Comb-shaped graft copolymers with cellulose side-chains prepared via click chemistry

Article abstract of DOI:10.1016/j.carbpol.2011.10.055

Comb-shaped copolymers with cellobiose acetate or cellulose triacetate (CTA) side-chains, PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), were prepared by grafting N-(15-azidopentadecanoyl)-2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O- acetyl-β-d-glucopyranosyl)-β-d-glucopyranosylamine (CTA2-C15-N ₃) and N-(15-azidopentadecanoyl)-tri-O-acetyl-β-cellulosylamine (CTA13-C15-N₃, number average degree of polymerization (DP _n) = 13) onto poly(2-propyn-1-yl methacrylate) (PPMA, weight average degree of polymerization (DP_w, X + Y = 5.59 × 10²)) via "click chemistry". The copolymers were characterized by ¹H, ¹³C and two-dimensional NMR and size exclusion chromatography-multi-angle laser light scattering (SEC-MALS) measurements. The numbers of CTA side-chains (X) of PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15) were calculated as 4.03 × 10² and 2.45 × 10², respectively. Copolymers with cellulosic side-chains, PPMA-g-(CELL2-C15) and PPMA-g-(CELL13-C15), were successfully obtained after deacetylation of PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), respectively. X-ray diffraction measurements revealed that PPMA-g-(CELL13-C15) showed crystalline pattern of cellulose II, which is believed to have anti-parallel orientation.

Full text of DOI:10.1016/j.carbpol.2011.10.055

Carbohydrate Polymers 87 (2012) 2237–2245
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Comb-shaped graft copolymers with cellulose side-chains prepared via click
chemistry
Yukiko Enomoto-Rogers, Hiroshi Kamitakahara∗, Arata Yoshinaga, Toshiyuki Takano
Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Comb-shaped copolymers with cellobiose acetate or cellulose triacetate (CTA) side-chains,
PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), were prepared by grafting N-(15-azidopentadecanoyl)-
2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-␤-d-glucopyranosyl)-␤-d-glucopyranosylamine
(CTA2-C15-N3) and N-(15-azidopentadecanoyl)-tri-O-acetyl-␤-cellulosylamine (CTA13-C15-N3, num-
ber average degree of polymerization (DPn) = 13) onto poly(2-propyn-1-yl methacrylate) (PPMA, weight
average degree of polymerization (DPw, X + Y = 5.59 × 102)) via “click chemistry”. The copolymers were
characterized by 1H, 13C and two-dimensional NMR and size exclusion chromatography–multi-angle
laser light scattering (SEC–MALS) measurements. The numbers of CTA side-chains (X) of PPMA-g-(CTA2-
C15) and PPMA-g-(CTA13-C15) were calculated as 4.03 × 102 and 2.45 × 102, respectively. Copolymers
with cellulosic side-chains, PPMA-g-(CELL2-C15) and PPMA-g-(CELL13-C15), were successfully obtained
after deacetylation of PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), respectively. X-ray diffraction
measurements revealed that PPMA-g-(CELL13-C15) showed crystalline pattern of cellulose II, which is
believed to have anti-parallel orientation.
Received 3 September 2011
Received in revised form 20 October 2011
Accepted 20 October 2011
Available online 28 October 2011
Keywords:
Cellulose
Reducing-end
Comb-shaped graft copolymer
Cellulose side-chain
Click-chemistry
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
strategy (Enomoto-Rogers, Kamitakahara, Nakayama, Takano, &
Nakatsubo, 2009; Enomoto-Rogers, Kamitakahara, Takano,
&
Cellulose is
a
linear (1→4)-␤-glucopyranan having three
Nakatsubo, 2009). The number of cellulose graft chains per main-
chain was, however, only 3.86, because of low reactivity of a
cellulose macromonomer, while the weight average degree of
polymerization of poly(methyl methacrylate) main-chain was
4.14 × 102. It has been reported that the semi-flexible or rigid
macromonomers such as methacrylate-end capped poly(n-hexyl
isocyanate) (Kawaguchi, Mihara, Kikuchi, Lien, & Nagai, 2007; Se
& Aoyama, 2004) are hard to polymerize, whereas other flexi-
ble macromonomers such as poly(ethylene oxide) (Ito, Tomi, &
Kawaguchi, 1992) or poly(dimethylsiloxane) (Shinoda, Miller, &
Matyjaszewski, 2001) are not.
