Side chain impacts on pH- and thermo-responsiveness of tertiary amine functionalized polypeptides

Article abstract of DOI:10.1002/pola.27048

The systemic investigation of the structural impacts of side chains on the pH- and thermo-responsiveness of tertiary amine functionalized poly(l-glutamate)s (TA-PGs) was carried out. The TA-PGs polymers were effectively synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition click reaction of azido tertiary amines with poly(γ-propargyl-l-glutamate) (PPLG). Turbimetric measurements were performed to characterize the pH- and temperature-induced phase transition of TA-PGs in aqueous solution, which suggested a structural dependence of the properties on the N-substituted groups and the "linkers" between 1,2,3-triazole ring and the tertiary amine groups in the side chains. In detail, the pH responsive properties of TA-PGs were basically determined by the hydrophobicity of the N-substituted groups in the side chains and the pH transition point (pH_t) decreased as the increasing hydrophobicity of the N-substituted groups, while the temperature-responsiveness of TA-PGs were affected by either the N-substituted groups or the "linkers." TA-PGs with a moderate N-substituted amine group (e.g., DEA, PR, and PD) or a branched "linker" (e.g., iso-propylene and 2-methylpropylene group) were more likely to express the LCST-type phase transition tuned by pH variation. These structure-property relationships revealed in this study would help to develop the applications of TA-PGs in smart drug delivery systems. Copyright

Full text of DOI:10.1002/pola.27048

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ARTICLE
Side Chain Impacts on pH- and Thermo-Responsiveness of Tertiary
Amine Functionalized Polypeptides
Chunsheng Xiao,1 Yilong Cheng,1,2 Yu Zhang,1,2 Jianxun Ding,1 Chaoliang He,1 Xiuli Zhuang,1
Xuesi Chen1
1Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun
130022, People’s Republic of China
2University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
Correspondence to: X. Chen (E-mail: xschen@ciac.ac.cn)
Received 5 July 2013; accepted 2 December 2013; published online 22 December 2013
DOI: 10.1002/pola.27048
ABSTRACT: The systemic investigation of the structural impacts
of side chains on the pH- and thermo-responsiveness of tertiary
amine functionalized poly(L-glutamate)s (TA-PGs) was carried
out. The TA-PGs polymers were effectively synthesized by Cu(I)-
catalyzed azide-alkyne cycloaddition click reaction of azido terti-
ary amines with poly(c-propargyl-L-glutamate) (PPLG). Turbimet-
ric measurements were performed to characterize the pH- and
temperature-induced phase transition of TA-PGs in aqueous
solution, which suggested a structural dependence of the prop-
erties on the N-substituted groups and the “linkers” between
1,2,3-triazole ring and the tertiary amine groups in the side
chains. In detail, the pH responsive properties of TA-PGs were
basically determined by the hydrophobicity of the N-substituted
groups in the side chains and the pH transition point (pHt)
decreased as the increasing hydrophobicity of the N-substituted
groups, while the temperature-responsiveness of TA-PGs were
affected by either the N-substituted groups or the “linkers.” TA-
PGs with a moderate N-substituted amine group (e.g., DEA, PR,
and PD) or a branched “linker” (e.g., iso-propylene and 2-
methylpropylene group) were more likely to express the LCST-
type phase transition tuned by pH variation. These structure–
property relationships revealed in this study would help to
develop the applications of TA-PGs in smart drug delivery sys-
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tems.
