage parameters were employed: 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. Molecular weights and molecular weight
distributions were determined through gel permeation chromatog-
raphy (GPC) using a Waters 510 high-performance liquid
chromatography (HPLC) system equipped with a 410 differential
refractometer and three Ultrastyragel columns (100, 500, and 103 Å)
connected in series, with DMF as the eluent (flow rate: 0.4 mL/min).
DSC was performed using a TA-Q20 instrument operated at a scan
rate of 10 °C/min over the temperature range from 25 to 220 °C
under a N2 atmosphere. FTIR spectra of the polymer films were
recorded using a Bruker Tensor 27 FTIR spectrophotometer and the
conventional KBr disk method; 32 scans were collected at a spectral
EXPERIMENTAL SECTION
■
Materials. L-Tyrosine was purchased from MP Biomedicals.
Triphosgene (TCI), propargylamine (Acros, 99%), P4VP (Aldrich,
60 000 Da), DMF (Echo, 99.5%), MeCN (Acros, 99.5%), hexane
(Acros), MeOH (Acros), tetrahydrofuran (THF, Tedia) were
purchased commercially. All reaction mixtures were magnetically
stirred under Ar. Solvents were distilled from an appropriate drying
agent prior to use.
L-Tyrosine N-carboxyanhydride (Tyr-NCA).47 Triphosgene
(5.30 g, 17.7 mmol) was added to a solution ofL-tyrosine (8.00 g,
44.2 mmol) in MeCN (300 mL) heated under reflux (70 °C) under a
N2 atmosphere. After continuing refluxing for 4 h, the solution cooled
to 0 °C. The solvent was removed through rotary evaporation and the
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Scheme 1. Synthesis of PTyr Using Propargylamine as the Initiator
1837 and 1765 cm−1, corresponding to the CO units a and
b, respectivelyconfirm the formation of the NCA ring.48
After ring-opening polymerization of the NCA monomer, the
FTIR spectrum revealed the absence of the absorbances from
the CO groups a and b of the NCA ring and the appearance
of new signals at 1655, 1630 (c), and 1545 (d) cm−1
representing the amide bonds in the polymer backbone.48
Table 2. Characteristic and Thermal Properties of PTyr/
P4VP Complexes
PTyr/P4VP
feed composition
bulk composition
PTyr
P4VP
PTyr
P4VP
yield (wt %)
Tg (°C)
20
40
50
60
80
80
60
50
40
20
37
41
45
52
60
63
59
55
48
40
35
42
55
64
38
183
186
184
184
183
1
Figure 2a presents the H NMR spectrum of the tyrosine
monomer in d6-DMSO. We assign the singlet at 9.34 ppm to
the proton on the nitrogen atom, the signal at 9.04 ppm to the
proton of the phenolic OH group, the doublets at 6.97 and 6.66
ppm to the protons of the benzyl ring, and the triplet at 4.66
and doublet at 2.49 ppm to the alkyl CH2 and CH protons.
resolution of 1 cm−1. Because polymers containing OH groups are
hygroscopic, pure N2 gas was used to purge the spectrometer’s optical
box to ensure dry sample films. X-ray diffraction (XRD) data were
collected on the wiggler beamline BL17A1 of the National
Synchrotron Radiation Research Center (NSRRC), Taiwan. A
triangular bent Si (111) single crystal was employed to obtain a
monochromated beam having a wavelength (λ) of 1.33001 Å. The
XRD patterns were collected using an imaging plate (IP; Fuji BAS III;
area = 20 × 40 cm2) curved with a radius equivalent to the sample-to-
detector distance (280 mm). The two-dimensional (2D) XRD
patterns of the samples (typical diameter: 10 mm; thickness: 1 mm)
were circularly averaged to obtain one-dimensional (1D) diffraction
profiles I(Q). The values of Q were calibrated using standard samples
of silver behenate and Si powder (NBS 640b).
