Controlled polymerization of N-substituted glycine N-thiocarboxyanhydrides initiated by rare earth borohydrides toward hydrophilic and hydrophobic polypeptoids

Article abstract of DOI:10.1021/ma501131t

N-substituted glycine N-thiocarboxyanhydrides (NTAs) are alternative monomers to prepare polypeptoids with large-scale producing potential compared to the corresponding N-carboxyanhydrides (NCAs) due to their easily synthetic approach and stability during

Full text of DOI:10.1021/ma501131t

Article
pubs.acs.org/Macromolecules
Controlled Polymerization of N‑Substituted Glycine
N‑Thiocarboxyanhydrides Initiated by Rare Earth Borohydrides
toward Hydrophilic and Hydrophobic Polypeptoids
Xinfeng Tao, Yangwei Deng, Zhiquan Shen, and Jun Ling*
MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang
University, Hangzhou 310027, China
*
S Supporting Information
ABSTRACT: N-substituted glycine N-thiocarboxyanhydrides
NTAs) are alternative monomers to prepare polypeptoids
(
with large-scale producing potential compared to the
corresponding N-carboxyanhydrides (NCAs) due to their
easily synthetic approach and stability during purification and
storage. Novel monomer N-butylglycine NTA (NBG-NTA)
has been synthesized and well characterized for the first time.
Rare earth borohydrides [RE(BH ) (THF) , RE = Sc, Y, La,
4
3
3
Nd, Dy, and Lu] have been first applied in the polymerization
of sarcosine NTA (Sar-NTA) and NBG-NTA to achieve high
molecular weight (MW) hydrophilic and hydrophobic polypeptoids. Polysarcosines (PSars), poly(N-butylglycine)s (PNBGs),
and their copolymers with high yields, high MWs, and moderate MW distributions are synthesized at 60 °C by using
RE(BH ) (THF) initiators. MWs of polypeptoids are controlled by feed molar ratios. For instance, PSar with an absolute M of
4
3
3
n
27.7 kDa (DP = 390) and PDI of 1.14 is produced successfully from Sar-NTA. Thermoresponsive random copolypeptoids
poly(sarcosine-r-N-butylglycine)s [P(Sar-r-NBG)s] have reversible phase transitions (cloud point temperature) in aqueous
solution and minimal cytotoxicity comparable to PEG and PSar, which is promising in various biomedical and biotechnological
applications. Thermal properties of homo- and co-polypeptoids are investigated by TGA and DSC measurements.
INTRODUCTION
in aqueous solution. Luxenhofer and co-workers carried out
living ROP of N-substituted glycine NCAs initiated by primary
■
Poly(α-peptoid)s, or polypeptoids, are biomaterials possessing
N-substituted polyglycine backbones with side groups on the
nitrogen atoms. Though having similar structure compared to
polypeptides, polypeptoids lack hydrogen bonding along the
backbones because of N-substituents, which makes them
soluble in many common solvents and amenable to thermal
1
,19
20
amines,
as well as surface-initiated and solid support-
21
initiated polymerizations, to synthesize a series of homo-
polypeptoids as well as di- and multiblock co-polypeptoids.
They investigated the solubility, thermal property, and
biodegradability of the products to enhance comprehensive
understanding. Water-soluble and biodegradable polypeptoids
were regarded as a competitive alternatives of PEG. Schlaad
1
4
10
1
−5
processing.
The conformations of polypeptoids are con-
1
trolled by steric hindrance and electronic properties of side
groups rather than hydrogen bonding, exhibiting random coils
or well-defined secondary structures. Oligopeptoids with
designed structure have enhanced membrane permeability and
proteolytic stability to enzymes compared with oligopeptides
but still can be biodegradable through oxidative pathways.
Because of the challenge of synthesis and potential in
biomedical applications, researchers have been paying increas-
ing attention to polypeptoids.
Polypeptoids are mainly prepared by ring-opening polymer-
reported the thermoresponsive poly(N-C3 glycine)s with Tcps
effected by structural and electronic properties of the side
groups. Crystalline microparticles of poly(N-(n-propyl)glycine)
and poly(N-allylglycine) were observed during long-term
6
,7
8
9
13
10
annealing in aqueous solution.
NCAs must be synthesized and polymerized in extremely
anhydrous and anaerobic solutions and apparatuses due to their
sensitivity to moisture and heat. Moreover, they cannot be
stored for a long period of time and have to be specially purified
6
,11
2
2,23
before use.
(NTAs),
Amino acid N-thiocarboxyanhydrides
ization (ROP) of N-substituted glycine N-carboxyanhydride
2
4−27
1,12−14
the thio-analogues of NCAs, are much more
(
NCA) monomers.
