Using hydrogen-bonding interactions to control the peptide secondary structures and miscibility behavior of poly(l -glutamate)s with phenolic resin

Article abstract of DOI:10.1021/ma200721e

We synthesized three low-molecular-weight poly(glutamate)s-poly(γ- methyl l-glutamate) (PMLG), poly(γ-ethyl l-glutamate) (PELG), and poly(γ-benzyl l-glutamate) (PBLG)-through living ring-opening polymerization of their α-amino acid-N-carboxyanhydride derivatives and then blended them with phenolic resin to control the secondary structures of these polypeptides. Each of the three binary blends exhibited a single glass transition temperature (differential scanning calorimetry) and a single-exponential decay of proton spin-lattice relaxation times in the rotating frame [T_1ρ^H; solid state nuclear magnetic resonance (NMR) spectroscopy], characteristic of a miscible system. The strength of the interassociative interactions depended on the nature of the hydrogen bond acceptor groups, increasing in the order phenolic/PELG > phenolic/PMLG > phenolic/PBLG, as evidenced through analyses using Fourier transform infrared (FTIR) spectroscopy and the Painter-Coleman association model. The fractions of α-helical conformations (measured using FTIR and solid-state NMR spectroscopy) of PMLG and PELG decreased initially upon increasing the phenolic content but increased thereafter; in contrast, the fraction of α-helical conformations of PBLG increased continuously upon increasing the phenolic contents. Using variable-temperature infrared spectroscopy to investigate the changes in the conformations of the secondary structures of the peptide segments in these three binary blends, we found that the α-helical conformation in these three blend systems correlated strongly with the rigidity of side-chain groups, the strength of the intermolecular hydrogen bonding with the phenolic resin, the compositions of phenolic resin, and the temperature. More interestingly, the content of α-helical conformations of the polypeptides in these phenolic/PBLG blends increased upon increasing the temperature.

Full text of DOI:10.1021/ma200721e

ARTICLE
pubs.acs.org/Macromolecules
Using Hydrogen-Bonding Interactions To Control the Peptide
Secondary Structures and Miscibility Behavior of Poly(L-glutamate)s
with Phenolic Resin
Shiao-Wei Kuo* and Chi-Jen Chen
Department of Materials and Optoelectronic Science, Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University,
Kaohsiung, Taiwan
S Supporting Information
b
ABSTRACT: We synthesized three low-molecular-weight poly-
(glutamate)s—poly(γ-methyl L-glutamate) (PMLG), poly(γ-ethyl
L-glutamate) (PELG), and poly(γ-benzyl L-glutamate) (PBLG)—
through living ring-opening polymerization of their R-amino acid-
N-carboxyanhydride derivatives and then blended them with
phenolic resin to control the secondary structures of these poly-
peptides. Each of the three binary blends exhibited a single glass
transition temperature (differential scanning calorimetry) and a
single-exponential decay of proton spinꢀlattice relaxation times in
the rotating frame [T1FH; solid state nuclear magnetic resonance
(NMR) spectroscopy], characteristic of a miscible system. The
strength of the interassociative interactions depended on the nature
of the hydrogen bond acceptor groups, increasing in the order phenolic/PELG > phenolic/PMLG > phenolic/PBLG, as evidenced through
analyses using Fourier transform infrared (FTIR) spectroscopy and the PainterꢀColeman association model. The fractions of R-helical
conformations (measured using FTIR and solid-state NMR spectroscopy) of PMLG and PELG decreased initially upon increasing the
phenolic content but increased thereafter; in contrast, the fraction of R-helical conformations of PBLG increased continuously upon
increasing the phenolic contents. Using variable-temperature infrared spectroscopy to investigate the changes in the conformations of the
secondary structures of the peptide segments in these three binary blends, we found that the R-helical conformation in these three blend
systems correlated strongly with the rigidity of side-chain groups, the strength of the intermolecular hydrogen bonding with the phenolic
resin, the compositions of phenolic resin, and the temperature. More interestingly, the content of R-helical conformations of the polypeptides
in these phenolic/PBLG blends increased upon increasing the temperature.
