synthesis and thermoreversible gelation of diblock methylcellulose analogues via huisgen 1,3-dipolar...
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ORIGINAL PAPER
Synthesis and thermoreversible gelation of diblockmethylcellulose analogues via Huisgen 1,3-dipolarcycloaddition
Atsushi Nakagawa • Hiroshi Kamitakahara •
Toshiyuki Takano
Received: 12 January 2012 / Accepted: 9 April 2012 / Published online: 22 April 2012
� Springer Science+Business Media B.V. 2012
Abstract A novel synthetic method to link acety-
lated cellulose derivatives with methylated cellulose
derivatives via Huisgen 1,3-dipolar cycloaddition was
developed to produce 1,2,3-triazole-linked diblock
copolymers consisting of hydrophilic cellobiose or
low-molecular-weight cellulose and a hydrophobic
2,3,6-tri-O-methyl-cellulose. Huisgen 1,3-dipolar cyclo-
addition had the advantage over glycosylation reaction
of being able to connect a hydrophilic block having
higher molecular weight than cellobiose with a
hydrophobic 2,3,6-tri-O-methyl-cellulose block. As a
consequence, 2.0 wt% aqueous solutions of the 1,2,3-
triazole-linked diblock methylcellulose analogues
exhibited the thermoreversible gelation in water at
around 25 �C as same as that of b-(1 ? 4)-linked
diblock methylcellulose. Differential scanning calo-
rimetry measurements of 2.0 wt% aqueous solutions of
the diblock copolymers revealed that an important
structural factor for its thermoreversible gelation
was not a b-(1 ? 4)-glycosidic linkage between
hydrophilic and hydrophobic blocks of diblock meth-
ylcellulose, but a sequence of anhydro 2,3,6-tri-O-
methyl-glucopyranosyl units and that of unmodified
glucopyranosyl ones.
Keywords Diblock copolymer �Methylcellulose � Click chemistry � Triazole �Thermoreversible gelation � DSC
Introduction
This paper describes the important structural factor for
thermoreversible gelation via the self-assembly of the
amphiphilic diblock copolymers consisting of only
saccharide chains in both hydrophilic and hydrophobic
blocks. We have focused on the chemical structure of
crosslinking points of a methylcellulose (MC) gel, and
synthesized cooligomers and copolymers having
blocky functionalization patterns as models of indus-
trially produced MC (Kamitakahara et al. 2006, 2007,
2008, 2012; Kamitakahara and Nakatsubo 2010;
Nakagawa et al. 2011a, b, c).
The cross-linking points of MC play an important
role in thermoreversible gelation (Hirrien et al. 1998).
It is of academic significance to clarify the nature and
structure of the reversible cross-linking points respon-
sible for the solution behavior of MC to tune the
properties of the gel. To gain insight into the structure
of the cross-linking points, light scattering and X-ray
scattering studies have been undertaken on industri-
ally produced MCs with heterogeneous functionaliza-
tion pattern and laboratory-produced MCs with
relatively uniform functionalization pattern (Bodvik
et al. 2010; Zhou et al. 2008). Kato et al. (1978) have
reported that the crystalline regions consisting of 4–8
A. Nakagawa � H. Kamitakahara (&) � T. Takano
Graduate School of Agriculture, Kyoto University,
Kitashirakawa-Oiwake-cho, Sakyo-ku,
Kyoto 606-8502, Japan
e-mail: [email protected]
123
Cellulose (2012) 19:1315–1326
DOI 10.1007/s10570-012-9703-7
of 2,3,6-tri-O-methyl-glucopyranosyl units act as
‘crosslinking loci’ on heating. However, isolation of
well-defined MC fragments containing such a
sequence of 2,3,6-tri-O-methyl-glucopyranosyl units
from MC with heterogeneous functionalization pattern
were very difficult since they represent only a small
fraction of total MC (Adden et al. 2006).
Based on our model synthesis strategy, a diblock
MC copolymer consisting of hydrophilic cellobiosyl
and hydrophobic 2,3,6-tri-O-methyl-cellulosyl blocks
(GG-236MC) was synthesized via glycosylation reac-
tion of 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-(1 ? 4)-
2,3,6-tri-O-acetyl-a-D-glucopyranosyl 2,2,2-trichloro-
acetimidate with methyl 2,3,6-tri-O-methyl celluloside
having a hydroxyl group at C-4 of non-reducing end
and subsequent deacetylation to investigate thermal
aggregation behavior and thermoreversible gelation of
industrially produced MC (Nakagawa et al. 2011b).
As a result, 2.0 wt% aqueous solution of the diblock
copolymer, GG-236MC, formed a hydrogel at room
temperature, accompanied by the formation of hydro-
phobic environment (Nakagawa et al. 2011a). Our
synthesis approach to the structure–property relation-
ships achieved the results that a sequence of 2,3,6-tri-
O-methyl-glucopyranosyl units caused thermorevers-
ible gelation of MC, and that the length of hydrophobic
block influenced the gelation temperature. However,
the diblock copolymers having unmodified cello-
oligosaccharides with higher DP than cellobiose still
remain important synthetic targets to investigate the
influence of hydrophilic block on gelation.
It is a key determinant for synthesis of diblock
copolymer through glycosylation method whether a
glycosyl donor is actually available. Glycosylation of
the glycosyl donors having higher molecular weight
than cellobiose remains difficult. We therefore
planned to synthesize the diblock copolymers having
linkages other than glycosidic bond between hydro-
philic and hydrophobic blocks, and investigated the
influence of the linkage on the hydrogelation of the
diblock copolymers.
