Synthesis, Characterization, and thermoresponsive properties of Helical Polypeptides Derivatized from Poly(γ-4-(3-chloropropoxycarbonyl)benzyl-L-glutamate)

Article abstract of DOI:10.1002/pola.27708

A series of side-chain-functionalized α-helical polypeptides, i.e., poly(γ-4-(3-chloropropoxycarbonyl)benzyl-_L-glutamate) (6) have been prepared from n-butylamine initiated ring-opening polymerization (ROP) of γ-4-(3-chloropropoxycarbonyl)benzyl-_L-glutamic acid-based N-carboxyanhydride. Polypeptides bearing oligo-ethylene-glycol (OEG) groups or 1-butylimidazolium salts were prepared from 6 via copper-mediated [2+3] alkyne-azide 1,3-dipolar cycloaddition or nuleophilic substitution, respectively. CD and FTIR analysis revealed that the polymers adopt α-helical conformations both in solution and the solid state. Polymers bearing OEG (m = 3) side-chains showed reversible LCST-type phase transition behaviors in water while polymers bearing 1-butylimidazolium and I^- counter-anions exhibited reversible UCST-type transitions in water. Variable-temperature UV-vis analysis revealed that the phase transition temperatures (T_pts) were dependent on the main-chain length and polymeric concentration.

Full text of DOI:10.1002/pola.27708

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Synthesis, Characterization, and Thermoresponsive Properties
of Helical Polypeptides Derivatized from
Poly(c24-(3-chloropropoxycarbonyl)benzyl-L-glutamate)
Yong Deng, Xi Wang, Qiulin Yuan, Mengxiang Zhu, Ying Ling, Haoyu Tang
Key Laboratory of Polymeric Materials and Application Technology of Hunan Province, Key Laboratory of Advanced
Functional Polymer Materials of Colleges and Universities of Hunan Province, College of Chemistry, Xiangtan University,
Xiangtan, Hunan 411105, China
Correspondence to: H. Tang (E-mail: htang@xtu.edu.cn) or Y. Ling (E-mail: yingling0202@gmail.com)
Received 13 March 2015; accepted 16 May 2015; published online 23 June 2015
DOI: 10.1002/pola.27708
ABSTRACT: A series of side-chain-functionalized a-helical poly-
peptides, i.e., poly(c-4-(3-chloropropoxycarbonyl)benzyl-L-gluta-
mate) (6) have been prepared from n-butylamine initiated
ring-opening polymerization (ROP) of c-4-(3-chloropropoxycar-
bonyl)benzyl-L-glutamic acid-based N-carboxyanhydride. Poly-
peptides bearing oligo-ethylene-glycol (OEG) groups or 1-
behaviors in water while polymers bearing 1-butylimidazolium
and I2 counter-anions exhibited reversible UCST-type transitions
in water. Variable-temperature UV-vis analysis revealed that the
phase transition temperatures (Tpts) were dependent on the main-
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butylimidazolium salts were prepared from
6 via copper-
mediated [213] alkyne-azide 1,3-dipolar cycloaddition or
nuleophilic substitution, respectively. CD and FTIR analysis
revealed that the polymers adopt a-helical conformations both in
solution and the solid state. Polymers bearing OEG (m 5 3)
side-chains showed reversible LCST-type phase transition
KEYWORDS: conformational analysis; functionalization of poly-
mers; liquid-crystalline polymers (LCP); ring-opening polymer-
ization; stimuli-sensitive polymers; water-soluble polymers
Thermoresponsive polymers with lower critical solution
transition temperatures (LCSTs) or upper critical solution
temperatures (UCSTs) have been extensively studied for their
potential applications in delivery of therapeutic molecules
and tissue engineering.27–29 However, most thermorespon-
sive polymers are derivatized from nonbiodegradable polya-
crylates or polyacrylamides, which restricted their use in
biomedical applications. For example, high molecular weight
polymethacrylamides used in long-circulating delivery
systems were not able to eliminate from the organisms (e.g.,
kidney), thus, they accumulated in the body and lead to toxic
side effects.30 Recently, thermoresponsive polypeptides have
gained significant attention due to their promising properties
including biodegradability, biocompatibility and considerable
chemical diversity with regard to the numerous amino acids
(e.g., -glutamic acid, -lysine, and -cysteine).5,6,9,29,31–35 The
INTRODUCTION Polypeptides are mimics of natural peptides
with many attractive properties, such as biocompatibility,
unique secondary structures (e.g., a-helix and b-sheet), and
controlled polymerization methods which allow for large-
scale production.1,2 Polypeptides and polypeptide-containing
polymers with different functional side chains (e.g., oligosac-
charides,3,4 poly/oligo-ethylene-glycols,5,6 guanidinium, and
imidazolium7–9) and diverse architectures (e.g., block copoly-
mers,10,11 multiarm star polymers,12,13 and molecular bottle
brushes14,15) have been prepared for versatile uses, includ-
ing tissue-engineering scaffolds,16 drug-delivery carriers,17
cell-penetrating agents,7 modifiers for nanoparticles, and
elastomers.18–21 Research efforts that focus on the synthesis
of polypeptides with clickable side chains have gained signif-
icant attention due to the high grafting efficiency, specificity,
mild reaction conditions, and tolerance of functional
groups.22–24 For example, Hammond and co-workers
reported the synthesis of poly(c-propargyl-L-glutamate) and
side-chain grafting of azido-terminated polyethylene glycols
with high grafting efficiency.25 Tang and Zhang reported the
quantitative synthesis of mannose-polypeptide conjugates
using poly(c-azidopropyl-L-glutamate).26
L
L
L
covalent modification of polymers using poly(ethylene glycol)
(PEG), namely peglation, has achieved interesting properties,
such as nontoxicity and thermoresponsive property.36,37
Thus, thermoresponsive polypeptides have been developed
by incorporation of short PEG segments. For example, Li and
co-workers reported the LCST-type phase transition
Additional Supporting Information may be found in the online version of this article.
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behaviors of peglated poly(L-glutamates) or poly(L-cysteine)
prepared from ring-opening polymerization of peglated N-
carboxyanhydrides (NCAs).5,32 Our recent work has demon-
strated that helical polypeptides bearing 1-butylimidazolium
groups and I– or BF24 counter anions exhibited UCST-type
transitions in water.9 Nevertheless, the structure–property
relationship between thermoresponsive polypeptides and
their solution phase transition behaviors still remains
unclear.
min21. Narrowly distributed polystyrene standards in the
molecular weight range of 0.5–7.5 3 107 g mol21 (PSS,
Mainz, Germany) were utilized for calibration. Polymer solu-
tions for the GPC test with a concentration of 5 mg mL21 in
DMF/0.01M LiBr were prepared by directly mixing and shak-
ing at room temperature. FTIR spectra were recorded on a
Thermo Scientific Nicolet 6700 FTIR spectrometer equipped
with an attenuated total reflection (ATR) sample holder.
Solid samples were placed on the diamond crystal window
and pressed with a metal probe. Spectral measurements
were carried out in the transmittance mode (scan
range 5 4000–600 cm21, resolution 5 2 cm21, number of
scans 5 2, T 5 25 8C). Differential scanning calorimetry (DSC)
experiments were carried out on a TA DSC Q100 calorimeter
with a programmed heating and cooling procedure under
nitrogen at a heating/cooling rate of 10 8C min21 in the
range from 260 to 150 8C. The temperature and heat flow
were calibrated with benzoic acid and indium. The samples
were encapsulated in hermetically sealed aluminum pans
with a typical sample weight of ꢀ4 mg. The crystalline and
liquid crystalline textures of the samples were studied under
In this contribution, we report the synthesis and thermores-
ponsive properties of a-helical polypeptides bearing oligo-
ethylene-glycol (OEG) groups or 1-butylimidazolium salts.