’ INTRODUCTION
followed nature’s strategies for producing supramolecular bioactive
assemblies.13ꢀ24 The non-peptide blocks have often been used as
macroinitiators; for example, polystyrene,20 poly(ethylene oxide),25
poly(dimethylsiloxane),26,27 poly(ε-caprolactone),28 poly(N-iso-
propylacrylamide),29 poly(butadiene),30 poly(isoprene),31 poly(2-
ethyl-2-oxazoline),32 and polyfluorene.33 Klok et al. reported that the
Fourier transform infrared (FTIR) spectra of poly(styrene-b-γ-
benzyl L-glutamate) (PS-b-PBLG) copolymers revealed significant
stabilization of the R-helical secondary structure relative to those of
the corresponding PBLG oligomers.20 We have previously combined
the well-defined macromolecular architectures of polyhedral oligo-
meric silsesquioxane (POSS) and PBLG to generate polymeric
building blocks (POSS-b-PBLG), and we have incorporated POSS
moieties at the chain ends of PBLG units to allow intramole-
cular hydrogen bonding to occur between the POSS and PBLG
units, thereby enhancing the latter’s R-helical conformations in the
The secondary structures of peptide chains influence the forma-
tion of well-defined tertiary structure of proteins.1 Polypeptides
form hierarchically ordered structures containing R-helices, which
can be regarded as a rigid rods stabilized through intramolecular
hydrogen-bonding interactions, and β-sheets, stabilized by inter-
molecular interactions, as fundamental secondary motifs.2 From a
synthetic point of view, the R-helical structures of polypeptides [e.g.,
poly(γ-benzyl L-glutamate) (PBLG)] cause them to behave as rigid-
rod-like polymers in solution and in the solid state,3ꢀ5 providing
unique bulk (e.g., thermotropic liquid crystalline ordering6,7) and
solution (thermoreversible gelation8ꢀ10) behavior. The β-sheet
secondary structure is also an important feature in the development
of several neurodegenerative disorders (e.g., prion diseases).11 As a
result, conformational studies of model polypeptides are important
steps toward mimicking the biological activity of more-complex
proteins.12
For several decades, most methods for synthesizing poly-
(peptide-b-non-peptide) (rod/coil) block copolymers, with
potential applications in tissue engineering and drug delivery, have
Received: March 28, 2011
Revised:
July 25, 2011
Published: August 18, 2011
r
2011 American Chemical Society
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solid state.35ꢀ37 The synthesis of diblock copolymers is, however, a
difficult and time-consuming method for varying the secondary struc-
ture of a polypeptide. From practical and economical points of view,
physical blending is a simpler and more effective method of modifying
polypeptides and other useful materials, with greater versatility and
flexibility, than is the development of new polymers.38ꢀ42
Here, we report that the secondary structures of polypeptides can
be altered through blending with other random-coil non-peptide
oligomers, mediated by hydrogen-bonding interactions. Painter
et al. used infrared spectroscopy (IR) and optical microscopy to
study the phase behavior of blends of three polyglutamates [poly(γ-
methyl L-glutamate) (PMLG), poly(γ-ethyl L-glutamate) (PELG),
and PBLG] with the random-coil polymer poly(vinylphenol)
(PVPh).38 In that study, each of these three polypeptides with
flexible side chains adopted an R-helical rigid-rod conformation
because of its high molecular weights: PMLG (46 000 g/mol),
PELG (160 000 g/mol), PBLG (248 000 g/mol), and PVPh
(9000ꢀ11 000 g/mol).2,43 The presence of intermolecular hydro-
gen bonds between the CdO groups on the side chains of PMLG
and PELG and the OH groups of PVPh, but not between PBLG and
PVPh, indicated that the latter system was immiscible and phase-
separated.38 At a low degree of polymerization (e.g., DP < 18 for
PBLG), both secondary structures (R-helix, β-sheet) are present;
when the DP increases, however, the R-helical secondary structure is
favored.2,43 In addition, high DPs cause the entropic term to become
small, thereby decreasing the miscibility of polymer blend systems.44
As a result, for this study we synthesized three low-molecular-weight
(both R-helical and β-sheet secondary structures) polyglutamates
(PMLG, PELG, and PBLG) through living ring-opening polymer-
ization of R-amino acid-N-carboxyanhydride (NCA) derivatives
and then blended them with phenolic resin (Mn = 500 g/mol) as a
means of altering their secondary structures. In addition, we
expected the various side-chain groups of PMLG, PELG, and PBLG
to affect the secondary structures by influencing the degrees of
hydrogen bonding with the phenolic resin. We have used differential
scanning calorimetry (DSC), FTIR spectroscopy, and solid-state
nuclear magnetic resonance (NMR) spectroscopy to investigate the
miscibility behavior, hydrogen-bonding interactions, and secondary
structures of these phenolic/polyglutamates blends. We have also
used variable-temperature IR spectroscopy to investigate the con-
formational changes that occur within the polypeptide segments
upon blending with phenolic resin.
Figure 1. 1H NMR spectra of the (a) MLG, (c) ELG, and (e) BLG
monomers in CDCl3 and the (b) PMLG, (d) PELG, and (f) PBLG
polypeptides in CDCl3 containing 15 wt % TFA.
polymer mixture was stirred for 6ꢀ8 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.
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 as the internal
standard. High-resolution solid-state NMR spectra were recorded at
room temperature using a Bruker DSX-400 spectrometer operated at
resonance frequencies of 399.53 and 100.47 MHz for 1H and 13C nuclei,
respectively. The 13C CP/magic angle sample spinning (MAS) spectra
were measured with a 3.9 μs 90ꢀ 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 cross-polarization
pulse sequence. An MAS rate of 5.4 kHz was used to avoid absorption
overlapping. The proton spinꢀlattice relaxation time in the rotating
frame (T1FH) was determined indirectly via carbon observation using a
90ꢀꢀτꢀspin lock pulse sequence prior to cross-polarization. The data
acquisition was performed via 1H decoupling and delay times (τ)
ranging from 0.1 to 12 ms with a contact time of 1.0 ms. Thermal
analysis through DSC was performed using a DuPont 910 DSC-9000
controller operated at a scan rate of 10 ꢀC/min over a temperature range
from ꢀ50 to +120 ꢀC under a N2 atmosphere. FTIR spectra of the
polymer films were recorded using the conventional KBr disk method.
