194
O. Sallouh, R. Weberskirch / Polymer 86 (2016) 189e196
phase over MgSO4 and removal of the solvent under reduced
pressure (840 mg, 0.9 mmol, 63%) of product was obtained.
that is in good agreement with the values based on the NMR and
GPC data (1.09 mmol azide functionalities/g P1 and 0.89 mmol TPP
functionalities/g P2). The exact quantification of azide and triphe-
nylphosphine groups can then be used to tune the formation of
hydrogels by the total polymer concentration or the stoichiometric
ratio of the azide and triphenylphosphine functional groups. Gels
were formed by simple mixing the precursor polymers P1 and P2 at
defined ratios of azide to triphenylphosphine functionalities and
overall polymer concentration.
1H-NMR (300 MHz, DMSO):
d
¼ 1.80 (s, 3H, CH3), 3.44 (m, 2H,
CH2), 3.62 (s, 3H, CH3), 4.12 (m, 2H, CH2), 5.61 (s, 1H, CH), 5.96 (s,
1H, CH), 7.17 (m, 4H, CH), 7.36 (m, 6H, CH), 7.53 (m, 1H, CH), 7.99 (m,
1H, CH), 8.69 (m, 1H, CH). 13C NMR (75.47 MHz, DMSO):
d
¼ 18.01,
25.1, 47.49, 53.37, 124.15, 128.69, 128.77, 129.01, 133.35, 133.63,
164.89, 167.19. 31P (162.02 MHz, DMSO):
d
¼ ꢀ4.62, ESI-MS:
Mcalculated ¼ 476.1549; Mmeasured ¼ 476.1632 [MþH]þ.
The gels G2 e G6 that were formed by this method were all
transparent and a typical example can be seen Fig. 4 for G2. The
FTIR of G2 shows that all important signals from the two precursor
copolymers P1 and P2 can be found in the gel (see Fig. 4). The
strong absorption bands at 1662 cmꢀ1 (ʋO]CeNH2, 2) was associ-
ated with the acrylamide co-monomer and 1547 cmꢀ1 (ʋO]CeNH, 3,
Amide II) was associated with the TPP-comonomer. Moreover, the
signals at 1725 cmꢀ1 (ʋO]CeO, 1) and at 1137 cmꢀ1 (ʋC-O-C, 4) were
associated with the ester groups of AEMA, AzeMA and TPP-MA
comonomers.
The results of oscillatory rheometry for G1 e G6 prepared with
precursor polymers P1 and P2 containing varying ratios of azide
and triphenylphosphine groups are shown in Fig. 3A and Table 2. In
the first set of experiments, we analyzed the formation of hydrogels
by using equal concentration of the azide and triphenylphosphine
functional groups but changing the total polymer concentration
from 2.5 to 10 wt%. As can be seen from the results in Table 2, no
chemically crosslinked gel G1 was formed at the lowest polymer
concentration of 2.5 wt%, however, chemically crosslinked gels
were formed at higher total polymer concentration of 5 wt% (G2)
and 10 wt% (G3) with G0-values of 1630 65 Pa and 2120 20 Pa,
respectively. Moreover, when changing the ratio of azide to tri-
phenylphoshine functional groups from 1:1 (G2) to 1: 5 (G6) the G0-
values decreased from 1630 65 Pa (G2) to 27 6 Pa (G6), see
Table 2. Thus, the results show that hydrogel properties can be
easily modified by the copolymer concentration and ratio of func-
tional groups.
2.1.6. Polymer synthesis
All polymerizations were carried out in Schlenk tubes under
inert atmosphere using freshly distilled and freeze, pump, thaw
degassed DMSO. A typical procedure for P2 was as follows:
In a Schlenk tubes were placed (0.24 g, 3.3 mmol, 1 eq.) acryl-
amide, (69 mg, 0.42 mmol, 0.125 eq.) AEMA, (199 mg, 0.42 mmol,
0.125 eq.) TPP-MA and (6.4 mg, 0.02 mmol, 0.006 eq.) V70 and then
they were solved in 10 ml DMSO. The resulting solution was
degassed by a freeze-thaw process under vacuum and stirred in an
oil bath at 30 ꢁC for 20 h. After that the polymer solution was
precipitated in 300 ml of diethyl ether. The precipitated polymer
was centrifuged and then dried under vacuum to give 0.49 g of a
white powder of P2 with 85% yield.
