Design of functionalized cellulosic honeycomb films: Site-specific biomolecule modification via "click chemistry"

Article abstract of DOI:10.1021/bm201364r

Value-added materials from naturally abundant polymers such as cellulose are of significant importance. In particular, cellulosic open-framework structures with controlled chemical functionality of the internal surface have great potential in many biosens

Full text of DOI:10.1021/bm201364r

Article
pubs.acs.org/Biomac
Design of Functionalized Cellulosic Honeycomb Films: Site-Specific
Biomolecule Modification via “Click Chemistry”
William Z. Xu, Xinyue Zhang, and John F. Kadla*
Advanced Biomaterials Chemistry Laboratory, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
*
S Supporting Information
ABSTRACT: Value-added materials from naturally abundant polymers such as cellulose are of significant importance. In
particular, cellulosic open-framework structures with controlled chemical functionality of the internal surface have great potential
in many biosensor applications. Although various cellulose derivatives can form porous honeycomb structured materials,
solubility issues and problems with film formation exist. To address this, we have generated robust cellulosic open-framework
structures that can be post-functionalized through site-specific modification. Regioselectively modified amphiphilic cellulose
azides, 3-O-azidopropoxypoly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl cellulosics, were synthesized, and honeycomb-
patterned films were readily produced by the simple breath figures method. Changing the degree of polymerization (DP) of the
pendent ethylene glycol (EG ) groups from 22 to 4 increased the corresponding honeycomb film pore diameters from ∼1.2 to
DP
∼
2.6 μm, enabling the potential tuning of pore size. Moreover, these novel azido-functionalized honeycomb films were easily
functionalized using Cu(I)-catalyzed alkyne−azide [2 + 3] cycloaddition reaction; biotin was “clicked” onto the azide
functionalized cellulosic honeycomb films without any effect to the film structure. These results indicate this system may serve as
a platform for the design and development of biosensors.
8,9
INTRODUCTION
chemical functionality of the internal surface or pore lining.
■
Such materials offer considerable internal surface area, often
highly reactive and ideal for molecular recognition applications
such as shape selective catalysis, molecular sieving, selective
Bioactive paper is a relatively new field receiving increasing
global interest. With increasing concerns over food and water
1
safety, the spread of disease, and the threat of bioterrorism
impacting all human life, there is great need for the
development of inexpensive bioactive packaging, assays, and
10−15
adsorption/separation, and chemical/biological sensing.
Micropatterned or honeycomb pore structures can be
formed by various methods including lithography, colloidal
crystal templating, emulsion droplet templating, and biotem-
2
sensors. Cellulose, the most abundant biopolymer on earth, is
biocompatible and has long been an ideal support material for
bioactive systems. Cellulose and its derivatives have served as
support matrixes for numerous biomolecules which can be
entrapped or encapsulated within the cellulosic matrix or
chemically/biochemically coupled. Cellulose-based materials
offer great potential for the development of pathogen detecting
biosensors.
16−19
plating.
However, these methods are relatively complex
and expensive. One of the simplest and most robust methods is
20,21
the so-called “breath figures” method.
In this method a thin
3
−7
layer of polymer solution is placed under a humid air stream,
which leads to microsized water droplets condensing onto the
Porous materials with tailored pore sizes and shapes are of
growing technological and scientific interest; particularly the
development of open-framework structures with controlled
Received: September 30, 2011
Revised: November 28, 2011
Published: December 8, 2011
©
2011 American Chemical Society
350
dx.doi.org/10.1021/bm201364r | Biomacromolecules 2012, 13, 350−357

Biomacromolecules
Article
cooling surface of the polymer solution. Due to capillary forces,
the water droplets rearrange into an ordered hexagonal
template, where the polymer precipitates, encapsulating the
40 °C (polymers). Chemical shifts were referenced to tetramethyl
1
15
silane (TMS; 0.0 ppm). 2D HMQC H/ N correlation NMR
spectrum was measured using a Bruker AVANCE-600 spectrometer at
room temperature. Chemical shifts were referenced to tetramethyl
22
water droplets and preventing their coalescence. Numerous
synthetic polymers, including star, linear, and block copoly-
1
silane (TMS; 0.0 ppm) for H and nitromethane (CH NO ; 0.0 ppm)
3
2
for 15N. Infrared spectra were obtained with a Perkin-Elmer Spectrum
One FT-IR spectrometer (ATR was employed for insoluble samples).
Spectra were recorded at a resolution of 4 cm and a total of 32 scans.
2
2−26
27,28
mers,
as well as polymer−particle systems
and semi-
2
9,30
−1
synthetic biopolymers,
have been used to form well-
Elemental analysis was measured using a Perkin-Elmer Series II
CHNS/O analyzer. Polymer molecular weight was determined by
GPC (Agilent 1100) equipped with RI and multiangular light
scattering (Wyatt - DAWN HELEOS II) detectors and calibrated
with polystyrene standards (columns, Stryagel HR-4 and HR-1;
temperature, 35 °C; eluting solvent, THF; flow rate, 0.5 mL/min;
sample concentration, 1.6 mg/mL; injection volume, 100 μL). The
surface morphology of the cast films was observed with a Hitachi
S-2600N scanning electron microscope (SEM).
structured porous materials using this technique.
Recently, we showed that micropatterned honeycomb films
with ordered pore structures of ∼2 μm diameter could be
produced using an amphiphilic regioselective cellulose deriva-
tive 3-O-poly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl
3
0
cellulose. In this system, the amphiphilicity of the poly-
mer was critical for the self-assembly and formation of uni-
31
form micropatterned films; the poly(ethylene glycol) seg-
ments preferentially interact with the condensing water dro-
plets to direct the self-assembly and, likely, preferentially alloc-
ate around the edges of the honeycomb pores. These unique
nanostructured materials may serve as a perfect macro-
molecular platform for the immobilization of bioactive com-
pounds and, therefore, the development of specific recognition
systems (e.g., biodefense, pathogen capture, detection, and des-
truction).