On the other hand, “grafting onto” (attachment of side-chains to
the backbone) strategy is known as another way to synthesize graft
copolymers (Hourdet, L’AIIoret, & Audebert, 1997; Kamitakahara
et al., 1998; Poe, Jarrett, Scales, & McCormick, 2004). Thus, the
“grafting onto” strategy was investigated to introduce more cel-
lulose molecules as side-chains compared to our former study, and
to control the orientation of cellulose chains.
hydroxyl groups per anhydroglucose unit. There have been several
studies on cellulose graft copolymers, and a variety of poly-
mers have been grafted onto the cellulose main-chain through its
hydroxyl groups at C2, C3, and C6 positions to alter properties of
cellulose or cellulosic materials (Dou & Jiang, 2007; Kang et al.,
2006; Nishio, 2006). On the other hand, there have been many
reports on glycopolymers with mono-, di-, or oligo-saccharides
as pendent groups binding at the reducing-end on a main-chain
(Kamitakahara et al., 1998; Ladmiral et al., 2006; Narumi, Miura,
et al., 2006; Narumi, Otsuka, et al., 2006; Ohno, Fukuda, & Kitano,
1998; Ohno, Tsujii, & Fukuda, 1998). However, to our knowledge,
there has been no report on a graft copolymer having cellulose side-
chains. A comb-shaped copolymer with cellulose side-chains could
achieve parallel orientation of cellulose molecules, and might have
unique unknown properties such as crystallinity.
We have therefore prepared a novel graft copolymer with
cellulose side-chains via free radical copolymerization of
a
cellulose macromonomer with methyl methacrylate, based on
“graft through” (homo- or co-polymerization of macromonomers)
Recently, the concept of “click chemistry” invented by Sharp-
less and coworkers (Kolb, Finn, & Sharpless, 2001; Wu et al., 2004)
has been introduced into the synthesis of polymeric materials
with well-defined and complex chain architectures. Specifically,
Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition between organic
azides and terminal alkynes for the transformation of 1,2,3-
triazoles has been received much attention as a highly efficient
∗
Corresponding author. Tel.: +81 75 753 6255; fax: +81 75 753 6300.
E-mail address: hkamitan@kais.kyoto-u.ac.jp (H. Kamitakahara).
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.10.055

2238
Y. Enomoto-Rogers et al. / Carbohydrate Polymers 87 (2012) 2237–2245
and stereoselective reaction coupled with an excellent compati-
bility for functional groups. Ladmiral et al. (2006) have reported
the preparation of glycopolymers by attaching azide containing
mono-saccharides onto the alkyne containing polymer main-chain
via “click chemistry”.
In the present study, based on “grafting onto” strategy and “click
chemistry”, we prepared comb-shaped copolymers with cellulose
side-chains, by grafting cellulose derivatives carrying a single azide
group onto the alkyne containing polymer main-chain. Chemical
structures of the synthesized graft copolymers and their crystalline
patterns were investigated.
methanol, filtered, and dried in vacuo to give an amorphous solid
(158.6 mg, 58.7% yield). 1H NMR (CDCl3): ı 0.20 (CH3 (TMS)), 0.95,
1.20 (CH3), 1.85, 1.95 (CH2), 4.60 (–O–CH2–). 13C NMR (CDCl3): ı
−0.24 (CH3 (TMS)), 17.4, 19.0 (CH3), 44.8, 45.1 (–CH2–C (CH3)–),
53.0 (–O–CH2–), 54.3 (–CH2–C (CH3)–), 92.3 (–C C-TMS), 98.4, 98.7
(–C C-TMS), 176.1, 176.5 (C O).
2.4. Poly(2-propyn-1-yl methacrylate) (PPMA)
To a solution of TMS-PPMA (128 mg) and acetic acid (50 ␮l.
1.5 eq. to PMA monomer unit) in tetrahydrofuran (THF) (10 ml),
TBAF·3H2O was added dropwise at −20 ◦C. The mixture was
stirred at room temperature for 3 h under nitrogen. The reac-
tion mixture was poured into methanol. The precipitate was
collected by centrifugation at 1000 rpm for 3 min, and dried in
vacuo to give an amorphous solid (78.1 mg, 96.4% yield). The
2. Experimental
2.1. Materials
number and weight average molecular weights (Mn,PS, Mw,PS
)
N-(15-Azidopentadecanoyl)-2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-␤-d-glucopyranosyl)-␤-d-glucopyranosylamine
(CTA2-C15-N3) (M = 874.97) and N-(15-azidopentadecanoyl)-
estimated by polystyrene standards were Mn,PS = 1.92 × 104 and
M
w,PS = 3.54 × 104, respectively. The absolute molecular weight
(Mw) was calculated as 6.94 × 104 by MALS measurements. The
weight average degree of polymerization (DPw) was calculated as
5.59 × 102 from Mw(PPMA)/M(PMA) = 6.94 × 104/124.21. The DPw
of PPMA is described as DPw = X + Y: X and Y are the numbers
of cellulosic graft chains and PMA units of the graft copoly-
mer (see Fig. 1). 1H NMR (CDCl3): ı 0.94, 1.09 (CH3), 1.90,
1.98 (CH2), 2.51 (–C CH), 4.62 (–O–CH2–). 13C NMR (CDCl3): ı
16.9, 18.9 (CH3), 44.8, 44.9 (–CH2–C (CH3)–), 52.2 (–O–CH2–),
53.9 (–CH2–C (CH3)–), 75.5 (–C CH), 77.2 (–C CH), 175.5, 176.7
(C O).