These polymers were derivatized from poly(c24-(3-chloro-
propoxycarbonyl)benzyl-L-glutamate) by two different post-
polymerization reactions. The polymers have been
characterized by a combination of spectroscopic and chro-
matographic techniques (e.g., NMR, FTIR, and GPC). Their
thermoresponsive phase transition behaviors were highly
related to the polarity of the solvents, the types of the pend-
ant groups and counter-anions, the length of OEG groups
and backbones, and polymeric concentrations.
a
polarized optical microscope (POM, Leica DM-LM-P)
coupled with a Mettler–Toledo hot stage (FP82HT). The films
for POM test were prepared by drop casting from polymer/
DMF solutions onto glass slides and dried at room tempera-
ture inside a drybox. One-dimensional wide-angle X-ray scat-
tering (1D WAXS) measurements were performed on a
Bruker D8 Advance diffractometer equipped with an
SSD160’celerator detector in the reflection mode. The pow-
der samples were placed in tinfoil slots (size: 12 mm 3
8 mm) with a typical sample weight of 70–80 mg. Ultravio-
let–visible (UV–vis) spectra were measured using a Agilent
Cary 100 spectrometer. The polymer aqueous solutions were
prepared by directly mixing and stirring at temperatures
below respective LCST-type transition temperatures or above
respective UCST-type transition temperature, and then placed
in a quartz cell with a path length of 1.0 cm. The transmit-
tances of solutions were collected at the wavelength of
600 nm. DI H2O or methanol at 25 8C was set to be 100% of
transmittance. The solutions were heated from low tempera-
ture to high temperature for LCST-type transitions and
cooled from high temperatures to low temperatures for
UCST-type transitions with initial stabilization of 20 min. The
solution phase transition temperature (Tpt) was determined
at 50% of transmittance in the heating cycle for LCST-type
transition or in the cooling cycle for UCST-type transition.
For ionic strength dependent studies, the salt concentration
was adjusted by the addition of NaX (X 5Cl or I). Circular
dichroism (CD) measurements were carried out on an AVIV
410 CD spectrometer (Biomedical, Lakewood, NJ). The poly-
mer aqueous solutions were prepared at concentrations of
1 mg mL21 by directly mixing and stirring at room tempera-
ture for the samples without phase transitions or at the tem-
EXPERIMENTAL
Materials
Anhydrous tetrahydrofuran (THF), hexane, n-butylamine and
triethylamine were dried over calcium hydride and distilled.
Anhydrous N,N-dimethylformamide (DMF) was dried over
molecular sieves before use. 3-Chloro-1-propanol (99%), p-
toluic acid (98%), N-bromosuccinimide (98%), propargyl bro-
mide (99%), 1-butylimidazole (98%), N,N,N0,N0-tetramethyl-
guanidine (TMG, 98%), NaI (98%), and triphosgene (99.5%)
were purchased from Energy Chemical. Sodium azide (99.5%)
was purchased from Sigma-Aldrich. L-Glutamic acid (L-Glu,
99%), benzoyl peroxide (BPO, 99%), and other chemicals
were purchased from Aladdin and used as received. Deionized
water (DI H2O) was obtained from Aquapro AR1–100L-P11
water-purification-system (Ever Young Enterprises Develop-
ment, P. R. China). -Glutamic acid copper salt and propargyl
L
terminated oligo-ethylene-glycols (Pr-OEGm, m 5 2 and 3)
were synthesized by reported procedures.33,38,39
Instrumentation
1H and 13C NMR spectra were recorded on a BRUKER
ARX400 MHz spectrometer at room temperature. Chemical
shifts (d) were reported in the units of ppm and referenced
to the protio impurities and solvent 13C resonances in deu-
terated solvents. The polymer solution with concentrations
of ꢀ15 mg mL21 for H NMR test and ꢀ44 mg mL21 for 13C
1
NMR test were prepared by directly mixing and shaking at
room temperature. Gel permeation chromatography (GPC)
measurements were performed on
a PL-GPC120 setup
equipped with a column set consisting of two PL gel 5 lm
MIXED-D columns (7.5 3 300 mm, effective molecular
weight range of 0.2–400.0 kg mol21) and PL-RI differential
refractive index (DRI) detector. DMF containing 0.01M LiBr
was used as the eluent at 80 8C at a flow rate of 1.0 mL
peratures
below
respective
LCST-type
transition
temperatures (TLs) or above UCST-type transition tempera-
tures (TUs). Then, the above solutions (1 mg mL21) were
diluted to 0.05 mg mL21 for CD measurements (note:
PBLG40-OEG3, PBLG40-ImCl, and PBLG40-ImI were readily
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dissolved in DI H2O at 0.05 mg mL21 at 25 8C). The solution
was placed in a quartz cell with a path length of 0.2 cm. CD
data were collected with the high tension voltage (i.e., the
voltage applied to the photomultiplier) less than 600 V. Two
scans were conducted and averaged between 185–250 nm
with a resolution of 0.5 nm. The data were processed by
subtracting the solvent (i.e., DI H2O) background and
smoothing with FFT-filter method with points of window of
8. The CD spectra were reported in mean residue ellipticity
(MRE) (unit: degꢁcm2 dmol21) which was calculated by the
equation [h]k 5 MRW 3 hk/10 3 d 3 c,40 where MRW is the
mean residue weight (MRW 5 the molecular weight of poly-
peptide repeating unit), hk is the observed ellipticity (mdeg)
at the wavelength k (i.e., 222 nm), d is the path length (mm),
and c is the concentration (mg mL21). The fractional helicity
(fH) of the polypeptides was calculated using the equation
fH 5 (2[h]222 1 3,000)/39,000 to allow for a quantitative
comparison of the relative helical content, where [h]222 is
the mean residue ellipticity at 222 nm.41
copper salt (2.0 g, 4.08 mmol), DMF (7.6 mL), and DI H2O
(1.3 mL). The mixture became dark blue after addition. After
all solids were dissolved (ꢀ2 h), additional DMF (5.9 mL)
was added, followed by addition of 3 (5.0 g, 17.2 mmol) in
one portion. The reaction solution became darker and was
stirred at 40 8C for 48 h. Then, acetone (200 mL) was added
to the mixture and stirred until a fine precipitate was
obtained (ꢀ1 h). The violet solid was collected by filtration,
followed by addition of freshly prepared EDTA (3.6 g)/
sodium bicarbonate (2.1 g) aqueous solution (30 mL), and
further stirring for 24 h. Filtration, washing with DI H2O,
and drying under vacuum afforded a white solid (4, 3.1 g,
53% yield). 1H NMR (DMSO-d, d, ppm): 7.96 (d, 1H,
ACOArH), 7.48 (d, 1H, ACH2ArH), 5.17 (s, 2H, ACH2Ar), 4.57
(t, 1H, ACHNH2), 4.37 (t, 2H, ACH2OCOA), 3.78 (t, 2H,
ACH2Cl), 2.16 (m, 2H, ACH2CH2CH2Cl), 1.96 (m, 2H,
ACH2CHNH2). FTIR (neat, cm21): 2967, 1710, 1573, 1280,
1170, 756.