FTIR spectra were recorded using a Bruker Tensor 27 FTIR spectro-
’ EXPERIMENTAL SECTION
Materials. Butylamine was purchased from Tokyo Kasei Kogyo,
Japan. γ-Methyl L-glutamate N-carboxyanhydride, γ-ethyl L-glutamate
N-carboxyanhydride, and γ-benzyl L-glutamate N-carboxyanhydride
monomers were prepared according to a literature procedure45 and
stored at ꢀ30 ꢀC. The phenolic was synthesized via a sulfuric acid-
catalyzed condensation reaction, producing an average molecular weight
(Mn) of 500, using a procedure described previously.46
PMLG, PELG, and PBLG. In a typical experiment, the NCA
monomer (2 g) was weighed in a glovebox under pure Ar, placed in a
flame-dried Schlenk tube, and dissolved in anhydrous N,N-dimethylfor-
mamide (DMF, 40 mL). The solution was stirred for 10 min, and then
butylamine (50 μL) was added using a N2-purged syringe. After stirring
the solution for 40 h at room temperature, the polymer was recovered
through precipitation in diethyl ether and dried under high vacuum.
Blend Preparations. Blends of phenolic/PMLG, phenolic/PELG,
and phenolic/PBLG at various blend compositions were prepared
through solution casting. A DMF solution containing 5 wt % of the
photometer; 32 scans were collected at a spectral resolution of 1 cmꢀ1
.
Because polymers containing OH groups are hygroscopic, pure N2 gas
was used to purge the spectrometer’s optical box to ensure dry sample
films. IR spectra of samples at elevated temperatures were recorded
using a cell mounted within the temperature-controlled compartment of
the spectrometer.
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Scheme 1. Syntheses of PMLG, PELG, and PBLG, Using Butylamine as the Initiator
Table 1. Self- and Interassociation Equilibrium Constants
and Thermodynamic Parameters of the Phenolic/PMLG,
Phenolic/PELG, and Phenolic/PBLE Blends at 25 ꢀCa
’ RESULTS AND DISCUSSION
Synthesis of PMLG, PELG, and PBLG. Figure 1 presents the 1H
NMR spectra of the MLG, ELG, and BLG monomers in CDCl3.
We assign each singlet at 6.4 ppm to the proton on the ring nitrogen
atom; the signals at 3.83, 4.18, and 5.12 ppm to the COOCH2R
protons of MLG, ELG, and BLG, respectively; the multiplets
between 2.0 and 2.6 ppm to the alkyl CH2 protons; and, for
BLG, the signals at 7.31ꢀ7.38 ppm to the benzyloxy ring protons.
Figure 1 also displays the 1H NMR spectra of PMLG, PELG, and
PBLG in CDCl3 containing 15% TFA. The signals of the protons on
the nitrogen atoms of PMLG, PELG, and PBLG resonated at 7.9
ppm (singlet); the signals of the butyl groups were located at 0.9
(CH3C3H6, triplet), 1.3 (CH3CH2C2H4, multiplets), 1.5 (C2H5-
CH2CH2, multiplets), and 3.2 ppm (C3H7CH2NH, triplet), and for
PBLG, the signals of the aromatic protons appeared as multiplets at
7.31ꢀ7.38 ppm. Scheme 1 presents assignment for all of the other
signals in the 1H NMR spectra of PMLG, PELG, and PBLG. We
determined the molar masses of PMLG, PELG, and PBLG from
their 1H NMR spectra using the equation47,48
equilibrium constant
polymer
V
Mw
Δ
DP
K2
KB
KA
phenolic
PMLG
PELG
84.0 105 12.0
105.5 143 11.5
122.0 157 11.0
165.9 219 11.2
6
10
9
23.3
52.3
30.0
50.0
9.0
PBLG
7
a V = molar volume (mL/mol),43 Mw = molecular weight (g/mol), δ =
solubility parameter (cal/mL)1/2 43
, DP = degree of polymerization, K2 =
dimer self-association equilibrium constant, KB = multimer self-associa-
tion equilibrium constant, and KA = interassociation equilibrium
constant.
revealed the absence of the absorbances from the CdO groups
a and b of the NCA rings and the appearance of new absorbance
at 1655, 1627 (d), and 1543 cmꢀ1 (e), representing amide
1
bonds in the polymer backbone.49 Our H NMR and FTIR
IbMLG
Mn, PLG
¼
þ Mbutylamine
spectroscopic analyses confirmed that the successful syntheses
of PMLG, PELG, and PBLG.
Ig
where Ib and Ig are intensities of the signals of the methylene protons b
on the side chains of PMLG, PELG, and PBLG and the methylene
protons g of the butylamine initiator, respectively. Mbutylamine is the
molar mass of the butylamine initiator. Table 1 lists the DPs of PMLG,
PELG, and PBLG as determined using 1H NMR spectroscopy.
