Thermoresponsive xylan hydrogels via copper-catalyzed azide-alkyne cycloaddition

Article abstract of DOI:10.1016/j.carbpol.2013.11.058

In the present work, hydrogels of birch wood xylan and thermoresponsive poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol) (PEG-PPG-PEG) were prepared using copper catalyzed alkyne-azide cycloaddition (CuAAC) in aqueous reaction conditions. First, reactive azide groups were introduced on the backbone of xylan by etherification of 1-azido-2,3- epoxypropane in alkaline water/isopropanol-mixture at ambient temperature, providing degree of substitution (DS) values up to 0.28. On the second step, the azide groups were reacted with propargyl bifunctional PEG-PPG-PEG utilizing CuAAC, leading to formation of crosslinked hydrogels. The novel xylan derivatives were characterized with liquid and solid state nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FT-IR) and elemental analysis (EA). The temperature controlled swelling behavior of the developed hydrogels was evaluated in the range of 7-70 C by water absorption and compressive stress-strain measurements, which showed a reduction in water content and change in stiffness with increasing temperature. The morphology of the hydrogels at different temperatures was studied by scanning electron microscopy (SEM), which showed a reduction in pore size with increasing temperature.

Full text of DOI:10.1016/j.carbpol.2013.11.058

Carbohydrate Polymers 102 (2014) 637–644
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Thermoresponsive xylan hydrogels via copper-catalyzed azide-alkyne
cycloaddition
∗
Nikolaos Pahimanolis, Anni Sorvari, Nguyen Dang Luong, Jukka Seppälä
Department of Biotechnology and Chemical Technology, Aalto University School of Chemical Technology, Kemistintie 1, 02150 Espoo, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present work, hydrogels of birch wood xylan and thermoresponsive poly(ethylene glycol)-b-
poly(propylene glycol)-b-poly(ethylene glycol) (PEG-PPG-PEG) were prepared using copper catalyzed
alkyne-azide cycloaddition (CuAAC) in aqueous reaction conditions. First, reactive azide groups were
introduced on the backbone of xylan by etherification of 1-azido-2,3-epoxypropane in alkaline
water/isopropanol-mixture at ambient temperature, providing degree of substitution (DS) values up
to 0.28. On the second step, the azide groups were reacted with propargyl bifunctional PEG-PPG-PEG
utilizing CuAAC, leading to formation of crosslinked hydrogels. The novel xylan derivatives were char-
acterized with liquid and solid state nuclear magnetic resonance spectroscopy (NMR), Fourier transform
infrared spectroscopy (FT-IR) and elemental analysis (EA). The temperature controlled swelling behavior
Received 27 September 2013
Received in revised form 30 October 2013
Accepted 27 November 2013
Available online 6 December 2013
Keywords:
Xylan
Hydrogel
Thermoresponsive
Etherification
Click chemistry
Modification of polysaccharides
◦
of the developed hydrogels was evaluated in the range of 7–70 C by water absorption and compressive
stress–strain measurements, which showed a reduction in water content and change in stiffness with
increasing temperature. The morphology of the hydrogels at different temperatures was studied by scan-
ning electron microscopy (SEM), which showed a reduction in pore size with increasing temperature.