Synthesis of c-4-(3-Chloropropoxycarbonyl)
benzyl-L-glutamic Acid Based N-Carboxyanhydride (5)
4 (1.0 g, 2.8 mmol), triphosgene (0.42 g, 1.4 mmol), and
anhydrous THF (15 mL) were mixed in a 50 mL round-
bottom flask. The mixture was stirred at 50 8C for ꢀ2 h until
all solid were dissolved. Then, the reaction was stirred at
room temperature for 15 h. The solvent was removed under
vacuum. The product was recrystallized from ethyl acetate/
hexane 3 times to afford a white solid (5, 0.81 g, 76% yield).
1H NMR (CDCl3, d, ppm): 8.04 (d, 1H, ACOArH), 7.41 (d, 1H,
ACH2ArH), 6.30 (s, 1H, ANHA), 5.19 (s, 2H, ACH2Ar), 4.48
(t, 2H, ClCH2CH2CH2A), 4.39 (t, 1H, ACHNHA), 3.69 (t, 2H,
ClCH2CH2CH2A), 2.64 (t, 2H, AOCOCH2A), 2.31(m, 2H,
ClCH2CH2CH2A), 2.24 (m, 2H, AOCOCH2CH2A).13C NMR
(100 MHz, CDCl3): d 172.17, 169.44 (2C), 166.04, 151.95,
140.50, 129.97, 127.91, 66.22, 61.93, 56.84, 41.30, 31.72,
29.61, 26.89. FTIR (neat, cm21): 3303, 2964, 1857, 1788,
1719, 1655, 1615, 1547, 1111, 756.
Synthesis of 4-Methylbenzoic Acid 3-Chloropropyl Ester
(2)
p-Toluic acid (20.0 g, 0.147 mol) and thionyl chloride
(100 mL) were mixed in a 250 mL round-bottom flask. The
mixture was heated slowly to ꢀ80 8C and stirred at that
temperature for 3 h. Excess thionyl chloride was removed at
50 8C under vacuum. The residue was dissolved in anhy-
drous THF (40 mL) and transferred to a constant pressure
drop funnel. In an ice-water bath, the above THF solution
was added dropwise to a 250 mL round-bottom flask con-
taining an anhydrous THF solution (40 mL) of 3-chloro-1-
propanol (22 mL, 0.263 mol) and triethylamine (40 mL,
0.287 mol). The reaction was stirred at room temperature
for 24 h. The precipitate (i.e., triethylamine hydrochloride)
was removed by filtration and the solvent was removed at
40 8C under vacuum. The residue was dissolved in dichloro-
methane (50 mL), followed by washing with 1N HCl solution
(50 mL 3 2), K2CO3 aqueous solution (50 mL 3 2), and
brine (50 mL 3 2). Drying over anhydrous sodium sulfate,
filtration, and evaporation afforded a light yellow liquid. (2,
28.3 g, 91% yield). 1H NMR (CDCl3, d, ppm): 7.91 (d, 1H,
ACOArH), 7.23 (d, 1H, CH3ArH), 4.46 (t, 2H, AOCH2A), 3.69
(t, 2H, ACH2CH2CH2A), 2.43 (s, 3H, ACH3), 2.24 (m, 2H,
ACH2CH2CH2A). FTIR (neat, cm21): 2964, 1770, 1735, 1504,
1270, 1142, 825, 688.
Synthesis of Poly(c-4-(3-chloropropoxycarbonyl)
benzyl-L-glutamate) (6)
A representative ring-opening polymerization of 5 is as fol-
lows. 5 (1.0 g, 2.6 mmol) was dissolved in anhydrous DMF
(10 mL) in a vial under nitrogen, followed by addition of n-
butylamine/DMF stock solution (CI 5 0.1 mol L21, 520 mL,
52.0 mmol) with a syringe. The polymerization solution was
stirred at room temperature for 48 h. Then, it was slowly
added to a tenfold cold methanol to precipitate the product.
Filtration and drying afforded the production as a white solid
Synthesis of c-4-(3-Chloropropoxycarbonyl)
benzyl-L-glutamate (4)
1
(6, 0.75 g, 85% yield). H NMR (CDCl3, d, ppm): 7.88 (d, 1H,
2 (5.0 g, 23.6 mmol), N-bromosuccinimide (NBS, 5.0 g, 28.1
mmol), BPO (0.23 g, 0.95 mmol), and carbon tetrachloride
(50 mL) were mixed in a 100 mL round-bottom flask. The
mixture was stirred at 70 8C for 90 min and at 80 8C for
another 90 min. Filtration and evaporation afforded the
product as a yellow viscous liquid (3, 6.5 g, 95% yield).
ACOArH), 7.25 (d, 1H, ACH2ArH), 5.00 (m, 2H, ACH2Ar), 4.38
(t, 2H, ClCH2CH2CH2A), 4.05 (t, 1H, ACOCHNHA), 3.62 (t, 2H,
ClCH2CH2CH2A), 2.70 (m, 2H, ACHCH2CH2COOA), 2.40 (t, 2H,
ACHCH2CH2COOA), 2.15 (m, 2H, ClCH2CH2CH2A). 13C NMR
(100 MHz, CDCl3): d 171.25 (2C), 166.08, 141.89, 130.03,
127.68, 65.78 (2C), 61.89, 40.99, 31.21, 30.58 (2C). GPC:
Mn 5 19700, PDI 5 1.12, DPGPC 5 58; Mn 5 13600, PDI 5 1.25,
DPGPC 5 40; Mn 5 8800, PDI 5 1.14, DPGPC 5 26. FTIR (neat,
cm21): 3286, 2962, 1716, 1651, 1615, 1546, 1106, 754.
In a 500 mL round-bottom flask, N,N,N0,N0-tetramethylguani-
dine (2.1 mL, 1.67 mmol) was added slowly to a stirred mix-
ture of L-glutamic acid (1.2 g, 8.2 mmol), L-glutamic acid
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Synthesis of Poly(c-4-(3-azidopropoxycarbonyl)
Synthesis of Poly(c-benzyl-L-glutamate)-N-
benzyl-L-glutamate) (7)
butylimidazolium-chloride Conjugate (10, PBLG-ImCl)
A representative postpolymerization is as follows. 6 (40.0 mg,
0.12 mmol of chlorine) was dissolved in DMF (1.0 mL) under
nitrogen, followed by addition of acetonitrile (1.0 mL) solution
of NaI (54 mg, 0.36 mmol) and 1-butylimidazole (74.5 mg,
0.60 mmol). The mixture was stirred at 80 8C for 48 h. Then,
brine (4.0 mL) was added to the reaction solution at room
temperature. The solution was then stirred at room tempera-
ture for 3 h to promote ion exchange. The product was purified
by dialysis against NaCl aqueous solution (0.31 mol/L) in a
dialysis bag with a cutoff molecular weight of 3000 g mol21
for 24 h and against DI water for 48 h. Removing the solvent
under vacuum afforded a glassy solid (41.4 mg, 76% yield). 1H
NMR (DMSO-d6, d, ppm): 9.27 (s, 1H, ANCHNA), 7.88 (d, 1H,
ACOArH), 7.80A7.87 (m, 2H, ANCHCHNA), 7.47 (d, 1H,
ACH2ArH), 5.15 (s, 2H, ArCH2OA), 4.32A4.37 (m, 4H,
AOCH2CH2CH2NA), 4.11 (m, 3H, ANCH2CH2A and
ACOCHNHA), 2.31 (m, 2H, AOCH2CH2CH2NA), 1.86-2.01 (m,
4H, ACOCH2CH2A), 1.71 (m, 2H, ACH2CH2CH3), 1.24 (m, 2H,
ACH2CH2CH3), 0.88 (t, 3H, ACH2CH2CH3). 13C NMR of PBLG-
ImCl (100 MHz, DMSO-d): d 171.31 (2C), 166.13, 148.46 (2C),
130.08, 128.15, 126.77, 122.87 (2C), 62.51 (3C), 48.52, 47.51,
31.76, 29.01 (2C), 19.28, 14.38. GPC of PBLG-ImCl:
Mn 5 27100, PDI 5 1.12, DPGPC 5 58; Mn 5 18700, PDI 5 1.25,
DPGPC 5 40; Mn 5 12100, PDI 5 1.14, DPGPC 5 26. FTIR (neat,
cm21): 3294, 2960, 1716, 1653, 1614, 1549, 1115, 756.