Several cellulosic diblock copolymers have been
synthesized by introduction of long alkyl groups into
the reducing end of cellulose. Kamitakahara et al. have
reported the synthesis of a cellulosic diblock copoly-
mer by introduction of long alkyl chain via amide
linkage, which was stable under alkaline condition for
removal of acetyl groups of cellulose triacetate block
(Kamitakahara and Nakatsubo 2005; Kamitakahara
et al. 2005). In this synthetic route for the cellulosic
diblock copolymer, cellulose triacetate having an
azide group at C-1 of reducing end has been synthe-
sized as a key synthetic intermediate by stepwise
modification of cellulose (Enomoto-Rogers et al.
2009, 2012).
Recently, a Cu(I)-catalyzed chemoselective cou-
pling between organic azides and terminal alkynes for
the formation of 1,2,3-triazoles has attracted attention
owing to its convenient, quick, and quantitative
reaction (Hasegawa et al. 2006). Among the multiple
reactions that could be termed ‘click chemistry’, the
Huisgen 1,3-dipolar cycloaddition has been success-
fully applied for the synthesis of glycosyl triazoles
(Neto et al. 2010; Wilkinson et al. 2006; Chittaboina
et al. 2005), oligosaccharide analogues (Marmuse et al.
2005; Hotha and Kashyap 2005), and polysaccharide
derivatives (Negishi et al. 2011; Zhang et al. 2008;
Elchinger et al. 2011; Liebert et al. 2006; Koschella
et al. 2011; Enomoto-Rogers et al. 2012). Most of the
reports regarding polysaccharides have focused on the
application of ‘click chemistry’ to synthesize graft type
copolymers of alkyne or azide compounds onto
polysaccharide chains such as 6-azide-6-deoxycellu-
lose (Negishi et al. 2011; Liebert et al. 2006; Pohl et al.
2008, 2009), 6-azide-6-deoxycurdlan (Hasegawa et al.
2006), and 3-O-propargyl-cellulose (Fenn et al. 2009).
As far as we know, there have been no reports on the
synthetic method for linking cellulose derivatives
having an azide group at C-1 of reducing end and an
alkyne group at C-4 of non-reducing end via ‘click
chemistry’ (Schatz and Lecommandoux 2010). Thus,
we describe here the synthesis of 1,2,3-triazole-linked
diblock copolymers via ‘click’ reaction and their
thermoreversible gelation properties in water. Essen-
tial factor for the thermoreversible gelation of 1,2,3-
triazole-linked diblock MC analogues will be
discussed.
Results and discussion
Basic design for diblock MC analogues
Figure 1 illustrates our synthesis strategy for preparation
of diblock methylcellulose analogues via Huisgen 1,3-
dipolar cycloaddition. Methyl 2,3,6-tri-O-methyl-cellu-
loside having a propargyl group at C-4 of non-reducing
end (2: 2a, 2b) was synthesized by propargylation of a
1316 Cellulose (2012) 19:1315–1326
123
hydroxyl group of methyl 2,3,6-tri-O-methyl-cellulo-
side prepared according to a methanolysis published in
our recent paper (Nakagawa et al. 2011b). Peracetyl b-
cellobiosyl azide (3) and peracetyl b-cellulosyl azide (6)
(DPn = 6.9, Mw/Mn = 1.83) were converted from
cellobiose and cellulose, respectively (Gyorgydeak
et al. 1993; Kamitakahara and Nakatsubo 2005; Kami-
takahara et al. 2005). Copper(I)-catalyzed Huisgen 1,3-
dipolar cycloaddition of compounds 2a and 2b (terminal
alkyne) to glycosyl azides 3 and 6 gave compounds 4
and 7 and subsequent removal of acetyl groups afforded
1,2,3-triazole-linked diblock MC analogues 5 and 8,
respectively, as shown in Fig. 3.
Synthesis of diblock MC analogue 5 via Huisgen
1,3-dipolar cycloaddition
Synthesis of alkyne compound 2 and azide compound 3
Propargylation of compound 1 using 3-bromopropyne
in the presence of NaH in DMF gave compound 2a in
54 % yield, as shown in Fig. 2. In the 1H-NMR
spectrum of compound 2a measured in CDCl3, the
resonance of a methyne proton of a propargyl group
appeared at 2.46 ppm as triplet. The molecular
weights and polydispersity index of compound 2 (2a,
2b) were summarized in Table 1. MALDI-TOF MS
spectrum of compound 2a will be shown in later
section. Cellobiose octaacetate was converted into
peracetyl b-cellobiosyl azide (3) by using trimethyl-
silylazide, according to the literature (Kamitakahara
and Nakatsubo 2005) in 80 % yield in two reaction
steps.
Cycloaddition of alkyne compound 2a and azide
compound 3
Diblock MC analogue 5 was synthesized according to
the reaction sequence, as shown in Fig. 3. The amount
of peracetyl b-cellobiosyl azide (3), Cu(I)Br, sodium
ascorbate, and N, N, N0, N00, N000-pentamethyldiethy-
lenetriamine (PMDETA) were calculated using the
number-average molecular weight (Mn) of compound
2a estimated by GPC analysis, since compound 2a was
a polydisperse mixture of compounds having different
molecular weights.