Figure 2 displays FTIR spectra of the MLG, ELG, and BLG
monomers and their corresponding polymers, recorded at
room temperature. Two typical CdO stretching bands—at
1873 and 1789 cmꢀ1, corresponding to the CdO units a and b,
respectively—confirm the formation of the NCA ring;49 the
CdO stretching bands at 1711 cmꢀ1 correspond to free CdO
units c of the MLG, ELG, and BLG NCA monomers. After ring-
opening polymerization of the NCA monomers, FTIR spectra
Figure 3 presents scale-expanded FTIR spectra of the CdO and
amide stretching regions of PMLG, PELG, and PBLG, recorded at
room temperature. We split the CdO and amide stretching
frequencies of these three polypeptides split into four bands: at
1734ꢀ1741, 1690ꢀ1695, 1653ꢀ1656, and 1623ꢀ1627 cmꢀ1
,
corresponding to the free CdO groups (on the side chains) and
the random coil, R-helical, and β-sheet secondary structures of
the polypeptides, respectively.2 These four bands fitted well to
the Gaussian function; Figure 3 displays typical examples of the
separation. The fractions of R-helical secondary structures of the
polypeptides were similar (PMLG: 42.5%; PELG: 51.1%; PBLG:
51.2%). In this manner, we could readily distinguish the influence of
each of the side chain groups of PMLG, PELG, and PBLG on the
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Figure 3. Curve fitting of the signals in the FTIR spectra of (a) PMLG,
(b) PELG, and (c) PBLG.
Figure 2. FTIR spectra of the (a) MLG, (c) ELG, and (e) BLG
monomers and the (b) PMLG, (d) PELG, and (f) PBLG polypeptides
at room temperature.
secondary structure, mediated through hydrogen bonding with the
phenolic resin.
Thermal Analyses of Polymer Blends. Thermal character-
ization of polymer blends is a well-known method for determin-
ing the miscibility of polymer blends. Some miscible blends can
show a dual glass temperature with a relatively large Tg difference
between two homopolymers with weak intermolecular
interactions.50 In this study, the values of Tg of polypeptides
having rigid objects with well-defined secondary structures
(helices, sheets) were difficult to observe.51 Previous studies
reported that pure phenolic, PMLG, PELG, and PBLG were +55,
ꢀ11, ꢀ13, and +18 ꢀC, respectively.38,52 The Tg difference is not
large, and the hydrogen-bonding interaction is strong as evi-
denced by FTIR analyses in this study that will be discussed in
next section. As a result, the miscibility of any two polymers in the
amorphous state is characterized by the presence of a single glass
transition temperature (Tg).
Figure 4 presents the second heating runs in our DSC analyses
of phenolic resin blends with PMLG, PELG, and PBLG at
various compositions; all three blend systems exhibit a single
glass transition temperature over the entire range of composi-
tions, indicating that they were fully miscible blends exhibiting a
homogeneous amorphous phase. For PMGL and PELG, the
characteristics of the blends with phenolic resin were in general
agreement with those of their blends with the PVPh homopo-
lymer. In contrast, the PVPh/PBLG blends were immiscible and
phase-separated, presumably because of the (i) much lower
molecular weights of the PBLG (2000 g/mol) and the hydrogen
Figure 4. DSC traces (second heating run) of (A) phenolic/PMLG,
(B) phenolic/PELG, and (C) phenolic/PBLG blends.
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Figure 5. FTIR spectra (4000ꢀ2700 cmꢀ1) recorded at room temperature for the (A) phenolic/PMLG, (B) phenolic/PELG, and (C) phenolic/PBLG
blends.
Figure 6. FTIR spectra (1800ꢀ1580 cmꢀ1) recorded at room temperature for the (A) phenolic/PMLG, (B) phenolic/PELG, and (C) phenolic/PBLG
blends.
bond donor polymer [500 g/mol (phenolic)] used in this study,
relative to those [248 000 (PBLG) and 11 000 (PVPh) g/mol]
used previously, with the lower DPs increasing the entropic
terms and thereby enhancing the miscibility of the polymer blend
system,44 and (ii) stronger interassociation hydrogen bonding of
PBLG with the phenolic resin than with the PVPh,53,54 as has
been reported in previous studies,55,56 contributing significantly
to the enthaplic term.44
Hydrogen-Bonding Interactions and Conformations of
the Peptide Segments after Blending with Phenolic Resin.
IR spectroscopy can provide information regarding the specific inter-
actions between polymers, both qualitatively and quantitatively.
The OH stretching range in an IR spectrum is sensitive to the
degree of hydrogen bonding. Figure 5 displays FTIR spectra (in
the range 2700ꢀ4000 cmꢀ1) of phenolic/PMLG, phenolic/
PELG, and phenolic/PBLG blends, recorded at room tempera-
ture. The spectrum of pure phenolic features two distinct bands
in the OH stretching region: a very broad band centered at
3320 cmꢀ1 representing the wide distribution of hydrogen-
bonded OH groups and a sharp band at 3525 cmꢀ1 representing
the free OH groups. For the pure polypeptides, we observe a
sharp band at 3290 cmꢀ1, representing NH stretching (primary
amine) vibrations of the polypeptides. The intensity of the
signal for the free OH groups decreased upon increasing the
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polypeptide content, as would be expected. Meanwhile, the
broad signal of the hydrogen-bonded OH groups shifted to
higher frequency upon increasing the polypeptide content,
whereas the sharp band representing the NH stretching vibra-
tions of the polypeptides remained unchanged. All these ob-
served changes suggest a switch from strong intramolecular
OH OH (phenolic/phenolic) hydrogen bonds into weak
3 3 3
intermolecular OH CdO (phenolic/polypeptides) hydro-
3 3 3
gen bonds,57,58 with no hydrogen-bonding interactions between
OH groups of phenolic and the NH groups of the polypeptides.