©
2013 Elsevier Ltd. All rights reserved.
1
. Introduction
Iqbal, Hussain, Koschella, & Heinze, 2008; Saghir, Iqbal, Koschella,
Heinze, 2009; Schwikal & Heinze, 2007; Schwikal, Heinze,
&
Hemicellulose is the most abundant polysaccharide family next
Ebringerová, & Petzold, 2006; Vincendon, 1998) and development
of bifunctional derivatives using acrylamide (Ren, Peng, & Sun,
2008). In addition, esterification reactions typically employing
anhydrides or activated carboxylic acids (Buchanan et al., 2003;
Daus & Heinze, 2010; Fundador, Enomoto-Rogers, Takemura, &
Iwata, 2012; Hansen & Plackett, 2011; Hesse, Liebert, & Heinze,
2006; Hettrich et al., 2006; Salam, Pawlak, Venditti, & El-tahlawy,
2011) and sulfating agents (Daus et al., 2011; Hettrich et al., 2006)
have been used to alter the chemical properties and introduce new
functionality to xylans. In order to tailor the properties of natural-
based polymers, the copper catalyzed azide-alkyne cycloaddition
(CuAAc), often referred as “click”-reaction, offers a straightforward
and efficient way to build new molecular complexity (Kolb, Finn,
& Sharpless, 2001; Rostovtsev, Green, Fokin, & Sharpless, 2002;
Tornoe, Christensen, & Meldal, 2002). Characteristics for click-type
reactions is that they can be performed at mild reaction conditions
and have high tolerance toward functional groups, oxygen and
water, which makes the use of this chemistry tempting for the
modification of natural polymers. The CuAAC offers extensive pos-
sibilities to tailor polymer properties (Binder & Sachsenhofer, 2007,
to cellulose, present as a component in most plant cell walls. In
hardwood species, the main hemicellulose type is xylan, which
predominantly consists of d-xylopyranoside units connected by
␤
-(1→4)-linkages along with acetyl-, glucuronic acid and species
dependent side groups (Deutschmann & Dekker, 2012). Among
other methods, xylan can be conveniently extracted from biomass
by alkali extraction (Ebringerová & Heinze, 2000; Glasser, Kaar, Jain,
&
Sealey, 2000) where the acetyl groups are also hydrolyzed ren-
dering the backbone hydroxyl groups available for derivatization
reactions.
Several chemical modifications targeting the hydroxyl groups
have been reported for xylan, including etherification using
epoxides and alkyl halides in alkaline media (Bigard et al., 2011;
Ebringerová, Hromádková, Kacuráková,
& Antal, 1994; Fang,
Fowler, Tomkinson, & Hill, 2002; Jain, Sjöstedt, & Glasser, 2001;
Kataja-aho, Haavisto, Asikainen, Hyvärinen, & Vuori, 2008; Laine
et al., 2013; Petzold, Günther, Kötteritzsch, & Heinze, 2008;
Petzold, Schwikal, & Heinze, 2006; Ren, Sun, & Liu, 2007; Saghir,
2
008; Fournier, Hoogenboom, & Schubert, 2007; Meldal, 2008;
∗ Corresponding author. Fax: +358 9 47022622.
E-mail addresses: jukka.seppala@aalto.fi, jukka.seppala@tkk.fi (J. Seppälä).
Lallana, Riguera, & Fernandez-Megia, 2011; Lallana, Fernandez-
Trillo, Sousa-Herves, Riguera, & Fernandez-Megia, 2012; Lallana,
0
144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.11.058

6
38
N. Pahimanolis et al. / Carbohydrate Polymers 102 (2014) 637–644
Sousa-Herves, Fernandez-Trillo, Riguera,
&
Fernandez-Megia,
the chemical structure of the synthesized products. The temper-
ature controlled swelling behavior of the developed hydrogels
was evaluated in the range of 7–70 C, which showed a reduction
in water absorption with increasing temperature. The hydrogels
could have applications as drug delivery systems, or work as part
of separation, fractionation or self-cleaning membranes (Klouda
& Mikos, 2008; Vermonden, Censi, & Hennink, 2012; Wandera,
Wickramasinghe, & Husson, 2010).
2
012; Kempe, Krieg, Becer, & Schubert, 2012). The utilization of
◦
CuAAC on the modification of polysaccharides has been reported
in several publications (Bernard, Save, Arathoon, & Charleux, 2008;
De Geest et al., 2008a, 2008b; Eissa, Khosravi, & Cimecioglu, 2012;
Elchinger, Montplaisir, & Zerrouki, 2012; Hafrén, Zou, & Córdova,
2
006; Hasegawa et al., 2006; Koschella, Richter, & Heinze, 2010;
Liebert, Hänsch, & Heinze, 2006; Pohl, Schaller, Meister, & Heinze,
008; Ritter, Knudsen, Mondrzik, Branscheid, & Kolb, 2012; Schatz,
2
Louguet, Le Meins, & Lecommandoux, 2009; Tankam, Müller,
Mischnick, & Hopf, 2007; Xu, Zhang, & Kadla, 2012; Zhang, Xu,
Wu, Zhang, & Zhuo, 2009). However, studies on the modification
of xylan using the CuAAC-reaction is limited to a recent paper,
where propargyl end-functionalized polylactide was grafted on
azide-containing xylan (Enomoto-Rogers & Iwata, 2012).