In the case of glycosylation for the synthesis of
diblock MC copolymers exemplified with methyl b-D-
glucopyranosyl-(1 ? 4)-b-D-glucopyranosyl-(1 ? 4)-
2,3,6-tri-O-methyl-celluloside, the excess amount of the
glycosyl donor, 2,3,4,6-tetra-O-acetyl-b-D-glucopyran-
osyl-(1 ? 4)-2,3,6-tri-O-acety-a-D-glucopyranosyl
2,2,2-trichloroacetimidate (20 equivalents), was required
to obtain a glycosylated product. In contrast, ‘click’
cycloaddition requires less amount of hydrophilic build-
ing block, peracetyl cellobiosyl azide (3), compared with
glycosylation reaction. Two equivalents of peracetyl
b-cellobiosyl azide (3) was reacted with compound 2a
(1.0 equivalent) in the presence of Cu(I)Br (10 equiva-
lent), sodium ascorbate (20 equivalents), and PMDETA
(10 equivalents) for 24 h at r.t. The cycloaddition product
was purified by silica gel chromatography (eluent:
EtOAc) to give compound 4 in 85 % yield.
The chemical structure of ‘click’ cycloaddition
product was supported by NMR spectroscopy, as
shown in Fig. 4. The characteristic resonances of a
methyne proton (2.46 ppm as triplet) of compound 2a
and H-1 (4.62 ppm as doublet with coupling constant
J = 9.0 Hz) of compound 3 disappeared in 1H-NMR
spectrum. The resonance corresponding to the ano-
meric center of the glucopyranose residue attached to
the triazole unit was observed at 5.82 ppm as a doublet
with J = 9.0 Hz. The triazole proton appeared at
7.69 ppm as a singlet, indicating the formation of a
single isomer. The molecular weight and polydisper-
sity index of compound 4 were summarized in
Table 1. The DPn of compound 4 (DPn = 27.3) was
higher than compound 2a (DPn = 25.9), resulting
Fig. 1 Syntheses of diblock
methylcellulose analogues 4and 7 via Huisgen 1,3-
dipolar cycloaddition
between terminal alkyne 2and peracetyl glycosyl
azides 3 and 6
Cellulose (2012) 19:1315–1326 1317
123
from the connection of peracetyl cellobiosyl block
with hydrophobic 2,3,6-tri-O-methyl-cellulosyl block.
Acetyl groups of compound 4 were removed using
NaOMe in MeOH and THF at r.t. to afford compound
5 in 99 % yield. Figure 5 shows 1H-NMR spectrum of
compound 5 measured in D2O. The triazole proton
resonance appeared at 8.31 ppm as a singlet. In
addition, the resonance corresponding to the anomeric
center of the glucopyranose residue attached to the
triazole unit was visible at 5.79 ppm as a doublet with
J = 9.0 Hz, indicating that deacetylation had no
influence on the resonance of H-1(b) proton. The1H-NMR spectrum of compound 5 revealed that acetyl
groups were successfully removed without any
cleavage of triazole linkage under alkali reaction
condition to give a diblock MC analogue 5.
The MALDI-TOF MS spectra of compounds 2a, 4,
and 5 were measured using 2,5-dihydroxybenzoic acid
(DHB) as a matrix in positive linear mode, as shown in
Fig. 6. The observed molecular weights of compounds
were in good agreement with their calculated molecular
weights, indicating that reactions proceeded as
expected. The regular side peaks a and b (shown in
insets in Fig. 6) could be assigned to be the mass peaks
of compound 1 and methyl 2,3,6-tri-O-methyl-cellu-
loside having a methyl group at C-4 of non-reducing
end derived from original 2,3,6-tri-O-methyl-cellu-
lose, respectively.
a3-Bromopropyne / NaH / DMF/ 70 °C / 48 h
Fig. 2 Synthetic rote for
compound 2a
Table 1 Molecular weights and DPs of compounds 2, 4, 6 and 7
Compound Mn (9103) Mw (9103) Mw/Mn DPnc mc nc
2a (Alkyne) 5.3 8.3 1.56 25.9 – 25.9
2b (Alkyne) 5.8 8.1 1.39 28.4 – 28.4
6 (Azide) 2.0 3.7 1.85 6.9 6.9 –
4a 5.8 8.6 1.48 27.3 2 25.3
7b 7.7 11.0 1.42 35.0 6.5 28.5
a Peracetylated cellobiosyl azide (3) ? 2a, b 6 ? 2b, c calculated by 1H-NMR spectroscopy
m Degree of polymerization of hydrophilic block
n Degree of polymerization of hydrophobic block
a Cu(I)Br / Sodium ascorbate / PMDETA / 20% MeOH / CH2Cl2 / r.t. / 24 h; b 28% NaOCH3 in MeOH / MeOH / THF / r.t. / 12 h.
Fig. 3 Synthetic route
for diblock MC analogues
5 and 8
1318 Cellulose (2012) 19:1315–1326
123
Cycloaddition of alkyne compound 2b and azide
compound 6
Figure 3 shows synthetic route for diblock MC
analogue 8 having low-molecular-weight cellulose as
a hydrophilic block. Compound 7 was synthesized
according to the synthetic procedure of compound 4.