We recorded FTIR spectra at room temperature to obtain
information regarding the hydrogen-bonding interactions and
secondary structures of the phenolic/PMLG, phenolic/PELG,
and phenolic/PBLG blends (Figure 6). Analyzing these spectra
using the second-derivative technique,13 we observed seven major
peaks, representing the free CdO groups (1734ꢀ1741 cmꢀ1);38
the hydrogen-bonded CdO groups of the side chains (1710ꢀ
1720 cmꢀ1);38 the secondary structures of the amide I groups
in random coil (1690ꢀ1695 cmꢀ1),13 R-helical (1653ꢀ
1656 cmꢀ1),20ꢀ22 and β-sheet (1623ꢀ1627 cmꢀ1 20ꢀ22
)
confor-
mations;andthe stretching ofbenzeneunits (1610 and 1594 cmꢀ1).
Here, we ignore the amide I group in random coil at 1640ꢀ1650
and 1660ꢀ1670 cmꢀ1 since many bands were difficult to calculate
the quantitative area fraction of secondary structures.59 For decon-
volution, we fitted a series of Gaussian distributions to quantify the
fraction of each of the peaks (Figure 7). Table 2 and Figures 8 and 9
summarize the curve fitting data for the amide I groups of the
β-sheet, R-helical, and random coil structures and for the free and
hydrogen-bonded CdO units of the side chains.
Figure 8 displays the fraction of hydrogen-bonded CdO
groups of PMLG, PELG, and PBLG plotted with respect to
the phenolic content at room temperature; it indicates that the
fraction of hydrogen-bonded CdO groups on the side chains of
the polypeptides increased upon increasing the phenolic resin
contents. Furthermore, the degree of hydrogen bond formation
in the blends with phenolic resin increased in the order phenolic/
Figure 7. Curve fitting of the signals in the FTIR spectra of the (A)
phenolic/PMLG = 60/40, (B) phenolic/PELG = 60/40, and (C)
phenolic/PBLG = 60/40 blends.
Table 2. Curve Fitting Data for the CdO and Amide Groups in the Phenolic/Polypeptide Blends at 25 ꢀC
carbonyl group in polypeptide
amide group in polypeptide
free CdO
H-bond CdO
ν (cmꢀ1
Af (%)
random coil
ν (cmꢀ1
Af (%)
R-helix
β-sheet
blend
ν (cmꢀ1
)
Af (%)
)
)
ν (cmꢀ1
)
Af (%)
ν (cmꢀ1
)
Af (%)
phenolic/PMLG
0/100
1741
1739
1739
1739
1738
100
0
1693
1693
1693
1692
1692
13.5
9.3
8.0
5.8
4.9
1655
1653
1653
1653
1654
42.5
41.1
35.2
39.6
46.8
1626
1625
1625
1623
1623
44.0
49.6
56.8
54.6
48.3
20/80
70.4
61.3
37.8
28.3
1720
1718
1718
1717
29.6
38.7
62.2
71.7
40/60
60/40
80/20
phenolic/PELG
0/100
1735
1735
1733
1733
1733
100
0
1695
1692
1692
1692
14.7
9.3
6.2
4.5
0
1655
1655
1655
1655
1655
51.1
48.8
56.0
59.1
100
1627
1626
1623
1623
34.2
41.9
37.8
36.4
0
20/80
68.1
43.1
28.7
17.6
1713
1710
1709
1709
31.9
56.9
71.3
82.4
40/60
60/40
80/20
phenolic/PBLG
0/100
1736
1735
1735
1734
1734
100
0
1693
1690
1690
1691
18.2
12.6
10.4
5.2
1655
1655
1655
1655
1655
51.2
65.4
74.0
86.6
100
1625
1625
1624
1625
30.6
22.0
15.6
8.2
20/80
74.5
66.5
58.2
40.0
1711
1712
1710
1712
25.5
33.5
41.8
60.0
40/60
60/40
80/20
0
0
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PELG > phenolic/PMLG > phenolic/PBLG. Compared with the
data for blending with the PVPh homopolymer, the phenolic/
PMLG and phenolic/PELG exhibited trends similar to those for
the intermolecular hydrogen bonding that occurred between
PVPh and PMLG or PELG; the proportion of hydrogen-bonded
CdO groups of PVPh/PBLG was, however, negligibly small in
most of the compositions. In the phenolic/PBLG blends, we
observed relatively higher fractions of hydrogen-bonded CdO
groups of PBLG than we had found previously for the PVPh/
PBLG blends. Therefore, we confirmed that the phenolic/PBLG
blend was a miscible system due to the existence of intermole-
cular hydrogen bonds between the OH groups of the phenolic
resin and the CdO groups of PBLG. Next, we determined the
corresponding inter- and self-association equilibrium constants
for these systems. The self-association constants of phenolic
resin (K2 = 23.3; KB = 52.3) have been determined
previously.60 We determined the interassociation constants
KA directly using a least-squares fitting procedure based on
the fraction of hydrogen-bonded CdO observed experimen-
tally in the binary phenolic/PMLG, phenolic/PELG, and
phenolic/PBLG blends; we obtained values of 30, 50, and 9,
respectively. Table 1 lists all the parameters required by the
PainterꢀColeman association model43 to estimate the thermo-
dynamic properties for these polymer blends. The interassocia-
tion equilibrium constants and relative ratios of KA/KB
increased in the order phenolic/PELG > phenolic/PMLG >
phenolic/PBLG. Therefore, the chemical structure of the
group accepting the hydrogen bonds has great impact on these
values, as has been discussed in detail previously.55,56
Figure 9 summarizes the fractions of the secondary structures
of PMLG, PELG, and PBLG with respect to the phenolic
content, at room temperature. The fractions of the random coil
structures of all three binary blends decreased upon increasing
the phenolic content, indicating that the presence of the phenolic
resin stabilized the secondary structures of the polypeptides.