Allyloxypoly(ethylene glycol) (1a−c). Allyloxypoly(ethylene
glycol) (1a−c) was synthesized from poly(ethylene glycol) MW =
2
00 (EG ), poly(ethylene glycol) MW = 600 (EG ), and poly-
4 13
(ethylene glycol) MW = 1000 (EG ). Sodium hydride (0.15 mol, 1.5
22
equiv) was first washed three times (3 × 100 mL) with freshly distilled
anhydrous THF. Poly(ethylene glycol) (EG ; EG ; EG ; 0.15 mol,
4
13
22
1
.5 equiv) was then added slowly and stirred in 10 mL of anhydrous
THF in an ice bath for 1 h, followed by dropwise addition of allyl
bromide (0.1 mol, 1 equiv). The reaction took place at room
temperature for 2 days and then at 50 °C for 2 days. After the reaction
mixture had cooled to room temperature, 10 mL of distilled water was
added, followed by removal of solvent through rotary evaporation.
A total of 100 mL of distilled water was then added, followed by
extraction with 3 × 70 mL of chloroform. The combined organic
phases were washed with 100 mL of 0.1 M Na S O solution, 5 ×
Site-specific modification of biomolecules is an important
and challenging area of research. One of the most widely used
techniques is the Cu(I)-catalyzed alkyne−azide [2 + 3]
3
2,33
cycloaddition or “click” reaction.
This azide−alkyne
cycloaddition offers good reproducibility and a high degree of
specificity and is compatible with water, which makes it
potentially appropriate for a variety of in vitro and in vivo
2
2
3
1
1
00 mL distilled H O, 100 mL saturated NaHCO solution, and finally
2
3
00 mL saturated NaCl solution. The organic phase was subsequently
3
4−37
applications.
Thus far the use of Cu(I)-catalyzed cyclo-
dried over anhydrous MgSO and the solvent was removed by rotary
4
addition to functionalize micropatterned films has been limited
but offer great potential to control the properties of surfaces
evaporation. Yield: 1a (from EG ) 72%; 1b (from EG ) 67%; 1c
4
13
1
(from EG ) 66%. H NMR (CDCl , 300 MHz) δ (ppm): 2.88
22
3
38
while not impacting the bulk properties of the film. Herein,
we demonstrate site-specific functionalization of cellulosic micro-
patterned films using Cu(I)-catalyzed cycloaddition reactions.
Biotin, selected as a model bioactive compound, was functional-
ized with an alkyne linker and “clicked” onto the 3-O-
azidopropoxypoly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl
cellulosic micropatterned film.
(s, 1H), 3.42−3.78 (m, 16.5/52.9/89.3H), 3.94 (d, J = 5.26 Hz, 2H),
.09 (d, J = 10.08 Hz, 1H), 5.19 (d, J = 17.10 Hz, 1H), 5.77−5.90
m, 1H). 13C NMR (CDCl , 75.4 MHz) δ (ppm): 61.4 (O−CH -
5
(
3
2
CH -OH), 69.2 (CH -O-CH -CHCH ), 70.1, 70.3, 71.9 (O-CH -
2
2
2
2
2
CHCH ), 72.3 (O-CH -CH -OH), 116.8 (O-CH -CHCH ),
2
2
2
2
2
1
34.5 (O-CH -CHCH ).
2
2
Allyloxypoly(ethylene glycol) Tosylate (2a−c). Allyloxypoly-
(
(
ethylene glycol) tosylate (2a−c) was synthesized from allyloxypoly-
ethylene glycol) (1a−c). Allyloxypoly(ethylene glycol) (0.066 mol)
EXPERIMENTAL SECTION
Materials. Poly(ethylene glycol) MW = 200 (EG ), poly(ethylene
was dissolved in 10/40 mL distilled H O/THF. KOH (0.092 mol, 1.4
■
2
equiv) was added and the solution was cooled in an ice bath. A tosyl
chloride (0.073 mol, 1.1 equiv) solution in 50 mL of THF was added
dropwise and reacted at 0−5 °C for 4 h and then at room temperature
for 18 h. The solvent was then removed by rotary evaporation, and
4
glycol) MW = 600 (EG ), poly(ethylene glycol) MW = 1000 (EG ),
13
22
dimethylthexylsilyl chloride (TDMSCl, 95%), imidazole, anhydrous
N,N-dimethylacetamide (DMA), anhydrous N,N-dimethylformamide
(
DMF), anhydrous dimethyl sulfoxide (DMSO), p-toluenesulfonyl
100 mL of distilled H
70 mL chloroform. The combined organic phases were washed with 2 ×
100 mL distilled H O, 100 mL of 0.1 N HCl, 100 mL of saturated
NaHCO solution, and 100 mL of saturated NaCl solution. The
organic phase was subsequently dried over anhydrous MgSO and
the solvent (chloroform) was removed by rotary evaporation. Yield:
2
O was added, followed by extraction with 3 ×
15
chloride (tosyl chloride), sodium azide (NaN ), sodium azide-1- N
3
15
(
Na N ), sodium hydride (60% dispersion in mineral oil), sodium
2
3
thiosulfate (pentahydrate), triphenylphosphine (PPh ), 9-borabicyclo-
3
3
(
3.3.1)nonane (9-BBN, 0.5 M in THF), hydrogen peroxide (35%),
carbon tetrachloride, (+)-biotin N-hydroxysuccinimide (biotin-NHS,
98%), propargylamine (98%), triethylamine (≥99%), and tetra-n-
4
1
≥
2a 85%; 2b 86%; 2c 86%. H NMR (CDCl , 300 MHz) δ (ppm):
3
butylammonium iodide (TBAI) were purchased from Sigma-Aldrich
and used as received. Potassium hydroxide, sodium hydroxide,
anhydrous magnesium sulfate, potassium iodide, toluene (HPLC
grade), chloroform (ACS certified), sodium chloride (ACS certified),
sodium bicarbonate (ACS certified), L(+)-ascorbic acid, and cupric
sulfate pentahydrate (ACS certified) were purchased from Fisher
Scientific and used as received. Tetrahydrofuran (THF, Fisher
Scientific), acetone (ACS certified, Fisher Scientific), cellulose
2.38 (s, 3H), 3.43−3.70 (m, 14.5/50.9/87.3H), 3.96 (d, J =
5.26 Hz, 2H), 4.09 (t, J = 4.82 Hz, 2H), 5.11 (d, J = 10.08 Hz, 1H),
5.21 (d, J = 17.10 Hz, 1H), 5.78−5.91 (m, 1H), 7.28 (d, J = 8.33
Hz, 2H), 7.73 (d, J = 7.89 Hz, 2H). 13C NMR (CDCl
, 75.4 MHz)
3
δ (ppm): 21.4 (C H -CH ), 68.4 (O−CH -CH -O-tosyl), 69.1,
6
4
3
2
2
69.2 (CH -O-CH -CHCH ), 70.4, 70.5 (O-CH -CH -O-tosyl),
2
2
2
2
2
72.0 (O-CH -CHCH ), 116.8 (O-CH -CHCH ), 127.7
2
2
2
2
(C H -CH ), 129.6 (C H -CH ), 132.8 (C H -CH ), 134.6 (O-CH -
6
4
3
6
4
3
6
4
3
2
(
Fluka, Avicel PH-101, ∼50 μm particle size), and anhydrous lithium
CHCH ), 144.6 (C H -CH ).
