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Generic Method for Modular Surface Modification of Cellulosic Materials in Aqueous Medium by Sequential “Click” Reaction and Adsorption Ilari Filpponen,† Eero Kontturi,*,† Sami Nummelin,*,‡ Henna Rosilo,‡ Erkki Kolehmainen,§ Olli Ikkala,‡ and Janne Laine*,† †

Department of Forest Products Technology, School of Chemical Technology, Aalto University, P.O. Box 16300, 00076 Aalto, Finland ‡ Department of Applied Physics, School of Science, Aalto University, P.O. Box 15100, 00076 Aalto, Finland § Laboratory of Organic Chemistry, Department of Chemistry, University of Jyväskylä, P.O. Box 35, 400014, Finland S Supporting Information *

ABSTRACT: A generic approach for heterogeneous surface modification of cellulosic materials in aqueous medium, applicable for a wide range of functionalizations, is presented. In the first step, carboxymethyl cellulose (CMC) modified with azide or alkyne functionality, was adsorbed on a cellulosic substrate, thus, providing reactive sites for azide−alkyne cycloaddition click reactions. In the second step, functional units with complementary click units were reacted on the cellulose surface, coated by the click-modified CMC. Selected model functionalizations on diverse cellulosic substrates are shown to demonstrate the generality of the approach. The concept by sequentially combining the robust physical adsorption (“physical click”) and robust chemical reaction (“chemical click”) allows versatile, simple, and environmentally friendly modification of a cellulosic substrate with virtually any azide- or alkyne-modified molecule and even functionalization with several types of units.

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catalyzed azide−alkyne cycloaddition (CuAAC)11−16 and thiol−ene click chemistry.17−20 On the other hand, irreversible adsorption of certain polysaccharides, such as carboxymethyl cellulose (CMC), onto cellulose surface is a well-established phenomenon.21−23 Indeed, the robustness of the process might even warrant denotation as “physical click reaction”. Here, we describe how to combine the above two concepts sequentially for a generic modular platform, by first physically binding the modified CMC chains with clickable functional groups on the cellulose surface in aqueous medium, followed by the actual click reaction where the desired molecule is covalently attached to the modified CMC in situ as already adsorbed on the surface (Scheme 1). The method allows covalent addition of virtually any azide- or alkyne-modified molecule on the surface of a cellulosic material. Presently, commercially available “click” libraries encompassing a large variety of functionalized molecules add to the appeal of this method. To demonstrate the generic nature of the approach, we have used a variety of diverse molecules to modify a range of cellulosic substrates: a ubiquitous protein (bovine serum albumin), a fluorescent probe (dansyl), and a broadly applied synthetic polymer (poly-

INTRODUCTION Cellulose is the principal structural component of plants and the contemporary strive for sustainable environment has amounted to a renewed scientific interest as a source for commodity chemicals and new materials,1 particularly in the form of mechanically excellent nanofibrillated cellulose (NFC).2 These pave the way toward new advanced applications beyond paper and textiles.3−8 Yet many of them require a range of different chemical modifications of the cellulosic material in question. In principle, the hydroxyl groups of the anhydroglucose unit of cellulose suggest to use etherification and esterification, but cellulose rarely follows the norms of common organic chemistry and its reaction behavior is often counterintuitive.9,10 Even more, to preserve the native cellulose structure, the chemical reactions cannot be made in the dissolved homogeneous state. In addition, the nanofibers are prone to aggregation if nonaqueous reaction medium is used. Therefore, a generic platform for heterogeneous chemical reactions of cellulose, and in particular native cellulose nanofibers, in aqueous medium, allowing surface modification with a wide range of functional groups, is called for but has remained a challenge. Over the recent years, robust, quick, and high fidelity chemical reactions tolerating both water and oxygen have been developed under the context of click chemistry, that is, the Cu1© 2012 American Chemical Society

Received: November 23, 2011 Revised: January 16, 2012 Published: January 18, 2012 736

dx.doi.org/10.1021/bm201661k | Biomacromolecules 2012, 13, 736−742

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Scheme 1. Schematic Representation of the Sequential Modification of Cellulosic Materialsa