6 (0.2 g, 0.59 mmol of chlorogroups), sodium azide (0.38 g, 5.8
mmol), and DMF (6 mL) were mixed in a vial, followed by stir-
ring at 40 8C for 48 h. The suspended inorganic salts were
removed by centrifugation. The DMF solution was slowly added
to a 10-fold NaCl aqueous solution (2 mol L21) to precipitate
the production. Filtration and drying under vacuum afforded
the product as a white solid (7, 0.15 g, 75% yield). 1H NMR
(CDCl3, d, ppm): 7.88 (d, 1H, ACOArH), 7.26 (d, 1H, ACH2ArH),
5.00 (m, 2H, ACH2Ar), 4.32 (t, 2H, N3CH2CH2CH2A), 4.05 (t,
1H, ACOCHNHA), 3.40 (t, 2H, N3CH2CH2CH2A), 2.72 and 2.20
(m, 2H, ACHCH2CH2COOA), 2.41 (t, 2H, ACHCH2CH2COOA),
1.98 (m, 2H, N3CH2CH2CH2A). 13C NMR (100 MHz, CDCl3): d
171.31 (2C), 165.91, 141.87, 129.98, 127.63, 65.66 (2C), 61.03,
48.12, 30.62 (2C), 28.31. GPC: Mn 5 18800, PDI 5 1.12,
DPGPC 5 55; Mn 5 12900, PDI 5 1.27, DPGPC 5 38; Mn 5 8000,
PDI 5 1.15, DPGPC 5 23. FTIR (neat, cm21): 3286, 2954, 2096,
1716, 1653, 1614, 1546, 1100, 754.
Synthesis of Poly(c-benzyl-L-glutamate)-oligo-ethylene-
glycol conjugates [8 (PBLG-OEG2) and 9 (PBLG-OEG3)]
A representative copper-mediated [2 1 3] alkyne-azide 1,3-
dipolar cycloaddition is as follows. 7 (25 mg, 0.074 mmol of
azido groups), Pr-OEG2 (21 mg, 0.133 mmol), CuBr (10.5 mg,
0.069 mmol), and PMDETA (15 mL, 0.062 mmol) were mixed
in DMF (4 mL) under nitrogen. The mixture was stirred at
room temperature for 24 h. The product was purified by dialy-
sis against distilled water (pH 5 5–6) in a dialysis bag with a
cutoff molecular weight of 3000 g mol21 for 3 days. Removing
the solvent under vacuum afforded a glassy solid. (PBLG-
Synthesis of Poly(c-benzyl-L-glutamate)-N-
butylimidazolium-iodide Conjugate (11, PBLG-ImI)
PBLG-ImCl (20 mg, 0.043 mmol of imidazolium) was dissolved
in DI H2O (5 mL) and mixed with NaI (68 mg, 0.45 mmol). The
reaction was stirred at room temperature for 5 min. The prod-
uct was precipitated immediately and collected by centrifuga-
tion. The product was repeated stirring in the aqueous
solution (5 mL) of NaI (68 mg, 0.45 mmol) to further perform
the ion-exchange reaction. Then, cold DI H2O was used to
wash the product to remove any salt residue. Removing the
solvent under vacuum afforded a glassy solid (21.4 mg, 90%
yield). 1H NMR (DMSO-d6, d, ppm): 9.23 (s, 1H, ANCHNA),
7.88 (d, 1H, ACOArH), 7.80A7.87 (m, 2H, ANCHCHNA), 7.47
(d, 1H, ACH2ArH), 5.15 (s, 2H, ArCH2OA), 4.32–4.37 (m, 4H,
AOCH2CH2CH2NA), 4.11 (m, 3H, ANCH2CH2A and
ACOCHNHA), 2.31 (m, 2H, AOCH2CH2CH2NA), 1.86–2.01 (m,
4H, ACOCH2CH2A), 1.71 (m, 2H, ACH2CH2CH3), 1.24 (m, 2H,
ACH2CH2CH3), 0.88 (t, 3H, ACH2CH2CH3). 13C NMR of PBLG-
ImI (100 MHz, DMSO-d): d 171.28 (2C), 166.15, 148.44 (2C),
129.99, 128.17, 126.78, 122.86 (2C), 62.51 (3C), 48.53, 47.51,
31.78, 29.03 (2C), 19.29, 14.40. GPC of PBLG-ImI: Mn 5 32400,
PDI 5 1.12, DPGPC 5 58; Mn 5 22300, PDI 5 1.25, DPGPC 5 40;
Mn 5 14500, PDI 5 1.14, DPGPC 5 26. FTIR (neat, cm21): 3292,
2960, 1714, 1651, 1614, 1545, 1107, 754.
1
OEG2, 28.7 mg, 78% yield). H NMR (CDCl3, d, ppm): 7.87 (d,
1H, ACOArH), 7.67 (s, 1H, ANCH@CA), 7.26 (d, 1H,
ACH2ArH), 5.10 (m, 2H, ACH2Ar), 4.62 (s, 2H, ACCH2OCH2A),
4.48 (t, 2H, ACOOCH2CH2A), 4.27 (t, 2H, ACH2CH2NA), 4.07
(t, 1H, ACOCHNHA), 3.63A3.51 (m, 8H, ACH2CH2OA), 3.33
(s, 3H, ACH2OCH3), 2.74 (m, 2H, ACHCH2CH2COOA), 2.49 (t,
2H, ACHCH2CH2COOA), 2.33 (m, 2H, ACOOCH2CH2A). 13C
NMR of PBLGAOEG2 (100 MHz, CDCl3): d 171.22 (2C), 166.02,
145.11, 141.25, 129.95, 127.52, 126.09, 71.77 (2C), 70.62
(2C), 69.97, 65.51 (2C), 61.88, 58.35, 47.41, 29.92 (3C). GPC of
PBLG-OEG2: Mn 5 29200, PDI 5 1.12, DPGPC 5 58; Mn 5 20200,
PDI 5 1.25, DPGPC 5 40; Mn 5 13100, PDI 5 1.14, DPGPC 5 26.