2
6
4
3
chloride (Sigma-Aldrich) were dried prior to use.
Characterization. H and C NMR spectra were measured using
a Bruker AVANCE-300 spectrometer at 25 °C (small molecules) or
Allyloxypoly(ethylene glycol) Iodide (3a−c). Allyloxypoly-
(ethylene glycol) iodide (3a−c) was synthesized from allyloxypoly-
(ethylene glycol) tosylate (2a−c). Allyloxypoly(ethylene glycol)
1
13
3
51
dx.doi.org/10.1021/bm201364r | Biomacromolecules 2012, 13, 350−357

Biomacromolecules
Article
a
Scheme 1. Synthetic Scheme for the Preparation of 3-N EG-2,6-TDMS Cellulose
3
a
Note: n = 4 for a, n = 13 for b, and n = 22 for c. Reagents and conditions: (i) allyl bromide, NaH, THF; (ii) tosyl chloride, KOH, H O/THF;
2
(
(
iii) KI, acetone; (iv) TDMSCl, imidazole, DMA, LiCl; (v) 3a−c, NaH, TBAI, THF; (vi) 9-BBN, NaOH, H O , THF; (vii) PPh , CCl , DMA;
2
2
3
4
viii) NaN , THF, DMA.
3
tosylate (0.056 mol) was dissolved in 224 mL of dry acetone, followed
by addition of potassium iodide (0.224 mol, 4 equiv). The mixture was
refluxed for 18 h and then cooled to room temperature. The solvent
3-O-Allyloxypoly(ethylene glycol)-2,6-di-O-thexyldimethyl-
silyl Cellulose (3-AllyloxyEG-2,6-TDMS Cellulose; 5a−c). 3-O-
Allyloxypoly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl cellulose
(3-allyloxyEG-2,6-TDMS cellulose; 5a−c) was synthesized from 2,6-
TDMS cellulose (4) and allyloxypoly(ethylene glycol) iodide (3a−c).
Sodium hydride (45 mmol, 10 equiv) was first washed three times
was removed by rotary evaporation and 100 mL of distilled H O was
2
then added, followed by extraction with 3 × 70 mL chloroform. The
combined organic phases were washed with 100 mL of 0.1 M Na S O
2
2
3
(3 × 40 mL) with freshly distilled anhydrous THF, and then 2,6-
solution, 3 × 100 mL of distilled H O, 100 mL of saturated NaHCO3
2
TDMS cellulose (2.0 g, 4.5 mmol) and TBAI (0.45 mmol, 0.1 equiv)
were added and stirred in 40 mL of anhydrous THF in an ice bath for
1 h, followed by dropwise addition of 3a−c (45 mmol, 10 equiv). The
reaction took place at room temperature for 5 days. It was then
quenched with 40 mL of methanol. The reaction mixture was
precipitated into 300 mL of pH 7 buffer solution. The crude product
solution, and 100 mL of saturated NaCl solution. The organic phase
was subsequently dried over anhydrous MgSO and the solvent was
4
1
removed by rotary evaporation. Yield: 3a 95%; 3b 95%; 3c 82%. H
NMR (CDCl , 300 MHz) δ (ppm): 3.19 (t, J = 6.80 Hz, 2H), 3.47−
3
3.64 (m, 12.5/48.9/85.3H), 3.69 (t, J = 7.02 Hz, 2H), 3.95 (d, J = 5.26
Hz, 2H), 5.10 (d, J = 10.52 Hz, 1H), 5.20 (d, J = 17.10 Hz, 1H), 5.78−
5
.91 (m, 1H). 13C NMR (CDCl , 75.4 MHz) δ (ppm): 2.8 (O-CH -
was subsequently isolated by filtration and washed with H O, ethanol,
2
3
2
and methanol, respectively. Further purification was done by dissolving
the crude product in chloroform and precipitating in methanol to
CH -I), 69.2 (CH -O-CH -CHCH ), 70.0 (O-CH -CH -I), 70.3,
2
2
2
2
2
2
7
1.7, 71.9 (O-CH -CHCH ), 116.7 (O-CH -CHCH ), 134.5
2
2
2
2
1
afford a white powder. Purified product: DS by H NMR: 5a, 0.47; 5b,
(
O-CH -CHCH ).