a

(A) Any unmodified cellulose surface is (B) exposed to aqueous solution containing carboxymethyl cellulose (CMC) modified with alkyne or azide moieties (step 1) and (C) any molecule R with alkyne or azide modification can be attached to the already adsorbed CMC by a click reaction (step 2). Chemical structures of (D) alkyne (left) and azide (right) modified carboxymethyl cellulose (CMC), (E) alkyne and azide modified functional groups, and (F) final surface modified product with clicked functional groups on the modified CMC attached on a cellulose surface. duplicated. Only the changes in the normalized frequencies of the fifth overtone are presented to make the illustration simpler. Atomic Force Microscopy (AFM). The surface morphology of samples was investigated with an AFM instrument Nanoscope IIIa Multimode scanning probe microscopy (Digital Instruments Inc., Santa Barbara, CA, U.S.A.) equipped with E-scanner. The images were scanned using tapping mode in air with silicon cantilevers (NSC15/ AIBS from Ultrasharp μmasch, Tallinn, Estonia). The scan sizes of images were 5 × 5 μm2 and 1 × 1 μm2. No image processing except flattening was done and at least three different areas on each sample were measured. X-ray Photoelectron Spectroscopy (XPS). The surface chemical composition of samples was investigated via X-ray photoelectron spectroscopy (XPS). Prior to the experiments the samples were evacuated in prechamber overnight and a specified in situ reference (100% cellulose) was measured with each sample batch, in order to verify satisfactory experimental vacuum conditions during the analysis. The measurements were done using a Kratos Analytical AXIS 165 electron spectrometer and monochromatic Al Kα X-ray irradiation at 100 W. All spectra were collected at an electron takeoff angle of 90°. Both elemental wide-region data (1 eV intervals, 80 eV pass energy) and high resolution spectra (0.1 eV intervals, 20 eV pass energy) of carbon (C 1s), nitrogen (N 1s), oxygen (O 1s), and sulfur (S 2p) regions were collected. All spectra were recorded at three different locations on each sample; the area and depth of analysis was 1 mm2 and less than 10 nm, respectively. No sample degradation due to ultrahigh vacuum or X-rays was observed during the XPS measurements. Elemental Analysis. The percent carbon (C), hydrogen (H), and nitrogen (N) contents (%) were performed using a Perkin-Elmer 2400 Series II CHNS equipment. The remaining sample was assumed to be oxygen (O).

(ethylene glycol)) were covalently deposited on the surfaces of regenerated cellulose, NFC, and native cotton fibers (filter paper).

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MATERIALS AND METHODS

Bovine serum albumin (BSA, M w 66000 g mol −1 ), CMC (carboxymethyl cellulose, Mw 250000 g mol−1, DS = 0.7), and methoxypolyethylene glycol azide (OMe-PEG-N3, Mw = 20000) were purchased from Sigma-Aldrich. CuSO4 × 5H2O (98+%) and Lascorbic acid (99%) were purchased from Acros Organics. Trimethylsilylcellulose (TMSC) was prepared from cellulose powder from spruce (Fluka), as described elsewhere.24 Filter paper was Schleicher and Schuell brand 589. All other reagents were used as received. The water used in all solutions was deionized and further purified with a Millipore Synergy UV unit (18.2 MΩ cm). The QCMD crystals were AT-cut quartz crystals supplied by Q-Sense AB (Västra Frölunda, Sweden). The fundamental frequency (f 0) was 5 MHz and the sensitivity constant (C) was 17.7 ngHz−1 cm−2. CMC (0.5 g/L) was dissolved in 25 mM CaCl2 at pH 6. BSA was dissolved in PBS buffer (pH 7.2) at a concentration of 100 μg/mL. CuSO4 × 5H2O/Lascorbic acid solution was prepared by dissolving 40 mg of CuSO4 × 5H2O and 140.9 mg of L-ascorbic acid in 50 mL of PBS buffer. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). The adsorptions of CMC and modified CMCs on cellulose were studied with a QCM-D E4 apparatus from Q-Sense (Västra Frölunda, Sweden). The basic principles of the QCM-D technique have been described by Rodahl et al.25 and Höök et al.26 The QCM-D measurements were conducted at the fundamental frequency of 5 MHz and its overtones 15, 25, 35, 45, 55, and 75 MHz at 25 °C with the constant flow rate of 0.1 mL/min. The cellulose surfaces were allowed to swell overnight in the appropriate buffer solution in prior to the QCM-D measurements. Each measurement was at least 737


