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Heterogeneous Modification of Cellulose Nanocrystals and Surface Assemblies
Ilari Filpponena,b, Ingrid Hoegera, Lucian Luciaa, Janne Laineb and Orlando J. Rojasa,b
a Department of Forest Biomaterials, NCSU, Raleigh, NC, United States b Department of Forest Products Technology, Aalto University, Espoo, Finland
The modification of polysaccharides plays a central role in the field of sustainable chemistry.i By the virtue of their huge abundancy and the structural and superstructural diversity polysaccharides are ideal starting materials for defined modifications and specific applications. The chemical modification of polysaccharides provides a versatile route for the structure and property design of such materials.ii Due to the chemical functionality of polysaccharides (bearing hydroxyl and/or carboxylic acid groups) the esterification and etherification are the most common approaches for the modification reactions of polysaccharides. Moreover, the oxidation and homogenous nucleophilic substitution reactions are applied but to a lesser extent. Cellulose and dextran are the most commonly used starting materials for the creation of highly engineered nanoparticles.iii,iv,v
In general, 1,3-dipolar cycloaddition reactions have long been popular in the generation of carbohydrate mimetics in homogeneous reaction environment.vi More precisely, the thermally induced cycloaddition (Huisgen reaction) occurs between an azide and a triple bond and is nowadays often referred as a member of the click-reaction family because of its robustness.vii The reaction has gained increasing attention after discovering that the 1,3-dipolar cycloaddition between azides and terminal alkynes can be catalysed by Cu(I) salts.viii,ix,x,xi In fact, the Huisgen reaction has become the most popular click reaction to date by the virtue of its high yields, rapidity, high regio- and stereoselectivity, mild reaction conditions and experimental simplicity. Several authors have described the use of this novel click-chemistry concept for the generation of carbohydrate mimetics and derivatives.xii,xiii,xiv In this communication a method for the grafting of amine-terminated monomers onto the reducing end-groups of cellulose nanocrystals (CNCs) followed by the click-chemistry reaction is demonstrated. Initially the reducing end groups in cellulose nanocrystals were functionalized by 4-hydrazinobenzoic acid via hydrazone linkages as anchor groups.xv In the next step, amino-terminated compounds were grafted on to the activated CNCs via the carbodiimide-mediated formation of an amide linkage between the amine and the carboxylic groups on the reducing ends of activated CNCs. Subsequently, two sets of CNCs were prepared, containing on their reducing end an azide derivative and an alkyne derivative, respectively. Finally the click-chemistry reaction, i.e., the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition between the azide and the alkyne activated CNCs was employed, bringing together the nanocrystalline materials in a regularly packed arrangement. The produced linked nanomaterials were characterized by elemental analyses, 1H and 31P NMR spectroscopies, size exclusion chromatography and transmission electron microscopy (TEM).
NNH
OOH
CellOO
OHOHHNHN N
H OOH
Cell OO
OH OH HNH
OO
ON3
NNH
O OHCellO
O
OHOHHNHN N
H OOHCell O
O
OH OH HNH
OO
ON N
N
CNC-HBA-AZ CNC-HBA-PR
CNC-HBA-Click
CuSO4 x 5H2O
Ascorbic acid
Figure 1. Schematic representation of the click-reaction between the reducing-end modified cellulose nanocrystals.
i Heinze, T.; Liebert, T. Prog. Polym. Sc. 2001, 26, 1689-1762. ii Klemm, D. K.; Heublein, B.; Fink, H. P.; Bohn, A. Angew. Chem., Int. Ed. 2005, 44, 3358-3393. iii Huang, J.; Kunitake, T. J. Am. Chem.Soc. 2000, 125, 11834-11835. iv Liebert, T.; Hornig, S.; Hesse, S.; Heinze, T. J. Am. Chem. Soc. 2005, 127, 10484-10485. v Huang, J.; Ichinose, I.; Kunitake, T. Angew. Chem., Int. Ed. 2006, 118, 2949-2952. vi Gallos, J. K.; Koumbis, A. E. Curr. Org. Chem. 2003, 7, 397–426. vii Huisgen, R. Proc. Chem. Soc. 1960, 357–369. viii Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–3064. ix Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Green, M. G.; Fokin, V. V.;
Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 1053-1057. x Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. 2001, 113, 2056-2075. xi Iha, R. K.; Wooley, K. L.; Nyström, A. M.; Burke, D. J.; Kade, M. J.; Hawker, G. J. Chem. Rev. 2009, 109, 5620-
5686. xii Huisgen, R. Pure Appl. Chem. 1989, 61, 613−628. xiii Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004−2021. xiv Wu, P.; Feldman, A. K.; Nugent, A. K.; Hawker, C. J.; Scheel, A.; Voit, B.; Pyun, J.; Fréchet, J. M. J.; Sharpless,
K. B.; Fokin, V. V. Angew. Chem., Int. Ed. 2004, 43, 3928 −3932. xv Sipahi-Sağlam, E.; Gelbrich, M.; Gruber E. Cellulose 2003, 10, 237–250.
