heterogeneous modification of cellulose nanocrystals and surface

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



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



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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NanoCell R

Cell Polymer

Cell Protein

Cell Antibody

CompositesPolymer R

Protein R

Antibody R

Composites

Biomedical applications

Wet strength additives



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

heterogeneous modification of cellulose nanocrystals and surface

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