1
Figure 2b displays the H NMR spectrum of PTyr in d6-
DMSO. The signal of the protons on the nitrogen atoms of
PTyr appeared at 8.05 ppm (singlet); the signal of the phenolic
OH proton appeared at 9.24 ppm; the singlets at 2.30 and 3.83
ppm correspond to the CC−H and CCCH2 protons,
respectively; and the signals of the aromatic protons appeared
as 6.97 and 6.66 ppm. Figure 2 provides assignments for all of
1
the other signals in the H NMR spectrum of PTyr. We
determined the molar mass of PTyr from its 1H NMR spectrum
using the equation49,50
IeMTyr
Mn,PTyr
=
+ Mpropargylamine
2Ig
RESULTS AND DISCUSSION
where Ie and Ig are the intensities of the signals of the methine
protons e on the main chain of PTyr and the methylene
protons g of the propargylamine initiator, respectively, and
Mpropargylamine is the molar mass of the propargylamine initiator.
■
Synthesis of PTyr. Figure 1 displays FTIR spectra of the
tyrosine monomer and corresponding polymer, recorded at
room temperature. Two typical CO stretching bandsat
Figure 1. FTIR spectra of the (a) tyrosine NCA monomer and (b) PTyr polypeptide at room temperature.
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Figure 4. DSC traces (second heating run) of (A) PTyr/P4VP blends
and (B) PTyr/P4VP complexes.
1
Figure 2. (A) H and (B) 13C NMR spectra of the (a, c) tyrosine
NCA monomer and (b, d) PTyr polypeptide.
Figure 5. Plots of Tg with respect to composition, based on the Kwei
equation, for PTyr/P4VP blends and complexes.
carbon atom (NHCOC) appear at 34.7 and 58.2 ppm,
respectively. Figure 2d displays the 13C NMR spectrum of
PTyr in d6-DMSO; the signals for the amide carbon atoms
appear at 170 and 162 ppm, while those of the alkyne carbon
atoms (derived from the macroinitiator propargylamine)
appear at 72 and 80 ppm. The signals for the amino acid α-
carbon atoms (NHCOC) appear at 54.3 ppm. Figure 2(d)
provides assignments for the remaining signals of the carbon
atoms of PTyr. Figure 3 presents the MALDI−TOF mass
spectrum of PTyr. The mass difference between all adjacent
peaks for PTyr is m/z 164, as expected for a tyrosine repeating
Figure 3. MALDI−TOF mass spectrum of the PTyr polypeptide.
Here, we chose propargylamine as the macroinitiator because it
provided the potential ability to synthesize block copolymers
using click reactions18,19 (for brevity, we will discuss such
materials in a future paper).
Figure 2c presents the 13C NMR spectrum of the tyrosine
monomer in d6-DMSO. The signals for the CO carbon
atoms appear at 171.2 and 151.2 ppm; the signal for the
phenolic C−OH carbon atom appears at 156.6 ppm; those of
the phenyl ring appear at 130.3, 124.4, and 114.8 ppm. The
signals for the benzylic carbon atom and the amino acid α-
1
unit. Taken together, the H NMR, 13C NMR, FTIR, and
MALDI−TOF mass spectra confirmed the successful synthesis
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Figure 6. IR spectra, recorded at room temperature, displaying the
region between 1030 and 970 cm−1, for (A) PTyr/P4VP blends and
(B) PTyr/P4VP complexes.
Figure 8. FTIR spectra (1750−1580 cm−1) recorded at room
temperature of (A) PTyr/P4VP blends and (B) PTyr/P4VP
complexes.
Table 3. Fractions of Hydrogen-Bonded Components in
PTyr/P4VP Blends and Complexes at Room Temperature,
Determined from Curve Fitting of FTIR Spectral Data
hydrogen-bonded pyridyl
free pyridyl rings
rings
ν,
W1/2
,
ν,
W1/2,
blend
cm−1
cm−1
Af, %
cm−1
cm−1
Ab, % fb, %
20/80
40/60
50/50
60/40
80/20
993
993
994
994
994
10
10
10
9
70.0
48.4
43.0
33.7
22.0
1005
1005
1006
1006
1006
11
11
11
11
12
30.0
51.6
57.0
66.3
78.0
30.0
51.6
57.0
66.3
78.0
9
Complex
40/60
48/52
55/45
59/61
63/37
993
993
994
994
994
8
8
8
8
8
25.3
22.8
20.9
20.4
17.8
1003
1003
1003
1003
1004
11
11
11
11
11
74.7
77.2
79.1
79.6
82.2
74.7
77.2
79.1
79.6
82.2
Figure 9. Curve fitting of the signals in the FTIR spectra of (a) pure
PTyr, (b) the PTyr/P4VP =50/50 blend, and (c) the PTyr/P4VP =
55/45 complex.