Heterogeneous modification of
cellulose nanocrystals and surface
assemblies
Ilari Filpponen2, Ingrid Hoeger1, Lucian A. Lucia1, Orlando J. Rojas1,2 and Janne Laine2
1Colloids and Interfaces Group Department of Forest Biomaterials, NCSU2 Department of Forest Products Technology, Aalto University
Cellulose Nanocrystals (CNCs)
Cellulose is the most abundant natural biopolymer which upon acid hydrolysisyields highly crystalline rod-like rigid hydrophilic particles having nanoscaledimensions
Acid hydrolysis of cellulose to form cellulose nanocrystals
Native cellulose
Acid hydrolysis
Amorphous regions
Crystalline regions
Individual cellulose polymer + Glucose
Revol et al., Int. J. Biol. Macromol. 14, 170-172, 1992
Individual cellulose nanocrystals
Starting Material: Whatman #1
filter paper (cotton, 98% α-cellulose,
80% crystallinity)
Treatment: Hydrolysis with 2.5 M
Hydrochloric acid (3 hours at 100°C)
Purification: Centrifugation and dialysis
Never-dried CNCs were used for
the further derivatization reactions
Experimental conditions
Architecture of the Cotton Crystal
The cotton cellulose crystals are of a rectangular shape with average
dimensions of 40 ± 18 Å
the amount of individual cellulose chains within a cotton crystallite can be
calculated using the two lattice parameters of cellulose Iβ unit cell, a =
0.801 nm and b = 0.817 nm, respectively
This model corresponds to a minimum of 4 x 4 and a maximum of 8 x 8
packing (using 40 ± 18 Å as the dimensions) of cellulose chains within a
crystallite. Therefore, a crystal can contain 16 to 64 chains of cellulose
Leppänen, K.; Andersson, S.; Torkkeli, M.; Knaapila, M.; Kotelnikova, N.; Serimaa, R. Cellulose 2009, 16, 999-1015
= individual cellulose chain
8 x 8 packing model
Reducing End Aldehyde Groups
= Reducing end aldehyde group
Parallel: Antiparallel:
The arrangement of individual cellulose chains inside the crystals; parallel vs.
antiparallel (cellulose I vs. cellulose II)
Derivatization only on the one end of the crystal vs. derivatization on the both
ends of the crystal
Antiparallel arrangement can be achieved by the mercerization of cellulose I
Huisgen Cycloaddition (Click chemistry)
The Huisgen Cycloaddition is the reaction of a dipolarophile with a 1,3-
dipolar compound that leads to 5-membered (hetero)cycles.
Huisgen, R. Proc. Chem. Soc. 1960, 357–369.
Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Green, M. G.; Fokin, V. V.; Sharpless.
K. B. Angew. Chem., Int. Ed. 2002, 41, 1053-1057.
Liebert, T.; Hänsch, C.; Heinze, T. Macromol. Rapid Commun. 2006, 27, 208-213.
Hafrén, J.; Zou, W.; Córdova, A. Macromol. Rapid Commun. 2006, 27, 1362–1366.