2 2
0
.37; 5c, 0.45. Yield: 5a, 2.01 g, 81%; 5b, 1.62 g, 53%; 5c, 1.75 g, 43%.
2
,6-Di-O-thexyldimethylsilyl Cellulose (2,6-TDMS Cellulose;
). 2,6-Di-O-thexyldimethylsilyl cellulose (2,6-TDMS cellulose; 4)
−1
FTIR (cm ): 3508 (vOH), 2957 (vCH3(as)), 2869 (vCH3(s)), 1648
), 1465 (δCH3,CH2(as)), 1377 (δCH3(s)), 1252 (δSi−C), 1118
4
3
9
(v
was synthesized according to the procedure of Koschella et al. with a
slight modification.
CC
30,40
(v
,
), 1080, 1037 (vC−O−C(AGU)), 834, 778
Cellulose (2.0 g, 0.012 mol) was suspended in
Si−O−C
C−O−C(EG)
1
(
vSi−C/Si−O‑C). H NMR (CDCl , 300 MHz) δ (ppm): 0.13−1.65
100 mL of DMA and stirred at 120 °C for 2 h. The slurry was then
3
(
TDMS group), 3.31−4.38 (AGU), 3.65 (EG), 4.03 (d, 2H) 5.18 (d,
cooled to 100 °C, and lithium chloride (6.0 g, 0.14 mol) was added
and allowed to stir for 30 min. The mixture was then cooled to room
temperature and stirred overnight to afford a clear viscous solution.
Imidazole (4.0 g, 0.060 mol) was added to the solution and stirred for
1
3
J = 9.21 Hz, 1H), 5.28 (d, J = 17.10 Hz, 1H), 5.86−5.99 (m, 1H).
C
NMR (CDCl , 75.4 MHz) δ (ppm): −3.5−34.3 (TDMS group), 69.6,
3
7
0.7 (EG), 72.2 (CH -CHCH , 116.9 (CH -CHCH ), 134.9
2
2
2
2
(CH -CHCH ).
2 2
1
h, followed by dropwise addition of TDMSCl (12.5 mL, 0.060 mol).
3-O-Hydroxypropoxypoly(ethylene glycol)-2,6-di-O-thexyl-
The solution was stirred for an additional 1 h before being heated to
dimethylsilyl Cellulose (3-HOEG-2,6-TDMS Cellulose;
6
methylsilyl cellulose (3-HOEG-2,6-TDMS cellulose; 6a−c) was
synthesized from 5a−c. Compounds 5a−c (0.75 mmol allyloxy
group; 5a, 880 mg; 5b, 1.37 g; or 5c, 1.50 g) were dissolved in
7
1
0 °C and stirred for 4 h. The reaction mixture was then heated to
00 °C and stirred for 24 h. After that, the reaction mixture was cooled
a−c). 3-O-Hydroxypropoxypoly(ethylene glycol)-2,6-di-O-thexyldi-
to room temperature and poured slowly into 150 mL of pH 7 buffer
solution. The crude product was subsequently isolated by filtration and
washed with H O, ethanol, and methanol, respectively. Dissolving
2
3
1
7
6 mL of freshly distilled anhydrous THF, followed by the addition of
8 mL of 0.5 M 9-BBN (12 equiv) in THF. The mixture was stirred at
5 °C for 3 h and subsequently cooled down to 0−5 °C with an ice
the crude product in chloroform and precipitating in methanol
afforded the purified product as a white powder. Yield: 4.9 g, 89%.
Elemental analysis: DS 1.92; calculated for DS = 2: C, 59.14%; H,
bath. Distilled water was then added dropwise to eliminate unreacted 9-
BBN (until no bubbling was observed). Subsequently, 18 mL of 3 M
NaOH solution and 15.6 mL of 35% H O were added dropwise,
−1
1
0.38%. Found: C, 58.92%; H, 10.32%. FTIR (cm ): 3508 (vOH),
2
957 (vCH3(as)), 2871 (v
(s)), 1465 (δ
,CH2(as)), 1378 (δ
CH
3
(s)), 1252
CH
3
CH
3
2
2
(
δSi−C), 1118 (vSi−O−C), 1078, 1037 (vC−O−C(AGU)), 835, 778
respectively. The reaction mixture was further stirred at room
temperature for 16 h. THF was then removed by rotary evaporation
13
(vSi−C/Si−O−C). C NMR (C D , 313 K, 75.4 MHz) δ (ppm):
6 6
−
3.5−34.8 (TDMS group), 60.9 (C6), 73.1−77.4 (C5∼C2),
and 90 mL of distilled H O was added. The crude product was filtered
off and washed with distilled water and methanol, respectively. Further
2
1
02.8 (C1).
3
52
dx.doi.org/10.1021/bm201364r | Biomacromolecules 2012, 13, 350−357

Biomacromolecules
Article
1
Figure 1. H NMR spectra of the synthesized poly(ethylene glycol)
derivatives 1a, 2a, and 3a.
1
Figure 3. H NMR spectra of the synthesized 2,6-TDMS cellulose
derivatives 4, 5a, 6a, 7a, and 8a.
purification of the product was carried out by dissolving crude product
in chloroform and precipitating in methanol. Yield: 6a, 687 mg, 77%;
1
3
3.29−4.36 (AGU), 3.66 (EG). C NMR (CDCl , 75.4 MHz)
3
−
1
6
(
1
(
(
3
b, 1.08 g, 78%; 6c, 1.30 g, 86%. FTIR (cm ): 3510 (vOH), 2957
vCH3(as)), 2870 (vCH3(s)), 1465 (δCH3,CH2(as)), 1378 (δCH3(s)), 1252 (δSi−C),
118 (v ), 1080, 1037 (vC−O−C(AGU)), 834, 778
δ (ppm): −3.5−25.2, 34.3 (TDMS group), 32.8 (-O-CH -CH -CH -Cl),
2
2
2
41.9 (-O-CH -CH -CH -Cl), 67.7 (-O-CH -CH -CH -Cl), 70.6 (EG).