Zhang and co-workers prepared
3,12
stable monomers for polypeptoid synthesis with the potential
of large-scale production. Kricheldorf and co-workers reported
cyclic homo- and block-polypeptoids, as well as cyclic brush-
1
5
like polypeptoids by using N-heterocyclic carbenes (NHCs)
16
and investigated the polymerization mechanism and physical
3
,5
properties detailedly. They also synthesized thermorespon-
Received: May 31, 2014
Revised: August 19, 2014
Published: September 10, 2014
17
sive linear and cyclic random copolypeptoids and bottlebrush
1
8
copolypeptoids with tunable cloud point temperatures (T s)
cp
©
2014 American Chemical Society
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a pioneer work on the polymerization of NTAs initiated by
Synthesis of Sarcosine NTA (Sar-NTA). Sarcosine (22.27 g, 0.25
mol), XAA (45.02 g, 0.25 mol), and NaOH (20.82 g, 0.52 mol) were
dissolved in 300 mL of water and reacted for 72 h at 25 °C with
stirring. Then the reaction mixture was acidified by concentrated
hydrochloric acid. The product N-ethoxythiocarbonylsarcosine was
extracted with chloroform. The organic phase was washed with
aqueous citric acid (5 wt %) and concentrated under reduced pressure
after drying over Na SO . The remaining product was dissolved in 300
2
8
primary amine. They thought that it was too hard to prepare
high molar mass polypeptides from NTAs by using primary
amine initiators because of the low activity of NTA monomers.
In our previous work, polysarcosines (PSars) with quantitative
yields (>94%), low polydispersity indices (PDIs) (<1.2), and
predictable molecular weights (MWs) (when DP < 100) were
synthesized from sarcosine NTA (Sar-NTA) initiated by
poly(ethylene glycol)amine, which created a more controllable
2
4
mL of dry chloroform in an argon atmosphere. PBr (30.3 mL, 0.318
3
mol) was added dropwise in 15 min at 0 °C. The reaction mixture was
29
synthetic method toward polypeptoids. Synthesis of high
MWs (DP > 100) polypeptoids from N-substituted glycine
NTAs was still a significant and challenging issue requiring
highly active initiators.
stirred for an additional 10 min at 0 °C and 1 h at room temperature.
After being washed by saturated solution of NaHCO and deionized
3
water, the chloroform solution was dried with MgSO4 and
concentrated in a vacuum. The crude product was purified by column
chromatography (ethyl acetate:petroleum ether = 1:3). Light yellow
Rare earth compounds exhibit excellent catalytic properties
oil was obtained (14.0 g, yield 42.7%) and stored under an argon
30−35
36,37
38−40
in ROP of lactones,
cyclic ethers,
lactides,
and NCAs as well.
polymerization of NCAs initiated by rare earth borohydrides
cyclic carbonates,
1
atmosphere. H NMR (CDCl /TMS) δ: 3.11 ppm (s, 3H), 4.21 ppm
4
1−43
44−47
3
We have reported
(
s, 2H).
Synthesis of N-Butylglycine Hydrochloride. Glyoxylic acid
[
(
RE(BH ) (THF) ] with quantitative yields and high MWs
M = 86.2 kDa). The high catalytic activity of rare earth
n
monohydrate (72.88 g, 0.792 mol) and n-butylamine (39.0 mL, 0.396
mol) were added to 900 mL of CH Cl . After being stirred at 25 °C
for 24 h, the solvent was evaporated and 800 mL of hydrochloric acid
2 mol/L) was added. The reaction mixture was refluxed for 12 h.
4
3
3
4
7
2
2
compounds makes them promising initiators for ROP of NTAs
to produce high-MW polypeptoids.
In this work, we investigate the controlled ROP of Sar-NTA
and N-butylglycine NTA (NBG-NTA) initiated by RE-
(
(
Then the solvent was concentrated. The yellow crude product was
recrystallized in methanol/ethyl ether to obtain white crystals (42.77 g,
1
yield 64.4%). H NMR (DMSO-d /TMS) δ: 0.88 ppm (t, 3H), 1.31
6
BH ) (THF) (RE = Sc, Y, La, Nd, Dy, and Lu) to produce
4 3 3
ppm (m, 2H), 1.61 ppm (m, 2H), 2.89 ppm (t, 2H), 3.83 ppm (s,
high-MW hydrophilic and hydrophobic polypeptoids. The
polymerizations are carried out in selected solvents resulting in
high yields, moderate PDIs, and predictable MWs by tuning
feed molar ratio of monomer to initiator. Random copolymers
poly(sarcosine-r-N-butylglycine)s [P(Sar-r-NBG)s] are also
synthesized by ROP of Sar-NTA with NBG-NTA. They are
thermoresponsive polymers with reversible phase transitions,
2
H), 9.26 ppm (s, 2H).
Synthesis of N-Butylglycine NTA (NBG-NTA). NBG-NTA was
synthesized by the same procedure of Sar-NTA from N-butylglycine
hydrochloride. The crude product was purified by column
chromatography (ethyl acetate:petroleum ether = 1:7). Yellow oil
was obtained (8.5 g, yield 36.1%) and stored under an argon
atmosphere. H NMR (CDCl /TMS) δ: 0.97 ppm (t, 3H), 1.38 ppm
1
3
1
3
(m, 2H), 1.61 ppm (m, 2H), 3.54 ppm (t, 2H), 4.19 ppm (s, 2H). C
known as cloud point temperature (T ), in aqueous solution.
cp
NMR (CDCl /TMS) δ: 13.65 ppm (CH CH CH CH −), 19.88 ppm
3
3
2
2
2
The biocompatibility of the copolypeptoid P(Sar-r-NBG) is
assessed by cell viability assay. Thermal properties of homo-
and co-polypeptoids are investigated and compared with the
data in the literature.
(
(
CH CH CH CH −), 29.49 ppm (CH CH CH CH −), 44.02 ppm
3
2
2
2
3
2
2
2
CH CH CH CH −), 59.89 ppm (−CH CO), 164.73 ppm (−NCO-
3
2
2
2
2
SCO−), 194.26 ppm (−NCOSCO−).