tri-O-acetyl-␤-cellulosylamine
(CTA13-C15-N3,
DPn = 13)
(Mn = 4.12 × 103, Mw = 5.33 × 103) were prepared as described
in our previous articles (Kamitakahara, Enomoto, Hasegawa,
&
Nakatsubo, 2005; Kamitakahara & Nakatsubo, 2005). The
cellulose and cellulose triacetate derivatives were abbreviated
as CELL or CTA with the number of the degree of polymeriza-
tion (DPn) of the cellulosic chain (2 or 13), followed by the
abbreviation of the pentadecanoyl group (C15), and the end
group (N3). MALDI-TOF MS measurements revealed that the
molecular weights of CTA13-C15-N3 with each DP value agreed
well with theoretical values, and quantitative substitution of
the reducing-end was confirmed. 2,2ꢀ-Azobis(isobutyronitrile)
(AIBN) was crystallized from ethanol before use. 3-(Trimethyl
silyl)-2-propyn-1-ol, methacryloyl chloride, triethylamine (Et3N),
N,N,Nꢀ,Nꢀ,Nꢀꢀ-pentamethyldiethylenetriamine (PMDETA), cop-
per(I)bromide (Cu(I)Br), tetrabutyl ammonium fluoride trihydrate
(TBAF·3H2O), and all other reagents were commercially obtained
and used without further purification.
2.5. PPMA-g-(CTA2-C15)
CTA2-C15-N3 (17.6 mg, 1 eq. to PMA monomer), PPMA
(2.5 mg), and PMDETA (7 ␮l, 2 eq. to PMA monomer) in N,N-
dimethylformamide (DMF) (0.3 ml) were loaded into a glass flask,
and degassed by three freeze-pump-thaw cycles. The flask was
purged with nitrogen, Cu(I)Br (5.7 mg, 2 eq. to PMA monomer)
was added, and the flask was degassed again and sealed. The
mixture was stirred at room temperature for 24 h. After comple-
tion of the reaction, the mixture was extracted with chloroform,
washed with water and brine, dried with Na2SO4, and concen-
trated to dryness. The crude product was analyzed by SEC–MALS
measurements. The compound was dried in vacuo to give an
amorphous solid PPMA-g-(CTA2-C15) (17.8 mg, 88.6% yield). The
2.2. 3-(Trimethylsilyl)-2-propyn-1-yl methacrylate (TMS-PMA)
3-(Trimethylsilyl)-2-propyn-1-yl methacrylate (TMS-PMA) was
prepared according to the previous article (Ladmiral et al., 2006;
Scarpaci et al., 2009). To a solution of 3-(trimethylsilyl)-2-propyn-
1-ol (1.0 ml, 1 eq.) and Et3N (1.5 ml, 1.5 eq.) in dichloromethane
(2.0 ml), methacryloyl chloride (1.0 ml, 1.5 eq.) was added. The
mixture was stirred at room temperature for 0.5 h under nitro-
gen. After completion of the reaction, the mixture was extracted
with ethyl acetate, washed with water and brine, dried with
Na2SO4, and concentrated to dryness. Crude product was puri-
fied by open preparative column chromatography (eluent:ethyl
acetate/n-hexane (1:19, v/v)) to give colorless syrup (1.12 g,
81.1% yield). 1H NMR (CDCl3): ı 0.20 (CH3 (TMS)), 1.98 (CH3),
4.78 (–O–CH2–), 5.64, 6.20 (C CH2). 13C NMR (CDCl3): ı −0.35
(CH3 (TMS)), 18.3 (CH3), 52.9 (–O–CH2–), 91.9 (–C C-TMS),
99.1 (–C C-TMS), 126.4 (CH2 C–CH3), 135.7 (CH2 C–CH3), 166.5
(C O).
number and weight average molecular weights (Mn,PS, Mw,PS
)
estimated by polystyrene standards were Mn,PS = 7.58 × 104 and
M
w,PS = 2.24 × 105, respectively. The absolute molecular weight
(Mw) was determined to be 4.22 × 105 by MALS measurements.
Number of graft chains (X) was calculated as X = 4.03 × 102
from
X = (Mw(PPMA-g-(CTA2-C15)) − Mw(PPMA))/M(CTA2-
C15-N3) = (4.22 × 105–6.94 × 104)/874.97.