P1: 1H-NMR (300 MHz, D2O): 0.95, 1.09, 2.10, 3.13, 3.57, 4.11.
P2: 1H-NMR (300 MHz, D2O): 0.92, 1.07, 2.12, 3.12, 3.66, 4.04,
7.21, 7.39, 7.57.
2.1.6.1. Preparation of the hydrogels. A typical procedure of hydro-
gel preparation was as follows:
18 mg of the azide-containing copolymer P1 was dissolved in
300
m
l PBS-buffer (pH ¼ 7.4) and 32 mg TPP-containing copolymer
P2 was dissolved in 700
ml PBS-buffer (pH ¼ 7.4). The two polymer
solutions were mixed together and poured in a round Teflon form
for 20 h at 37 ꢁC.
2.2. Characterization of the copolymers
To evaluate the efficiency of the Staudinger ligation, the reaction
between the azide group in P1 and the triphenylphosphine unit of
P2 was monitored using quantitative FT-IR spectroscopy. Different
concentration of P1 and P2 were mixed and incubated at 20 ꢁC
between FT-IR readings to monitor conversion of the azide group
over the course of 10 h for G2 to G4. As can be seen from Fig. 5, the
conversion of the azide functionality depends on the ratio of azide
to triphenylphosphine functionalities for G2 and G4 as well as the
overall copolymer concentration of 5 or 10 wt% for G2 and G3,
respectively.
In a copolymer mixture with equal number of azide and tri-
phenylphosphine functional groups but different total polymer
concentration of 5 wt% and 10 wt% for G2 and G3, respectively, 40%
of the azide chemical groups were consumed after 30 min for G3
compared to 45 min for G2. Moreover, when the ratio of azide to
triphenylphosphine functional groups was changed from 1:1 (G2)
to 1:1.5 (G4), 40% azide conversion was detected after 59 min for G4
compared to 45 min for G2. While the kinetics of azide conversion
in the first hour of the gel formation was quite similar major dif-
ferences could be observed after around 90 min when the azide
consumption slowed considerably. After 200 min azide consump-
tion was 41% for G4, 49% for G2 and 56% for G3 suggesting that
overall copolymer concentration and the ratio of functional groups
have a large effect on the kinetics and efficacy of hydrogel forma-
tion. While Stabler et al. reported around 40% azide consumption
for their alginate-N3 polymer after crosslinking with the TPP-
modified PEG our precursor polymers indicate higher azide con-
sumption of up to 56% when the overall polymer concentration is
increased to 10 wt% for G3.
The copolymers P1 and P2 were of high purity as demonstrated
by proton NMR. Gel permeation chromatography yielded a Mn
value of 56,8 and 34.5 kDa for P1 and P2, respectively, with PDI
values of 2.1 and 1.6 typical for a free radical polymerization. Both
polymers displayed water-solubility as an important prerequisite
for the subsequent gel formation experiments. A typical example of
copolymer characterization by 1H NMR spectroscopy is depicted in
Fig. 2. As can be seen, the methylene groups 2 and 3 near to the
azide functionality and the ammonium chloride group gave distinct
signals to determine the azide and the AEMA content while the
acrylamide content can be calculated based on the signals 5 and 6.
The experimental results are summarized in Table 1. Moreover, 1H
NMR analysis confirmed the successful introduction of the azide
monomer AzeMA in P1 and the TPP-MA monomer in P2 with 10
and 12 mol %, respectively, which is in excellent agreement with the
theoretically expected values of 10 mol %. The FTIR spectra of the P1
displayed a characteristic band at 2160e2120 cmꢀ1 (ʋN3) that can
be assigned to the azide functionality of the AzeMA comonomer.
Moreover, the peak at 1547 cmꢀ1 (ʋO]CeNH, 3, Amide II) was
associated with the TPP-MA comonomer in P2 (see Fig. 4).
2.3. Hydrogel formation and characterization
In the next step we analyzed the hydrogel formation with the
two water-soluble precursor polymers P1 and P2. Therefore, UV/
Vis analysis of the copolymers P1 and P2 was carried out to
determine the amount of azide and TPP functional groups. We
obtained 1.11 mmol azide/g for P1 and 0.643 mmol TPP/g for P2