Dipolarophiles: alkenes, alkynes, carbonyls and nitriles
1,3-dipolar compounds: azides, nitril oxides, ozone and diazoalkanes
Recently applied for the generation of carbohydrate mimetics and derivatives
in heterogeneous media
Overall Objectives
Selective grafting of the reducing end groups of
cellulose nanocrystals (CNCs)
Building of nano blocks by means of the click
chemistry
“Click”
= Reducing end aldehyde group
Experimental Part
Synthesis
1. Introduction of a carboxylic acid functionality
2. Click precursors via amidation
3. Copper (I) catalyzed Click reaction between modified CNCs
Characterization Elemental analysis
1H and 31P Nuclear magnetic resonance spectroscopy (NMR)
Transmission electron microscopy
Gel permeation chromatography
The Amount of Reducing End Aldehydes
The reducing end group content of cellulose nanocrystals were
determined by the classical BCA (bicinchoninic acid) colorimetric
assay developed by Johnston et al
The reducing end group content was found to be in the range of 36
to 45 µmol/g. This data is compatible with the results obtained by
Kongruang et al. for commercial microcrystalline cellulose
Johnston, D. B., Shoemaker, S. P., Smith, G. M., and Whitaker, J. R. (1998), J. Food Biochem. 22, 301–319
Kongruang, S., Han, M.J., Breton, C. I. G., and Penner, M. H., Applied Biochemistry & Biotechnology, Vol. 113–116, 2004
CellO
OH
OH
OH O
OH
H
CNCs
CellO
OH
OH
OH O
OH
H NH
NH2
O
OH
N NH
O
OH
OH
CellO
O
OHOH
H
+
CNCs
CNC-HBA
4-hydrazinobenzoic acid
Borate buffer pH 935C, 48 hr
Introduction of a COOH-group
For a comparison the treatment were repeated at the same conditions without any
addition of 4 hydrazinobenzoic acid (Control sample: CNC-HBA-ref)
E. Sipahi-Sağlam, M. Gelbrich & E. Gruber. Cellulose 10: 237–250, 2003.
1H NMR of Acetylated CNCs
a) 1H NMR spectrum of acetylated CNC-HBA-ref and b) 1H NMR spectrum of acetylated CNC-HBA
The COOH-moiety necessary for the further derivatization reactions was installed
Aromatic signals
a) b)
CNC-HBA showed the aromatic signals from the grafted 4-hydrazinobenzoic acid
Phosphitylation of Cellulose in Ionic Liquid
Reaction is carried out in Ionic Liquid, 1-allyl-3-methylimidazolium chloride ([amim]Cl)
Phosphitylation of hydroxyls with tetramethyl-1,3,2 dioxaphospholanyl moieties makes cellulose 31P NMR detectable (31P label)
The phosphitylated OH- and COOH-groups in cellulose can then be quantitatively assessed against an internal standard
NN
Cl+
Cell O H Cl PO
OP
O
O
OCell+ + HCl
2-chloro-4,4,5,5-tetramethyl-1,3,2-
dioxaphospholane
King, A. W. T. Kilpeläinen, I., Heikkinen, S., Jarvi, P., and Argyropoulos, D.S. Biomacromolecules , 2009, 10, 458–463
King, A. W. T., Zoia, L., Filpponen, I., Olszewska, A., Xie, H., Kilpeläinen, I., and Argyropoulos, D. S. J. Agric. Food
Chem. 2009, 57, 8236–8243
Quantitative 31P NMR Spectroscopy in Ionic Liquid
R-COOH: 45 µmol/g
Internal
standard
Internal
standard
R-OH
R-OH
(a) 31P NMR spectra of phosphitylated control cellulose nanocrystals (CNC-HBA-ref) and (b) phosphitylated
4-hydrazinobenzoic acid modified cellulose nanocrystals (CNC-HBA)
a)
b)
136138140142144146148150152 ppm
0.6
6
10.7
6
1.0
0136138140142144146148150152 ppm
32.5
7
1.0
0
The amount of -COOH found in CNC-HBA correlates well with the amount
of reducing end groups (recall 36-45 µmol/g from the BCA assay)
N NH
O
OHOH
Cell OO
OHOH
H
N NH O
OH
Cell OO
OHOH
H
NH
NH2
MES-buffer, pH 4
EDC/NHSR.T., 24hr
+
CNC-HBA-PR
Propargylamine (PR)CNC-HBA
Amidation-Precursor for the Click-Reaction
For a comparison the treatment was repeated at the same conditions