Number
of
PMA units (Y) was calculated as Y = 1.56 × 102 from
Y = DPw(PPMA) − X = 5.59 × 102–4.03 × 102. 1H NMR (CDCl3):
ı
1.24 (br. s, aliphatic-H), 1.99, 2.01, 2.03, 2.04, 2.09, 2.11 (CH3–CO),
3.72 (C5ꢀ-H, C5-H), 3.78 (C4-H), 4.01–4.17 (C6ꢀ-Hb, C6-Hb), 4.04,
4.16, 4.40, 4.44, 4.46 (C6ꢀ-Ha, C6-Ha), 4.57 (C1ꢀ-H), 4.84 (C2-H),
4.93 (C2ꢀ-H), 5.09 (C4ꢀ-H), 5.17 (C3ꢀ-H), 5.23 (C1-H), 5.27 (C3-H),
6.67 (NH), 7.93 (triazole). 13C NMR (CDCl3): ı 18.3 (CH3), 20.4,
20.6, 20.8 (CH3–CO–), 25.1 (C1–NH–CO–CH2–CH2–), 26.5, 29.2,
29.6, 30.3 (aliphatic-C), 36.4 (C1–NH–CO–CH2–), 44.7 (CH2–C),
50.4 (CH2–O), 58.3 (CH2–C), 61.5 (C6ꢀ), 61.9 (C6), 67.8 (C4ꢀ), 70.8
(C2), 71.5 (C2ꢀ), 71.8 (C5ꢀ), 72.5 (C3), 72.9 (C3ꢀ), 74.6 (C5), 77.9 (C1),
100.6 (C1ꢀ), 124.4 (CH (triazole)), 141.6 (C (triazole)), 169.1, 169.3,
169.5, 170.2, 170.3, 170.5, 171.0 (CH3–CO–), 173.7 (C1–NH–CO–),
176.1, 177.1 (CO).
2.3. Poly (3-(trimethylsilyl)-2-propyn-1-yl methacrylate)
(TMS-PPMA)
TMS-PMA (0.3 ml, 1 eq.) and AIBN (2.5 mg, 0.01 eq.) in ben-
zene (0.3 ml) were loaded into a glass tube, and degassed by three
freeze–pump–thaw cycles. The tube was sealed under vacuum, and
placed in an oil bath at 75 ◦C for 1 day. The tube was cooled to room
temperature, and opened. The reaction mixture was poured into

Y. Enomoto-Rogers et al. / Carbohydrate Polymers 87 (2012) 2237–2245
2239
Fig. 1. Preparation of: (a) poly (2-propyn-1-yl methacrylate) (PPMA), (b) PPMA-g-(CELL2-C15) and PPMA-g-(CELL13-C15).
(C6-Hb), 4.37–4.43 (C6-Ha, C1-H), 4.81 (C2-H), 5.08 (C3-H). 13C
NMR (CDCl3): ı 20.5 (CH3–CO–), 29.5 (aliphatic-C), 62.0 (C6), 71.7
(C2), 72.4 (C5, C3), 100.5 (C1), 169.3, 169.7, 170.2 (CH3–CO– of C2,
C3, C6, respectively).
2.6. PPMA-g-(CTA13-C15)
CTA13-C15-N3 (42.2 mg, 1 eq. to PMA monomer) and PPMA
(1.3 mg) were treated in the same manner with that for PPMA-g-
(CTA2-C15) using PMDETA (18.2 ␮l, 10 eq. to PMA monomer) and
Cu(I)Br (15.0 mg, 10 eq. to PMA monomer) in DMF (0.3 ml). The
remaining CTA13-C15-N3 in the crude product was removed by gel
permeation chromatography with chloroform using a Shimadzu
SEC system equipped with a fraction collector to give an amor-
phous solid PPMA-g-(CTA13-C15) (11.6 mg, 26.6% yield). The area
ratio (A(%)) of the high molecular fraction to the total area including
the remaining CTA13-C15-N3 in chromatogram (RI) of the crude
product was A(%) = 43.8%. Number of graft chains (X) was calculated
as 2.45 × 102 from X = DPw(PPMA) × A(%) = 5.59 × 102 × 43.8(%),
on the assumption that the initial molar ratio of CTA13-C15-N3
to PMA monomer unit was 1.0. The number of PMA units (Y)
in PPMA-g-(CTA13-C15) was calculated to be 3.14 × 102 from
Y = DPw(PPMA) − X = 5.59 × 102–2.45 × 102. The molecular weight
of PPMA-g-(CTA13-C15) was estimated to be 1.38 × 106 from
M(PPMA-g-(CTA13-C15)) = Mw(PPMA) + X × Mw,PS(CTA13-C15-
N3) = 6.94 × 104+2.45 × 102 × 5.33 × 103. 1H NMR (CDCl3): ı 1.25
(m, aliphatic-H), 1.9–2.1 (CH3–CO–), 3.56 (C5-H), 3.72 (C4-H), 4.06
2.7. PPMA-g-(CELL2-C15)
To
a
solution
of
PPMA-g-(CTA2-C15)
(25 mg)
in
methanol/chloroform (1/4, v/v, 1 ml), sodium methoxide (50 ␮l,
0.03 mmol) was added at room temperature, and stirred for
4 h under nitrogen. The precipitated compound was filtered
and washed by methanol to give an amorphous solid, PPMA-
g-(CELL2-C15) (14.9 mg, quantitative). 1H NMR (DMSO-d6): ı
1.21, 1.46, 1.79, 2.08 (br. s, aliphatic-H), 3.0–3.2, 3.7, 4.2, 4.6,
5.0 (ring-H), 4.74 (C1-H), 8.14 (triazole), 8.32 (NH). 13C NMR
(DMSO-d6): ı 25.9 (C1–NH–CO–CH2–CH2–), 29.1 (aliphatic-C),
35.4 (C1–NH–CO–CH2–), 44.4 (CH2–C–CH3), 49.4 (CH2–O), 57.7
(CH2–C), 60.3 (C6ꢀ), 61.1 (C6), 70.0 (C4ꢀ), 72.1 (C2), 73.3 (C2ꢀ), 75.8
(C5ꢀ), 76.4 (C3, C3ꢀ), 76.8 (C5), 79.2 (C4), 80.4 (C1), 103.2 (C1ꢀ),
125.0 (CH (triazole)), 140.8 (C (triazole)), 172.9 (C1–NH–CO–),
176.0 (CO).