2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A:
Polym. Chem. 2014, 52, 671–679
KEYWORDS: click chemistry; drug delivery systems; polypep-
tides; stimuli-responsive polymers; structure-property relations
interest due to the differences of pH and temperature
between normal tissues and tumor tissues, as well as intra-
cellular compartments.11,12 Synthesis of the pH and thermo
dual responsive (co)polymers can be readily achieved by
block, random, or grafting copolymerization of the pH-
sensitive and thermo-sensitive monomers, oligomers, or
polymers. For example, Chen et al. have prepared the pH and
thermo dual sensitive polymers based on the thermo-
sensitive poly(N-isopropylacrylamine) (PNIPAM) and the pH
sensitive poly(L-glutamic acid) via block or grafting copoly-
merization.13,14 Schubert et al. have demonstrated the syn-
thesis of tunable pH- and temperature-sensitive copolymers
by random copolymerization of N,N-dimethylaminoethyl
methacrylate (DMAEMA) and poly(ethylene glycol) methyl
ether methacrylate (PEGMA).15 Besides the copolymerization
methods, the homopolymers can also undergo pH and
thermo dual responsive behavior by introducing both the pH
and thermo stimuli responsive moieties in the single side
chain. For instance, Li et al. introduced a new kind of pH
and thermo dual sensitive homopolymer containing an
INTRODUCTION The rapid development of smart drug deliv-
ery technologies has evoked the increasing need of stimuli-
responsive polymers. In the past decade, a majestic amount
of stimuli-responsive polymers that respond to various stim-
uli, such as pH, thermo, light, enzyme, glucose, and magnetic
field, have been extensively investigated for a diverse of drug
delivery applications.1–3 However, it is increasingly recog-
nized that the drug delivery systems trigged by only one
stimulus are less satisfactory for overcoming the complicated
bio-barriers and achieving the ideal therapeutic goals. To
address this issue, the polymeric drug delivery systems have
been engineered to response to dual or multiple stimuli,
such as pH and redox,4,5 pH and thermo,6–8 photo and pH,9
so forth, which may be beneficial for drug loading, targeted
transport, and programmed site-specific drug release.10,11
Thus, the design and synthesis of dual or multiresponsive
(co)polymers have attracted more and more attention.
Among these multiresponsive (co)polymers, pH and thermo
dual responsive (co)polymers have received principal
Additional Supporting Information may be found in the online version of this article.
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2013 Wiley Periodicals, Inc.
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aspartic acid derivative (i.e., with a carboxyl and a N-isopro-
paryl group) in the side chains.16 The lower critical solution
temperatures (LCSTs) of the resultant polymers were tested
to be dependent on the solution pH values and the polymer
molecular weights. Poly(N,N-dimethylaminoethyl methacry-
late) (PDMAEMA) is another example of homopolymer pos-
sessing pH tunable thermo-responsiveness.17 The cloud
point temperature (CPT) of PDMAEMA could be varying
from 31 to 72 ꢀC as the pH value decreased from 11.0 to
7.0.18 To achieve a more applicable pH and temperature
responsive points that were located in the physiological
range, Elvira et al. synthesized a methacrylate monomer
bearing pyrrolidine instead of N,N-dimethyl group in the side
chains, the resulting polymer exꢀhibited phase transition tem-
perature varying from 13 to 30 C as the pH value decreased
from 7.4 to 4.0.19 Inspired by this, one can anticipate that
the pH and thermo dual responsiveness of the tertiary amine
functionalized polymers can be easily tuned by varying the
N-substituted groups.20–22 However, up to now, no system-
atic investigation on the structure–property relationships has
been reported.
stirring in glacial acetic acid and washing with ethanol then
ethyl ether before drying under vacuum. N,N,N0,N00,N00-Pentam-
ethyldiethylenetriamine (PMDETA, 98%, Alfa Asear) was dis-
tilled before use. N,N0-Dimethylformamide (DMF) was stored
over calcium hydride (CaH2) and distilled under nitrogen.
Benzyl amine (99%, Aladdin) was vacuum-distilled over
potassium hydroxide (KOH) and stored under nitrogen.
All the other reagents and solvents were purchased from
Sinopharm Chemical Reagent Co. Ltd., China, and used as
obtained.
Characterization
1H NMR spectra were recorded on Bruker AV 300 (300 MHz)
and Bruker AV 600 (600 MHz) NMR spectrometer in CDCl3,
D2O, or CF3COOD (d-TFA). Fourier transform infrared (FT-IR)
spectra were recorded on a Bio-Rad Win-IR instrument using
a KBr method. Molecular weight (Mw) and polydispersity
index (PDI5 Mw/Mn) were determined by gel permeation
chromatography (GPC) equipped with Waters 515 HPLC
pump and Waters Styragel HT3 column (500–30,000) plus
HT4 column (5000–600,000), using OPTILAB DSP Interfero-
metric Refractometer (Wyatt Technology) as the detector.