of PTyr. Table 1 lists the DP of PTyr used in this study, as
determined from its 1H NMR and MALDI−TOF mass spectra.
Thermal Analyses of PTyr/P4VP Blends and Com-
plexes. Generally, only a single glass transition can be
observed if the components of blends and complexes are
thermodynamically miscible. DSC is a convenient method for
observing the thermal characteristics that arise from the
different interactions of miscible polymer blends and
complexes. Here, we increased the feed composition up to
the bulk composition, similar to the method reported for
poly(methacrylic acid) (PMAA)/P4VP complex systems.51 The
Figure 7. Fraction of pyridyl groups of P4VP hydrogen bonded to the
polypeptide, plotted with respect to the PTyr content in the blend and
complex systems.
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Figure 10. Secondary structures in the (A) PTyr/P4VP blends and
(B) PTyr/P4VP complexes at various P4VP contents.
Figure 13. WAXD patterns of the (A) PTyr/P4VP blends and (B)
PTyr/P4VP complexes.
DSC thermograms of various PTyr/P4VP blends and
complexes in Figure 4 reveal that all of the blends and
complexes exhibited only a single glass transition, suggesting
that these PTyr/P4VP blends and complexes were fully
miscible with a homogeneous amorphous phase. Meanwhile,
we observed a single value of Tg that was higher than that of
either individual polymer. A large positive deviation in the value
of Tg indicates a strong interaction between the two
components. This result is similar to the glass transition
behavior of PVPh/P4VP miscible blend systems obtained from
DMF solutions. The Tg composition curves of the miscible
blends and complexes in Figure 5 reveal that the single values
of Tg of all blends and complexes were significantly higher than
those predicted by the Fox or linear rule. Generally, if the Tg−
composition relationship deviates substantially, neither the
linear relationship nor the ideal Fox rule is applicable.52 In
general, the Kwei equation53 is a more appropriate fit to the
Tg−composition relationship for highly deviated miscible block
copolymer or polymer blends:
Figure 11. FTIR spectra (600−460 cm−1) recorded at room
temperature for the (A) PTyr/P4VP blends and (B) PTyr/P4VP
complexes.
WT + kW T
1
g1
2 g2
T =
+ qWW
g
1 2
W + kW
(2)
1
2
where w1 and w2 are the weight fractions of the compositions,
Tg1 and Tg2 are the corresponding glass transition temperatures,
and k and q are fitting constants. Figure 5 displays the
dependence of the value of Tg on the composition of the
miscible blends and complexes of PTyr/P4VP, where the
maximum deviations of the highest value of Tg were obtained
when the blend compositions were PTyr/P4VP = 50/50−60/
40. In this study, we obtained values of k and q of 1 and 135,
respectively, for the complex and 1 and 100, respectively, for
the blends, determined from the nonlinear least-squares best
fits in Figure 5. Here, q is a parameter corresponding to the
strength of hydrogen bonding in the system; it reflects a
balance between the breaking of self-association hydrogen
bonds and the forming of interassociation hydrogen bonds.
Furthermore, the observed difference in the value of q from
these two systems implies that the strength of the
interassociation interaction within the PTyr/P4VP complex
was greater than that in the corresponding PTyr/P4VP blend,
consistent with our previous findings.38 It is generally believed
Figure 12. 13C CPMAS spectra (recorded at room temperature) for
the (A) PTyr/P4VP blends and (B) PTyr/P4VP complexes.