2240
Y. Enomoto-Rogers et al. / Carbohydrate Polymers 87 (2012) 2237–2245
2.8. PPMA-g-(CELL13-C15)
To
a
solution of PPMA-g-(CTA13-C15) (4.8 mg) in
methanol/chloroform (1/4, v/v, 0.3 ml), sodium methoxide
(50 ␮l, 0.03 mmol) was added at room temperature, and stirred for
4 h under nitrogen. The precipitated compound was collected by
centrifugation at 1000 rpm for 3 min to give an amorphous solid,
PPMA-g-(CELL13-C15) (2.4 mg, 88.9% yield).
2.9. General measurements
1H, 13C, and two-dimensional NMR spectra including H–H
COSY (correlation spectroscopy), HSQC (heteronuclear single quan-
tum coherence) were recorded with a Varian INOVA300 FT-NMR
(300 MHz) or a JEOL JNM-A500 FT-NMR (500 MHz) spectrometer in
CDCl3 or DMSO-d6 with tetramethylsilane (TMS) as internal stan-
dard. Chemical shifts (ı) and coupling constants (J) are reported
in (ppm) and (Hz), respectively. Fourier transform infrared (FT-
IR) spectra were recorded on a FTIR-4000 spectrophotometer
equipped with ATR attachment (Durasampl IR II).
Fig. 2. The chromatograms (RI) and the molecular weight (Mw) plots of: (a) PPMA
and (b) PPMA-g-(CTA2-C15).
2.10. SEC–MALS measurement
Size exclusion chromatography–multi-angle laser light scatter-
ing (SEC–MALS) measurements were carried out at 25 ◦C using
a Shimadzu SEC system (CBM-10A, SPD-10A, SIL-10A, LC-10AT,
FCV-10AL, CTO-10A, RID-10A, and FRC-10, Shimadzu, Japan) and
MALS detector (DAWN EOS, Wyatt Technology Co. Ltd., U.S.A.)
(ꢀ = 690 nm). Chloroform was used as eluent. The flow rate was
1.0 ml/min. The photometer was calibrated with pure toluene.
Shodex columns (K802, K802.5, and K805) were used. Number
and weight average molecular weights (Mn,PS, Mw,PS) and polydis-
persity index (PDI, Mw,PS/Mn,PS) were estimated using polystyrene
standards (Shodex). An absolute molecular weight (Mw) was
determined by MALS measurements using Zimm plots. Refrac-
tive index (RI) increments (dn/dc) were calculated assuming 100%
recovery of injected mass. The dn/dc values were 0.047 for PPMA-
g-(CTA2-C15) and 0.064 for poly(2-propyn-1-yl methacrylate)
(PPMA).
N-(15-azidopentadecanoyl)-2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-␤-d-glucopyranosyl)-␤-d-glucopyranosylamine
(CTA2-C15-N3), was grafted onto the PPMA main-chain at the
molecular ratio [PMA monomer]/[CTA2-C15-N3] = 1/1, via click
chemistry using Cu(I)Br and PMDETA in DMF (Fournier & Du Prez,
2008), as described in Fig. 1b.
The chromatograms (RI) and plots of the molecular weight
determined by MALS (Mw) of PPMA and PPMA-g-(CTA2-C15) are
shown in Fig. 2. No peak corresponding to a free CTA2-C15-N3
was observed in its chromatogram as shown in Fig. 2b. The char-
acteristics of the copolymers are summarized in Table 1. In 1H
NMR spectrum of PPMA-g-(CTA2-C15) in Fig. 3a, the resonances
assigned to the triazole appeared at 7.93 ppm and that assigned to
the alkyne proton of the PPMA main-chain disappeared. In Fig. 3b,
13C resonances assigned to the triazole were observed at 124.4
and 141.6 ppm. The heteronuclear connectivity (CH) of the tria-
zole was confirmed by its HSQC spectrum (data not shown). The
relative integral area of the triazole (1H) to the ring-H (14H) was
1.12–14, indicating a quantitative formation of the triazole. In the
FT-IR spectrum of PPMA-g-(CTA2-C15) in Fig. 4c, disappearance
of the N3 absorbance at 2120 cm−1 after click reaction supports
a quantitative formation of the triazole.