Polymerizations of Sar-NTA and NBG-NTA Initiated by
RE(BH ) (THF) . All polymerizations were performed using the
4
3
3
Schlenk technique, and all polymerization tubes were predried and
purged with argon.
As a typical homopolymerization, Sar-NTA (0.729 g, 5.56 mmol)
was dissolved in 11.5 mL of dry acetonitrile, followed by 0.26 mL of
EXPERIMENTAL SECTION
■
Materials. Sarcosine (98%, Shanghai Adamas Reagent, China), n-
butylamine (99.5%, J&K Chemical Reagent, China), glyoxylic acid
monohydrate (98%, J&K Chemical Reagent, China), carbon disulfide
Y(BH ) (THF) in THF solution (0.0542 mol/L). The tube was
4 3 3
(
(
99%, Sinopharm Chemical Reagent, China), sodium chloroacetate
98%, Shanghai Jingchun, China), and phosphorus tribromide (98.5%,
sealed and placed in a 60 °C oil bath for 48 h. The polymer was
isolated by precipitation from diethyl ether and dried in a vacuum
(0.387 g, 98%).
Sinopharm Chemical Reagent, China) were used as received. Dioxane
and tetrahydrofuran (THF) were refluxed before use over sodium/
benzophenone ketyl and potassium/benzophenone ketyl, respectively.
NBG-NTA (0.372 g, 2.15 mmol) was dissolved in 4.5 mL of dry
THF, followed by 0.18 mL of Lu(BH ) (THF) in THF solution
4 3 3
N,N-Dimethylformamide (DMF) was stirred over CaH and followed
(0.0572 mol/L). The tube was sealed and placed in a 60 °C oil bath
for 48 h. The polymer was isolated by precipitation from diethyl ether
and dried in a vacuum (0.190 g, 78.1%).
As a typical random copolymerization, Sar-NTA (0.297 g, 2.26
mmol) and NBG-NTA (0.268 g, 1.55 mmol) were dissolved in 7.6 mL
2
by distillation under reduced pressure. Acetonitrile was stirred over
CaH and distilled. RE(BH ) (THF) (RE = Sc, Y, La, Nd, Dy, and
2
4
3
3
Lu) was prepared according to the procedure described in the
30
47
literature and our previous report.
Synthesis of S-Ethoxythiocarbonylmercaptoacetic Acid
XAA). NaOH (30.0 g, 0.75 mol) was dissolved in 150 mL of ethanol
of dry THF, followed by 0.34 mL of Lu(BH ) (THF) in THF
4 3 3
(
solution (0.0572 mol/L). The tube was sealed and placed in a 60 °C
oil bath for 48 h. The polymer was isolated by precipitation from
diethyl ether and dried in a vacuum (0.221 g, 55.5%).
and 375 mL of water followed by bubbling argon through the solution
for 15 min. Then carbon disulfide (54 mL, 0.90 mol) was added
dropwise with stirring at 0 °C. The mixture was reacted for 2 h in an
argon atmosphere. Afterward, an aqueous solution of sodium
chloroacetate (87.36 g, 0.75 mol) was added to the reaction mixture
and stirred at 25 °C. After 24 h, the mixture was acidified by adding
concentrated hydrochloric acid and extracted three times with 200 mL
Measurements. Molecular weights (MWs) and polydispersity
indices (PDIs) were determined by size-exclusion chromatography/
multiangle laser light scattering (SEC/MALLS) which consisted of a
Waters 1515 isocratic high performance liquid chromatograph pump, a
Wyatt DAWN DSP MALLS detector, a Wyatt Opitlab DSP
interferometric refractometer (RI), and a column of PLgel 5 μm
MIXED-C. DMF containing 0.05 mol/L LiBr was used as the eluent
with a flow rate of 1.0 mL/min at 60 °C. MWs and PDIs were
determined by MALLS data and conventional SEC analysis with RI
and calibration curve, respectively. Commercial poly(methyl
of chloroform each. The extracts were dried with Na SO4 and
2
concentrated in a vacuum. The products were obtained by crystallizing
twice in chloroform/hexane and isolated by filtration, which were light
1
yellow crystals (48.7 g, yield 36.1%). H NMR (CDCl /TMS) δ: 1.43
3
ppm (t, 3H), 3.98 ppm (s, 2H), 4.66 ppm (q, 2H).
6
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methacrylate)s were used as the calibration standards. The refractive
index increment (dn/dc) of the polypeptoids was determined by a
Brookhaven BI-DNDC differential refractometer and BI-DNDCW
software. The dn/dc of PSar and PNBG in 0.05 mol/L LiBr/DMF at
shelf-time make NTA monomers suitable commercial products
to prepare polypeptoids.
Polymerization Features. In order to synthesize high-MW
PSars from Sar-NTA, we have to solve two problems, i.e., low
25 °C was determined as 0.0931 and 0.0786 mL/g, respectively.
activity of Sar-NTA monomer and poor solubility of PSar
Nuclear magnetic resonance (NMR) spectra were recorded on a
1,29
1
polymer in most common organic solvents.