FTIR (neat, cm21) of PBLG-OEG2: 3286, 2923, 1716, 1651,
1614, 1546, 1103, 754. 1H NMR of PBLG-OEG3 (CDCl3, d,
ppm): 7.87 (d, 1H, ACOArH), 7.68 (s, 1H, ANCH@CA), 7.26 (d,
1H, ACH2ArH), 5.11 (m, 2H, ACH2Ar), 4.63 (s, 2H,
ACCH2OCH2A), 4.49 (t, 2H, ACOOCH2CH2A), 4.27 (t, 2H,
ACH2CH2NA), 4.07 (t, 1H, ACOCHNHA), 3.62A3.52 (m, 8H,
ACH2CH2OA), 3.33 (s, 3H, ACH2OCH3), 2.73 (m, 2H,
ACHCH2CH2COOA), 2.48 (t, 2H, ACHCH2CH2COOA), 2.32 (m,
2H, ACOOCH2CH2A). 13C NMR of PBLG-OEG3 (100 MHz,
CDCl3): d 171.23 (2C), 166.04, 145.14, 141.22, 130.01, 127.51,
126.12, 71.83 (2C), 70.65 (4C), 69.99, 65.49 (2C), 61.90,
58.28, 47.39, 29.93 (3C). GPC of PBLG-OEG3: Mn 5 31800,
PDI 5 1.12, DPGPC 5 58; Mn 5 21900, PDI 5 1.25, DPGPC 5 40;
Mn 5 14200, PDI 5 1.14, DPGPC 5 26. FTIR (neat, cm21) of
PBLG-OEG3: 3290, 2917, 1716, 1653, 1614, 1549, 1099, 754.
RESULTS AND DISCUSSION
Synthesis and Characterization
Two types of thermoresponsive polypeptides bearing oligo-
ethylene-glycol (OEG) groups or 1-butylimidazolium salts
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SCHEME 1 The synthetic route of poly(c24-(3-chloropropoxycarbonyl)benzyl-L- glutamate) (6).
were designed to achieve water-soluble and a-helical poly-
peptides with lower critical solution transition temperatures
(LCSTs) or upper critical solution temperatures (UCSTs),
respectively. The target polypeptides were derivatives of
poly(c-benzyl-L-glutamate) (PBLG) which adopted a-helical
conformation in many organic solvents and processed liquid
crystalline phases both in the solid-state and solutions.42,43
The presence of benzyl groups not only facilitated the crys-
tallization of N-carboxylanhydride monomers, but also stabi-
lized the a-helical conformation by hydrophobic interactions.
In order to systematically investigate the structure–property
relationship, polypeptides with various main-chain lengths,
OEG lengths, and counter-anions were prepared. The specific
synthetic route was discussed as follows.
propanol in the presence of triethylamine to afford the corre-
sponding esters (2, Scheme 1). Bromination of 2 with N-bro-
mosuccinimide in carbon tetrachloride afforded the
brominized esters (3) by following the previous literature.44
Monoesterification of L-Glutamic acid copper salt with 3 in
DMF and water afforded 4 which were purified by stirring in
EDTA/sodium bicarbonate solution and repeatedly washing
with water. Cyclization of 4 with triphosgene afforded the
crystalline N-carboxylanhydride monomers (5) which were
purified by recrystallization in ethyl acetate/hexane at 220
8C. The molecular structures of 2, 4, and 5 were verified by
NMR (Supporting Information Figs. S1 and Fig. 1) and FTIR
spectroscopy (Supporting Information Figs. S2 and Fig. 2),
respectively.
p-Tolulic acid was reacted with thionyl chloride to afford the
p-toluoyl chloride followed by reacting with 3-chloro-1-
Ring-opening polymerization of 5 was conducted at room
temperature in DMF using n-butylamine as the initiators.
FIGURE 1 (a) 1H and (b) 13C NMR spectra of c24-(3-chloropropoxycarbonyl)benzyl-L-glutamic acid based N-carboxyanhydride (5)
in CDCl3.
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FIGURE 2 FTIR spectra of 5, 6, 7, PBLG-OEG2, PBLG-OEG3, PBLG-ImCl, and PBLG-ImI in the solid-state.
The polymerization was quenched by precipitation with cold
methanol until conversion was above 95%, which was moni-
tored by FTIR spectroscopy. Poly(c-4-(3-chloropropoxycar-
bonyl)-benzyl-L-glutamate)s (6) with different molecular
weights and narrow molecular weight distribution (PDI)
were readily obtained by adjusting the initial monomer to
initiator ratios. The molecular structure and molecular
weight of the resulting polymer (6), were confirmed by 1H
NMR [Fig. 3(a)], FTIR (Fig. 2) and GPC (Supporting Informa-
1
tion Fig. S3), respectively. The chemical shifts in the H NMR
spectrum were consistent with the structure of 6. In the
FTIR spectrum,
6 (Mn 5 19700, PDI 5 1.12, DPGPC 5 58)
exhibited characteristic peaks at 3286, 1716, 1651, 1546,
and 1615 cm21, corresponding to vNAH, vCAO, amide I, amide
II, and vphenyl, respectively. The sample adopted a-helical con-
formation in the solid-state based on the amide I and amide
II bands.45–47 Additionally,
6 (Mn 5 19,700, PDI 5 1.12,
DPGPC 5 58) showed good thermal stability with a decompo-
sition temperature at 274 8C as revealed by TGA [Supporting
Information Fig. S4(a)]. 6 with various main-chain lengths
(DPGPC 5 26, 40, and 58) showed glass transition tempera-
tures at ꢀ15 8C as revealed by DSC [Supporting Information
Fig. S4(b)]. POM result [Supporting Information Fig. S4(c)]
suggested that 6 possessed nematic liquid crystalline phase
in the temperature range of 25–274 8C. No liquid crystalline
phase to isotropic phase transition was observed due to
their rigid helical backbones. In the WAXS diffractograms
[Supporting Information Fig. S4(d)], 6 exhibited a principal
diffraction at q 5 3.93 nm21 (d 5 1.60 nm) in the tempera-
ture range of 20–200 8C together with higher order reflec-
tions at 31/2q and 41/2q, suggesting hexagonally packed
cylinder structures. The liquid crystalline textures and
molecular packing of 6 are similar to that of PBLG.47,48
FIGURE 3 1H NMR spectra of (a) 6, (b) 7, (c) PBLG-OEG3 in
CDCl3 and (d) PBLG-ImCl, (e) PBLG-ImI in DMSO-d6.
Poly(c24-(3-azidopropoxycarbonyl)benzyl-L-glutamate) (7)
bearing azido groups was prepared by treatment 6 with
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SCHEME 2 The synthetic route of polypeptides bearing oligo-ethylene-glycol or 1-butylimidazolium salt.