2.11. X-ray diffraction measurements
X-ray diffraction measurements were carried out with a Rigaku
diffractometer Ultima IV. A Nickel-filtered CuK␣ radiation was used
at 40 kV and 30 mA. Cellulose microcrystalline (Avicel, Merck) and
low-molecular-weight cellulose (Isogai & Usuda, 1991) was used to
obtain reflection pattern of cellulose I and cellulose II, respectively.
The peak of X-ray diffraction curves are resolved to four peaks
due to (1 −1 0), (1 1 0), (2 0 0) and amorphous reflections. Crys-
tallinity index was calculated from the ratio of the sum of integrated
intensities of (1 −1 0), (1 1 0), (2 0 0) reflections to the sum of inte-
grated intensities of the four peaks (Ishikawa, Okano, & Sugiyama,
1997).
3.2. Structural characterization of PPMA-g-(CTA2-C15)
Conformational structure of PPMA-g-(CTA2-C15) in chloro-
form was investigated by means of SEC–MALS measurements.
The absolute molecular weight (Mw) of PPMA chain was deter-
mined to be 6.94 × 104 by MALS measurements. The weight average
degree of polymerization of the PPMA (DPw, X + Y) was calcu-
lated as 5.59 × 102 from Mw(PPMA)/M(PMA), where the molecular
3. Results and discussion
weight of PMA monomer is 124.21. The molecular weight (Mw,PS
)
3.1. Preparation and characterization of PPMA-g-(CTA2-C15)
(7.58 × 104) of PPMA-g-(CTA2-C15) was underestimated by PS
standards, compared to the absolute molecular weight (Mw
)
Poly(2-propyn-1-yl methacrylate) (PPMA) was obtained via free
radical polymerization of trimethylsilyl methacrylate monomer
using AIBN and subsequent deprotection of trimethylsilyl group
using TBAF·3H2O according to the previous article (Ladmiral et al.,
2006), as described in Fig. 1a. We have prepared cellulose deriva-
tives with a ␻-azidopentadecanoyl group at the reducing-end in
previous work (Enomoto, Kamitakahara, Takano, & Nakatsubo,
2006; Kamitakahara et al., 2005; Kamitakahara & Nakatsubo, 2005).
(42.2 × 104) determined by MALS measurements. It is well known
that SEC, when calibrated only with linear standard polymers
such as polystyrene (PS), severely underestimates the molecu-
lar weight of a branched polymer which has a more compact
molecular volume in solution than that of a corresponding linear
polymer with the same molecular weight (Ito et al., 1992). This
fact supports successful grafting of CTA2-C15-N3 onto PPMA and
the branched structures of the copolymers with CTA side-chains as

Y. Enomoto-Rogers et al. / Carbohydrate Polymers 87 (2012) 2237–2245
2241
Fig. 3. (a) 1H NMR spectrum of PPMA, (b) 1H and (c) 13C NMR spectra of PPMA-g-(CTA2-C15), (d) 1H and (e) 13C NMR spectra of PPMA-g-(CELL2-C15) (f) 1H NMR spectrum
of PPMA-g-(CTA13-C15).

2242
Y. Enomoto-Rogers et al. / Carbohydrate Polymers 87 (2012) 2237–2245
Table 1
Characteristics of PPMA, PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15).
d
d
d
f
Polymers
A(%)a
Yield (%)c
Mn,PS (10−4
)
Mw,PS (10−4
)
Mw,PS/Mn,PS
Mw (10−4
)
X + Y (10−2
)
X (10−2
)
Y (10−2
)
PPMA
PPMA-g-(CTA2-C15)
PPMA-g-(CTA13-C15)
1.92
7.58
22.9
3.54
22.4
87.5
1.84
2.95
3.81
6.94e
42.2e
5.59
5.59
5.59
5.59
b
88.6
26.6
4.03g
2.45i
1.56h
3.14h
43.8
137.57 j
a
Peak area ratio (%) of the graft copolymer to total peak area of the graft copolymer and remaining CTA-C15-N3 calculated from SEC(RI) elution curves.
b
c
d
e
f
Quantitative.
Yield of the graft copolymers.
Estimated by polystyrene standards.
Determined by MALS measurements.
DPw of PPMA main-chain calculated as X + Y = Mw(PPMA)/M(PMA).
g
h
i
Number of CTA2-C15 chains calculated from X = (Mw(PPMA-g-(CTA2-C15)) − Mw(PPMA))/Mw,PS(CTA2-C15-N3). M(PMA) = 124.21. M(CTA2-C15-N3) = 874.97.
Number of PMA units calculated from Y = (X + Y) − X.
Number of CTA13-C15 graft chains calculated from X = DPw(PPMA) × A(%).
j
Molecular weight calculated from Mw = Mw(PPMA) + X × Mw,PS (CTA13-C15-N3). Mn,PS and Mw,PS of CTA13-C15-N3 = 4.12 × 103 and 5.33 × 103.
discussed in our previous articles (Enomoto-Rogers, Kamitakahara,
Nakayama, et al., 2009; Enomoto-Rogers, Kamitakahara, Takano,
et al., 2009). The PPMA-g-(CTA2-C15) has the same PPMA main-
chain with the same DPw value. The number of CTA chains (X)
of PPMA-g-(CTA2-C15) was calculated as 4.03 × 102 from X = (Mw
(PPMA-g-(CTA2-C15)) − Mw (PPMA))/M(CTA2-C15-N3). The num-
ber of PMA monomers (Y) was calculated as 1.56 × 102 from
Y = (X + Y) − X.