2
. Experimental
2.1. Materials
Birch wood xylan (xylose content ≥90%, degree of acety-
lation less than 4% determined with 1H NMR), Mn = 11700 g/mol,
Hydrogels, three-dimensional networks of hydrophilic poly-
mers capable of retaining large amount of water, have a broad
field of applications, and the research on such materials is an
ongoing task. Applications such as drug-delivery systems, arti-
ficial muscles and sensors are being developed (Calvert, 2008;
Coviello, Matricardi, Marianecci, & Alhaiqu, 2007; Klouda & Mikos,
PDI = 2.02 determined by GPC against PEG standards) was pur-
chased from Sisco Research Laboratories Pvt. Ltd. and used as
received.
Propargyl bromide (80 wt% in toluene) and HNO3 (65%) were
obtained from Fluka Chemicals. l-Ascorbic acid (99%), CuSO ·5H O
4
2
(99%), epichlorohydrin (99%), ethylenediaminetetraacetic acid
2
008). Polysaccharide based hydrogels have some advantages over
tetrasodium salt dihydrate (EDTA, 99%), 2-propanol (99.8%), NaH
synthetic polymers, since in addition to their abundant avail-
ability, they are biologically compatible and degradable (Coviello
et al., 2007). Xylans are interesting candidates for such materi-
als also due to their bioactive properties (Cipriani et al., 2008;
Ebringerová & Heinze, 2000; Ebringerová, Kardosová, Hromádková,
(
(
95%) and NaNO (97%) were purchased from Sigma–Aldrich. NaN3
99%), acetic acid (99.8%) and NaOH (99%) were from Merck. All
2
chemicals were used as received. Anhydrous grade tetrahydrofuran
THF) was purchased from VWR and stored over molecular sieves.
Poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide)
(
Malovíková,
& Hribalová, 2002). For example, xylan based-
(
PEO–PPO–PEO) triblock copolymers were from BASF (trade name
hydrogels have been developed from methacrylated xylan and
subsequent radical polymerization of hydroxyethyl methacry-
late for drug release studies (Silva, Habibi, Colodette, & Lucia,
©
Pluronic PE6100 and PE 6400 both having a central PPO block
of 1750 g/mol and 10% or 40% of PEO in molecule respectively).
Poly(ethylene glycol) 2000 g/mol was from Fluka. The polymers
2
011). In addition, xylan-rich hemicelluloses grafted with acrylic
◦
were vacuum-dried at 40 C for 48 h before use.
acid in the presence of N,N-methylene-bis-acrylamide crosslinker
yielded hydrogels with multistimulus response properties (Peng,
Ren, Zhong, Peng, & Sun, 2011). Allylated xylan derivatives
were successfully crosslinked by UV induced radical crosslink-
ing with and without N,N’-diallylaldardiamides yielding novel
bio-based hydrogels (Pohjanlehto, Setälä, Kammiovirta, & Harlin,
2
.2. Preparation of 1-azido-3-chloro-propanol
The synthesis of 1-azido-3-chloropropanol was done starting
from epichlorohydrin. The ring-opening reaction of the epox-
ide with azide-ion was done according to a modified method
2
011). The xylan hydrogel formation has also been reported on
enzymatically aided method (Chimphango, van Zyl, & Görgens,
012), by physical (Hettrich & Fanter, 2010) or ionic interac-
(Fringuelli, Piermatti, Pizzo, & Vaccaro, 1999; Pahimanolis et al.,
2
010; Yang, Shao, Li, Wang, & Zhang, 2011). Isopropanol (109.0 ml)
2
and acetic acid (7.2 ml, 125.8 mmol) were mixed with a solution
of NaN3 (8.177 g, 125.8 mmol) in 74.0 ml of water. Epichlorohy-
drin (6.6 ml, 84.2 mmol) was then added under stirring and the
tions (Gabrielii & Gatenholm, 1998; Gabrielii, Gatenholm, Glasser,
Jain, & Kenne, 2000). The utilization of the CuAAC reaction for
the synthesis of polysaccharide hydrogels has been reported
for azide and alkyne derivatized hyaluronan and the obtained
hydrogels were demonstrated to serve as drug reservoirs and
scaffolds (Crescenzi, Cornelio, Di Meo, Nardecchia, & Lamanna,
◦
1
reaction was continued at 30 C for 24 h, until H NMR analysis
showed complete consumption of the epoxide. A water solution
of NaNO2 (14.4 ml, 41.6 mmol) was then added, followed by the
dropwise addition of HNO3 (5.76 ml, 83.8 mmol) to eliminate any
excess azide-ions. The stirring was continued for 24 h at room
temperature, by which time the formation of nitrous oxides had
ceased. To concentrate the solution, 12 g of NaCl was added and
the separated propanol phase was collected. The aqueous phase
was extracted once with 50 ml of diethyl ether and the organic
phases were combined. This resulted in additional phase separa-
tion of water, which was discarded. The organic phase was further
concentrated by removing diethyl ether by rotary evaporation. The
obtained propanol solution of 1-azido-3-chloropropanol (58 ml,
concentration 1.34 mmol/ml, yield 92% by 1H NMR analysis, acetic
acid content 0.61 mmol/ml) was stored in dark at room tempera-
ture and used without further purification.