with the CNC-HBA-ref
NH2
OO
ON
3N N
H
O
OHOH
OO
OHOH
H
Cell
N NH O
OH
Cell OO
OHOH
H
NH
OO
ON
3
MES-buffer, pH 4
EDC/NHSR.T., 24hr
11-azido-3,6,9-trioxaundecan-1-amine (AZ)
+
CNC-HBA-AZ
CNC-HBA
Amidation-Precursor for the Click-Reaction
For a comparison the treatment was repeated at the same conditions
with the CNC-HBA-ref
NNH
OOH
CellOO
OHOH
H
NH
N NH O
OH
Cell OO
OHOH
H
NH
OO
ON
3
NNH
OOH
CellOO
OHOHH
NH
N NH O
OH
Cell OO
OH OHH
NH
OO
ON N
N
CNC-HBA-AZ CNC-HBA-PR
CNC-HBA-Click
CuSO4 x 5H2O
Ascorbic acid
Click-Reaction
For a comparison the treatment was repeated at the same conditions
with the CNC-HBA-ref
Quantitative 31P NMR Spectroscopy in Ionic Liquid
Internal
standardR-OH
No apparent signals in COOH-region
The absence of COOH-groups points toward successful click-reaction
31P NMR spectra of phosphitylated “clicked” cellulose nanocrystals (CNC-HBA-Click)
Sample % C % H % N % Oa
CNC-HBA 42.56 5.97 0.44 51.03
CNC-HBA-AZ 42.30 6.22 0.97 50.51
CNC-HBA-PR 42.19 6.11 0.69 51.01
CNC-HBA-Click 42.64 6.12 1.12 50.12
CNC-HBA-Click-ref 42.57 6.18 0.08 51.17
Elemental Analysis
Click-precursors and Click-product contained elevated amount nitrogen when compared
to the control sample (CNC-HBA-Click-ref)
aO = 100 % - C (%) – H (%) – N (%)
Benzoylation of Cellulose; Allowing the
Visualization of Mol. Weight Distribution
Cell-OH + Benzoyl-Cl Cell-Bz
Reaction is carried out in Ionic Liquid
UV – active benzoyl group
Benzoylated cellulose is completely soluble in THF (compatible with
GPC)
NN
Cl+
Xie, H.; King, A.; Kilpelainen, I.; Granstrom, M.; Argyropoulos, D. S. Biomacromolecules 2007, 8, 3740–3748.
GPC-Molecular Weight Distribution
CNC-HBA-Click showed elevated molecular weight when compared to the control
sample (CNC-HBA-Click-ref)
Sample Mn (1 x103 gmol-1) MW (1 x103 gmol-1) Mp (1 x103 gmol-1) PD
CNC-HBA-Click-ref 18 70 51 4.1
CNC-HBA-Click 22 201 79 9.0
Transmission Electron Microscopy
(a) TEM image of control cellulose nanocrystals (CNC-HBA-Click-ref) and (b) TEM image of
modified cellulose nanocrystals (CNC-HBA-Click)
b)
The length-wise growth apparent in Figure b) points toward the linking of individual
cellulose nanocrystals via their reducing end groups – However, aggregation of
cellulose chains was also observed (Figures a and b)
a)
Conclusions
Click chemistry was successfully utilized for linking the
individual cellulose nanocrystals via their reducing end
groups – provides opportunities for different applications
(biomedical, papermaking, composites)
The length-wise growth of linked cellulose nanocrystals
points toward the possibility of using click chemistry for
manipulating cellulosic materials in a nano-scale level
Specific Applications Using Modified Cellulose to
introduce new chemistry onto cellulose surfaces
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Podsiadlo, P. et al. (2005). Biomacromolecules, 6(6), 2914-2918
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Tan, W. et al. (2004). Medicinal Research Reviews, 24(5), 621–638
NanoCell R
Cell Polymer
Cell Protein
Cell Antibody
CompositesPolymer R
Protein R
Antibody R
Composites
Biomedical applications
Wet strength additives
Biomedical applications
Biomedical applications
R = N3 or alkyne
Please remember to turn in your
evaluation sheet...
Thank youIlari Filpponen2, Ingrid Hoeger1, Lucian A. Lucia1, Orlando J. Rojas1,2 and Janne Laine2
Heterogeneous modification of
cellulose nanocrystals and surface
assemblies
1Colloids and Interfaces Group Department of Forest Biomaterials, NCSU2 Department of Forest Products Technology, Aalto University