1
NaN3 in DMF at 40 8C for 2 days (Scheme 2). H NMR analy-
sis of the reaction mixture reveals a quantitative conversion
of chlorogroups into azido groups [Fig. 3(b)]. The presence
Pr-OEGm (m 5 2, 3, and 7). Nevertheless, polypeptides with
OEG7 pendants were water soluble and showed no solution
phase transition in water below 100 8C. Thus, those samples
were removed from this work. Additionally, the reagents and
conditions of the copper-mediated [2 1 3] alkyne-azide 1,3-
dipolar cycloaddition were followed by previous litera-
tures.14,26 The molecular structures of polymers bearing OEG
groups were verified by 1H NMR [Fig. 3(c) and Supporting
Information Figure S6], GPC (Supporting Information
Fig. S5), and FTIR (Fig. 2). For examples, in the 1H NMR
spectra [Fig. 3(c)], the appearance of chemical resonance
(Hj) at 7.68 ppm suggests the formation of triazole groups
after the copper-mediated [2 1 3] alkyne-azide 1,3-dipolar
of azide group is evident in FTIR spectrum of
7
(mN@N@N 5 2096 cm21, Fig. 2). Additionally, 6 and 7 showed
similar GPC curves (Supporting Information Fig. S5), suggest-
ing that no polymeric degradation occurred during the reac-
tion with NaN3. Poly(c-benzyl-L-glutamate)-oligo-ethylene-
glycol conjugates (i.e., PBLG-OEG2 and PBLG-OEG3, Table 1)
were prepared by a copper-mediated [2 1 3] alkyne-azide
1,3-dipolar cycloaddition between 7, and propargyl termi-
nated oligo-ethylene-glycols (Pr-OEGm, m 5 2 and 3) (Scheme
2). Initially, we have designed and explored three types of
TABLE 1 Molecular Parameters of Polypeptides Bearing Oligo-ethylene-glycol or 1-Butylimidazolium Salt
h
Tpt (8C)
me
X–f
Tg (8C)
TL
TU
a
b
c
d
g
Name
Mn
Mw/Mn
DPGPC
DPNMR
PBLG26-OEG2
PBLG40-OEG2
PBLG58-OEG2
PBLG26-OEG3
PBLG40-OEG3
PBLG58-OEG3
PBLG26-ImCl
PBLG40-ImCl
PBLG58-ImCl
PBLG26-ImI
13,100
20,200
29,200
14,200
21,900
31,800
12,100
18,700
27,100
14,500
22,300
32,400
1.14
1.25
1.12
1.14
1.25
1.12
1.14
1.25
1.12
1.14
1.25
1.12
26
40
58
26
40
58
26
40
58
26
40
58
20
33
48
20
33
48
20
33
48
20
33
48
2
2
2
3
3
3
–
–
–
–
–
–
–
8.4
–
–
–
5.2
–
–
–
23.2
22.2
24.0
28.6
15.7
34.6
35.9
39.3
38.4
37.8
–
–
–
41.5
–
–
24.1
–
–
19.1
–
Cl–
Cl–
Cl–
I–
I–
I–
–
–
–
–
–
–
–
–
–
66.8
67.6
69.2
PBLG40-ImI
PBLG58-ImI
a
e
Number-average molecular weight calculated from the degree of poly-
merization (DPGPC, which was determined by GPC) of the precursors
(i.e., 6) plus respective molecular weight of polypeptide repeating units.
DP of OEG groups.
Counter anions.
f
g
h
Glass transition temperatures determined by DSC.
Solution phase transition temperature in DI H2O (2 mg mL21 for
b
Molecular weight distribution of the precursors determined by GPC.
c
Degree of polymerization of the precursors calculated from the GPC
PBLG-OEG3 and 10 mg mL21 for PBLG-ImI): TL 5 LCST type-transition
temperatures; TU 5 UCST-type transition temperature.
results (Mn).
d
Degree of polymerization of the precursors calculated from the 1H
NMR results.
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TABLE 2 Representative Solubility of Polypeptides Bearing Oligo-ethylene-glycol or 1-Butylimidazolium Salt
Polymer
Chloroform
THF
Acetone
Ethanol
Methanol
H2O
DMSO
PBLG58-OEG2
PBLG58-OEG3
PBLG58-ImCl
PBLG58-ImI
S
S
I
U*
U*
U*
U
U
U
S
I
S
S
S
S
S
I
S
I
L*
S
U
S
I
I
I
U
U
S 5 soluble; I 5 insoluble; U 5 UCST-type phase transition; L 5 LCST-type phase transition (concentration 5 10 mg mL21). Note: U* or L* mean that
the polymers were partially soluble in the solvents and the solutions exhibited UCST- or LCST-type phase transitions.
cycloaddition. In the GPC chromatographs (Supporting Infor-
mation Fig. S5), a noticeable peak shift can be observed by
tion Figs. S7 and S8) and decomposition temperature (Sup-
porting Information Fig. S9) as suggested by POM
(Supporting Information Fig. S10) and WAXS results (Sup-
porting Information Fig. S11), suggesting that the hexagonal
molecular packing of poly(c24-(3-chloropropoxycarbonyl)-
benzyl-L-glutamate) (6) was disrupted by conjugating with
flexible OEG groups or cationic 1-butylimidazolium groups.
For the polypeptides bearing the same OEG side-groups, the
Tg decreased as the main-chain length increased likely due
to the increased free volume in the samples with longer
backbone length (Supporting Information Fig. S8). For the
polypeptides samples with the same main-chain length, the
Tg also decreased as increasing the length of OEG groups
due to the increased side-chain flexibility. Polypeptides bear-
ing 1-butylimidazolium groups showed higher Tg than the
ones bearing OEG groups (Supporting Information Fig. S8).
The samples with I– counter-anions showed higher Tg than
the ones bearing Cl– counter-anions, suggesting increased
main-chain mobility when bearing I– counter-anions (Sup-
porting Information Fig. S8).
comparing 7 (DPGPC 5 58) with PBLG58-OEG2 and PBLG58
-
OEG3, suggesting an increase of molecular weight after graft-
ing OEG groups. In the FTIR spectra (Fig. 2), the increased
peaks intensity (compare to vC@O of ester) at 2923 and
1099 cm21 was resulted from the vibration of C–H (vCAH
)
and ether bond (vCAOAC), respectively. The absence of
mN@N@N in the FTIR spectra (Fig. 2) and the 1:2 integration
ratio of Hj and Hf in the 1H NMR spectrum [Fig. 3(c)] sug-
gested the successful grafting of OEG groups to the polypep-
tide backbones with nearly quantitative grafting density
(99%). FTIR analysis also indicated the a-helical conforma-
tion of the polymers bearing OEG side-chains in the solid-
state based on the amide I and amide II bands (Fig. 2).
Poly(c-benzyl-L-glutamate)-N-butylimidazolium-chloride con-
jugate (PBLG-ImCl, Table 1) was prepared via a postpolyme-
rization (i.e., nuleophilic substitution) between 6 and 1-
butylimidazole in DMF at 80 8C (Scheme 2). Sodium iodide
(NaI) was used to promote the reaction. The product was
purified by firstly dialyzing against NaCl aqueous solution,
and then dialyzing against DI water to remove excess NaCl.
Solubility and Solution Conformation
1
The solubility of the polypeptides bearing different OEG
groups or 1-butylimidazolium salts was tested in various sol-
vents (Table 2). Polypeptides with phase transition behaviors
in organic solvents have been rarely reported. It is interest-
ing to observe that polypeptide samples bearing OEG groups
showed a UCST-type transition in ethanol and methanol. The
samples with short OEG length (m 5 2) also showed UCST-
type transition in THF and acetone while the samples with
longer OEG length (m 5 3) showed typical LCST-type transi-
tion in DI water. For the polymers bearing 1-butylimidazolim
salts, they showed poor solubility in less polar solvents such
as toluene, chloroform and THF while they showed good sol-
ubility in highly polar solvent (i.e., DMSO). The types of
counter-anions played an important role to show solution
phase transition behaviors. For example, the samples with
Cl– counter-anions showed good solubility and no solution
phase transitions in ethanol, methanol, and DI water, never-
theless, the samples with I– counter-anions showed UCST-
type phase transitions in these solvents.
The chemical shifts in the H NMR spectrum [Fig. 3(d)] were
consistent with the structure of PBLG-ImCl. The grafting den-
sity was ꢀ90%, which was calculated based on the integra-
tion of 1H NMR resonances [Fig. 3(d)] of He and Hj. In the
FTIR spectrum (Fig. 2), the amide I band at 1653 cm21 sug-
gested the a-helical conformation of PBLG-ImCl in the solid-
state.
Poly(c-benzyl-L-glutamate)-N-butylimidazolium-iodide (PBLG-
ImI, Table 1) was synthesized by an ion-exchange reaction
from PBLG-ImCl. Specifically, PBLG-ImCl was stirred in NaI
aqueous solutions at room temperature and purified by
washing with NaI aqueous solutions and cold DI H2O. 1H
NMR [Fig. 3(e)] analysis confirmed the molecular structures
of PBLG-ImI as evidenced by the chemical shift of Hj of imi-
dazolium shifted from 9.27 ([Fig. 3(d)] to 9.23 ppm [Fig.