We carried out
Bruker Avance DMX 500 spectrometer ( H: 500 MHz) with DMSO-
Sar-NTA ROP using Y(BH ) (THF) as initiator in various
d6 or CDCl3 as solvent and tetramethylsilane (TMS) as internal
reference. Electrospray ionization mass spectrometry (ESI-MS)
spectrum was measured by Varian MS-500 liquid chromatograph
mass spectrometer. The hydrodynamic diameters of the nanoparticles
were measured by dynamic light scattering (DLS) using a particle size
analyzer (Zetasizer Nano Series, Malvern Instruments) at 25 °C. The
measurements were made at a fixed angle at 90° and a wavelength of
4 3
3
solvents to select optimal reaction medium (Table S1).
Although DMF is a standard reaction solvent for polymer-
izations of NCAs, it is not suitable for NTAs. Sar-NTA
polymerizations in DMF at 20 °C (S1 in Table S1) result in
only low-MW products with a low yield of 43.2%, which is
consistent with our previous report of primary amine-initiated
657 nm. UV−vis spectra were recorded on a UV-2550 UV−vis
29
polymerizations of Sar-NTA. It is necessary to increase
spectrophotometer (Shimadzu) equipped with a TCC-240A temper-
ature controlled cell holder (Shimadzu). Differential scanning
calorimetry (DSC) analyses were performed on a TA Q200
instrument. PSars were heated to 160 °C at a rate of 10 °C/min
under a nitrogen purge, held for 3 min to erase the thermal history,
cooled to 25 °C at a rate of 20 °C/min, and finally subjected to a
second scan. PNBGs and P(Sar-r-NBG)s were heated from −25 to
temperature due to the low reactivity of NTA, but it does not
work in this case. When the polymerization temperature is
elevated to 60 °C (S2 in Table S1), the yield is still low
(
48.1%) and MW of the product is too low to be distinguished
from the solvent in SEC. DMF can react with growing peptide
chains to form aldehyde groups, which terminates the
polymerization. DMF also slightly decomposes at 60 °C to
dimethylamine and then initiates the polymerization to yield
49
250 °C at a rate of 10 °C/min under a nitrogen purge, held for 3 min
to erase the thermal history, cooled to −30 °C at a rate of 20 °C/min,
and finally subjected to a second scan. Thermogravimetric analysis
2
6,27,49
low-MW products.
Sar-NTA polymerizations in dioxane
(
TGA) was performed on a TA Q500 instrument. Samples were
(
S3−S6 in Table S1) achieve a more acceptable yield (>70%)
heated to 500 °C at a rate of 10 °C/min.
but not in a controlled way as the M s of PSars increase slightly
n
Cytotoxicity Assay. HepG2 cells were seeded in 96-well plates
with the initial density of 10 000 cells per well and cultured overnight
to 80% cell confluence. All polymers were dissolved in 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and
diluted to appropriate concentrations (14−80 μg/mL). The polymer/
HEPES buffer solution was added to the wells, and the cells were
incubated in the incubator for 48 h. The polymer concentration in the
well was in the range from 14 to 80 μg/mL. Cell viability was tested
with methylthiazolyltetrazolium (MTT) assay. Briefly, the medium
was replaced by solution containing 20 μL of MTT, and the cells were
incubated for 4 h. Then, the medium was removed, and 200 μL of
dimethyl sulfoxide (DMSO) was added to dissolve the generated
formazan. The plates were shaken for 15 min before the detection with
a microplate reader (550, Bio-Rad) at 570 nm. A total of five replicates
were conducted for each treatment.
from 10.4 to 13.5 kDa when feed molar ratios of monomer to
initiator ([Sar]/[Y]) increase 8 times from 49 to 406. The
uncontrollability is attributed to the poor solubility of PSar in
dioxane, which leads to precipitation during the polymerization.
The heterogeneous reaction in dioxane confines the active
chain end to react with monomers, limiting MW of PSar and
broadening PDI. What encourages us is that PSars initiated by
Y(BH ) (THF) show higher MWs than those by primary
4
3
3
28,29
amine initiators.
In short, neither DMF nor dioxane is an
appropriate reaction solvent for Sar-NTA polymerization.
Acetonitrile, a good solvent for PSar, is an appropriate
polymerization medium. The results of Sar-NTA ROPs
initiated by Y(BH ) (THF) in acetonitrile (Scheme 1) are
4 3
3
summarized in Table 1. Without precipitation or turbidness,
polymerizations of Sar-NTA initiated by Y(BH ) (THF) at 60
4
3
3
RESULTS AND DISCUSSION
■
°C for 48 h with various feed molar ratios of [Sar]/[Y] from 98
to 516 (1−4 in Table 1) exhibit quantitative yields (>90%) and
moderate PDIs (1.26−1.40). All SEC traces of the products are
Monomer Synthesis. Sar-NTA is prepared according to
2
8
the procedure described in the literature and our previous
2
9
work (Scheme S1 in Supporting Information) and confirmed
1
by H NMR (Figure S1). We report NBG-NTA for the first
time (Scheme S2) from N-butylglycine hydrochloride. The
Scheme 1. Polymerization of Sar-NTA and NBG-NTA
Initiated by RE(BH ) (THF)
48
4
3
3
1
structure of NBG-NTA monomer is well characterized by H
1
3
1
1
1
13
NMR, C NMR, H− H COSY, and H− C HMQC (Figures
S2−S5) spectra. The signal of methyl protons of the n-butyl
side group is found at 0.97 ppm (t, H ), and methylene protons
a
b
c
on side group appear at 1.38 ppm (m, H ), 1.61 ppm (m, H ),
d
e
and 3.54 ppm (t, H ). The signal at 4.19 ppm (s, H ) is
assigned to methylene protons on five-membered ring. The
a
b
methyl signal H is coupled with signal H (inset C in Figure
b
c
S4). Likewise, two methylene signals H and H on the n-butyl
side group are coupled with protons on adjacent carbons
(
insets A and B in Figure S4). ESI-MS spectrometry of NBG-
+
NTA shows a peak at 174.0 m/z generated by (NBG-NTA)H .