2 2 2 2 2 2
3
-O-Azidopropoxypoly(ethylene glycol)-2,6-di-O-thexyldi-
Si−O−C,C−O−C(EG)
1
methylsilyl Cellulose (3-N EG-2,6-TDMS Cellulose; 8a−c). 3-O-
3
vSi−C/Si−O‑C). H NMR (CDCl , 300 MHz) δ (ppm): 0.07−1.65
TDMS group), 1.83 (q, J = 5.48 Hz, 2H, -O-CH -CH -CH -OH),
3
Azidopropoxypoly(ethylene glycol)-2,6-di-O-thexyldimethyl-
2
2
2
silyl cellulose (3-N EG-2,6-TDMS cellulose; 8a−c) was synthesized
13
3
.32−4.37 (AGU), 3.65 (EG). C NMR (CDCl , 75.4 MHz) δ
3
from 7a−c. Compounds 7a−c (0.40 mmol chloro group; 7a, 482 mg;
(
ppm): −3.5−25.2, 34.3 (TDMS group), 32.2 (-O-CH -CH -CH -
2
2
2
7
b, 746 mg; or 7c, 816 mg) were dissolved in 8 mL of freshly distilled
OH), 60.7 (-O-CH -CH -CH -OH), 70.4, 70.7 (EG).
2
2
2
anhydrous THF, followed by the addition of 40 mL of DMA and
20 mg NaN or Na N (8.0 mmol, 20 equiv). The mixture was
stirred at 80 °C for 18 h and subsequently cooled to room temperature
and precipitated into 160 mL of distilled water. The crude product was
filtered off and washed with distilled water and then methanol. Further
purification was done by dissolving the crude product in chloroform
3-O-Chloropropoxypoly(ethylene glycol)-2,6-di-O-thexyldi-
15
5
3 3
methylsilyl Cellulose (3-Cl-EG-2,6-TDMS Cellulose; 7a−c). 3-O-
Chloropropoxypoly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl cel-
lulose (3-Cl-EG-2,6-TDMS cellulose; 7a−c) was synthesized from
6
a−c. Compounds 6a−c (0.50 mmol hydroxypropoxy group; 6a,
595 mg; 6b, 924 mg; or 6c, 1.02 g) were dissolved in 6 mL of CCl ,
4
and precipitating in methanol. Yield: 8a, 420 mg, 86%; 8b, 589 mg,
79%; 8c, 765 mg, 93%. 8a, M = 257 kDa, PDI = 1.70. FTIR (cm ):
followed by the addition of 24 mL DMA and 524 mg PPh (2 mmol, 4
−1
3
w
equiv). The mixture was stirred at 90 °C for 18 h and subsequently
cooled to room temperature and precipitated into 150 mL of
methanol. The crude product was filtered off and washed with
methanol. Further purification was done by dissolving the crude
product in chloroform and precipitating in methanol. Yield: 7a, 518
3
514 (vOH), 2957 (vCH3(as)), 2869 (vCH3(s)), 2098 (vN3(as)), 1465
(δCH3,CH2(as)), 1378 (δCH3(s)), 1251 (δSi−C), 1118 (vSi−O−C,C−O−C(EG)), 1079,
1
1
037 (vC−O−C(AGU)), 835, 778 (vSi−C/Si−O−C). H NMR (CDCl , 300
3
MHz) δ (ppm): 0.05−1.63 (TDMS group), 1.86 (q, J = 6.36 Hz,
2H, -O-CH
−1
2
-CH
2
-CH
2
-N
3
3
), 3.28−4.36 (AGU), 3.40 (t, J = 6.58 Hz,
mg, 86%; 7b, 678 mg, 73%; 7c, 956 mg, 93%. FTIR (cm ): 3502
13
2
H, -O-CH -CH -CH -N ), 3.66 (EG). C NMR (CDCl , 75.4 MHz) δ
2
2
2
3
(vOH), 2957 (vCH3(as)), 2870 (vCH3(s)), 1465 (δCH3,CH2(as)), 1378 (δCH3(s)),
(
ppm): −3.5−25.2, 34.3 (TDMS group), 29.3 (-O-CH -CH -CH -N ),
2
2
2
3
1
8
1
252 (δSi−C), 1118 (vSi−O−C, C−O−C(EG)), 1080, 1037 (vC−O−C(AGU)),
48.6 (-O-CH -CH -CH -N ), 67.9 (-O-CH -CH -CH -N ), 70.6
2
2
2
3
2
2
2
3
1
34, 778 (vSi−C/Si−O−C). H NMR (CDCl , 300 MHz) δ (ppm): 0.05−
3
.65 (TDMS group), 2.03 (q, J = 6.14 Hz, 2H, -O-CH -CH -CH -Cl),
2
2
2
Figure 2. 13C NMR spectra of the synthesized poly(ethylene glycol)
derivatives 1a, 2a, and 3a.
Figure 4. 13C NMR spectra of the synthesized 2,6-TDMS cellulose
derivatives 4, 5a, 6a, 7a, and 8a.
3
53
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Biomacromolecules
Article
RESULTS AND DISCUSSION
■
Recently we demonstrated that open-framework structures with
uniform pores of ∼2 μm diameter could be produced from
amphiphilic 3-O-poly(ethylene glycol) monomethyl ether-2,6-
30
di-O-thexyldimethylsilyl cellulose by breadth figures method.
Increasing the chain length of the pendent poly(ethylene glycol)
monomethyl ether (EGM) groups dramatically improved film
formation and pore uniformity. However, the degree of EGM
substitution (DSEGM) was very low, ∼0.1−0.2 (theoretical 1.0),
likely due to the poor reactivity of the tosylated-EGM reagent.