3.3. Preparation and characterization of PPMA-g-(CTA13-C15)
Under conditions the same as those for CTA2-C15-N3,
N-(15-azidopentadecanoyl)-tri-O-acetyl-␤-cellulosylamine
(CTA13-C15-N3, DPn = 13) was grafted onto the PPMA main-chain
with the molecular ratio [PMA monomer]/[CTA13-C15-N3] = 100%
to obtain PPMA-g-(CTA13-C15), as described in Fig. 1b. In chro-
matogram of PPMA-g-(CTA13-C15) before purification, the two
Fig. 4. FT-IR spectra of: (a) PPMA, (b) CTA2-C15-N3, (c) PPMA-g-(CTA2-C15), (d) PPMA-g-(CELL2-C15), (e) CTA13-C15-N3, (f) PPMA-g-(CTA13-C15) and (g) PPMA-g-(CELL13-
C15).

Y. Enomoto-Rogers et al. / Carbohydrate Polymers 87 (2012) 2237–2245
2243
methacrylate studied previously (Enomoto-Rogers, Kamitakahara,
Takano, et al., 2009). Thus, “graft onto” strategy and “click reac-
tion” were efficient methods to obtain a copolymer with cellulosic
side-chains.
3.4. Preparation and characterization of PPMA-g-(CELL2-C15)
and PPMA-g-(CELL13-C15)
PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15) were treated
with sodium methoxide to remove acetyl groups, as described
in Fig. 1b, and the deprotected copolymers, PPMA-g-(CELL2-C15)
and PPMA-g-(CELL13-C15), were obtained. Completion of deacety-
lation was supported by a strong OH absorbance appeared at
ꢁ = 3446 cm−1 in FT-IR spectrum as shown in Fig. 4d and g. The
PPMA-g-(CELL2-C15) was soluble in DMSO, and insoluble in water.
The PPMA-g-(CELL13-C15) was insoluble in water and other com-
mon solvents, such as DMSO and DMF. In 1H NMR spectrum of
PPMA-g-(CELL2-C15) in DMSO-d6 (Fig. 3d), the resonances of acetyl
protons disappeared, and those of ring-protons of cellobiose moi-
eties remained. In addition, the resonances at 8.14 and 8.32 ppm
were assigned to the triazole and amide protons (NH), respectively.
In the 13C NMR spectrum (Fig. 3e), the resonances assigned to two
carbons of the triazole were observed at 124.4 and 141.6 ppm. The
carbonyl carbons of the ester linkage (O–CO) of PPMA and the amide
linkage (C1-NH-CO) remained at 176.0 and 172.9 ppm, respec-
tively. These data indicate that the acetyl groups of the copolymers
were selectively removed without cleavage of the triazole, ester
and amide linkages. The comb-shaped copolymers having cellu-
lose side-chains with “head-to-tail” orientation were successfully
obtained.
Fig. 5. Chromatograms (RI) of PPMA-g-(CTA13-C15) (a) before and (b) after purifi-
cation.
peaks consisting of a remaining CTA13-C15-N3 and the fraction
with higher molecular weight were observed, as shown in Fig. 5a,
indicating a formation of graft copolymer with CTA side-chains.
The high-molecular-weight fraction was collected by SEC system
equipped with a fraction collector, and PPMA-g-(CTA13-C15) was
obtained in 26.6% yield as shown in Fig. 5b. In both FT-IR and 1H
NMR spectra of purified PPMA-g-(CTA13-C15), peaks assigned to
CTA chains were observed in Figs. 3f and 4f, indicating that the
compounds are composed of CTA chains. The resonances of the
triazole and PPMA of PPMA-g-(CTA13-C15) were however not
identified in both NMR and FT-IR spectra, because of the low PPMA
content of the copolymer.
The Mn,PS and Mw,PS of PPMA-g-(CTA13-C15) were estimated
to be 2.29 × 105 and 8.75 × 105, respectively by SEC analysis using
PS standards. The molecular weight of the branched copolymer
should be underestimated by PS standards compared to the abso-
lute molecular weight determined by MALS analysis, as discussed
in the previous section. It was, however, impossible to measure an
absolute molecular weight of PPMA-g-(CTA13-C15) by MALS anal-
ysis because of high scattering intensity of the compounds (see
supplemental data). This high intensity is due to small amount
of insoluble fraction with a large radius, which could not be
removed and not dispersed at low concentration or after long-
term storage in chloroform. The molecular weight was calculated
as 1.2 × 107, and overestimated compared to an possible maxi-
mum value (3.03 × 106), which was calculated by initial molar ratio
[CTA13-C15-N3]/[PMA] = 1.0 and 100% grafting rate.