All the syntheses are conducted in open air. It is much easier to
prepare N-substituted glycine NTA than NCA analogues. The
liquid NTA monomers can be stored in an argon atmosphere at
room temperature for several months to a year without any
decomposition or oligomerization. Easy synthesis and long
6
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a
Table 1. Polymerization of Sar-NTA in Acetonitrile Initiated by Y(BH ) (THF)
3
4
3
b
c
c
d
d
sample
[Sar]/[Y]
temp (°C)
yield (%)
Mn,theo (kDa)
Mn,RI (kDa)
PDI
Mn,LS (kDa)
PDI
1
2
3
4
5
6
98
202
395
516
168
180
60
60
60
60
25
40
84.1
92.4
98.0
>99
5.9
13.3
27.5
36.7
2.4
6.7
14.6
27.1
34.6
3.3
1.34
1.40
1.30
1.26
1.18
1.25
12.2
22.5
27.7
1.12
1.09
1.14
20.5
68.7
8.8
6.3
a
b
c
Polymerization conditions: [Sar] = 0.5 mol/L, 48 h in acetonitrile. Theoretical M , M
= [Sar]/[Y] × yield × 71. Determined by SEC
n
n,theo
d
measurement with interferometric refractometer. Determined by SEC measurement with MALLS detector (dn/dc = 0.0931 mL/g).
unimodal as shown in Figure 1 and Figure S6 detected by RI
and MALLS detectors, respectively. According to SEC/MALLS
Figure 1. SEC traces of 1−4 detected by interferometric
refractometer.
1
Figure 2. H NMR spectra of 1 (A), 7 (B), and 13 (C) in DMSO-d
6
analyses, products 2−4 with feed molar ratio of 202, 395, and
(
∗: water).
5
16 have absolute M s of 12.2, 22.5, and 27.7 kDa, respectively.
n
The absolute M s of products 2−4 are lower than the
n
corresponding theoretical ones because the Y(BH ) (THF)
traces of the products (Figure S8) suggest that every reaction
has only one active species during polymerization.
4
3
3
3
0,47
can initiate more than one propagating chain.
The structure
1
of the obtained product has been confirmed by H NMR in
We expand rare earth borohydrides to synthesize poly-
peptoid from another monomer N-butylglycine N-thiocarbox-
yanhydride (NBG-NTA). It is the first report of this new
monomer despite its NCA analogue (NBG-NCA) having been
a
DMSO-d (Figure 2A). The protons in PSar backbone (H ′:
6
b
2
.65−3.05 ppm; H ′: 3.85−4.45 ppm) are well identified. This
is the first time to produce hydrophilic PSars with M s higher
n
1
,12,13
than 10 kDa by Sar-NTA which can replace those obtained
from Sar-NCA and suitable for application as biopoly-
noticed recently.
The corresponding poly(N-butylglycine)
(PNBG) product is hydrophobic and soluble in many organic
solvents, e.g., THF and chloroform, differing from PSar.
Polymerizations of NBG-NTA initiated by Lu(BH ) (THF)
7
,11,14
mers.
When polymerization temperature decreases to 40 and 25 °C
5 and 6 in Table 1), PSar yields decrease to 68.7% and 20.5%
4
3
3
(
were carried out in THF at 60 °C for 48 h (Scheme 1) with the
results summarized in Table 2. The yields are higher than 75%,
and PDIs of PNBGs are moderate (1.22−1.42) (7−9 in Table
and M s drop to 6.3 and 3.3 kDa (Figure S7), respectively.
n
Therefore, it is necessary to carry out NTA polymerization at
6
0 °C to reach quantitative yields, in accordance with the case
2). M s increase from 3.8 to 10.8 kDa detected by SEC
n
29
of primary amine initiators. The other five rare earth
measurement where RI detector underestimates the M s in
n
borohydride complexes, i.e., RE(BH ) (THF) where RE =
Sc, La, Nd, Dy, and Lu, are investigated as initiators for Sar-
Table 2. When [NBG]/[Lu] increases to 381 (10 in Table 2),
4
3
3
the yield decreases to 65.3% and M (10.7 kDa) remains the
n
NTA ROP. All of them are more efficient than primary
same as sample 9, which suggests a good controllability of
NBG-NTA ROP only when [NBG]/[Lu] is no higher than
300. All SEC traces of the products are shown in Figure 3 and
Figure S9. According to SEC/MALLS analyses, products 8−10
with feed [NBG]/[Lu] ratio of 209, 306, and 381 have absolute
2
8,29
amines.