Using the corresponding EGM-iodide improved the DSEGM
to ∼0.5 after optimization of reaction conditions (5 equiv
40
EGM-I; 10 equiv NaH; TBAI ; 4 days). Unfortunately, these
cat
conditions are not suitable for derivatization using ethylene
glycols; condensation of the HO-EG-I occurred, rapidly
consuming the reagent and resulting in very low degrees of
substitution (DSEG < 0.1). Therefore, a protection scheme to
maximize EG substitution as well as enable subsequent
conversion to the desired cellulosic azide for the site-specific
azide−alkyne cycloaddition was developed and is outlined in
Scheme 1.
1
15
Figure 5. 2D HMQC long-range H/ N correlation J
= 10 Hz
NH)
(
NMR spectrum of 8a synthesized using 15N-labeled NaN3.
EG). 1 N NMR (CDCl , 600 MHz) δ (ppm): −309.4 (-CH -N
5
(
3
2
+
−
+
−
+
−
N N ), −169.4 (-CH -NN N ), −131.0 (-CH -NN N ).
2
2
Alkynated Biotin (9). Alkynated biotin (9) was synthesized
41
A key reagent in this multistep synthesis is the allyloxypoly-
according to the procedure of Lin et al. with a simplified workup
procedure. To a solution of biotin-NHS (100 mg, 0.29 mmol) in 6 mL
of DMF, 30 μL of propargylamine (26 mg, 0.46 mmol), and 90 μL of
triethylamine (60 mg, 0.60 mmol) were added. After stirring at room
temperature for 24 h, the reaction mixture was placed under reduced
pressure to remove volatile molecules, leaving a dried crude product
114 mg). H NMR (DMSO-d , 300 MHz) δ (ppm): 1.20−1.70
6H), 2.08 (t, J = 7.23 Hz, 2H), 2.50 (1H), 2.57 (d, J = 12.28 Hz, 1H),
.82 (dd, J = 12.28, 4.82 Hz, 1H), 3.07 (2H), 3.83 (2H), 4.13 (1H),
.30 (1H), 6.34 (1H), 6.41 (1H), 8.21 (1H). 13C NMR (DMSO-d6,
5.4 MHz) δ (ppm): 25.1, 27.7, 28.0, 28.2, 34.8, 39.8, 55.4, 59.4, 61.0,
2.8, 81.3, 162.7, and 171.8.
-O-Biotinpoly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl
Cellulose (3-biotinEG-2,6-TDMS Cellulose; 10a−c). 3-O-
Biotinpoly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl cellulose
(
ethylene glycol) iodide (3a−c). It was prepared from
poly(ethylene glycol) via a three-step conversion (Scheme 1).
Accordingly, poly(ethylene glycol) (EG , EG13, and EG22) was
4
first protected by reacting allyl bromide with one of the EG
hydroxyl groups to form allyloxypoly(ethylene glycol) 1a−c.
The second hydroxyl group was then converted to the
corresponding tosylate 2a−c, followed by further conversion
to the iodide 3a−c. Although the reaction stoichiometry was
selected to allylate only one of the hydroxyl groups, a small
amount of poly(ethylene glycol) diallyl ether was formed
and some unreacted poly(ethylene glycol) remained after the
reaction. The unreacted poly(ethylene glycol) was removed
through intensive washing, while the diallyl ether was not
removed as it did not affect the subsequent reaction.
1
(
6
(
2
4
7
7
3
(
8
1
3-biotinEG-2,6-TDMS cellulose; 10a−c) was synthesized by reacting
Monoallylation of the EG and further conversion to the allyl-
a−c with 9. To a solution of 42 mg (0.034 mmol azido group) 8a in
1
13
EG-I was confirmed by H and C NMR. Figure 1 shows the
mL of THF was added a solution of the above synthesized alkynated
1
H NMR spectra for compounds 1a, 2a, and 3a, respectively.
biotin (114 mg, ∼0.29 mmol) in 1 mL of DMSO, followed by the
addition of 4.3 mg (0.017 mmol) cupric sulfate pentahydrate and 6 mg
0.034 mmol) L(+)-ascorbic acid. After being stirred at 40 °C for 3
days, the reaction mixture was cooled down to room temperature and
then precipitated into 20 mL of distilled water and subsequently
washed with water and methanol. The product was dried in vacuo.
Yield: 39.4 mg.
Preparation of Honeycomb Films of 3-O-Azidopropoxypoly-
ethylene glycol)-2,6-di-O-thexyldimethylsilyl Cellulose. Mi-
The multiplet centered at 5.82 ppm and the doublets at 5.19,
5
.09, and 3.94 ppm are assigned to the methyne proton
(
(
CH CH-CH ) and methylene protons (CH CH−CH
2
2
2
2
and CH CH-CH ) on the allyl group, respectively. The
2
2
shoulder at 2.88 ppm in the spectrum of 1a corresponds to the
free hydroxyl group of the allyloxypoly(ethylene glycol). After
tosylation (2a) the spectrum is significantly changed; new
peaks appear at 7.73, 7.28, and 2.38 ppm, attributed to the
(
cropatterned films were prepared by applying 10 μL of neat
solution (1% 8a−c in toluene) onto a glass slide in a humid
environment (flow rate, 700 mL/min; relative humidity, 70−80%;
aromatic ring (C H -) and methyl (C H -CH ) protons of
6 4 6 4 3
the tosyl group, the broad hydroxyl peak at 2.88 ppm has
disappeared, and the triplet associated with the methylene
protons adjacent to the formerly free hydroxyl group (O-CH2-
3
0
room temperature).