DMF containing 0.01 M LiBr was used as elute at 1.0 mL
In the present study, a systemic investigation on the struc-
tural impacts of the N-substituted groups and the “linkers”
between 1,2,3-triazole ring and terminal tertiary amino
group on the pH- and thermo-responsiveness of tertiary
amine functionalized poly(L-glutamate)s (TA-PGs) were care-
fully performed. The TA-PGs were readily synthesized by
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click
reaction of a variety of azido tertiary amines with poly(c-pro-
pargyl-L-glutamate) (PPLG). As is well known, the synthetic
polypeptides have many promising applications in biomedi-
cal fields due to the excellent biocompatibility and structural
mimics of the natural proteins.23–26 Especially, the synthesis
of polypeptides that respond to a variety of stimuli, such as
thermo,27–31 pH,32,33 glucose,34 redox,35–37 and photo,38 has
attracted more and more attention for biomedical uses.
Therefore, the structural insights into the structure–property
relationships would be beneficial for design and preparation
of smart drug delivery carriers or bio-imaging technologies
based on the tertiary amine functionalized polypeptide
materials.
min21 at 50 C. The synthesized tertiary amine functionalized
ꢀ
polypeptides were characterized by aqueous GPC equipped
with Waters 1515 Isocratic HPLC Pump, Waters Ultrahydro-
gelTM Linear Column (500–10 M), and Waters 2414 Refractive
Index Detector. About 0.5 M acetic acid/0.5 M sodium acetate
buffer solution was used as elute at 0.5 mL min21 at 35 C.
ꢀ
Synthesis of PPLG
The c-propargyl-L-glutamate N-carboxyanhydride (PLG-NCA)
was prepared as described in our previous report.27,39 Then,
PPLG was synthesized by the ring-opening polymerization of
PLG-NCA using benzyl amine as initiator. Briefly, in a flame-
dried flask, PLG-NCA (4.99 g, 23.6 mmol) was dissolved in
dry DMF (40 mL) followed by addition of benzyl amine (43
lL). The reaction mixture was stirred for 3 days at room
temperature before precipitating into excess amount of ethyl
ester/methanol mixture (v/v 5 4:1). The polymer was col-
lected by centrifugation, washed twice with ethyl ether and
dried under vacuum (2.6 g, 66% yield). 1H NMR (600 MHz,
CDCl3/d-TFA, d, ppm): 2.07 and 2.26 (ACHCH2CH2A), 2.50
(ACH2CCH), 2.60 (ACHCH2CH2A), 4.45 (ACH2AC6H5), 4.73
(ACHCH2CH2A, and ACH2CCH), 7.34 (AC6H5). Based on the
1H NMR spectrum, the degree of polymerization was calcu-
lated to be 61. Mn (NMR) 5 10,300 g mol21, Mw (GPC) 5
9400 g mol21, and Mw/Mn (GPC) 5 1.24.
EXPERIMENTAL
Materials
2-Chloroethylamine hydrochloride (98%, Aladdin), 2-
dimethylaminoethyl chloride hydrochloride (99%, Aladdin),
2-diethylaminoethyl chloride hydrochloride (99%, Aladdin), 3-
dimethylaminopropylchloride hydrochloride (98%, Alfa Asear),
2-dimethylaminoisopropyl chloride hydrochloride (98%, Alfa
Asear), 3-dimethylamino-2-methylpropyl chloride hydrochlor-
ide (98%, Alfa Asear), N-(2-hydroxyethyl)hexamethyleneimine
(95%, Alfa Asear), 2-methylaminoethanol (98%, Aladdin),
2-morpholinoethanol (98%, J&K), N-(2-hydroxyethyl)pyrroli-
dine (98%, J&K) 2-piperidinoethanol (99%, aldrich), and 2-
diisopropylaminoethanol (981%, Adamas) were used as
received. Copper (I) bromide (98%, Aladdin) was purified by
Synthesis of Azido Tertiary Amine Compounds
As typically showed in Scheme 1, the amino alcohol was first
transferred into amino chloride hydrochloride using thionyl
chloride (some amino chloride hydrochlorides are commer-
cially available which can eliminate this process) and subse-
quently azidation by sodium azide in water. For example, 1-
(2-hydroxylethyl)-pyridine (2.58 g, 20 mmol) was first dis-
solved in chloroform (30 mL) and stirred in an ice-cold bath,
to which 2.1 mL of thionyl chloride (30 mmol) in chloroform
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SCHEME 1 Synthesis of azido tertiary amines.