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Scheme 2. Possible Structures of (a) pure PTyr and (b) PTyr/P4VP Complexes
about specific interactions between polymers and the secondary
structures of polypeptides, both qualitatively and quantita-
tively.54,55 Some of the characteristic modes of P4VP are
sensitive to the presence of hydrogen bonds, and have been
widely discussed. For example, the signals of P4VP at 1590,
1050, 993, and 625 cm−1 shifted to 1600, 1067, 1011, and 634
cm−1, respectively, after hydrogen bonding with the carboxylic
acid groups of poly(ethylene-co-methacrylic acid).56 Unfortu-
nately, it was difficult for us to analyze the band at 1590 cm−1
for P4VP because of overlap with the band at 1600 cm−1 from
PTyr. Indeed, we could use only the band at 993 cm−1 to
analyze the hydrogen bonding interactions between the OH
groups of PTyr and the pyridyl groups of P4VP. Figure 6
presents room-temperature FTIR spectra in the range 970−
1030 cm−1 for the PTyr/P4VP blends and complex. Pure P4VP
provided a characteristic band at 993 cm−1, corresponding to
the free pyridyl rings; pure PTyr provided a band at 1015 cm−1.
These two bands were well resolved, without overlapping. A
new band appeared at 1003−1006 cm−1, which we assign to the
hydrogen-bonded pyridyl rings of P4VP in the PTyr/P4VP
blends and complexes. The FTIR spectra of all of the blend and
complex systems revealed the existence of hydrogen bonding
between the pyridyl and OH groups, but it was difficult to
quantify the degree of hydrogen bonding because of the
presence of three bands in this region. As a result, we
performed a digital subtraction of the signal for pure PTyr at
1015 cm−1, based on the weight fraction of P4VP in these
blends and complexes.38,57 All of the signals for the pyridyl
groups split into two bands that fit well to the Gaussian
function (Table 3). Figure 7 summarizes the results from curve
fitting; the fraction of hydrogen-bonded pyridyl rings increased
upon increasing the PTyr content in the blend and complex
systems, similar to our previously reported findings for
phenolic/P4VP and PVPh/P4VP blends.38,56 In addition, the
fraction of hydrogen-bonded pyridyl groups of P4VP at the
same PTyr weight fraction revealed that the degree of hydrogen
bonding in the complex was greater than that of the blends;
Scheme 3. Possible Structures for the Polymer Chains in the
(a) PTyr/P4VP Blends and (b) PTyr/P4VP Complexes
that interassociation between two polymer chains in a complex
reduces segmental mobility, thereby increasing their glass
transition temperatures. In addition, the positive value of q of
100 obtained for the PTyr/P4VP blend is similar to that for
PVPh/P4VP blends obtained from DMF solution.38
FTIR Spectroscopic Analyses of PTyr/P4VP Blends and
Complexes. FTIR spectroscopy can provide information
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these results are consistent with those determined using the
Kwei equation.
participate in hydrogen bonding interactions, they can compete
with P4VP for the OH groups in PTyr. MeOH is a relatively
weaker hydrogen bond acceptor than DMF. As a result, the
PTyr/P4VP blends also formed interpolymer complexes in
MeOH, but not in DMF, a solvent that has a great ability to
break hydrogen bonds.39−41 Furthermore, the values of Tg of
the PTyr/P4VP complexes obtained from MeOH solutions
were always higher than those of the miscible PTyr/P4VP
blends obtained from DMF solution casting. The random coils
were well separated for the PTyr/P4VP mixture in DMF
solution, with the polymer chains being quite extended prior to
solvent evaporation because interassociation hydrogen bonding
between PTyr and DMF was stronger than that between PTyr
and P4VP. As a result, the random coil conformation was
preferred in the PTyr/P4VP blend systems. In contrast, the
stronger hydrogen bonding between PTyr and P4VP in MeOH
tended to induce polymer complex aggregation, with a possible
mechanism involving the formation of interpolymer complexes
through interchain hydrogen bonding of two polymer chains.
As a result, the content of β-sheets stabilized through
intermolecular interactions increased in the PTyr/P4VP
complex systems. To confirm this hypothesis, we explored
the secondary structures using solid state NMR spectroscopy
and WAXD.