3(e)]. The FTIR spectra of PBLG-ImI showed the amide I
band at 1651 cm21, suggesting the a-helical conformation in
the solid-state.
The aqueous solution conformations of the polypeptides
bearing OEG groups or 1-butylimidazolium salts were char-
acterized by CD spectroscopy at 25 8C. All polypeptide sam-
ples adopted a-helical conformation in water, as verified by
While polypeptides bearing OEGs or 1-butylimidazolium salts
adopt a-helical conformation in the solid-state, they are
amorphous polymers in the temperature range between
respective glass transition temperature (Supporting Informa-
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FIGURE 4 CD spectra of (a) PBLG40-OEG3, (b) PBLG40-ImCl, and PBLG40-ImI in DI H2O (0.05 mg mL21).
the characteristic CD bands at 208 and 222 nm (Fig. 4). For
examples, PBLG40-OEG3 showed relatively low fractional hel-
icity (fH 5 66.1%) likely due to the dehydration of the OEG
side chains at 25 8C. PBLG40-ImCl and PBLG40-ImI bearing 1-
butylimidazolium salts showed fractional helicities of 85.3
and 91.5%, respectively. In other words, the resulting water-
soluble polypeptides may adopt heterogeneous solution con-
formations with 85.3–91.5% helices. The fH values was in
the sequence of I2 > Cl2, suggesting that counter-anions
with smaller ionic radius and higher charge density may fur-
ther increase the charge repulsions between adjacent imida-
zolium groups and subsequently destabilize the a-helical
conformation. This result was consistent with our previous
work.9
salts in various solutions were studied by variable-
temperature UV-vis spectroscopy (Fig. 5). In aqueous solu-
tions, PBLG-OEG3 with OEG groups (m 5 3) and different
main-chain lengths showed reversible LCST-type phase tran-
sition behaviors in the temperature range of 16.4–41.5 8C at
the concentration of 2 mg mL21 [Fig. 5(a,b)]. The mechanism
involves a balance between polymer–water interactions and
polymer–polymer interactions.37 Above LCST, the balance is
disrupted and polymer–polymer interactions are favoured as
compared to polymer–water interactions, resulting in solu-
tion phase separation. The hysteresis for PBLG-OEG3 samples
was less pronounced than other thermoresponsive polymers
with intra- and/or intermolecular H-bonds.49,50 The LCST-
type transition temperature (TL) was determined at the 50%
transmittance in the heating cycle. It decreased as the man-
chain length increased, suggesting that longer main-chain
length facilitated polymeric aggregations at the transition
temperatures. Additionally, TL decreased when PBLG-OEG3
Solution Phase Transition Behaviors
The thermoresponsive phase transition behaviors of the
polypeptides bearing OEG groups or 1-butylimidazolium
FIGURE 5 (a) Optical images of PBLG40-OEG3 aqueous solution at different temperatures. Variable-temperature UV–vis spectra of
(b) PBLG-OEG3/H2O (2 mg mL21) with various main-chain length, (c) PBLG26-OEG3, and (d) PBLG40-OEG3 at various concentrations.
(e) The LCST-type transition temperatures of PBLG26-OEG3 and PBLG40-OEG3 versus concentrations.
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FIGURE 6 (a) Optical images of PBLG40-ImI aqueous solution at different temperatures. Variable-temperature UV–vis spectra of (b)
PBLG-ImI/H2O (10 mg mL21) with various main-chain length, (c) PBLG26-ImI, and (d) PBLG40-ImI at various concentrations. (e) The
UCST-type transition temperatures of PBLG26-ImI and PBLG40-ImI versus concentrations.
concentration increased from 0.5 to 2.0 mg mL21 due to the
increased intermolecular aggregation [Fig. 5c–e)]. In compar-
ison, PBLG-ImI with 1-butylimidazolium groups and I–
counter-anions showed reversible UCST-type phase transition
behaviors in aqueous solutions [Fig. 6(a,b)]. The UCST-type
transition temperature (TU) was determined at the 50%
transmittance in the cooling cycle. The TUs were independ-
ent with the polymeric main-chain length and dramatically
FIGURE 7 (a) Optical images of PBLG58-OEG3/methanol solution at different temperatures. Variable-temperature UV–vis spectra of
(b) PBLG58-OEG2/methanol and PBLG58-OEG3/methanol (1 mg mL21), and (c) PBLG-OEG3 with various main-chain length and con-
centrations in methanol. (d) The UCST-type transition temperatures of PBLG-OEG3 versus main-chain length (i.e., DPGPC) and
concentrations.
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decreased at low concentration (e.g., 4 mg mL21), suggesting
a mechanism of the electrostatic interactions at the transi-
5 C. Chen, Z. Wang, Z. Li, Biomacromolecules 2011, 12, 2859–
2863.
tion temperatures [Fig. 6(b–e)].9,51,52 In methanol, PBLG58
-
6 X. Fu, Y. Shen, W. Fu, Z. Li, Macromolecules 2013, 46, 3753–
3760.
OEG2, and PBLG58-OEG3 with different OEG side-chains
showed reversible UCST-type phase transition behaviors at
40.2 and 33.1 8C, respectively, at the concentration of 1 mg
mL21 [Fig. 7(a,b)]. The mechanism of the phase transition
was likely due to the reversible hydrogen-bonding interac-
tions between OEG side-chains and methanol. The TUs of
PBLG-OEG3 solutions were increased as increasing the poly-
meric main-chain length or as the concentration increased
[Fig. 7(c,d)], suggesting that the mechanism of the phase
transitions originated from a reversible hydrogen-bonding
between OEG side-chains and methanol. Currently, we are
working on more thermoresponsive polypeptides with UCST-
type phase transition in organic solvents (e.g., methanol)
aiming to better understand their structure–property rela-
tionships and mechanisms.
7 H. Tang, L. Yin, K. H. Kim, J. Cheng, Chem. Sci. 2013, 4,
3839–3844.
8 Q. Hu, Y. Deng, Q. Yuan, Y. Ling, H. Tang, J. Polym. Sci. A:
Polym. Chem. 2014, 52, 149–153.
9 Y. Deng, Y. Xu, X. Wang, Q. Yuan, Y. Ling, H. Tang,
Macromol. Rapid Commun. 2015, 36, 453–458.
10 Q.-H. Zhou, J.-K. Zheng, Z. Shen, X.-H. Fan, X.-F. Chen, Q.-F.
Zhou, Macromolecules 2010, 43, 5637–5646.
11 F. Zhou, T. Ye, L. Shi, C. Xie, S. Chang, X. Fan, Z. Shen,
Macromolecules 2013, 46, 8253–8263.
12 A. Saꢀnchez-Ferrer, R. Mezzenga, Macromolecules 2009, 43,
1093–1100.
13 S. Junnila, N. Houbenov, A. Karatzas, N. Hadjichristidis, A.
Hirao, H. Iatrou, O. Ikkala, Macromolecules 2012, 45, 2850–2856.
14 H. Tang, Y. Li, S. H. Lahasky, S. S. Sheiko, D. Zhang,
Macromolecules 2011, 44, 1491–1499.
CONCLUSIONS
15 Y. Liu, P. Chen, Z. Li, Macromol. Rapid Commun. 2012, 33,
287–295.