(8 mL) was added dropwise for 10 min. After that, the mix-
ture was heated to reflux for 4 hr. The solvent was then
evaporated under reduced pressure and the left solid was re-
dissolved in ethanol and precipitated into diethyl ether. The
resultant light yellow solid was filtrated, washed with diethyl
ether, and used for the next reaction without further purifica-
tion. The obtained amino chloride hydrochloride product was
re-dissolved in deionized (DI) water (60 mL) and sodium
azide (3.9 g, 60 mmol) was added. The mixture was stirred at
3-azidopropyl-N,N-dimethylamine (Ap-DMA), yield 56%. 1H
NMR (300 MHz, CDCl3, d ppm): 3.29 (t, 2H, J 5 6.8 Hz), 2.29
(t, 2H, J 5 7.1 Hz), 2.17 (m, 6H), 1.70 (m, 2H).
3-Azido-2-methylpropylene-N,N-dimethylamine (Amp-DMA),
yield: 45%. 1H NMR (300 MHz, CDCl3, d ppm): 3.38 (dddd,
2H, J 5 5.2 Hz, J 5 11.2 Hz, J 5 18.5 Hz, J 5 41.5 Hz), 2.19
(m,7H), 1.93 (m, 2H), 0.96 (dd, 3H, J 5 6.5 Hz, J 5 14.1 Hz).
Click Reaction of PPLG with Azido Tertiary Amines
A typical synthesis procedure is shown as follows. PPLG
(150 mg, containing 0.9 mmol alkyne side chains), Ae-DMA
(205 mg, 1.8 mmol), and PMDETA (19 lL) were dissolved in
DMF (10 mL). The mixture was subjected to three cycles of
vacuum-pump-thaw and then the copper (I) bromide was
added under nitrogen. The reaction vessel was sealed and
stirred for 24 hr at room temperature. The DMF solvent was
evaporated under reduced pressure and diluted with 5 mL
of 3 M HCl. The mixture was transferred into a MWCO 5
3500 dialysis bag and dialyzed against DI water for 48 hr.
The product was obtained as white solid after lyophilization,
yield 73.6%. The yields of other tertiary amine functional-
ized polypeptides (TA-PGs) are given in the Table 1. The 1H
NMR spectra of TA-PGs were recorded on Bruker AV 600
(600 MHz) NMR spectrometer in D2O.
ꢀ
80 C in an oil bath for 24 hr. After cooling, the mixture was
adjusted to pH approximately 10 by addition of KOH and
extracted three times with diethyl ether (50 mL each time).
The combined extracts were dried over magnesium sulfide.
Finally, N-(2-azidoethyl)-pyridine (Ae-PD) was obtained after
evaporating the solvent under reduced pressure. Yield: 87%.
1H NMR (300 MHz, CDCl3, d ppm): 3.30 (t, 2H, J 5 6.2 Hz),
2.50 (t, 2H, J 5 6.3 Hz), 2.37 (d, 4H, J 5 4.4 Hz), 1.55 (m, 4H),
1.40 (dd, 2H, J 5 5.7 Hz, J 5 10.9 Hz).
2-Azidoethylamine (Ae-A), yield: 15%. (Caution: the Ae-A is
highly explosive) 1H NMR (300 MHz, CDCl3, d ppm): 3.26 (t,
2H, J 5 5.7 Hz), 2.77 (m, 2H).
2-Azidoethyl-N-methylamine (Ae-MA), yield: 56%. 1H NMR
(300 MHz, CDCl3, d ppm): 3.33 (m, 2H), 2.66 (m, 2H), 2.35
(s, 3H).
Potentiometric Titration
2-Azidoethyl-N,N-dimethylamine (Ae-DMA), yield: 56%. 1H
NMR (300 MHz, CDCl3, d ppm): 3.33 (t, 2H, J 5 6.1Hz), 2.47
(t, 2H, J 5 6.1Hz), 2.24 (s, 6H).