Solid State NMR Spectroscopic and WAXD Analyses
of PTyr/P4VP Blends and Complexes. Figure 12 displays
13C CP/MAS spectra of the PTyr/P4VP blends and complexes
at room temperature. The different 13C chemical shifts of the
Cα, Cβ, and amide CO carbon atom nuclei were related to
the different local conformations of the individual amino acid
residues, characterized by their dihedral angles and types of
intermolecular and intramolecular hydrogen bonds.59,60 In the
case of pure PTyr, the phenolic groups in the side chains could
stabilize the α-helical secondary structures; the chemical shifts
of the corresponding Cα, Cβ, and amide CO carbon atom
nuclei appeared at 58.0, 36.1, and 177.0 ppm, respectively. In
the β-sheet conformation, these chemical shifts (52.1, 39.3, and
169.6 ppm, respectively) were located upfield by approximately
3−7 ppm relative to those for the α-helical conformations.60
Other assignable signals were similar to those in Figure 2d. The
CO region of the spectrum of the PTyr/P4VP blend system
revealed that the contents of the α-helical and β-sheet
conformations of PTyr disappeared gradually upon increasing
the P4VP content. In contrast, the fraction of β-sheet
conformations remained almost unchanged upon increasing
the P4VP content in the PTyr/P4VP complex system, similar
to our findings from analyses of the amide VI bands using FTIR
spectroscopy.
Figure 8 presents FTIR spectra recorded at room temper-
ature in the range from 1750 to 1580 cm−1, providing
information regarding the secondary structures (amide I
group) of the PTyr/P4VP blends and complexes. Analyzing
these spectra using the second-derivative technique,7 we
observed eight major peaks for pure PTyr: for the ring
vibrations of tyrosine at 1597 and 1615 cm−1; the α-helical
conformation at 1655 cm−1; the β-sheet conformation at 1630
cm−1; the β-turn conformation at 1670 cm−1; and the random
coil conformation at 1643, 1683, and 1700 cm−1.58 After
blending with the P4VP homopolymer, the fraction of the β-
sheet conformation at 1630 cm−1 increased initially at lower
P4VP homopolymer contents but then decreased at higher
P4VP homopolymer contents in the complex system. For
deconvolution, we fitted a series of Gaussian distributions
(Figure 9) to quantify the fractions of each of the peaks. Figure
10 summarizes the curve-fitting data for the amide I groups of
the β-sheet, α-helical, and random coil structures of PTyr when
blended and complexed with the P4VP homopolymer. It has
been reported that the α-helix and β-sheet secondary structures
of PBLG are both present at low DPs (<18), but when the DP
increases, the α-helical secondary structure is favored.28 In this
study, we also observed both the α-helix and β-sheet
conformations for the PTyr oligomer at a DP of 8. When
blended with the P4VP, the fractions of the α-helix and β-sheet
conformations decreased, implying that the fraction of random
coil conformations increased, upon increasing the content of
the P4VP homopolymer. When complexed with P4VP, which
can form strong hydrogen bonds, the fractions of the random
coil and α-helix conformations were lower than that of pure
PTyr, implying that the fraction of β-sheet conformations was
higher than that in pure PTyr. Therefore, the secondary
structures for PTyr were highly dependent on the strength of
the intermolecular interactions and the chain behavior in
solution.
In addition to amide I bands, amide VI bands are also
sensitive to secondary structure modifications of polypeptides.
The relative contributions of α-helix and β-sheet conformations
are well correlated with the absorptions of the amide I bands.
Unfortunately, our analysis of the random coil structures were
difficult because the three major peaks contributing to the
amide I bands of the tyrosine residues in the random coil
conformation were strongly overlapped with the signals for the
ring vibrations (1597 and 1615 cm−1) of the α-helix and β-
sheet conformations.58 Figure 11 presents FTIR spectra
recorded at room temperature in the range from 600 to 460
cm−1, from which we obtained information regarding the
secondary structures (amide VI group) of the PTyr/P4VP
blends and complexes. We attribute the signal near 540 cm−1 to
a predominant β-sheet conformation and that at 512 cm−1 to an
α-helical conformation.58 The fraction of β-sheet conformations
decreased significantly upon increasing the P4VP content in the
PTyr/P4VP blend system; in contrast, the fraction of β-sheet
conformations remained almost unchanged upon increasing the
P4VP content in the PTyr/P4VP complex system, consistent
with our findings from analyses of the signals for the amide I
groups.