More interestingly, the fractions of the R-helical secondary
structures of these three different polypeptides exhibited differ-
ent trends upon increasing the phenolic resin content. For
PMLG and PELG, the fractions of R-helical conformations
decreased initially upon increasing the phenolic content but
increased thereafter; in contrast, the fraction of the R-helical
conformation of PBLG increased continuously upon increasing
the phenolic content.
It has been reported that at a low DP of PBLG (DP < 18) both
secondary structures (R-helix, β-sheet) are present, but when the
DP increases, the R-helical secondary structure is favored.2 In
this study, we also observed both the R-helical and β-sheet
conformations for the PBLG oligomer at a DP of 7; we found,
however, that the content of R-helical conformations increased
upon increasing the phenolic resin content. It has been proposed
that stacking of the side-chain benzene rings of PBLG plays a role
in stabilizing its various structures;38 indeed, among all of our
studied polypeptides, we found that PBLG at its lowest DP
featured the highest content of R-helical conformations. Jeon
et al. reported that the mobility of the side-chain groups of
polyglutamates also affects the R-helical conformation.61 Their
experimental data revealed that longer flexible side chains
Figure 8. Fraction of hydrogen-bonding CdO groups of the polypep-
tides plotted with respect to the phenolic content and the corresponding
interassociation equilibrium constants based on the PainterꢀColeman
association model.
Figure 9. Secondary structures in the (A) phenolic/PMLG, (B) phenolic/PELG, and (C) phenolic/PBLG blends at various phenolic resin contents.
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Scheme 2. Formation of r-Helical Conformations in Phe-
nolic/PBLG Blends
Table 3. Chemical Shifts (ppm) of the Signals in the 13C CP/
MAS/DD NMR Spectra of the Phenolic/Polypeptide Blends
phenolic
phenolic/PMLG
C-1
C-2
C-3
C-4
100/0
60/40
40/60
20/80
152.3
152.8
153.1
153.2
129.8
129.1
128.6
128.2
116.0
116.2
115.5
115.7
35.8
36.5
36.3
36.3
PMLG
C-5
C-6
CR
C-7
C-8
C-9
0/100
20/80
40/60
60/40
176.3
176.4
176.5
176.5
173.0
173.0
173.5
172.2
57.5/51.9
57.3/52.2
57.5/52.4
57.5/52.5
31.0
31.3
31.5
31.6
27.0
27.3
27.6
26.9
51.9
52.2
52.4
52.5
phenolic
phenolic/PELG
C-1
C-2
C-3
C-4
100/0
60/40
40/60
152.3
153.7
153.8
129.8
129.4
130.0
116.0
116.1
115.8
35.8
36.4
36.5
PELG
C-5
C-6
CR
C-7
C-8
C-9
C-10
0/100
40/60
60/40
176.3
176.6
175.9
172.1
172.7
172.8
57.8/52.5
57.8/53.1
57.4/52.8
31.1
32.2
31.7
21.7
21.3
20.5
60.9
61.8
62.0
14.7
14.9
14.3
phenolic
phenolic/PBLG
C-1
C-2
C-3
C-4
100/0
60/40
40/60
20/80
152.3
152.6
153.0
153.2
129.8
129.2
128.8
129.1
116.0
116.9
115.4
115.2
35.8
36.4
36.6
36.7
PBLG
C-5
C-6
CR
C-7 C-8 C-9 C-10 C-11
0/100 176.1 172.5 57.6/53.0 30.3 28.0 65.7 136.0 129.0
20/80 176.1 172.2 57.6/52.7 31.4 27.9 65.9 136.0 129.1
40/60 176.2 172.4 57.6/52.7 31.6 26.5 65.6 136.7 128.8
Figure 10. 13C CPMAS spectra recorded at room temperature of
the (A) phenolic/PMLG, (B) phenolic/PELG, and (C) phenolic/
PBLG blends.
60/40 176.2 172.3 57.6/ꢀ
31.6 26.0 66.2 136.8 129.2
copolymers as a result of a thermodynamic field created by the
enthalpic interactions of unlike blocks.12 It is well established
that the R-helical and β-sheet conformations are stabilized
by intra- and intermolecular hydrogen-bonding interactions,
respectively. In the miscible phenolic/PBLG blend, intermole-
cular hydrogen bonding of the PBLG segments initially dis-
rupted and then induced intramolecular hydrogen bond-
ing between the PBLG segments upon increasing the phenolic
resin content, as indicated in Scheme 2; as a result, the con-
tent of R-helical conformations increased upon increasing the
phenolic resin content.
induced weaker hydrogen bonds forming between the CdO
groups and the amide linkages of the R-helical conformation; a
corollary is that rigid benzene rings might enhance hydrogen
bonding between the CdO groups and the amide linkages of
the R-helical conformation. In the phenolic/PBLG blend,
hydrogen bonds formed between the CdO groups of PBLG
and the OH groups of the phenolic resin; their presence would
accentuate the rigidity of the side-chain groups of PBLG.