2
007, Huerta-Angeles et al., 2011; Huerta-Angeles et al., 2012).
In addition, networks based on cellulose (Koschella, Hartlieb,
Heinze, 2011, Pierre-Antoine, Francois, Rachida, 2012)
&
&
and thermoresponsive cellulose/poly(N-isopropylacrylamide-co-
hydroxyethyl methacrylate) hydrogels have been developed
(Zhang et al., 2009).
In our previous publication, we described a method for introduc-
ing azide-groups on the backbone of dextran using aqueous
reaction media (Pahimanolis, Vesterinen, Rich, & Seppala, 2010).
The azide functionalities provide a combinatorial approach to dis-
cover new materials, as a wide range of possible modifications
via CuAAC become available. In this paper, the modification is
applied to xylan. First, azide groups were introduced to the back-
bone of xylan using glycidyl azide under alkaline conditions. On
the second step, the novel azide modified xylan was crosslinked
with thermoresponsive alkyne end-functionalized polyethylene
glycol/polypropylene glycol/polyethylene glycol (PEG-PPG-PEG)
triblock copolymers using CuAAC, yielding temperature responsive
hydrogels. Elemental analysis, NMR and FT-IR were used to confirm
Warning! Low molecular weight organic azides are known to be
potentially explosive. For this reason, handling highly concentrated
solutions of these materials should be avoided.
1
H NMR (D O, ppm): ı = 3.36–3.54 (CH -Cl), 3.56–3.73 (CH -N ).
2
2
2
3
1
3
C NMR (D O, ppm): ı = 70.50 (C-OH), 53.92 (C-N ), 46.69 (C-Cl).
2
3

N. Pahimanolis et al. / Carbohydrate Polymers 102 (2014) 637–644
639
Table 1
The effect of different reaction conditions on the obtained degree of substitution (DS) in the etherification reaction of xylan with 1-azido-2,3-epoxypropane (AEP).
◦
DSa
Entry
Time (h)
Temperature ( C)
Molar ratio NaOH/AXU
Molar ratio AEP/AXU
Reaction efficiency (%)
1
2
4
5
6
7
8
9
24
24
24
24
24
24
24
24
6
30
30
30
30
30
30
30
30
55
55
30
30
0.26
0.26
0.79
0.79
0.79
0.79
1.32
1.32
1.32
1.32
2.64
2.64
0.50
2.02
0.50
1.01
2.02
3.01
0.50
2.02
0.50
1.98
0.58
1.98
0.06
0.14
0.08
0.14
0.17
0.28
0.08
0.18
0.06
0.23
0.07
0.18
12
7
16
14
8
9
16
9
12
12
12
9
10
11
12
13
6
24
24
a
Determined with elemental analysis.
2.3. Introducing azide groups to the backbone of xylan
2.4. Synthesis of alkyne end functionalized poly(ethylene
glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol)
The azide functionalization of xylan was done following a mod-
ified method (Pahimanolis et al., 2010; Yang et al., 2011): (Table 1,
entry 11) To a water solution of xylan (0.500 g in 10.0 ml of water)
Dry PEG-PPG-PEG (PE6400 2900 g/mol, 5.00 g, 1.7 mmol) was
dissolved in 5 ml of dry THF under argon. NaH (0.120 g, 5.0 mmol)
was added to the solution and the resulting dispersion was stirred
for 2 h, until the formation of hydrogen gas ceased. Propargyl
bromide (80% in toluene, 0.67 ml, 6.0 mmol) was added and the
suspension was stirred for 2 h, after which 5 ml of THF was added.