At comparable molar ratios of [Sar]/[RE] (S7−
S11 in Table S2), they produce PSars with M s between 5.2
n
and 7.0 kDa and the yields between 59.9% and 78.6%, which is
not as good as Y(BH ) (THF) though. This relates to the
4
3
3
differences of atomic radii and electronic structures of rare earth
metals, and yttrium is the most appropriate one. Unimodal SEC
M s of 13.7, 21.5, and 23.1 kDa, respectively, lower than the
n
corresponding theoretical ones due to Lu(BH ) (THF)
4
3
3
6
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a
Table 2. Polymerization of NBG-NTA in THF Initiated by Lu(BH ) (THF)
3
4
3
b
c
c
d
d
sample
[NBG]/[Lu]
yield (%)
Mn,theo (kDa)
Mn,RI (kDa)
PDI
Mn,LS (kDa)
PDI
7
8
9
98
209
306
381
74.6
78.1
80.2
65.3
8.3
18.4
27.7
28.1
3.8
7.4
1.22
1.40
1.42
1.51
13.7
21.5
23.1
1.04
1.13
1.10
10.8
10.7
1
0
a
b
c
Polymerization conditions: [NBG] = 0.5 mol/L, 48 h in THF at 60 °C. Theoretical M , M
= [NBG]/[Lu] × yield × 113. Determined by
n,theo
n
d
SEC measurement with interferometric refractometer. Determined by SEC measurement with MALLS detector (dn/dc = 0.0786 mL/g).
Figure 4. SEC traces of 11−13 detected by an interferometric
refractometer.
Figure 3. SEC traces of 7−10 detected by an interferometric
refractometer.
d
3
0,47
1
3.20 ppm (H ). The methylene protons on backbone of both
initiates more than one propagating chain.
The structure of
b
e
units (H ′ and H ) overlap in the range from 3.74 to 4.50 ppm.
the obtained product has been confirmed by H NMR analysis
in DMSO-d (Figure 2B). The protons of PNBG backbone
H : 0.84 ppm; H : 1.23 ppm; H : 1.39 ppm; H : 3.22 ppm;
H : 3.80−4.45 ppm) are clearly assigned. Thus, high-MW
hydrophobic PNBGs are synthesized successfully from NBG-
NTA using Lu(BH ) (THF) initiator.
Random copolymerizations of Sar-NTA with NBG-NTA are
carried out by RE(BH4) (THF) (RE = Y, La, and Lu)
initiators in THF (Scheme 1 and 11−13 in Table 3). To the
best of our knowledge, the polymer P(Sar-r-NBG) is
synthesized for the first time. Neither precipitation nor gelation
is observed during the polymerization, which indicates that
P(Sar-r-NBG) has good solubility in THF differing from PSar.
Copolymers are obtained with moderate yields (55.5−69.7%)
and moderate PDIs (1.30−1.40), which also reveals unimodal
SEC traces (Figure 4). H NMR analysis indicates that the
products are constituted by sarcosine and N-butylglycine units
based on the clearly distinguished signals (Figure 2C). Methyl
Based on the intensities of characteristic methyl proton signals
6
a
a
a
b
c
d
of H and H ′, the molar percentage of N-butylglycine units in
(
e
copolymer (NBG mol % in Table 3) is calculated by eq 1.
I(Ha)
NBG mol % =
× 100%
a′
4
3
3
a
I(H ) + I(H )
(1)
where I(H) is integral intensity of the corresponding proton.
NBG fractions calculated by NMR are close to the values in
feeding when yields are still low (55−70%), indicating Sar-NTA
and NBG-NTA have comparable activities. The random
structures are also proved by DSC and self-assembly behavior
below.
Polymerization Kinetics. We investigated polymerization
kinetics of Sar-NTA and NBG-NTA with the results
summarized in Figures S10 and S11. Polymerization kinetics
of Sar-NTA were measured at 60 °C with a feed molar ratio of
150 ([Sar-NTA]/[Y(BH ) (THF) ]) in both sealed tubes and
3
3
1
4
3
3
a
proton signals of sarcosine units (H ′) appear in a broad range
open systems under argon flow using acetonitrile as solvent and
Y(BH ) (THF) as initiator. H NMR spectra were collected
every 4 h by taking a small amount of reaction mixture and
dissolving in DMSO-d . Monomer conversions were calculated
1
from 2.67 to 3.06 ppm, while those of N-butylglycine units
4
3
3
a
(
H ) present at 0.85 ppm. The methylene proton signals on
side groups are detected at 1.23 ppm (H ), 1.39 ppm (H ), and
b
c
6
a
Table 3. Random Copolymerization of Sar-NTA with NBG-NTA Initiated by RE(BH ) (THF)
4
3
3
b
b
c
sample
initiator
[Sar]/[NBG]/[RE] (NBG mol %)
yield (%)
Mn,RI (kDa)
PDI
NBG (mol %)
11
12
13
Y(BH ) (THF)
3
124/79/1 (38.9)
124/95/1 (43.4)
117/80/1 (40.6)
62.3
69.7
55.5
4.0
3.9
5.8
1.30
1.34
1.40
40.7
38.6
40.6
4
3
La(BH ) (THF)
3
4
3
Lu(BH ) (THF)
3
4
3
a
b
Polymerization conditions: [Sar] + [NBG] = 0.5 mol/L, 48 h in THF at 60 °C. Determined by SEC measurement with interferometric
c
1
refractometer. Calculated by H NMR analyses.
6
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Macromolecules
Article
from the relative integration of the methylene proton resonance
around 98 °C (Figure 6A), which is lower than the reported
4
of Sar-NTA and PSar. Both ln(M /M ) vs time plots of the two
value (143 °C). It may attributed to that sample 2 has a
0
t
systems are linear with monomer conversions less than 60%.