Typically, TA-PGs were dissolved in DI water at a concentra-
tion of 1.0 mg mL21 and pH of the solution was adjusted to
approximately 3.0 using 0.5 M HCl. The titration was per-
formed by addition 20 lL of 0.1 M NaOH aqueous solution
each time and the pH value was recorded as the measured
value by keeping constant for 30 sec at each point. The
potentiomeric curve was obtained by plotting the measured
pH values against the volume of NaOH solution added.
2-Azidoethyl-N,N-diethylamine (Ae-DEA), yield: 90%. 1H
NMR (300 MHz, CDCl3, d ppm): 3.25 (t, 2H, J 5 6.3 Hz), 2.61
(t, 2H, J 5 6.3 Hz), 2.52 (q, 4H, J 5 7.1 Hz), 1.00 (t, 6H,
J 5 7.1 Hz).
2-Azidoethyl-N,N-diisopropylamine (Ae-DPA), yield: 87%. 1H
NMR (300 MHz, CDCl3, d ppm): 3.12 (t, 2H, J 5 6.6 Hz), 2.99
(qd, 2H, J 5 6.5 Hz, J 5 13.1 Hz), 2.63 (t, 2H, J 5 6.6 Hz), 0.99
(d, 12H, J 5 6.6 Hz).
Determination of pH and Temperature Induced Phase
Transition
N-(2-Azidoethyl)-pyrrolidine (Ae-PR), yield: 53%. 1H NMR
(300 MHz, CDCl3, d ppm): 3.33 (t, 2H, J 5 6.3 Hz), 2.62 (t,
2H, J 5 6.4 Hz), 2.49 (s, 4H), 1.73 (s, 4H).
Turbidimetric measurements were carried out on a UV–Vis
spectrometer (Shimadzu UV-2401PC) at 550 nm, equipped
with a temperature controller (Shimadzu S-1700). Polypep-
tides dissolved in DI water with a concentration of 2.0 mg
mL21 were used for both pH- and temperature-induced tur-
bidity measurements.
N-(2-Azidoethyl)-morpholine (Ae-MP), yield: 82%. 1H NMR
(300 MHz, CDCl3, d ppm): 3.69 (m, 4H), 3.32 (t, 2H, J 5 5.9
Hz), 2.56 (t, 2H, J 5 6.0 Hz), 2.47 (m, 4H).
1
N-(2-Azidoethyl)-hexaethylene (Ae-HEP), yield: 82%. H NMR
(300 MHz, CDCl3, d ppm): 3.22 (t, 2H, J 5 6.0 Hz), 2.69 (t,
2H, J 5 6.1 Hz), 2.64 (t, 4H), 1.58 (m, 8H).
To monitor the pH transition, the solution was first adjusted
to pH 4.0 to fully dissolve the TA-PGs, and then titrated with
0.5 or 0.1 M NaOH solution. The transmittance was recorded
at 20 ꢀC, as the pH gradually increased. The pH transition
point (pHt) was defined as the pH value at which the 50%
transmittance was observed. The temperature induced phase
transition was recorded at different pH values with a heating
2-Azido-N,N-dimethylisopropylamine
(Aip-DMA),
yield
80%.1H NMR (300 MHz, CDCl3, d ppm): 3.54 (qd, 1/2H,
J 5 6.4 Hz, J 5 12.9 Hz), 3.18 (ddd, 1H, J 5 6.5 Hz, J 5 12.5
Hz, J 5 18.3 Hz), 2.69 (qd, 1/2H, J 5 6.6 Hz, J 5 12.9 Hz),
2.35 (td, 1H, J 5 12.2 Hz, J 5 21.6 Hz), 2.20 (s, 6H), 1.05 (dd,
3H, J 5 6.6 Hz, J 5 67.7 Hz).