The salts were removed by centrifugation and the polymer was
1
.00 ml of 5 M NaOH solution was added and the mixture was
◦
stirred for 10 min at 55 C. The 1-azido-2,3-epoxypropane needed
for the etherification was prepared just prior to use, by adding
2
.18 ml of 5 M NaOH solution to 5.60 ml (7.5 mmol) of 1-azido-3-
chloropropanol and stirring for 5 min at room temperature. The
obtained epoxide solution was combined with the xylan solution
◦
precipitated in cold (−21 C) heptane. The centrifugation and pre-
◦
1
and the reaction was continued for 6 h at 55 C, by which time
H
cipitation was repeated two more times. The polymer was dried in
◦
NMR analysis showed complete consumption of the epoxide. The
solution was neutralized with 0.30 g of acetic acid and precipitated
in 60 ml of acetone, redissolved in a minimal amount of water and
precipitated twice in 70 ml of ethanol. The obtained azide function-
vacuum at 30 C for 48 h to obtain a yellow viscous liquid. The yield
was 3.20 g (64%).
The alkyne-end functionalization of PE6100 was done using
0.168 g (7.0 mmol) of NaH and 0.89 ml (8.0 mmol) of propargyl bro-
mide. The product was yellow viscous liquid and its yield was 1.24 g
(25%).
◦
alized xylan was lyophilized, dried in vacuum at 40 C overnight and
stored in the dark at room temperature for further use.
Fig. 1. Introducing azide groups on xylan (1), crosslinking of azide containing xylan with different polymers using CuAAC (2). PEG (A), PE6100 (B) and PE6400 (C).

6
40
N. Pahimanolis et al. / Carbohydrate Polymers 102 (2014) 637–644
Fig. 3. FT-IR spectra of hydroxypropyl azide xylan (A), crosslinked xylan/PE6100 (B)
and PE6100 (C).
Fig. 2. DEPT-135 and 13C NMR spectra of hydroxypropyl azide xylan.
The infrared-spectra were recorded with a Unicam Mattson
3
000 FTIR spectrometer from KBr pellets (for azide containing sam-
1
1
ples) or using a diamond ATR accessory.
Scanning electron microscopy (SEM) images were obtained
using a Zeiss Sigma VP instrument operating at 3 kV. Small gel sam-
H NMR (CDCl ): (ppm) 1.09, 2.43, 3.20–3.80, 4.15.
3
3
C NMR (CDCl ): (ppm) 17.4, 58.5, 68.5, 68.6, 69.0, 70.4, 70.5, 70.8,
3
7
2.9, 73.3, 74.6, 75.1, 75.3, 75.5, 77.4, 79.6.
◦
ples (approximately 0.1 g) were conditioned in water baths at 7 C
◦
or 70 C for 2 h, then frozen in liquid nitrogen and freeze-dried for
The alkyne-end functionalization of PEG was done as above,
4
8 h. The dried samples were cut with a sharp knife to expose fresh
◦
except that 40 C reaction temperature was used. The product was
brown waxy solid, and its yield was 4.38 g (88%).
surface and made conductive by Au/Pt sputtering.
The compressive stress–strain measurements were performed
using a TA Instruments AR-G2 rheometer equipped with a plate-
plate geometry (diameter 20 mm). The synthesized cylindrical gel
1
H NMR (CDCl ): (ppm) 2.43, 3.40–3.80, 4.16.
3
13
C NMR (CDCl ): (ppm) 58.4, 69.1, 70.4, 70.5, 74.7, 77.4, 79.6.
3
◦
samples were conditioned in a water bath at 7 or 70 C for 2 h.
The dimensions of each sample were measured and then placed
on the lower plate and compressed by the upper plate at a speed
of 10 ␮m/s. The diameters of the samples were between 20.73
and 12.75 mm and the heights were between 5.09 and 7.40 mm.
Compressive modulus was calculated for each sample from the
slope of the stress-strain curve in the beginning of the linear region.
For each material, two parallel samples were measured.