Cellulose triacetate with low molecular weight syn-
thesized by acetolysis of cellulose according to the
literature (Miller et al. 1960) was converted into
peracetyl b-cellulosyl azide (6) (DPn = 6.9, Mw/
Mn = 1.39) (Kamitakahara et al. 2005). Huisgen
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
Chemical Shift (ppm)
H2(d)
-COCH3
H1(d)
H3(d)
H5(d)
H6(d)
H4(d)
-OCH3
123
4 56
Triazole proton7.69 ppm
H1(b)5.82 ppmJ=9.0 Hz
(a) (b) (c) (d) (e)
123
4 56
Triazole proton
H3(b)H2(b)
H4(a)H2(a)
H1(a)H3(a)
78
9
H4(b)H5(b)
H6(a)H6(b)
H7
Fig. 4 1H-NMR spectrum
of compound 4 taken in
CDCl3
H2(d)
-OCH3
H1(d)
H3(d)H5(d)
H6(d)
H4(d)
Triazole proton8.31ppm
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
Chemical Shift (ppm)
123
4 56
H1(b)5.79 ppmJ=9.0 Hz
(b) (c) (d) (e)Triazole proton
5
123
4 56
78
9(a)
Fig. 5 1H-NMR spectrum
of compound 5 taken in D2O
Cellulose (2012) 19:1315–1326 1319
123
1,3-dipolar cycloaddition of compound 6 with com-
pound 2b using Cu(I)Br (10 equivalent), sodium
ascorbate (20 equivalent), and PMDETA (10 equiv-
alent) for 24 h at r.t. and subsequent purification by
silica gel chromatography (eluent: EtOAc) gave the
cycloaddition product 7 in 72 % yield. Two equiva-
lents of compound 6 to compound 2b were at least
enough to obtain the target compound 7. In 1H-NMR
spectrum of compound 7 as shown in Fig. 7, the
characteristic resonances of a methyne proton
(2.46 ppm as triplet) of compound 2b and H-1
(4.62 ppm as doublet) of compound 6 disappeared.
The resonance corresponding to the anomeric center
of the glucopyranose residue attached to the triazole
unit was observed at 5.81 ppm as a doublet with
J = 9.0 Hz. The triazole proton was visible at
7.70 ppm as a singlet.
The molecular weights, DPs, and the polydispersity
index (Mw/Mn) of compound 7 were estimated by GPC
measurements. The block lengths (m, n) of compound
7 were calculated using the resonances of H-1(c) at
5.81 ppm, H-2(e) at 2.95 ppm, and acetyl protons at
1.86–2.14 ppm. As shown in Table 1, the molecular
weight of compound 7 (Mn = 7.7 9 103) was the
approximate sum of the molecular weights of com-
pounds 2b (Mn = 5.8 9 103) and 6 (Mn = 2.0 9
103), indicating that the cycloaddition of compounds
2b and 6 proceeded successfully.
Acetyl groups of compound 7 were removed using
NaOMe in MeOH and THF at r.t. to give compound 8
in 96 % yield. Figure 8 shows 1H-NMR spectrum of
compound 8 measured in D2O. The triazole proton
resonance appeared at 8.31 ppm as a singlet. In
addition, the resonance corresponding to the anomeric
center of the glucopyranose residue attached to the
triazole unit was visible at 5.79 ppm as a doublet with
J = 9.0 Hz.
Thermal property of aqueous solution of diblock
methylcellulose analogues 5 and 8
We have reported that 2.0 wt% aqueous solution of the
diblock copolymer, methyl b-D-glucopyranosyl-(1 ? 4)-
b-D-glucopyranosyl-(1 ? 4)-2,3,6-tri-O-methyl-cell-
uloside (GG-236MC), were soluble in water at *0 �C
and formed a gel at *25 �C (Nakagawa et al. 2011a).
Compounds 5 and 8 were water-soluble with
concentration of 2.0 wt% at *0 �C, and were par-
tially soluble in water with concentration of 2.0 wt%
at *25 �C. The water-insoluble [5a (50.3 %) and 8a
(89.3 %)] and water-soluble fractions [5b (49.7 %)
and 8b (10.7 %)] of compounds 5 and 8 were collected
by centrifugation method at *25 �C. The molecular
weights, polydispersity index, DPns, and DS of
compounds 5a, 5b, 8a, 8b, and GG-236MC were
summarized in Table 2.
Figure 9 shows the photographs of 2.0 wt% aque-
ous solutions of compounds 5a, 8a, and GG-236MC.
The thermoreversible gelations of compounds 5a and
8a could be clearly observed at around 25 �C within
13
12DP=11
Positive linear mode, DHB matrix
14 1516
1718 19 20 21 22 23 24
13
1098DP=7
1112 13 14 15 16 17 18 19 20 21 22 23 24
34
25
12
11109DP=7
1415
1617 18 19 20 21 22 23 24 25 26
(a)
(b)
(c)
30002900
[M+Na]+
[M+Na]+
30002900
[M+Na]+
30002900
ab
a b
b
26 27 28 29 30 31 32 33 34 35
827 28 29 30 31 32
25 26 27 28 29 30 28 29 30 31 32 33
2000 3000 4000 5000 6000 7000 8000m/z
Fig. 6 MALDI-TOF MS
spectra of compounds 2a(a), 4 (b), and 5 (c). The
m/z values of sodium adduct
ions, [M ? Na]? of
compounds were observed.
DP values of compounds 4(b) and 5 (c) show the total
DP (DP = n?2). Insetenlarged spectra
corresponding to the mass
range m/z 2,850–3,050.
Series a: [M (compound
1) ? Na]?, series b:
[M (compound 1) ? 14
(Me) ? Na]?
1320 Cellulose (2012) 19:1315–1326
123
10 min after dissolving at *0 �C. Compounds 5a and
8a showed the same macroscopic hydrogel formation
as that of GG-236MC. Thus, we investigated thermal
properties of 2.0 wt% aqueous solutions of com-
pounds 5a, 5b, 8a, and 8b by means of DSC
measurements to compare with that of GG-236MC.
DSC analysis of aqueous solution of GG-236MC
showed endo- and exothermic peaks under heating and
cooling, respectively, depending on the molecular
weight and concentration (Nakagawa et al. 2011a).