We have demonstrated the synthesis of a new side-chain-
functionalized a-helical polypeptide, that is, poly(c24-(3-
chloropropoxycarbonyl)benzyl-L-glutamate) (6) from n-butyl-
amine initiated ring-opening polymerization (ROP) of c24-
(3-chloropropoxycarbonyl)benzyl-L-glutamic acid-based N-
carboxyanhydride (5). Two types of helical polypeptides
bearing oligo-ethylene-glycol (OEG) groups or 1-
butylimidazolium salts can be readily prepared from 6 via
different postpolymerizations, such as copper-mediated
[2 1 3] alkyne-azide 1,3-dipolar cycloaddition and nuleo-
philic substitution. Polymers bearing OEG (m 5 3) side-
chains showed reversible LCST-type phase transition behav-
iors in water and reversible UCST-type phase transition
behaviors in various organic solvents, such as methanol and
ethanol. The phase transition temperatures (Tpts) were
dependent on the main-chain length and polymeric concen-
tration. Polymers bearing 1-butylimidazolium and I– counter-
anions exhibited reversible UCST-type transitions in water,
methanol, and ethanol. The Tpts in water decreased as
decreasing the concentrations.
16 A. P. Nowak, V. Breedveld, L. Pakstis, B. Ozbas, D. J. Pine,
D. Pochan, T. J. Deming, Nature 2002, 417, 424–428.
17 E. P. Holowka, V. Z. Sun, D. T. Kamei, T. J. Deming, Nat.
Mater. 2007, 6, 52–57.
18 D. Neugebauer, Y. Zhang, T. Pakula, S. S. Sheiko, K.
Matyjaszewski, Macromolecules 2003, 36, 6746–6755.
19 H. Tang, D. Zhang, J. Polym. Sci. A: Polym. Chem. 2010, 48,
2340–2350.
20 H. Tang, Y. Ling, Y. Deng, D. Zhang, J. Polym. Sci. A:
Polym. Chem. 2014, 52, 1905–1915.
21 Y.-C. Lin, S.-W. Kuo, J. Polym. Sci. A: Polym. Chem. 2014,
52, 321–329.
22 C. R. Becer, R. Hoogenboom. U. S. Schubert, Angew. Chem.
Int. Ed. 2009, 48, 4900–4908.
23 M. A. Quadir, M. Martin, P. T. Hammond, Chem. Mater.
2013, 26, 461–476.
24 H. Lu, J. Wang, Z. Song, L. Yin, Y. Zhang, H. Tang, C. Tu, Y.
Lin, J. Cheng, Chem. Commun. 2014, 50, 139–155.
25 A. C. Engler, H. I. Lee, P. T. Hammond, Angew. Chem., Int.
Ed. 2009, 48, 9334–9338.
26 H. Tang, D. Zhang, Biomacromolecules 2010, 11, 1585–1592.
27 D. Roy, J. N. Cambre, B. S. Sumerlin, Prog. Polym. Sci.
2010, 35, 278–301.
ACKNOWLEDGMENTS
This work is supported by the National Natural Science Foun-
dation of China (Grant 21204075), Hunan Provincial Natural
Science Foundation of China (13JJ6042), Scientific Research
Fund of Hunan Provincial Education Department (13C908 and
14B175), and Xiangtan University start-up fund (12QDZ06).
28 V. Aseyev, H. Tenhu, F. Winnik, Adv. Polym. Sci. 2011, 242,
29–89.
29 C. He, X. Zhuang, Z. Tang, H. Tian, X. Chen, Adv. Healthcare
Mater. 2012, 1, 48–78.
30 M. Kissel, P. Peschke, V. Subr, K. Ulbrich, J. Schuhmacher,
J. Debus, E. Friedrich, PDA J. Pharm. Sci. Technol. 2001, 55,
191–201.
REFERENCES AND NOTES
31 C. Deng, J. Wu, R. Cheng, F. Meng, H.-A. Klok, Z. Zhong,
Prog. Polym. Sci. 2014, 39, 330–364.
1 T. J. Deming, Adv. Polym. Sci. 2006, 202, 1–18.
2 T. J. Deming, Prog. Polym. Sci. 2007, 32, 858–875.
32 X. Fu, Y. Ma, Y. Shen, W. Fu, Z. Li, Biomacromolecules
2014, 15, 1055–1061.
3 J. R. Kramer, T. J. Deming, J. Am. Chem. Soc. 2010, 132,
15068–15071.
33 Y. Wu, Y. Deng, Q. Yuan, Y. Ling, H. Tang, J. Appl. Polym.
Sci. 2014, 131, 41022.
4 J. R. Kramer, T. J. Deming, J. Am. Chem. Soc. 2012, 134,
4112–4115.
34 C. Xiao, Y. Cheng, Y. Zhang, J. Ding, C. He, X. Zhuang, X.
Chen, J. Polym. Sci. A: Polym. Chem. 2014, 52, 671–679.
WWW.MATERIALSVIEWS.COM
JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2015, 53, 2469–2480
2479

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
35 J. Yan, K. Liu, X. Zhang, W. Li, A. Zhang, J. Polym. Sci. A:
Polym. Chem. 2015, 53, 33–41.
44 D. Zhang, Y.-X. Liu, X.-H. Wan, Q.-F. Zhou, Macromolecules
1999, 32, 5183–5185.
36 G. Pasut, F. M. Veronese, Adv. Polym. Sci. 2006, 192, 95–
134.
45 R. D. B. Fraser, W. C. Price, Nature 1952, 170, 490–491.
46 G. Floudas, P. Papadopoulos, H. A. Klok, G. W. M. Vandermeulen,
J. Rodriguez-Hernandez, Macromolecules 2003, 36, 3673–3683.
37 J.-F. Lutz, J. Polym. Sci. A: Polym. Chem. 2008, 46, 3459–
3470.
47 P. Papadopoulos, G. Floudas, H. A. Klok, I. Schnell, T.
Pakula, Biomacromolecules 2004, 5, 81–91.
38 H. Yamamoto, T. Hayakawa, Int. J. Biolog. Macromol. 1982,
4, 116–120.
39 Y. Deng, Q. Hu, Q. Yuan, Y. Wu, Y. Ling, H. Tang,
Macromol. Rapid Commun. 2014, 35, 97–102.
48 H. Tang, D. Zhang, J. Polym. Sci. A: Polym. Chem. 2013, 51,
4489–4497.
40 S. M. Kelly, T. J. Jess, N. C. Price, Biochim. Biophys. Acta
2005, 1751, 119.
49 P. Kujawa, V. Aseyev, H. Tenhu, F. M. Winnik,
Macromolecules 2006, 39, 7686–7693.
41 J. A. Morrow, M. L. Segall, S. Lund-Katz, M. C. Phillips, M.
Knapp, B. Rupp, K. H. Weisgraber, Biochemistry 2000, 39,
11657–11666.
50 Z.-Y. Qiao, F.-S. Du, R. Zhang, D.-H. Liang, Z.-C. Li,
Macromolecules 2010, 43, 6485–6494.
51 H. Yoshimitsu, A. Kanazawa, S. Kanaoka, S. Aoshima,
Macromolecules 2012, 45, 9427–9434.
42 H. Block, Poly(c-benzyl-L-glutamate) and Other Glutamic
Acid Containing Polymers; Gordon and Breach Science
Publisher: New York, 1983.
52 E. Karjalainen, V. Aseyev, H. Tenhu, Macromolecules 2014,
47, 7581–7587.
43 I. Uematsu, Y. Uematsu, Adv. Polym. Sci. 1984, 59, 37–73.
2480
JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2015, 53, 2469–2480