The polymerization of Sar-NTA under open systems is faster
than that in sealed tubes. The liberation of COS gas has an
effect on the polymerization of Sar-NTA though it still remains
uncertain. The reason can be that the dissolved COS competes
with NTA monomer to react with the RE-NMe-polymer chain
end. A similar question is also under debate for NCA
polymerizations.
Polymerization kinetics of NBG-NTA were measured at the
same conditions using THF as solvent and Lu(BH ) (THF) as
4
3
3
initiator (Figure S11). The polymerization rate of NBG-NTA
in THF is lower than the case of Sar-NTA in acetonitrile
because of the lower polarity of THF. Similar to the Sar-NTA
cases, NBG-NTA polymerizations exhibit linear ln(M /M ) vs
0
t
time plots when the conversions are below 40%. The
polymerization rate is also faster under open systems. But the
difference is not as much as that of Sar-NTA, which may relate
to the solubility of COS in the solvents with difference
polarities. Polymerizations of Sar-NTA and NBG-NTA are
Figure 6. DSC profiles of PSar 2 (A), PBNG 7 (B), and P(Sar-r-NBG)
1
3 (C).
1
,16
much slower than the corresponding NCAs.
In addition, we measure low-MW products by FTIR analysis
to verify whether thiocarbonyl (COS) is still attached to
polymer chain ends or not. Two characteristic stretching
vibrations of Sar-NTA monomer can be distinguished in Figure
broader MW distribution than the reported PSar, and low-MW
chains act as plasticizer to decrease T . PNBG (sample 7)
exhibits two melting temperatures (T s) at 60 and 196 °C
g
m
(
Figure 6B) with corresponding melting enthalpies (ΔH s) of
m
−1
2
.3 and 35.6 J/g, respectively. T s of PNBG are slightly lower
m
4
S12A with the frequencies at 1741 cm assigned to a-CO and
700 cm− to b-CO. The reduced frequency of b-CO compared
1
than the data (70 and 225 °C) reported by Luxenhofer,
indicating a lower degree of crystallinity of our sample.
1
with a-CO is caused by the nitrogen electron pair delocalized
onto a-CO. This assignment has been demonstrated for NCA
However, they are quite different from the values (−65 and
5
4
7
2
(
27 °C) reported by Zhang. The second melting enthalpy
monomers. The low-MW PSar sample only arises a
characteristic stretching vibration of amide carbonyl (c-CO)
5
35.6 J/g) of our sample is lower than Zhang’s result (48 J/g),
with the frequency at 1663 cm−1 (Figure S12B). The
which also suggests a lower degree of crystallinity of our
sample. There is no detectable thermal transition temperature
disappearance of absorption bands of a-CO and b-CO indicates
that there is no thiocarbonyl (COS) attached to polymer chain
ends. The FTIR spectra of NBG-NTA and low-MW PNBG
sample show similar results (Figure S13). Therefore, we do not
have proof to conclude that the liberation of COS is slow.
Thermal Properties. We use TGA and DSC analyses to
investigate the thermal properties of homo- and co-poly-
(
(
T or T ) in the DSC profile of P(Sar-r-NBG) (sample 13)
Figure 6C), which further supports that P(Sar-r-NBG) is a
g m
random copolymer.
Response of P(Sar-r-NBG) to Thermal Stimuli. Homo-
and co-polypeptoids could be thermoresponsive materials with
1
3,17,18
tunable Tcps.
As an attractive water-soluble polypeptoid,
PSar does not exhibit detectable T by turbidity measurements
cp
peptoids. Decomposition temperatures (T s) of PSar (sample
d
below 100 °C and appears to be miscible with water in all ratios
2), PNBG (sample 7), and P(Sar-r-NBG) (sample 13) are 253,
1
(
for DP < 100). Contributing to the water-insoluble property
239, and 247 °C (Figure 5), respectively. DSC profiles show
of PNBG, P(Sar-r-NBG)s are thermally responsive and exhibit
reversible phase transitions with tunable Tcp in aqueous
solution by controlling NBG fraction in composition. Aqueous
solution of sample 13 becomes cloudy upon heating and gets
transparent again upon cooling illustrated by the photo in
Figure 7 inset, suggesting reversible phase transitions. Turbidity
of dilute aqueous solution of 13 is determined by variable UV−
that PSar (sample 2) has a glass transition temperature (Tg)
vis spectroscopy without stirring. The T (defined as the
cp
temperature at 50% UV−vis transmittance at the light
wavelength λ = 450 nm) of P(Sar-r-NBG) with a concentration
of 3.0 mg/mL is 37.4 °C (Figure 7). The transition temperature
window (ΔT) (corresponding to the temperature difference at
9
9% and 1% transmittance) is quite broad (22.6 °C) compared
to the random copolymers poly[2-(2-methoxyethoxy)ethyl
methacrylate-co-oligo(ethylene glycol) methacrylate] [P-
5
0
(
MEO MA-co-OEGMA)] and poly[(2-n-propyl-2-oxazo-
2
line)-r-(2-ethyl-2-oxazoline)] [P(nPrOx-r-EtOx)] (ΔT < 5
5
1,52
°
C),
while this result is similar to the cases of reported
Figure 5. TGA profiles of PSar 2 (A), PBNG 7 (B), and P(Sar-r-
NBG) 13 (C).
cyclic and linear P(N-ethylglycine-r-N-butylglycine)s [P(NEG-
r-NBG)s] (ΔT = 10−18 °C). Dependence of aggregation size
17
6
178
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Macromolecules
Article
Figure 7. Plots of transmittance at λ = 450 nm versus temperature for
the aqueous solution of P(Sar-r-NBG) 13 at a concentration of 3.0
mg/mL. Inset: photographs of sample 13 aqueous solution at 20 and
5
0 °C.