rate of 0.5 ꢀC min21
. The LCST was defined as the
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TABLE 1 Characterizations of Azido Amines and TA-PGs
PDIa
pHtb
DpH10%–90%
c
Azido Amines
pKa, app
TA-PGs
Yield (%)
pKa, app
Mna(kDa)
Ae-A
—
PP-A
ꢁ99
67.7
73.6
72.1
89.1
90.1
ꢁ99
78.5
89.4
89.8
85.6
81.4
6.8
7.1
6.2
6.4
6.2
6.7
6.2
4.7
6.1
7.2
6.0
6.8
5.6
5.1
5.1
4.3
6.0
6.1
—
1.30
1.19
1.18
1.18
1.18
1.28
—
—
—
Ae-MA
Ae-DMA
Ae-DEA
Ae-DPA
Ae-PR
9.1
8.4
8.9
9.4
8.9
8.7
6.2
9.0
9.3
8.7
9.1
PP-MA
—
—
PP-DMA
PP-DEA
PP-DPA
PP-PR
—
—
8.0
6.4
8.1
7.2
—
0.19
0.86
0.18
0.15
—
Ae-PD
PP-PD
Ae-MP
PP-MP
—
—
Ae-HEA
Ap-DMA
Aip-DMA
Amp-DMA
PP-HEA
PP-pDMA
PP-ipDMA
PP-mpDMA
5.0
8.1
8.6
8.3
1.12
1.22
1.22
1.22
6.5
—
0.78
—
—
—
—
—
a
c
Mn and PDI of TA-PGs were obtained from aqueous GPC (0.5 M acetic
DpH10%–90% was the pH range when the transmittance of polymer solu-
acid/0.5 M sodium acetate buffer solution).
tion decreased from 90% to 10%.
b
pHt was defined as the pH value at which the 50% transmittance was
observed.
temperature corresponding to 50% decrease of initial trans-
mittance at 550 nm.
titration. As typically shown in Figure 3, most of the samples
exhibited a buffering pH ranging from 5.0 to 8.0, which was
consistent with the pH gradient from neutral extracellular
environment to acidic intracellular compartments, indicating
the potential use of these pH responsive polypeptide materi-
als in intracellular drug delivery systems. In addition, based
on the titration curves, the apparent pKa (pKa,app) was
obtained, and the results are listed in Table 1. It was noted
that the pKa,app values of the primary and secondary amine
functionalized polypeptides were slightly lower than those of
other TA-PGs (6.2–6.8), which should be ascribed to the hin-
dering protonation of amine side chains caused by the N-
substituted groups.20 The morpholine bearing polypeptides
(PP-MP) showed the lowest pKa,app due to the relative lower
pKa,app of morpholine (6.2) as compared with other tertiary
amines (8.4–9.4). It should also be noted that the pKa,app of
PP-DMA and PP-DEA were a little lower than those of
poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and
poly(2-diethylaminoethyl methacrylate) (PDEAEMA), which
may be due to the electrophilic effect of 1,2,3-trizaole ring in
the side chains.33 In general, all TA-PGs had a lower pKa,app
than their corresponding azido amine compounds, which
was probably due to three reasons: (1) the electrophilic
effect of 1,2,3-trizaole ring in the side chains; (2) the hydro-
gen bond interaction between amine and carbonyl group in
the side chains, which might reduce the basic nature of
amine;40 (3) the dense protonated amine groups could
hinder the further protonation of the neighboring ones.20
RESULTS AND DISCUSSION
The TA-PGs were facilely synthesized by click coupling of
azido tertiary amines onto alkyne-functionlized poly(L-gluta-
mate) (PPLG) backbone (Scheme 2). The reactions were con-
ducted in DMF, at a molar ratio of alkyne/azide/CuBr/
PMDETA of 1:2:0.1:0.1, for 24 hr. Ten tertiary amines with
different N-substituted groups or “linkers” between amine
and azide groups were conjugated to the PPLG side chains. A
primary amine and a secondary amine functionalized poly-
peptides were also synthesized for comparison study. The
synthesized amino group bearing polypeptides were first
acidified with HCl before dialysis against DI water and lyoph-
ilization. The resultant polymers were well dissolved in
1
water and their structures were characterized by H NMR in
D2O. As typically shown in Figure 1, the 1H NMR spectrum
displays all the characteristic resonance peaks corresponding
to the structure of PP-DMA. Especially, the appearance of sin-
gle peak (d) from 1,2,3-triazole ring at 8.10 ppm demon-
strated the successful CuAAC click reaction. An almost 100%
click conjugation was further verified by comparing the inte-
gration between the peak (a) at 2.75 ppm and the peak (g)
at 2.55 ppm. The 1H NMR spectra of other TA-PGs are
showed in Figure S1 (in the Supporting Information). FT-IR
was also used to characterize the polymers before and after
click coupling, and the results are typically shown in Figure
2. The full disappearance of ACBCA stretch peak at 2130
cm21 for PP-DMA confirmed the complete reaction of side-
chain alkyne in PPLG with azido tertiary amines.