2
.5. Hydrogel preparation using CuAAC
The hydrogels were prepared as follows: 0.100 g of azide func-
tionalized xylan (Table 1, Entry 11, DS = 0.23) was dissolved in
.00 ml of water. The polymer used for crosslinking (0.100 g) was
4
added and allowed to dissolve for 5 h in an ice-bath under stirring
and overnight at 7 C. The stirring was continued in an ice bath for
◦
Swelling tests were done for three concurrent samples by
another 5 h. A freshly prepared water solution (0.63 ml) of copper
sulfate (1 equiv. to alkyne groups used) and ascorbic acid (2 equiv.)
was chilled in an ice bath and then added to the xylan/polymer
solution and allowed to stir for 30 s. 1.1 ml of the solution was then
pipetted on four cylindrical silicone molds (height 16.6 mm, diam-
weighting approximately 100 mg of gel preconditioned in water at
◦
7
C. The samples were then placed in capped vials of water and
◦
eter 16.0 mm) and allowed to gel at 7 C for 4 h. The obtained gels
were removed from the molds and submerged in 0.05 M EDTA solu-
◦
tion for 48 h at 7 C to complexate the copper ions. The solution was
replaced with distilled water and changed daily for a period of at
least a week.
2.6. Characterization
NMR spectra were recorded on a Bruker Avance-III 400 spec-
trometer in deuterium oxide (D O) or deuterated chloroform
2
1
13
(
CDCl ), using standard H, C and DEPT 135 pulse sequences.
3
Carbon-13 cross-polarization/magic angle spinning nuclear mag-
netic resonance (13C-CP/MAS NMR) spectra were recorded with
a double resonance 4 mm probe. Samples were spun in zirconia
rotors using a spinning rate of 10 kHz. A proton excitation pulse of
2
.95 ␮s and a CP contact time of 2.0 ms were used applying a lin-
ear ascending variable amplitude ramp, the acquisition time being
3.9 ms under high power decoupling. 15 k scans were accumu-
3
lated with a recycle delay of 5 s.
The degree of substitution (DS) was determined by elemental
analysis using a Perkin Elmer 2400 CHN equipment.
Fig. 4. 13C-CP/MAS spectra of crosslinked xylan/PE6100 (A), hydroxypropyl azide
xylan (B) and unmodified xylan (C).

N. Pahimanolis et al. / Carbohydrate Polymers 102 (2014) 637–644
641
◦
Fig. 5. Swelling ratio of hydrogels as a function of temperature (A) and swelling ratio over 5 min temperature cycles between 7 and 70 C (B). Swelling ratio as a function of
◦
◦
◦
◦
time at 70 C and 7 C, for gel samples preconditioned at 7 C and 70 C respectively (C).
◦
◦
Fig. 6. SEM images of freeze-dried hydrogels of xylans crosslinked with PEG, PE6100 and PE6400. The gel samples were conditioned at 7 C and 70 C before freeze-drying.

6
42
N. Pahimanolis et al. / Carbohydrate Polymers 102 (2014) 637–644
xylan. Low degree of functionalization is obtained with low sodium
hydroxide to anhydroxylose unit ratio (NaOH/AXU), which may be
due to insufficient activation of the xylan hydroxyl that undergo the
ring-opening of the epoxide. The obtained functionalization val-
ues are generally similar to those previously reported for dextran,
although lower amount of NaOH is needed for the reaction. This
may be due to structural differences between the polysaccharides,
or the fact that more concentrated epoxide solution was used in this
study (Pahimanolis et al., 2010). Increasing the NaOH/AGU ratio to
0
.79 yields slightly higher azide functionalization, however, a fur-
ther increase of the amount of NaOH does not significantly affect
the substitution values. The addition of higher amounts of epox-
ide increases the obtained DS value, although the functionalization
becomes increasingly difficult as can be noted from the lower reac-
◦
◦
tion efficiency. Raising the reaction temperature from 30 C to 55 C,
results in a faster reaction, consuming all of the epoxide within 6 h,
giving also a higher DS value with high epoxide feed. The modi-
fied xylans had increased water solubility, with the exception of
the highest substituted xylan of DS = 0.28, which formed turbid
solutions even when heated.
The functionalization of xylan with azide groups can be seen
−
1
from FT-IR spectra, showing the typical azide peak at 2110 cm
Fig. 3A). Furthermore, the structure of the ethers can be confirmed
(
from DEPT135 spectra of modified xylan, given in Fig. 2, where the
hydroxypropyl azide group can be identified. The substitution of
the hydroxyl groups at the positions 2 and/or 3, can be observed at
around 82 ppm and an upfield shift of carbon 1 peak affected by the
substitution, in accordance to previously reported xylan hydrox-
yalkyl ethers (Ebringerová et al., 1994; Kataja-aho et al., 2011;
Schwikal et al., 2006).