These endo- and exothermic peaks have suggested
that the dehydration of hydrated compounds and
AN-747-re
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
Chemical Shift (ppm)
123
4 56
H1(e)
H3(e)H5(e)
H6(e)
H4(e)
-COCH3
H2(e)
-OCH3
Triazole proton7.70 ppm
H1(c)5.81 ppm
J=9.0 Hz
(a) (b) (c) (d) (f)
Triazole proton
(e)
123
4 5
6
H3(c)H2(c)
H4(b)H2(b)
H1(b)H3(b)
H4(c)H5(c)
H6(b)H6(c)
H7
78
9
Fig. 7 1H-NMR spectrum
of compound 7 taken in
CDCl3
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
Chemical Shift (ppm)
H2(e)
-OCH3
H3(e)H5(e)
H6(e)
H4(e)
Triazole proton 8.31ppm
H1(c)5.79 ppmJ=9.0 Hz
(a) (b) (c) (d) (f)Triazole proton
(e)
123
4 56
123
4 56
78
9
H1(e)
Fig. 8 1H-NMR spectrum
of compound 8 taken in D2O
Cellulose (2012) 19:1315–1326 1321
123
rehydration of dehydrated compounds occurred upon
heating and cooling, respectively (Sarkar and Walker
1995).
DSC analysis of diblock MC analogues 5a, 5b, 8a,
8b and real diblock MC (GG-236MC) showed endo-
and exothermic peaks under heating and cooling
process, respectively. DSC curves at the second cycle
of GG-236MC and the diblock MC analogues 5a, 5b,
8a, 8b with a heating and cooling rate 3.5 �C/min are
shown in Fig. 10. In the case of GG-236MC having
Mn = 6.3 9 103, endothermic peaks appeared at
33 �C, whereas exothermic peaks appeared at 7 �C.
Compound 5a having Mn = 7.4 9 103 exhibited the
same endo- and exothermic peaks as GG-236MC. In
contrast, the endo- and exothermic peak temperatures
of compound 5b having Mn = 3.6 9 103 were higher
than those of compound 5a. The temperature of endo-
and exothermic peaks decreased with increasing the
hydrophobic saccharide chain length. The endother-
mic peak temperature of compound 8 increased from
35 to 37 �C with decreasing Mn from 8.2 9 103 to
6.5 9 103. Consequently, DSC measurement revealed
that the peak temperatures of diblock MC analogues
5a and 8a were almost the same as those of GG-
Table 2 The Mn, Mw/Mn, DPn of hydrophobic block, and endothermic peak of compounds 5a, 5b, 8a, 8b, and GG-236MC
Compound Mn (9103) Mw/Mn DPn DS Endothermic
peak (�C)Hydrophilic blocka Hydrophobic blocka
5a 7.4 1.39 2.0 33.0 2.80 33
5b 3.6 1.28 2.0 14.1 2.63 42
8a 8.2 1.35 9.0 26.7 2.23 35
8b 6.5 1.22 5.4 23.4 2.45 37
GG-236MC 6.4 1.27 2.0 26.2 2.76 33
a Calculated by 1H-NMR spectroscopy after acetylation
~0 ºC 25 ºC
DPn = 35.0, Mw/Mn = 1.39 , DS = 2.80
DPn = 35.7, Mw/Mn = 1.35 , DS = 2.28
5a
8a
n=33.0
n=26.7m=9.0
DPn = 28.2, Mw/Mn = 1.27, DS = 2.76
n=26.2GG-236MC
Fig. 9 Photographs of 2.0 wt% aqueous solutions of compounds 5a, 8a, and GG-236MC
1322 Cellulose (2012) 19:1315–1326
123
236MC, suggesting that the triazole between hydro-
philic and hydrophobic blocks did not affect the
hydration–dehydration phenomena obviously.
Conclusions
This paper represents the new synthesis method based
on ‘click chemistry’ for well-defined 1,2,3-triazole-
linked diblock MC analogues to unravel a key
structure for the thermoreversible gelation of diblock
copolymers consisting of only sugar chains in both
hydrophobic and hydrophilic blocks. Huisgen 1,3-
dipolar cycloaddition of peracetyl glycosyl azides of
cellobiose and low DP cellulose with terminal alkyne
derivatives converted from 2,3,6-tri-O-methyl-cellu-
lose afforded the desired 1,2,3-triazole-linked diblock
copolymers successfully. ‘Click chemistry’ was capa-
ble of reacting a polymeric terminal alkyne with
peracetylated cellulosyl azide having DPn = ca. 7.0.
Our synthetic method via ‘‘click chemistry’’ made it
possible not only to require less amount of a hydro-
philic building block, peracetyl cellobiosyl azide, but
also to link a hydrophilic cello-oligosaccharide block
having DPn of ca. 7.0 with a hydrophobic 2,3,6-tri-O-
methyl-cellulose block compared to glycosylation
reaction. Consequently, 2.0 wt% aqueous solutions
of the resultant diblock copolymers consisting of
hydrophilic cellobiose and low DP cellulose and
hydrophobic sequence of 2,3,6-tri-O-methyl-cellulo-
syl blocks formed hydrogels at r.t. DSC measurements
revealed that the triazole unit between hydrophilic
and hydrophobic blocks of such 1,2,3-triazole-linked
diblock copolymers had almost no influence on endo-
and exothermic peak temperatures caused by dehy-
dration and hydration, respectively. Our results
demonstrated that the diblock MC structure of hydro-
philic unmodified cello-oligosaccharides and a hydro-
phobic 2,3,6-tri-O-methyl-cellulose having DPn of
20–30 is sufficient condition for the thermoreversible
gelation of aqueous MC solution. The synthetic
strategy using click reaction reported in this paper
should help not only in controlling the thermal
properties of diblock MC analogues, but also in
understanding the relationship between structure of
commercial MC and thermoresponsive property.