The pH-induced phase transitions of TA-PGs were further
monitored by turbidimetric measurements at 20 ꢀC. As
shown in Figure 4, TA-PGs with N,N-dimethylamino (DMA)
side groups (PP-DMA, PP-pDMA, PP-ipDMA, and PP-mpDMA)
and morphiline side group (PP-MP) were well dissolved in
DI water, displaying no detectable pH transition as the
The buffering capabilities of the tertiary amine functionalized
polypeptides were then characterized by the potentiometric
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SCHEME 2 Synthesis of TA-PGs.
FIGURE 1 1H NMR spectrum of PP-DMA in D2O.
FIGURE 2 FT-IR spectra of PPLG and PP-DMA.
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lower pH and partially precipitation from solution at pH
exceeding pHt.
It is well documented that water-soluble polymers contain-
ing both hydrophilic and hydrophobic groups are likely to be
sensitive to temperature changes. The temperature-induced
phase transition is mainly governed by the balance of hydro-
philic effect (the hydrogen bonds between polar groups of
the polymers and the surrounding water molecules) and
inter- and intramolecular hydrophobic interactions of the
hydrophobic groups.17,41 So, it is expected that the as-
prepared TA-PGs would be sensitive to temperature changes
due to the existing of hydrophilic amine groups and hydro-
phobic polymer backbone and N-substituted groups. To fig-
ure out the thermo-sensitivity of TA-PGs with different side
chains, the turbidimetric measurements of the polypeptide
solutions at different pH values were performed and, the
results are shown in Figure 5. PP-A, PP-MA, PP-DMA, and
PP-MP, that displayed no pH transition in water, demon-
strated no temperature sensitiveness either, even at pH as
high as 10. By contrast, if the N-substituted groups became
more hydrophobic, such as PP-DEA and PP-PD, the tempera-
ture sensitivity could be observed to be dependent on the
pH value and LCSTs increased as the pH value decreased.
This is reasonable that the polymers will become more
hydrophilic due to the more amino groups protonated under
lower pH, which results in the increase of LCSTs. When the
side chains changed to more hydrophobic DPA and HEA, the
temperature induced phase transition, however, became
FIGURE 3 Typical titration curves of TA-PGs at 1.0 mg mL21
.
solution pH increased from 6 to 10. In contrast, PP-DEA, PP-
DPA, PP-PR, PP-PD, and PP-HEA with more hydrophobic N-
substituted groups than DMA exhibited sharp pH transitions
and the transition point (pHt) was measured to be depend-
ent on the hydrophobicity of N-substituted groups in the
side chains (data are listed in Table 1). The more hydropho-
bic N-substituted group led to lower pHt. However, the pH
transition processes (DpH10%–90%) of PP-DPA and PP-HEA
were longer than those of PP-DEA, PP-PR, and PP-PD. This
may be because that the more hydrophobic DPA and HEA
would result in phase transition occurred from relatively
FIGURE 4 Variation of transmittance versus solution pH for 2.0 mg mL21 aqueous solution of TA-PGs.