Formation of Honeycomb Membranes of 3-O-Biotinpoly-
ethylene glycol)-2,6-di-O-thexyldimethylsilyl Cellulose. Link-
CH -OH) has shifted downfield to 4.09 ppm. Finally, the
spectrum of the allyloxypoly(ethylene glycol) iodide 3a is
2
(
ing biotin to the surface of honeycomb membranes of 8a−c was
conducted by immersing the honeycomb membranes in a solution of
alkynated biotin 9 (57 mg, ∼0.14 mmol), 4.3 mg (0.017 mmol) cupric
sulfate pentahydrate, and 6 mg (0.034 mmol) L(+)-ascorbic acid in
marked by two new triplets at 3.70 (O-CH -CH -I) and
2
2
3
.19 ppm (O-CH -CH -I) along with the disappearance of the
2 2
peaks related to the tosyl group.
Similarly, the 13C NMR spectra (Figure 2) show the peaks
1
mL of DMSO at 45 °C for 3 days, followed by washing with distilled
associated with the allyl group at 134.5, 116.8, and 71.9 ppm.
water and methanol, and then drying before characterization with SEM
13
The tosylated product 2a, again has the additional C peaks
−1
and FTIR. FTIR (cm ): 2956 (v (as)), 2869 (v
(s)), 1700 (vCO
),
CH
3
CH
3
associated with the tosyl group at 144.6, 132.8, 129.6, 127.7,
and 21.4 ppm, and the peak of the methylene group now
next to the tosyl group (O-CH -CH -O-tosyl) has shifted from
1464 (δCH3,CH2(as)), 1377 (δCH3(s)), 1251 (δSi−C), 1115 (vSi−O−C,C−O−C(EG)),
1079, 1036 (vC−O−C(AGU)), 832, 779 (vSi−C/Si−O−C).
2
2
3
54
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Biomacromolecules
Article
Scheme 2. Synthetic Scheme for the Preparation of Alkynated Biotin (9) and Reaction with 3-N EG-2,6-TDMS Cellulose via
3
a
Cu-Catalyzed [2 + 3] Cycloaddition Reaction
a
Note: n = 4 for a, n = 13 for b, and n = 22 for c. Reagents and conditions: (i) propargylamine, triethylamine, DMF; (ii) cupric sulfate pentahydrate,
L(+)-ascorbic acid, DMSO.
As per Scheme 1, the allyloxypoly(ethylene glycol) iodides
(
3a−c) were then reacted with regioselective 2,6-di-O-TDMS
cellulose (4) to produce the 3-allyloxyEG-2,6-TDMS cellulose
1
13
(
(
5a−c). Again, using H NMR (Figure 3) and C NMR
Figure 4) spectroscopy the introduction of the allyloxyEG
group onto the C3-position of the 2,6-di-O-TDMS cellulose
was readily apparent. The methyne (5.86−5.99 ppm) and
methylene (5.28/5.18 and 4.03 ppm) protons associated with
the allyloxy group (Figure 3), as well as the corresponding
carbon signals at 134.9, 116.9, and 72.2 ppm, respectively
(
(
5
Figure 4), along with those associated with the AGU
anhydroglucopyranose unit) clearly confirm the formation of
1
a. Integrating the H NMR spectra determined the degree of
substitution to be ∼0.5, according to eq 1:
Figure 6. ATR-FTIR spectra for (a) 8a before and (b) 8a after the
surface “click reaction”; (c) 8c after the surface “click reaction”; and
(
d) 10a (solution “click reaction” using 8a).
A total of 7 is the number of protons associated with the AGU,
and n is the degree of polymerization of the poly(ethylene
glycol).
The terminal allyloxy group of the pendent EG chain was
subsequently oxidized to the corresponding hydroxypropoxy
group to afford 3-HOEG-2,6-TDMS cellulose (6a−c) in
1
relatively good yields (77−86%). Both the H NMR (Figure 3)
and 13C NMR (Figure 4) spectra show the disappearance of
the olefinic peaks and the concurrent appearance of the new
peaks associated with the hydroxypropoxy group; specifi-
cally, the methylene groups at 1.83/32.2 ppm (O-CH -CH -
2
2
CH -OH) and 3.80/60.7 ppm (O-CH -CH -CH -OH).
2
2
2
2
Further conversion to 3-ClEG-2,6-TDMS cellulose (7a−c)
by reaction with PPh and CCl lead to a slight change in
3
4
the chemical shift of the corresponding methylene protons
(
Figure 3). However, the 13C NMR (Figure 4) spectrum
clearly showed the successful transformation, with the
terminal carbon (-O-CH -CH -CH -Cl) shifting upfield to
Figure 7. SEM images of honeycomb membranes for 8a and 8c before
a and c) and after (b and d) the surface “click reaction”, respectively.
2
2
2
(
4
1.9 ppm. Finally the desired product 3-N EG-2,6-TDMS
3
cellulose (8a−c) was readily produced by reacting 7a−c
6
1.4 ppm (1a) to 68.4 ppm (2a). Conversion to the iodinated-
with NaN in THF/DMA. Conversion of the chloro group
3
allyl-poly(ethylene glycol) 3a sees complete disappearance
of the tosyl related peaks and the methylene peak adjacent
to the iodo group (O-CH -CH -I) is now shifted far upfield to
(7a) to the azido group (8a) caused a change in chemical
shift of the methylene quintet (O-CH -CH -CH -N ) from
2
2
2
3
2.03 ppm (7a) to 1.86 ppm (8a) along with a new triplet
2
2
2.8 ppm.
at 3.40 ppm associated with the methylene protons next
3
55
dx.doi.org/10.1021/bm201364r | Biomacromolecules 2012, 13, 350−357

Biomacromolecules
Article
−1
to the azido group (O-CH -CH -CH -N ). Likewise, the
change in the band associated with the azide group (2098 cm )
compared to that using 8c; the absorbance changing from 0.21
to 0.12 for 8a versus 0.11 to 0.03 for 8c. This implies that
significantly more azide groups on films prepared with the longer
azidopropoxypoly(ethylene glycol) side chain (N EG ) partici-
2
2
2
3
corresponding carbon signals shifted upfield to 29.3 ppm
O-CH -CH -CH -N ) and downfield to 48.6 (O-CH -CH -
(
2
2
2
3
2
2
CH -N ), respectively.