Floudas et al. reported that suppression of the β-sheet second-
ary structure of polyalanine (PALa) occurs in PBLG-b-PALa
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Figure 11. Scale-expanded solid-state 13C NMR spectra (displaying signals for the CdO and CR groups) of the (A) phenolic/PMLG, (B) phenolic/
PELG, and (C) phenolic/PBLG blends.
conformations. Because of the stronger hydrogen-bonding interac-
tions in the phenolic/PELG than in the phenolic/PMLG blends, the
PELG underwent an increase in its content of R-helical conforma-
tions at a relatively lower phenolic content. Therefore, the formation
of R-helical conformations for polyglutamates is strongly dependent
on the rigidity of the side chain groups, the strength of the
intermolecular hydrogen bonds with phenolic resin, and the com-
position of the phenolic resin.
We could also identify the secondary structures of the poly-
peptides on the basis of distinctly different resonances in their
solid-state NMR spectra. Figure 10 displays the 13C CP/MAS
spectra of the phenolic/PMLG, phenolic/PELG, and phenolic/
PBLG blends at room temperature. The peak at 153.2 ppm
represents the resonance of the phenolic carbon atom of pure
phenolic (C-1).62 The variations in this chemical shift of ca. 0.8,
1.5, and 0.7 ppm for the phenolic/PMLG, phenolic/PELG, and
phenolic/PBLG blends, respectively, indicate the existence of
intermolecular hydrogen bonding between the OH groups of the
phenolic and the CdO groups of the polypeptide segments, with
the strength of the intermolecular hydrogen bonds following the
order phenolic/PELG > phenolic/PMLG > phenolic/PBLG, a
trend that is similar to that deduced through FTIR spectroscopic
analysis. Specific interactions in polymer blends can affect the
chemical environments of neighboring molecules, resulting in
upfield or downfield shifts in their resonances.63ꢀ65 In addition,
the different 13C chemical shifts of the CR and amide CdO
resonances are related to the local conformations of the indivi-
dual amino acid residues, characterized by the dihedral angles
Figure 12. Semilogarithmic plots of the magnetization intensities of the
signals at 115 ppm with respect to the delay time for the phenolic/
PMLG, phenolic/PELG, and phenolic/PBLG blends.
In the phenolic/PMLG and phenolic/PELG blends, the fractions
of R-helical conformations of PMLG and PELG decreased initially
upon increasing the phenolic content (to 40 wt % for PELG and to
60 wt % for PMLG) but increased thereafter. This finding implies
that, at lower phenolic contents, intermolecular hydrogen bonding of
the PMLG and PELG segments increased to enhance the β-sheet
conformations or that the random coils changed to β-sheet
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Figure 13. FTIR spectra of the (A) phenolic/PMLG = 40/60, (B) phenolic/PELG = 60/40, and (C) phenolic/PBLG = 40/60 blends, recorded at
various temperatures.
Figure 14. Summary of fractions of R-helical conformations of the (A) phenolic/PMLG, (B) phenolic/PELG, and (C) phenolic/PBLG blends at
various temperatures.
and the types of intermolecular and intramolecular hydrogen-
bonding interactions.66,67 In the case of polyglutamate homo-
polymers, the side chains can stabilize the R-helical secondary
structure; correspondingly, the chemical shifts of the CR and
amide CdO resonances appear at 57.5 and 176 ppm. In the
β-sheet conformation, these chemical shifts (52.7 and 172 ppm,
respectively) are located upfield by ca. 4ꢀ5 ppm relative to those
for the R-helical conformations.2,43 Figure 10 provides assign-
ments for the other peaks; Table 3 summarizes the data.
Because the amide CdO resonance in the β-sheet conforma-
tion partially overlaps with the signal from the side-chain
ester moiety, distinctions between the two peptide secondary
structures are best performed from the distinctly different
CR resonances (57.5 ppm for R-helices, 52.7 ppm for β-sheets).2
Figure 11 presents scale-expanded (CdO and CR regions)
solid-state NMR spectra of phenolic/PMLG, phenolic/PELG,
and phenolic/PBLG blends at the same composition of
phenolic resin (40 wt %), recorded at room temperature.
From the CR region of the spectrum of the phenolic/PMLG
blend system, the content of R-helical conformations was
almost identical to that of pure PMLG; in the phenolic/PELG
and phenolic/PBLG systems, however, the contents of
R-helical conformations were greater than those of the pure
PELG and PBLG homopolymers. In addition, a new peak
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appeared at ca. 173ꢀ174 ppm (downfield to the signals of
both the CdO groups and β-sheet of the polypeptides) in the
CdO region of the spectra for all three blends, presumably
representing the hydrogen-bonded CdO groups.68,69 The
contribution of a second signal related to the hydrogen-
bonded CdO group. All of these results are consistent with
the features determined from the FTIR spectra.
systems strongly correlated with the phenolic resin content and
temperature. Similar to the results in Figure 9, the stronger
hydrogen-bonding interactions in the phenolic/PELG blend
than in the phenolic/PMLG blend caused PELG to more readily
transform to a greater fraction of R-helical conformations at a
relative lower phenolic content upon increasing the temperature.