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FIGURE 5 Thermo-responsiveness of TA-PGs with different N-substituted groups. The temperature-dependent transmittance of
PP-A, PP-MA, PP-DMA, and PP-MP in water were measured at pH 10.0, while that of PP-DEA, PP-DPA, PP-PR, PP-PD, and PP-HEA
were monitored at varied pHs.
undetectable again. It might be confused before the pKa,app
and pHt data were looked into. Most of the temperature sen-
sitive behaviors occurred at the pH value between pKa,app
and pHt of the polymers: pH 7.50–8.57 for PP-DEA
(pKa,app 5 6.4, pHt 5 8.0) and pH 6.96–7.21 for PP-PD
(pKa,app 5 6.2, pHt 5 7.2), while the pH gaps between pKa,app
and pHt of PP-DPA (pKa,app 5 6.2, pHt 5 6.4) and PP-HEA
(pKa,app 5 6.1, pHt 5 6.5) were relatively narrower. Further-
more, PP-DPA and PP-HEA would display marked transmit-
tance decrease at this pH region, and all of these made it
hard to find out a pH point for measuring the temperature
responsiveness. It is also interesting to note that the PP-DEA
and PP-PR with N-substituted groups containing the same
carbon atom numbers but in different forms (one is diethyl
group, the other is cyclic tetramethylene group) displayed
almost the same pHt but quite different thermo-sensitive
behaviors. This can be explained by the Saito’s theoretical
model,42–44 that is, the diethyl group that has a larger con-
tact area with water would be more hydrophobic than the
cyclic tetramethylene group upon heating.
After addressing the impact of N-substituted groups on the
thermo-sensitiveness, the influences of the “linker” structure
between 1,2,3-triazole ring and terminated amino groups
were also tested. Three kinds of TA-PGs, PP-pDMA, PP-
ipDMA, and PP-mpDMA, were synthesized in comparison
with PP-DMA. All of these TA-PGs took DMA as the same ter-
minal group in the side chains, while possessing different
“linkers.” Unlike the phenomenon described in the last para-
graph, that is, no pH-induced phase transitions accompanied
with no temperature-induced phase transitions, although all
the DMA bearing TA-PGs had no detectable pH-induced
phase transitions, the PP-ipDMA and PP-mpDMA showed
remarkable temperature-induced phase transitions at basic
pH (Fig. 6). It is notable that PP-pDMA and PP-ipDMA bear-
ing the structural isomers n-propylene and iso-propylene
FIGURE 6 Thermo-responsiveness of TA-PGs with different “linkers.” The linkers of PP-pDMA, PP-ipDMA, and PP-mpDMA were n-
propylene, iso-propylene, and 2-methylpropylene, respectively.
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groups as their “linkers,” respectively, exhibited the distinct
temperature-induced phase transitions. The n-propylene
“linker” of PP-pDMA should be more hydrophobic than the
iso-propylene “linker” of PP-ipDMA based on the Saito’s
theory.42–44 However, it was PP-ipDMA instead of PP-pDMA
whose temperature-induced phase transition was detectable.
This phenomenon indicated that the hydrophobicity of the
“linker” was not the decisive role for the temperature-
induced phase transition. Instead, the steric hindrance effect
of iso-propylene linker might be devoted to the temperature-
induced phase transition behavior by weakening the interac-
tion of amino groups with adjacent water molecules and
leading to the phase transition occurred at higher tempera-
ture.45 The same effect could also be seen for PP-mpDMA,
whose transition behavior was detectable from a lower pH
value due to the more hydrophobic 2-methylpropylene
“linker” as compared with the iso-propylene “linker” in PP-
ipDMA. And the pH tunable LCST pattern of PP-mpDMA was
similar to those of PP-DEA and PP-PD, that is, LCST
decreased as the increase of pH value. An extraordinary fact
is that the LCSTs of PP-ipDMA and PP-mpDMA at pH around
10.5 are relatively higher than those at pH at around 9.5,
this may be ascribed to the partial hydrolysis of the ester
bonds in the side chains at such a high pH level during the
test duration.33
basic pH, indicating that the steric hindrance effect of the
“linkers” played a critical role in the temperature responsive-
ness. In general, the insight into the structure–responsive-
ness relationships may be useful for designing and preparing
smart drug carriers based on the tertiary amine functional-
ized polypeptides.
ACKNOWLEDGMENTS
This research was financially supported by the National Natural
Science Foundation of China (Projects 51233004, 51273196,
51203153, 21174142 and 51021003) and the Ministry of Sci-
ence and Technology of China (International cooperation pro-
gram 2011DFR51090).
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