2
3
Further structural conformation of the azido product 8a (3-
3
22
O-azidopropoxypoly(ethylene glycol)-2,6-di-O-thexyldimethyl-
pated in the Cu(I)-catalyzed alkyne−azide [2 + 3] cycloaddition
1
15
silyl cellulose) was obtained from 2D HMQC long-range H/ N
than those with the corresponding N EG side chains. This may
3
4
15
correlation NMR (Figure 5) using the corresponding N-labeled
derivative. The 2D HMQC NMR clearly shows the azido func-
tionality and the correlation with the three methylene groups
be due to the longer N EG groups being more accessible; they
3
extend further into the reaction medium and, thereby, are more
easily modified.
15
+
15
−
(
O-CH -CH -CH - NN  N ) of the terminal propoxy
2
2
2
CONCLUSIONS
moiety.
■
Scheme 2 outlines the synthetic preparation of the alkynated
Regioselectively modified amphiphilic 3-O-azidopropoxy-
poly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl cellulosics
were synthesized and fully characterized using FTIR and
biotin (9) and its reaction with 3-N EG-2,6-TDMS cellulose via
3
Cu-catalyzed [2 + 3] cycloaddition reaction. To link biotin to
the synthesized intermediate 8a−c via “click chemistry”, biotin-
NHS was reacted with propargylamine to form the
1
13
1
15
NMR ( H, C, H/ N HMQC). Biotinylation via Cu(I)-
catalyzed alkyne−azide [2 + 3] cycloaddition reaction
produced an insoluble product prohibiting honeycomb film
formation. However, open-framework cellulosic structures
were readily produced from the regioselectively modified
amphiphilic cellulose azides using the simple breadth figures
method. Changing the DP of the pendent ethylene glycol
substituents had a dramatic effect on the pore structure
of the resultant honeycomb films; increasing the EG DP
from 4 to 22 decreased the average pore diameter from 2.61
(±0.21) to 1.2 (±0.29) μm. Moreover, these novel cellulosic
azide honeycomb films could be readily modified via a
heterogeneous surface “click reaction”. SEM analysis of the
films before and after site-specific biotinylation revealed no
change in film morphology, while ATR-FTIR analysis indica-
ted the films produced with the longer N EG side chains
41
corresponding alkynated biotin 9. As the 3-O-azidopropo-
xypoly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl cellulosics
(
8a−c) were not soluble in the reaction medium (DMSO),
they were first dissolved in a small amount of THF (∼42 mg/
mL) prior to being introduced into the reaction.
Surprisingly, the products (10a−c) had very poor solubility
in common solvents, including THF, DMSO, DMF, DMA,
benzene, toluene, and chloroform. As a result, these materials
were not able to be characterized using NMR. However,
successful conversion of 8a to 10a via “click chemistry” was
confirmed by ATR-FTIR (Figure 6a and 6d), with the azido
−1
stretching band at 2098 cm decreasing significantly while new
bands appear at 1700 and 1650 cm− corresponding to the
carbonyl and triazole groups associated with biotin.
1
3
22
The poor solubility of the 3-biotinEG-2,6-TDMS cellulosics
10a−c) precluded the casting of films. However, the precursor
azide molecules (8a−c) were readily soluble and easily cast to
form honeycomb films with uniform pore structures by the
breadth figures method. Figure 7 shows the surface morphology
of the honeycomb films obtained. Interestingly, increasing the
DP of the azidopropoxypoly(ethylene glycol) side chain from 4
were more reactive to the cycloaddition. Thus, robust open-
framework cellulosic structures capable of site-specific
modification can be produced using “click” chemistry,
opening the pathway to functional porous materials with
controlled chemical functionality.
(
ASSOCIATED CONTENT
Supporting Information
H NMR spectral data for sample 5a, 5b, and 5c, and EG DS
■
(
EG ) to 22 (EG ) substantially decreased the pore size of the
*
S
4
22
1
honeycomb film from an average diameter of 2.61 (±0.21) μm
to 1.2 (±0.29) μm (Figure 7a and c). This observation appears
to contradict our previous finding where low DP EG did not
calculation data. This material is available free of charge via the
Internet at http://pubs.acs.org.
30
form uniform honeycomb films. This apparent discrepancy is
likely due to the DS of the EG chains along the TDMS
cellulose backbone. In our prior work, the DS of the EG was
AUTHOR INFORMATION
Corresponding Author
Phone: (604) 827-5254. Fax: (604) 822-0661. E-mail: john.
kadla@ubc.ca.
■
*
∼
0.1, whereas the materials report here have a DS of ∼0.5. As it
is known that amphiphilicity dramatically affects honeycomb
film formation, it is likely the combination of higher DS with
the lower DP EG enabled uniform honeycomb films to be
obtained. We are currently investigating the effect of the EG
side chain length and the degree of substitution on the
formation of these honeycomb membranes.
ACKNOWLEDGMENTS
■
This work was financially supported by the SENTINEL
bioactive paper network. Special thanks go to Ms. Zorana
Danilovic, NMR facilities technician at the department of
chemistry, UBC, for assistance in the 2D HMQC H/ N NMR
measurement, and Mr. Derrick Horne, senior EM technician
at UBC bioimaging facility, for assistance in the SEM
measurements.
The 3-N EG-2,6-TDMS cellulosic (8a and c) micropatterned
1
15
3
films were then surface functionalized via the Cu-catalyzed [2 + 3]
cycloaddition reaction using the alkynated biotin 9. The reactions
were run for 3 days at 45 °C in DMSO. SEM analysis revealed no
effect of the site-specific biotinylation on the honeycomb film
morphology; Figure 7a vs b and c vs d. Confirmation of the
successful surface initiated “click reaction” was made by ATR-
FTIR. Again a decrease in the azido band at 2098 cm− was
observed accompanied by the appearance of the carbonyl and
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