The increased fraction of R-helical conformations upon increas-
ing the temperature at relatively high phenolic contents in all
three blend systems may have arisen from disruption of the
intermolecular hydrogen bonding between the CdO groups
of the polypeptides and the phenolic resin and subsequent
enhanced intramolecular hydrogen bonding of the polypeptides.
We also recorded FTIR spectra of a polystyrene/PBLG = 60/40
blend—where hydrogen bonding cannot occur between the PS
and PBLG segments—at different temperatures (Figure S3).
Clearly, the fraction of R-helical conformations decreased sig-
nificantly upon heating, different from the behavior of the
phenolic/PBLG system. This observation confirms that the
phenolic resin stabilized the R-helical conformations through
hydrogen-bonding interactions upon increasing the temperature.
We obtained additional information regarding the structures
of the phenolic/PMLG, phenolic/PELG, and phenolic/PBLG
blends from their proton relaxation behavior. We measured the
H
spinꢀlattice relaxation time in the rotating frame T1F to
examine the homogeneity of the polymer blends on the molec-
ular scale. The magnetization of resonance is expected to decay
according to the exponential function model
Mτ ¼ M0 expðꢀτ=T1FðHÞÞ
where τ is the delay time used in the experiment and Mτ is the
corresponding peak intensity. The value of T1FH can be obtained
from the slope of the plot of ln(Mτ/M0) with respect to τ. Figure 12
displays the T1FH relaxation behaviors of each of the three blends
H
(monitoring the phenolic signal at 115 ppm). A single value of T1F
’ CONCLUSIONS
appeared for the blends, indicating their good miscibility and
dynamic homogeneity. The maximum diffusive path length L can
be estimated using the approximate expression
We have synthesized three different polypeptide oligomers
through ROP of NCA monomers using butylamine as the
initiator; we then blended these polypeptides with phenolic resin
to induce changes in their secondary structures. DSC and solid-
state NMR spectroscopic analyses revealed that each blend
system was completely miscible in an amorphous phase over
the entire range of compositions. Using the PainterꢀColeman
association model to analyze the data from the FTIR spectra, the
strength of the hydrogen-bonding interactions increased in the
order phenolic/PELG > phenolic/PMLG > phenolic/PBLG
blends. The fraction of R-helical conformation in these three
blend systems correlated strongly with the rigidity of their side-
chain groups, the intermolecular hydrogen bonding strength
with the phenolic resin, the composition of phenolic resin blend,
and the temperature.
ÆL2æ ¼ 6DTi
H
For a value of T1F of 10 ms and an effective spin diffusion
coefficient, D, of 10ꢀ16 m2 sꢀ1, the dimensions of the features in
the phenolic/PMLG, phenolic/PELG, and phenolic/PBLG blends
were less than 2ꢀ3 nm in the amorphous phase, consistent with the
results from DSC analyses.62,63,70
Figure 13 presents FTIR spectra of the phenolic/PMLG,
phenolic/PELG, and phenolic/PBLG blends measured at tem-
peratures ranging from 25 to 150 ꢀC. The CdO stretching
frequency of each polypeptide split into two bands, one at
1734ꢀ1741 cmꢀ1 and the other at 1710ꢀ1720 cmꢀ1, corre-
sponding to free and hydrogen-bonded CdO groups, respec-
tively. The intensity of the signal for the hydrogen-bonded CdO
groups of the polypeptides decreased upon increasing the
temperature, as would be expected.55ꢀ58,70 In the phenolic/
PMLG = 40/60 blend, the content of β-sheet conformations
increased upon increasing the temperature, in agreement with
previous observations; the oligopeptide almost exclusively
adopted the R-helical conformation at 25 ꢀC.20,71 In the phe-
nolic/PELG = 60/40 blend, however, the fraction of R-helical
conformations decreased upon increasing the temperature from
25 to 75 ꢀC, increased upon increasing the temperature from 75
to 125 ꢀC, and then decreased upon increasing the temperature
from 125 to 150 ꢀC. More interestingly, the fraction of R-helical
conformations increased upon increasing the temperature in the
phenolic/PBLG = 40/60 blend. This trend is the exact opposite
of that observed for the phenolic/PMLG blend system. Figure 14
plots the fractions of R-helical conformations in the phenolic/
PMLG, phenolic/PELG, and phenolic/PBLG blends with re-
spect to the temperature. The fraction of the R-helical conforma-
tions decreased significantly upon heating each of the pure
PMLG, PELG, and PBLG oligomers. At a relatively high phenolic
content, the phenolic resin appears to stabilize the R-helical
conformation at higher temperature, especially in the phenolic/
PBLG blend system; however, the fractions of R-helical con-
formations in the phenolic/PMLG and phenolic/PELG blend
’ ASSOCIATED CONTENT
S
Supporting Information. Additional characterization
b
data (Figures S1ꢀS3). This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: kuosw@faculty.nsysu.edu.tw. Tel: 886-7-5254099.
’ ACKNOWLEDGMENT
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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