We also used WAXD to identify the changes in the
secondary structures of the PTyr/P4VP blends and complexes
(Figure 13). For pure PTyr at a DP of 8, the diffraction pattern
revealed the presence of β-sheet secondary structures. The first
peak, at a value of q of 0.54, reflects the distance (d = 1.15 nm)
between the backbones in the antiparallel β-pleated sheet
structure; the high-order diffraction maxima at scattering
vectors q of multiple integers relative to the position of the
first-order scattering maximum (q/qm = 1, 2, 3, and 4) also
suggested that the PTyr featured a lamellar microdomain
structure. The reflection at a value of q of 1.32 (d = 0.475 nm)
represents the intermolecular distance between adjacent
peptide chains within one lamella; the value of q of 1.45
originated mainly from the repeated residues of the polypeptide
chain (d = 0.445 nm).61 The features in the X-ray patterns
The solvent medium can play an important role in affecting
or controlling the type of complex that forms.39 PTyr/P4VP
yielded a complex precipitate in MeOH, but no precipitation
occurred in DMF. Because solvent molecules can also
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dx.doi.org/10.1021/ma301179t | Macromolecules 2012, 45, 6547−6556

Macromolecules
Article
gradually become a broad amorphous halo arising from the
random coil conformations upon increasing the P4VP content
in the PTyr/P4VP blend system, consistent with our FTIR and
solid state NMR spectroscopic analyses. In contrast, the signals
in the X-ray pattern at values of q of 0.54 and 1.32 remained
almost unchanged upon increasing the P4VP content in the
PTyr/P4VP complex system, indicating that the PTyr
maintained its β-pleated sheet structure. We expected that
the interactions of the P4VP homopolymer through hydrogen
bonding at the PTyr side chains should have expanded or
swollen the distance between the β-pleated sheets in the PTyr/
P4VP complex. Figure 13(B) reveals that the WAXD pattern of
the PTyr/P4VP complexes featured more than one strong
additional diffraction angle (at q = 0.178) relative to that of
pure PTyr. This broad peak, assumed to be a Bragg reflection,
corresponds to an ordered spacing of approximately 3.52 nm
for the PTyr/P4VP complexes. Increasing the P4VP homopol-
ymer content did not change the position of this diffraction
peak. Scheme 2 provides a schematic representation of the
organization of the β-pleated sheet structures for PTyr and the
PTyr/P4VP complex. As calculated from the WAXD data, the
β-pleated sheet lamellar structure for PTyr has a separation of
1.15 nm (Scheme 2a); the characteristic spacing of 3.52 nm in
Scheme 2b is consistent with a β-pleated sheet lamellar
structure with random coils of P4VP homopolymer chains.
Scheme 3 summarizes the possible morphologies, secondary
structures, and intermolecular interactions in the PTyr/P4VP
blends and complexes. The α-helical and β-sheet conformations
are stabilized by intra- and intermolecular hydrogen bonding
interactions, respectively. Upon blending with P4VP in DMF
solution, random coils of PTyr were well separated and the
polymer chains were quite extended prior to solvent
evaporation; FTIR spectra, solid state NMR spectra, and
WAXD analyses revealed that the PTyr/P4VP blend featured a
random coil conformation after solvent evaporation (Scheme
3a). The higher value of Tg for the PTyr/P4VP complex
relative to those for the PTyr/P4VP blends was probably due to
interpolymer complex aggregation through intermolecular
hydrogen bonding interactions, which stabilized the β-sheet
conformations (Scheme 3b).
AUTHOR INFORMATION
Corresponding Author
*E-mail: kuosw@faculty.nsysu.edu.tw. Telephone/Fax: 886-7-
5254099.
■
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
100-2221-E-110-029-MY3 and NSC100-2628-E-110-003.
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We have successfully synthesized a low-molecular-weight PTyr
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