Figure 9. Relative cell viability of PEG6K (A), PSar (sample 2) (B),
and P(Sar-r-NBG) (sample 13) (C) for HepG2 cells after 48 h
incubation at a concentration of 14, 20, 40, and 80 μg/mL.
versus temperature for aqueous solution of 13 (3.0 mg/mL) is
determined by DLS measurement as shown in Figure 8 and
by the additional N-butylglycine units. The cytotoxicity of
P(Sar-r-NBG) is similar to the copolypeptoid P(NEG-r-NBG)
17
reported by Zhang. However, when the concentration is
higher than 1 mg/mL, the copolypeptoid P(Sar-r-NBG)
exhibits a little cytotoxicity compared to PEG (M = 6000)
n
and PSar (sample 2) (Figure S15). In this case, sample 13
precipitates from the solution during the assay at high
concentration. The solid precipitates are responsible to the
increasing of cytotoxicity.
CONCLUSION
We have carried out a controlled ROP of N-substituted glycine
NTA initiated by RE(BH ) (THF) for the first time to
■
4
3
3
develop a versatile and practical synthetic method to prepare
high-MW hydrophilic and hydrophobic polypeptoids. Sar-NTA
polymerizations with high yields (>90%) succeeded by using
Y(BH ) (THF) in acetonitrile, and the MWs of PSar were
Figure 8. DLS profiles of P(Sar-r-NBG) 13 aqueous solution at
different temperatures at a concentration of 3.0 mg/mL.
4
3
3
designable by tuning the feed ratios of [Sar]/[Y]. PSar with an
absolute M of 27.7 kDa (DP = 390, PDI = 1.14) was
n
synthesized. This length was comparable to those prepared
from Sar-NCA. We prepared and characterized NBG-NTA and
used it as the monomer to produce PNBGs initiated by
Figure S14. P(Sar-r-NBG) chains are mainly unimers in dilute
aqueous solution at low temperature (Figure 8, 18 °C) with
partial aggregations. These aggregations existing below Tcp
broaden the effective particle sizes, which results in the broad
Tcp transition. When the temperature is elevated to 32 °C,
stable nanoparticles (135 nm, PDI = 0.127) are formed due to
the hydrophobic interaction-driving aggregation of P(Sar-r-
NBG) chains. Diameters of aggregations increase gradually with
the increasing temperature until copolymer chains precipitate
from water. When cooled down, the precipitate redissolves and
the aqueous solution turns back to being transparent.
Lu(BH ) (THF) in THF. P(Sar-r-NBG)s were also obtained
4 3
3
1
7
by copolymerization of Sar-NTA with NBG-NTA. Thermal
properties of homo- and co-polypeptoids were detailedly
investigated by TGA and DSC measurements. P(Sar-r-NBG)s
are thermoresponsive materials exhibiting reversible phase
transitions (T ) in aqueous solution. With low cytotoxicity as
cp
PEG and PSar, P(Sar-r-NBG)s are promising for various
biomedical and biotechnological applications. Easy accessibility
of NTA monomers paves the way for large-scale industrial
production of homo- and co-polypeptoids with high MW.
Furthermore, the biocompatibility of the copolypeptoid
P(Sar-r-NBG) is assessed by using MTT cell viability assay.
Human hepatoma (HepG2) cells are incubated for 48 h in
HEPES buffers containing sample 13 with different concen-
ASSOCIATED CONTENT
■
S
Supporting Information
trations. Sample 13 (3 mg/mL) has a T around human body
*
cp
1
13
1
1
temperature. The aggregation of sample 13 at 37 °C leads to
the changing of cytotoxicity. The copolypeptoid P(Sar-r-NBG)
exhibits negligible cytotoxicity at low concentrations (14−80
μg/mL) for the good solubility, which is comparable to
Polymerization data, H NMR, C NMR, H− H COSY, and
1
13
H− C HMQC spectra, SEC curves, polymerization kinetics
plots, FT-IR spectra, cytotoxicity assay, and plots of
aggregations size vs temperature measured by DLS. This
material is available free of charge via the Internet at http://
pubs.acs.org.
poly(ethylene glycol) (PEG, M = 6000) and PSar (sample 2)
n
(
Figure 9). The biocompatibility of PSar has not been hindered
6
179
dx.doi.org/10.1021/ma501131t | Macromolecules 2014, 47, 6173−6180

Macromolecules
Article
(
28) Kricheldorf, H. R.; Sell, M.; Schwarz, G. J. Macromol. Sci., Part A:
AUTHOR INFORMATION
■
*
Pure Appl. Chem. 2008, 45 (6), 425−430.
Corresponding Author
Tel +86-571-87953739; Fax +86-571-87951773; e-mail
lingjun@zju.edu.cn (J.L.).
(29) Tao, X. F.; Deng, C.; Ling, J. Macromol. Rapid Commun. 2014,
35 (9), 875−881.
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Notes
(
The authors declare no competing financial interest.
(
(
ACKNOWLEDGMENTS
■
(
Financial support from the National Natural Science
Foundation of China (21174122) is acknowledged. We thank
Prof. Youxiang Wang (Zhejiang University) for cytotoxicity
analysis.
(
2
(
2
(
2
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