◦
◦
Fig. 7. Xylan hydrogel crosslinked with PE6100 conditioned at 7 C and 70 C (A).
Compressive modulus of xylan hydrogels crosslinked with PEG, PE6100 or PE6400,
◦
◦
conditioned at 7 C and 70 C (B).
3.2. Crosslinking of xylan using CuAAC
The azide functionalized xylan was used to obtain crosslinked
hydrogels using poly(ethylene glycol) and thermo-responsive
◦
heated to the desired temperature (7–70 C). The samples were
then weighted after careful removal of excess water. The gel weight
was divided with the initial weight to obtain the relative swelling
ratio.
block copolymers of poly(ethylene glycol)/poly(propylene glycol)
◦
(
Fig. 1). The reaction was performed at 7 C in order to solubilize the
polymers. The gelation started within minutes after addition of the
copper catalyst, but it was allowed to take place for 4 h to ensure
maximum crosslinking. We found that longer reaction time weak-
ened the gels, eventually disintegrating and becoming solutions if
the crosslinking time was prolonged for several days. This is likely
because of the degradation of the polysaccharide and/or polymer
chain caused by the copper catalyst, an unfortunate drawback of
the CuAAC procedure (Lallana, Fernandez-Megia, & Riguera, 2009;
Lallana, Fernandez-Trillo, et al., 2012; Lallana et al., 2011; Uchida &
Kawakishi, 1986). For this reason, the copper catalyst was removed
by EDTA complexation and washing after the reaction.
3
. Results and discussion
3
.1. Introduction of azide functionalities to the backbone of xylan
The introduction of azide groups to the backbone of xylan was
done following a method described for the functionalization of dex-
tran and cellulose (Pahimanolis et al., 2010; Yang et al., 2011). The
1
-azido-2,3-epoxypropanol was prepared in a one-pot synthesis
procedure starting with the ring-opening of epichlorohydrin with
azide-ion in the presence of acetic acid. The obtained 1-azido-3-
chloropropanol was in turn converted to the epoxide-form with
alkaline treatment in high yield (Fringuelli et al., 1999). Further,
the etherification reaction of 1-azido-2,3-epoxypropane with the
hydroxyl groups of xylan was carried out under alkaline conditions
A typical FT-IR spectrum of the crosslinked product in Fig. 3B
−
1
shows a reduction in the intensity of the azide peak at 2110 cm
and the characteristic peaks of xylan and PEG-PPG-PEG. Moreover,
1
3
C-CP/MAS NMR spectrum (Fig. 4) shows the typical peaks of both
xylan and PEG-PPG-PEG along with unreacted excess azide groups
(56 ppm) and in addition, peaks at around 148 and 128 ppm show-
ing the presence of the triazole ring formed during the CuAAC
reaction. It must be noted thought, that the formation of intra-
chain loops and the presence of unreacted loose ends is possible in
these types of bifunctional crosslinking systems (Ossipov & Hilborn,
2006).
(
Fig. 1), a method similar to the cationization and hydroxyalkylation
of xylan and other polysaccharides using epoxides (Bigard et al.,
2
2
2
011; Ebringerová et al., 1994; Jain et al., 2001; Kataja-aho et al.,
011; Laine et al., 2013; Schwikal et al., 2006; Tomasik & Schilling,
004). In this way, azide-groups necessary for the subsequent
CuAAC-reaction were introduced in a simple one step reaction,
without solvent-exchange or drying steps involved in the synthesis.
Since low-molecular weight organic azides are potentially explo-
sive substances, the obtained 1-azido-2,3-epoxypropane solution
was used for the etherification reaction without further purification
or concentration.