Experimental details
Materials
SM-400 methylcellulose sample (methoxyl content:
27.5–31.5 %; viscosity of 2 % aqueous solution:
400 mPa s) was kindly provided by Shinetsu Chem-
ical Co., Japan. The products were purified on silica
Fig. 10 The second heating and cooling runs of 2.0 wt% aqueous solutions of compounds 5a, 5b, 8a, 8b, and GG-236MC at 3.5 �C/
min. a 5a; b 5b; c 8a; d 8b; e GG-236MC
Cellulose (2012) 19:1315–1326 1323
123
gel column chromatography (Wakogel C-200, Wako
Pure Chemical Industries or Silica Gel 60 N [spher-
ical, neutral], 100–210 lm, Kanto Chemical Co.).
General measurements
1H- and 13C-NMR spectra were recorded with a
Varian Inova 500 FT-NMR (500 MHz) spectrometer
in chloroform-d with tetramethylsilane as an internal
standard or in deuterium oxide with 3-(trimethylsilyl)-
1-propanesulfonic acid sodium salt as an external
standard. Chemical shifts (d) and coupling constants
(J) are given in d-values (ppm) and Hz, respectively.
Matrix assisted laser desorption/ionization time-of-
flight mass (MALDI-TOF MS) spectra were recorded
with a Bruker MALDI-TOF MS Autoflex III in the
positive ion and linear modes. For ionization, a
nitrogen laser was used. All spectra were measured
in the linear mode using external calibration. MALDI-
TOF MS spectra were measured with 2,5-dihydroxy-
benzoic acid (DHB) as a matrix. A Shimadzu liquid
chromatography injector (LC-10ATvp), a Shimadzu
column oven (CTO-10Avp), a Shimadzu ultraviolet
visible detector (SPD-10Avp), a Shimadzu refractive
index detector (RID-10A), a Shimadzu communica-
tion bus module (CBM-10A), a Shimadzu LC work-
station (CLASS-LC10), and Shodex columns (KF802,
KF802.5, and KF805) were used. Number and weight
averaged molecular weights (Mn, Mw) and polydis-
persity indices (Mw/Mn) were estimated using poly-
styrene standards (Shodex). A flow rate of 1 mL/min
at 40 �C was chosen. Chloroform was used as eluent.
Methyl 2,3,6-tri-O-methyl celluloside having
a propargyl group at C-4 of non-reducing end (2)
Methyl 2,3,6-tri-O-methyl celluloside (1) was synthe-
sized from industrially produced MC (SM-400)
according to the procedure in the literature (Nakagawa
et al. 2011b). To a solution of compound (1)
(1.0171 g, 4.98 mmol) in DMF (40 mL), NaH
(2.4898 g, 62.2 mmol, 60 % in mineral oil) was added
at r.t. Then 3-bromopropyne (4.5 mL, 60.1 mmol) was
added into the solution at 0 �C. The reaction mixture
was stirred at 70 �C for 48 h. MeOH (2.0 mL) was
added to the reaction mixture, and then the mixture
was poured slowly into MeOH (200 mL). The result-
ing precipitates were washed with MeOH (400 mL)
and n-hexane (100 mL) and dried to give compound
2a (0.5502 g, 54 % yield), 2b (56 % yield). Compound
2a: 1H-NMR (CDCl3):d 2.46 (CH2C:CH), 2.95 (H-2),
3.22 (H-3), 3.30 (H-5), 3.39 (C6–OCH3), 3.54 (C2–
OCH3), 3.58 (C3–OCH3), 3.62–3.66 (H-6a), 3.69 (H-4),
3.72–3.81 (H-6b), 4.33 (H-1), 4.29–4.43 (m, CH2C:CH). MALDI-TOF MS (positive linear mode; DHB as
matrix): the m/z with starting mass: DP = 7, Calcd. =
1,498; Found [M ? Na]? = 1,521, the increment
between DPn and DPn?1: 204, the range of detected
compounds: DP = 7–34.
Peracetylated glycosyl azides
Peracetylated glycosyl azides of cellobiose and low-
molecular-weight cellulose were synthesized from
cellobiose and cellulose, respectively, according to
the literature (Kamitakahara and Nakatsubo 2005;
Kamitakahara et al. 2005).
General method for Huisgen 1,3-dipolar
cycloaddition
To a solution of peracetylated glycosyl azide (2.0
equiv.) and methyl 2,3,6-tri-O-methyl celluloside hav-
ing terminal alkyne at C-4 of reducing end (1.0 equiv.) in
MeOH/CH2Cl2 (2 mL, 1/4, v/v), Cu (I) Br (10 equiv.),
sodium ascorbate (20 equiv. 4 M in H2O), and PMD-
ETA (10 equiv.) were added at r.t. The reaction mixture
was stirred at r.t. for 24 h. After 24 h, the mixture was
passed through silica gel chromatography column
eluted with 20 % MeOH/CH2Cl2 to give a crude
product. The crude product was purified by silica gel
column chromatography (eluent: EtOAc ? 20 %
MeOH/CH2Cl2) to give a reaction product.
Compound 4 (85 % yield): 1H-NMR (CDCl3): d1.87–2.10 (OCOCH3), 2.95 (H-2d), 3.21 (H-3d), 3.29
(H-5d), 3.38 (C6–OCH3), 3.65–3.78 (H-6d), 3.69 (H-
4d), 3.88–3.96 (H-4b, H-5b), 4.05–4.51 (H-6a, H-6b),
4.34 (H-1d), 4.56 (H-1a), 4.75–4.95 (H-7), 4.93
(H-2a), 5.09 (H-4a), 5.17 (H-3a), 5.38 (H-3b), 5.43
(H-2b), 5.82 (H-1b), 7.69 (H-9). MALDI-TOF MS
(positive linear mode; DHB as matrix): the m/z with
starting mass: DP = 11, Calcd. = 2,568; Found
[M ? Na]? = 2,590, the increment between DPn
and DPn ? 1: 204, the range of detected compounds:
DP = 11–35.