3.5. X-ray diffraction measurements
In general, cellulose I, the native form, is believed to have par-
allel orientation (Gardner & Blackwel, 1974; Sugiyama, Vuong, &
Chanzy, 1991). On the other hand, cellulose II, the regenerated
or mercellized form, is believed to have anti-parallel orienta-
tion (Kolpak & Blackwell, 1976; Langan, Nishiyama, & Chanzy,
1999). X-ray diffraction measurements were carried out to analyze
crystalline pattern of PPMA-g-(CELL2-C15) and PPMA-g-(CELL13-
C15). The cellobiose chains of PPMA-g-(CELL2-C15) showed no
crystalline pattern as shown in Fig. 6c. The cellulose chains of
PPMA-g-(CELL13-C15) had crystalline structure with crystallinity
index = 42.2%, as shown in Fig. 6e, but exhibited crystalline pat-
tern of cellulose II with three (1 −1 1), (1 1 0), and (2 0 0) reflections
located at d = 0.739, 0.451, and 0.407 nm, respectively (Isogai,
Usuda, Kato, Uryu, & Atalla, 1989). The reason for amorphous pat-
tern of cellulose is likely that crystallization of cellulose chains via
hydrogen bonding was inhibited by their covalent grafting on PPMA
chain at the longer interchain distances than those suitable for the
crystallization, as discussed in our previous work (Enomoto-Rogers,
Kamitakahara, Yoshinaga, & Takano, 2011b). Regarding the crys-
talline structure, there are some possibilities for the structures of
cellulose chains of PPMA-g-(CELL13-C15). Parallel cellulose chains
might show diffraction pattern of cellulose II, or anti-parallel cellu-
lose chains might be formed by interdigitation of cellulose chains
on the PPMA chain and give diffraction pattern of cellulose II. In
our previous study, nanoparticles consisting with cellulose chains
having radial “head-to-tail” orientation showed a crystalline pat-
tern of cellulose II (Enomoto-Rogers, Kamitakahara, Yoshinaga,
& Takano, 2011a). Thus, the XRD data of PPMA-g-(CELL13-C15),
which has cellulose side-chains with “head-to-tail” orientation,
suggested the possibility that not only anti-parallel but also “paral-
lel” orientations of cellulose chains might give a crystal structure of
cellulose II.
According to the chromatogram of refractive index (RI), the
area ratio of the high molecular fraction to the total area includ-
ing the remaining CTA13-C15-N3 was A(%) = 43.8%. Therefore, X
value of PPMA-g-(CTA13-C15) was calculated as 2.45 × 102 from
X = DPw(PPMA) × A(%) = 5.59 × 102 × 43.8(%), on the assumption
that the initial molar ratio of CTA13-C15-N3 to PMA monomer unit
was 1.0. The number of PMA units (Y) in PPMA-g-(CTA13-C15) was
calculated to be 3.14 × 102 from Y = DPw(PPMA) − X. The molecular
weight of PPMA-g-(CTA13-C15) was estimated to be 1.38 × 106
from
M(PPMA-g-(CTA13-C15)) = Mw(PPMA) + X × Mw,PS(CTA13-
C15-N3) = 6.94 × 104 + 2.45 × 102 × 5.33 × 103. The density of
cellulose side-chains per PPMA main-chain increased compared to
that of the copolymer PMMA-g-(CTA13-C15) (3.84 CTA side-chains
per PMMA main-chain with DPw of 4.14 × 102) obtained via radical
copolymerization of a cellulose macromonomer with methyl

2244
Y. Enomoto-Rogers et al. / Carbohydrate Polymers 87 (2012) 2237–2245
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4. Conclusions
Comb-shaped graft copolymers with cellulose triacetate (CTA)
side-chains, PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), were
prepared by grafting CTA2-C15-N3 and CTA13-C15-N3 (DPn = 13)
onto PPMA (DPw = 5.59 × 102), respectively, via “click chemistry”.
The numbers of CTA side-chains (X) of PPMA-g-(CTA2-C15) and
PPMA-g-(CTA13-C15) were calculated as 4.03 × 102 and 2.45 × 102,
respectively. The density of cellulose side-chains per main-chain
increased compared to that of the copolymers obtained via radical
copolymerization of a cellulose macromonomer. PPMA-g-(CELL2-
C15) and PPMA-g-(CELL13-C15) were successfully obtained by
deacetylation of PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15),
respectively. PPMA-g-(CELL13-C15) exhibited reflection pattern of
cellulose II, suggesting the possibility that not only anti-parallel but
also parallel orientations of cellulose chains might give a crystal
structure of cellulose II.
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Acknowledgements
We acknowledge Graduate School of Agricultural and Life Sci-
ences, the University of Tokyo, for 500-MHz NMR equipments. This
study was supported in part by a Grant-in-Aid from a Research Fel-
lowships of the Japan Society for the Promotion of Science (JSPS)
for Young Scientists (Y.E-R), and by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, and Culture of
Japan (Nos. 18688009 and 21580205).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
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