The thermal response of the hydrogels was investigated by
water absorption studies (Fig. 5). When PEG was used for the
crosslinking, the water absorption was affected only by a small
◦
amount when the temperature was increased from 7 to 70 C. On
the contrary, the thermoresponsive PEG-PPG-PEG gels, showed
a considerable drop in water content when heated. The hydro-
gel crosslinked with PE6100, also started losing water at lower
Table 1 shows the effect of the amount of added NaOH to
the obtained degree of substitution (DS) for the azide groups in

N. Pahimanolis et al. / Carbohydrate Polymers 102 (2014) 637–644
643
temperature, originating from the differences in cloud point, which
shows that the thermo-responsive properties can be adjusted by
the polymer used for crosslinking. The swelling/deswelling pro-
cess is also reversible, since cooling the gel causes a rise in swelling
ratio. The effect of temperature on the gels can be observed in the
SEM images of freeze-dried samples (Fig. 6), where the decrease in
water content results in a denser network structure as the pore size
Chimphango, A. F. A., van Zyl, W. H., & Görgens, J. F. (2012). In situ enzymatic aided
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Coviello, T., Matricardi, P., Marianecci, C., & Alhaiqu, F. (2007). Polysaccharide hydro-
gels for modified release formulations. Journal of Controlled Release, 119, 5–24.
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Biomacromolecules, 8, 1844–1850.
Daus, S., & Heinze, T. (2010). Xylan-based nanoparticles: Prodrugs for ibuprofen
release. Macromolecular Bioscience, 10, 211–220.
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◦
is reduced from approximately 10–20 ␮m at 7 C to around 2 ␮m
◦
at 70 C. No notable changes can be observed in the morphology
when PEG was used for the crosslinking.
◦
Fig. 7 shows the compressive moduli for the gels at 7 C and
◦ ◦
7
0 C. At 7 C, hydrogels crosslinked with PE6400 or PE6100 have
lower compressive moduli than the hydrogel crosslinked with PEG.
◦
This shows that at 7 C where PPG segments are water soluble, they
decrease the stiffness of the gels. Instead, the difference between
the PE6400 and PE6100 containing gels is not significant. The sit-
De Geest, B., Van Camp, W., Du Prez, F., De Smedt, S., Demeester, J., & Hennink, W.
(
2008b). Degradable multilayer films and hollow capsules via a ‘click’ strategy.
◦
uation changes, when the temperature is increased to 70 C. The
Macromolecular Rapid Communications, 29, 1111–1118.
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stiffness of PEG and PE6400 crosslinked gels drops slightly but
◦
◦
PE6100 crosslinked gel is clearly stiffer at 70 C than at 7 C. It
seems that the higher amount of PPG, which aggregates at high
temperature, makes the material stiffer. In addition, the length of
the polymer chain is shorter and crosslinking density higher for
PE6100, which may result in a decreased mobility of the network,
thus increasing the compressive modulus of the material.
1627–1640.
Ebringerová, A., & Heinze, T. (2000). Xylan and xylan derivatives – Biopolymers with
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(
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. Conclusions
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A simple, aqueous phase one-step synthesis route to prepare
azide functionalized xylan is presented. The azide functionalized
xylan is a valuable intermediate for broad modification possibilities
via the copper catalyzed azide-alkyne cycloaddition (CuAAc). The
azide groups were further reacted with alkyne bifunctional PEG or
PEG-PPG-PEG, yielding novel tunable thermoresponsive hydrogels.
Liquid and solid state NMR, FT-IR and elemental analysis were
used to characterize the obtained products. The hydrogels showed
reversible swelling at low temperature and deswelling at high tem-
perature. Depending on the length of the thermoresponsive PPG
segment, the compressive modulus either decreased or increased
Fang, J. M., Fowler, P., Tomkinson, J., & Hill, C. A. S. (2002). Preparation and character-
ization of methylated hemicelluloses from wheat straw. Carbohydrate Polymers,
4
7, 285–293.
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forward approach to novel macromolecular architectures. Chemical Society
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with sodium azide in water. A regioselective pH-controlled reaction. Journal of
Organic Chemistry, 64, 6094–6096.
◦
◦
at 70 C compared to 7 C. SEM images showed that the structure of
the PPG containing hydrogels was denser at high temperature than
without PPG or at low temperature.
Fundador, N. G. V., Enomoto-Rogers, Y., Takemura, A., & Iwata, T. (2012). Acetylation
and characterization of xylan from hardwood kraft pulp. Carbohydrate Polymers,
8
7, 170–176.
The obtained material might be of interest in, for instance, drug
release, particle separation or self-cleaning filtration applications.
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Acknowledgement
This work has been funded by the Graduate School for Biomass
Refining (Academy of Finland).
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