Compound 7 (72 % yield): 1H-NMR (CDCl3): d1.86–2.14 (OCOCH3), 2.95 (H-2d), 3.21 (H-3d), 3.29
(H-5d), 3.38 (C6–OCH3), 3.62–3.77 (H-6d), 3.69
1324 Cellulose (2012) 19:1315–1326
123
(H-4d), 3.88–3.94 (H-4c, H-5c), 4.02–4.52 (H-6b,
H-6c), 4.34 (H-1d), 4.42 (H-1b), 4.74–4.97 (H-7), 4.82
(H-2b), 5.03–5.17 (H-3b, H-4b), 5.34–5.43 (H-2c,
H-3c), 5.81 (H-1c), 7.70 (H-9).
General method for deacetylation
To a solution of acetylated MC analogue in MeOH
(1 mL) and THF (1 mL), NaOMe (10 equiv. per AGU)
were added at r.t. The mixture was stirred at r.t.
overnight. The solution was neutralized with DOWEX
H?. DOWEX H? was filtered off and washed with 20 %
MeOH/CH2Cl2. Combined filtrate and washings were
concentrated to dryness to give diblock MC analogue.
Compound 5 (99 % yield): 1H-NMR (D2O): d 3.14
(H-2d), 3.40 (C6–OCH3), 3.45 (H-3d), 3.56 (C2–
OCH3), 3.58 (C3–OCH3), 3.62–3.96 (H-4d, H-5d,
H-6d), 4.42 (H-1d), 8.31 (H-9). MALDI-TOF MS
(positive linear mode; DHB as matrix): the m/z
with starting mass: DP = 7, Calcd. = 1,457; Found
[M ? Na]? = 1,480, the increment between DPn and
DPn ? 1: 204, the range of detected compounds:
DP = 7–32.
Compound 8 (96 % yield): 1H-NMR (D2O): d 3.14
(H-2d), 3.41 (C6–OCH3), 3.45 (H-3d), 3.57 (C2–
OCH3), 3.59 (C3–OCH3), 3.65–3.96 (H-4d, H-5d,
H-6d), 4.43 (H-1d), 8.31 (H-9).
Differential scanning calorimetry (DSC)
measurement
DSC thermograms were recorded on a DSC823e
(Mettler Toledo, Zurich, Switzerland) under nitrogen
atmosphere during a heating/cooling cycle (0 ? 90
? 0 �C) with a heating and cooling rate 3.5 �C/min.
Each temperature cycle was sequentially repeated
three times in order to ensure and check the repro-
ducible response of the instrument.
Acknowledgments This investigation was supported in part
by a Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, and Culture of Japan (Nos. 18680009 and
21580205).
References
Adden R, Melander C, Brinkmalm G, Gorton L, Mischnick P
(2006) New approaches to the analysis of enzymatically
hydrolyzed methyl cellulose. Part 1. Investigation of the
influence of structural parameters on the extent of degra-
dation. Biomacromolecules 7(5):1399–1409
Bodvik R, Dedinaite A, Karlson L, Bergstrom M, Baverback P,
Pedersen JS, Edwards K, Karlsson G, Varga I, Claesson
PM (2010) Aggregation and network formation of aqueous
methylcellulose and hydroxypropylmethylcellulose solu-
tions. Colloids Surf A Physicochem Eng Aspects 354(1–3):
162–171
Chittaboina S, Xie F, Wang Q (2005) One-pot synthesis of tri-
azole-linked glycoconjugates. Tetrahedron Lett 46(13):
2331–2336
Elchinger P-H, Faugeras P-A, Boens B, Brouillette F, Mont-
plaisir D, Zerrouki R, Lucas R (2011) Polysaccharides: the
‘‘Click’’ chemistry impact. Polymers 3(4):1607–1651
Enomoto-Rogers Y, Kamitakahara H, Takano T, Nakatsubo F
(2009) Cellulosic graft copolymer: poly(methyl methac-
rylate) with cellulose side chains. Biomacromolecules
10(8):2110–2117
Enomoto-Rogers Y, Kamitakahara H, Yoshinaga A, Takano T
(2012) Comb-shaped graft copolymers with cellulose side-
chains prepared via click chemistry. Carbohydr Polym
87(3):2237–2245
Fenn D, Pohl M, Heinze T (2009) Novel 3-O-propargyl cellu-
lose as a precursor for regioselective functionalization of
cellulose. React Funct Polym 69(6):347–352
Gyorgydeak Z, Szilagyi L, Paulsen H (1993) Synthesis, struc-
ture and reactions of glycosyl azides. J Carbohydr Chem
12(2):139–163
Hasegawa T, Numata M, Sakurai K, Shinkai S (2006) ‘‘Click
chemistry’’ on polysaccharides: a convenient, general, and
monitorable approach to develop b-1,3-glucans with vari-
ous functional appendages. Carbohydr Res 341:35–40
Hirrien M, Chevillard C, Desbrieres J, Axelos MAV, Rinaudo M
(1998) Thermogelation of methylcelluloses: new evidence
for understanding the gelation mechanism. Polymer 39(25):
6251–6259
Hotha S, Kashyap S (2005) ‘‘Click Chemistry’’ inspired syn-
thesis of pseudo-oligosaccharides and amino acid glyco-
conjugates. J Org Chem 71(1):364–367
Kamitakahara H, Nakatsubo F (2005) Synthesis of diblock
copolymers with cellulose derivatives. 1. Model study with
azidoalkyl carboxylic acid and cellobiosylamine deriva-
tive. Cellulose 12(2):209–219
Kamitakahara H, Nakatsubo F (2010) ABA- and BAB-triblock
cooligomers of tri-O-methylated and unmodified cello-
oligosaccharides: syntheses and structure–solubility rela-
tionship. Cellulose 17(1):173–186
Kamitakahara H, Enomoto Y, Hasegawa C, Nakatsubo F (2005)
Synthesis of diblock copolymers with cellulose deriva-
tives. 2. Characterization and thermal properties of cellu-
lose triacetate-block-oligoamide-15. Cellulose 12(5):
527–541
Kamitakahara H, Nakatsubo F, Klemm D (2006) Block co-
oligomers of tri-O-methylated and unmodified cello-oli-
gosaccharides as model compounds for methylcellulose
and its dissolution/gelation behavior. Cellulose 13(4):
375–392
Kamitakahara H, Nakatsubo F, Klemm D (2007) New class of
carbohydrate-based nonionic surfactants: diblock co-olig-
omers of tri-O-methylated and unmodified cello-oligosac-
charides. Cellulose 14(5):513–528
Cellulose (2012) 19:1315–1326 1325
123
Kamitakahara H, Yoshinaga A, Aono H, Nakatsubo F, Klemm
D, Burchard W (2008) New approach to unravel the
structure–property relationship of methylcellulose. Cellu-
lose 15(6):797–801
Kamitakahara H, Murata-Hirai K, Tanaka Y (2012) Synthesis of
blockwise alkylated tetrasaccharide-organic quantum dot
complexes and their utilization for live cell labeling with
low cytotoxicity. Cellulose 19(1):171–187
Kato T, Yokoyama M, Takahashi A (1978) Melting tempera-
tures of thermally reversible gels IV. Methyl cellulose–
water gels. Colloid Polym Sci 256(1):15–21
Koschella A, Hartlieb M, Heinze T (2011) A ‘‘click-chemistry’’
approach to cellulose-based hydrogels. Carbohydr Polym
86(1):154–161
Liebert T, Hansch C, Heinze T (2006) Click chemistry with
polysaccharides. Macromol Rapid Commun 27(3):
208–213
Marmuse L, Nepogodiev SA, Field RA (2005) ‘‘Click chemis-
try’’ en route to pseudo-starch. Org Biomol Chem 3(12):
2225–2227
Miller GL, Dean J, Blum R (1960) A study of methods for
preparing oligosaccharides from cellulose. Arch Biochem
Biophys 91(1):21–26
Nakagawa A, Fenn D, Koschella A, Heinze T, Kamitakahara H
(2011a) Physical properties of diblock methylcellulose
derivatives with regioselective functionalization patterns:
first direct evidence that a sequence of 2,3,6-tri-O-methyl-
glucopyranosyl units causes thermoreversible gelation of
methylcellulose. J Polym Sci, Part B: Polym Phys 49(21):
1539–1546
Nakagawa A, Fenn D, Koschella A, Heinze T, Kamitakahara H
(2011b) Synthesis of diblock methylcellulose derivatives
with regioselective functionalization patterns. J Polym Sci
Part A: Polym Chem 49(23):4964–4976
Nakagawa A, Kamitakahara H, Takano T (2011c) Synthesis of
blockwise alkylated (1 ? 4) linked trisaccharides as
surfactants: influence of configuration of anomeric position
on their surface activities. Carbohydr Res 346(13):
1671–1683
Negishi K, Mashiko Y, Yamashita E, Otsuka A, Hasegawa T
(2011) Cellulose chemistry meets click chemistry: syn-
theses and properties of cellulose-based glycoclusters with
high structural homogeneity. Polymers 3(1):489–508
Neto V, Granet R, Krausz P (2010) Novel class of non-ionic
monocatenary and bolaform alkylglycoside surfactants.
Synthesis by microwave-assisted glycosylation and olefin
cross-metathesis or by ‘click-chemistry’: physicochemical
studies. Tetrahedron 66(25):4633–4646
Pohl M, Schaller J, Meister F, Heinze T (2008) Selectively
dendronized cellulose: synthesis and characterization.
Macromol Rapid Commun 29(2):142–148
Pohl M, Michaelis N, Meister F, Heinze T (2009) Biofunctional
surfaces based on dendronized cellulose. Biomacromole-
cules 10(2):382–389
Sarkar N, Walker LC (1995) Hydration–dehydration properties
of methylcellulose and hydroxypropylmethylcellulose.
Carbohydr Polym 27(3):177–185
Schatz C, Lecommandoux S (2010) Polysaccharide-containing
block copolymers: synthesis, properties and applications of
an emerging family of glycoconjugates. Macromol Rapid
Commun 31:1664–1684
Wilkinson BL, Bornaghi LF, Poulsen S-A, Houston TA (2006)
Synthetic utility of glycosyl triazoles in carbohydrate
chemistry. Tetrahedron 62(34):8115–8125
Zhang F, Bernet B, Bonnet V, Dangles O, Sarabia F, Vasella A
(2008) 2-Azido-2-deoxycellulose: synthesis and 1,3-dipo-
lar cycloaddition. Helv Chim Acta 91(4):608–617
Zhou J, Xu Y, Wang X, Qin Y, Zhang L (2008) Microstructure
and aggregation behavior of methylcelluloses prepared in
NaOH/urea aqueous solutions. Carbohydr Polym 74(4):
901–906
1326 Cellulose (2012) 19:1315–1326
123