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Fourier Transform Infrared Spectroscopy (FTIR). The spectra were collected using a Bio-Rad FTS 6000 spectrometer (Cambridge, MA) with a MTEC 300 photoacoustic detector (Ames, IA) at a constant mirror velocity of 5 kHz, 1.2 kHz filter, and 8 cm−1 resolution. First, a background spectrum with standard carbon black was measured. After collecting the background spectrum, the sample was put into a detection cell that was placed into the detector. After flushing with helium gas for 5 min, the cell was sealed, and the actual spectrum of the sample was recorded. The background measurement was carried out at the beginning of each set of measurements. For each measurement a minimum of 128 scans per spectrum were collected and processed using the Win-IR Pro 3.4 software (Digilab, Randolph, MA). NMR Spectroscopy. 13C cross-polarization magic angle spinning (CP/MAS) NMR measurements were recorded with a Bruker Avance 400 spectrometer equipped with a standard bore 4 mm dual CP/MAS probehead working at 100.62 MHz for carbon-13 and 400.13 MHz for proton, respectively. The sample was spun in a 4 mm outer diameter zirconia rotor at 10 kHz rate at 295 K. The spectral width was 300 ppm and the number of data points in the time domain was 2 K (giving an acquisition time of 34 ms), which was zero filled to 8 K prior to Fourier Transform (FT) giving 3.7 Hz digital resolution in the spectra. The FIDs were multiplied with an exponential window function of 10 Hz prior to FT and the chemical shifts were referenced to the CO resonance of glycine at 176.03 ppm. The contact time was 2 ms and the relaxation delay was 4 s. The number of scans after the overnight runs varied in the limits of 5000−10000. The 1H and 13C NMR spectra were recorded in CDCl3 or DMSOd6 on a Bruker Avance 400 spectrometer. The chemical shifts are reported in ppm relative to residual CHCl3 (δ 7.26) or residual DMSO-d6 (δ 2.50) for 1H NMR. For the 13C NMR spectra, the solvent peaks CDCl3 (δ 77.00) and DMSO-d6 (δ 39.50) were used as the internal standard. Preparation of Regenerated Amorphous Cellulose Model Films. Substrates for the spin-coated cellulose model film preparation were silicon dioxide (SiO2) covered QCM-D sensor crystals. Trimethylsilylcellulose (TMSC) was diluted in toluene and then spin-coated with a spinning speed of 4000 rpm.24 Prior to use in QCM-D, deposited TMSC layer on the SiO2 crystal was converted to cellulose by desilylation with HCl vapor according to a previously published method,27 resulting in a cellulose film with a predominantly amorphous structure.28 Synthesis of Azide-Modified CMC (CMC-EO-Azide). Synthesis of the modified CMCs was performed according the modified literature procedure. A 50 mg amount of CMC (DS = 0.7, Mw = 250000 g mol−1, 0.31 mmol of glycosyl units) was dissolved in 25 mL of NaOAc-buffer solution (10 mM, pH 5, fixed conductivity 3 mS/ cm). In typical synthesis, 240 mg (1.26 mmol) of EDC·HCl (N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) dissolved in 2.5 mL of NaOAc buffer solution, 144 mg (1.26 mmol) of NHS (Nhydroxysuccinimide) dissolved in 2.5 mL of NaOAc buffer solution, and 200 μL (1.00 mmol) of 11-azido-3,6,9-trioxaundecan-1-amine, respectively, were added to the CMC mixture (Scheme S1). The reaction was performed at room temperature under stirring for 24 h followed by the addition of ethanolamine (61 mg, 1.00 mmol) dissolved in 5 mL of Milli-Q H2O to remove the excess of the coupling reagents (pH was adjusted to 8.5 with 0.1 M HCl). Stirring was continued 12 h before the reaction mixture was dialyzed (MWCO = 30000 g mol−1) against distilled water for 3 days. Finally, the CMCEO-azide derivative (47 mg) was recovered by lyophilization as a white solid. FTIR of CMC-EO-azide reveals a new stretching band at 2120 cm−1 characteristic for azides and a stretching band at 1650 cm−1 characteristic for amides (Figure S1). See Table S1 for elemental analysis results. Synthesis of Alkyne-Modified CMC (CMC-Propargyl). A 50 mg of CMC (DS = 0.7, Mw = 250000 g mol−1, 0.31 mmol of glycosyl units) was dissolved in 25 mL of NaOAc buffer solution (10 mM, pH 5, fixed conductivity 3 mS/cm). In typical synthesis, 240 mg (1.26 mmol) of EDC·HCl (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) dissolved in 2.5 mL of NaOAc buffer solution,

144 mg (1.26 mmol) of NHS (N-hydroxysuccinimide) dissolved in 2.5 mL of NaOAc buffer solution, and 65 μL (1.00 mmol) of propargylamine, respectively, were added to the CMC mixture (Scheme S2). The reaction was performed at room temperature under stirring for 24 h followed by the addition of ethanolamine (61 mg, 1.00 mmol) dissolved in 5 mL of Milli-Q H2O to remove the excess of the coupling reagents (pH was adjusted to 8.5 with 0.1 M HCl). Stirring was continued 12 h before the reaction mixture was dialyzed (MWCO = 30000 g mol−1) against distilled water for 3 days. Finally, the CMC-propargyl derivative (45 mg) was recovered by lyophilization as a white solid. FTIR of CMC-propargyl reveals new stretching bands at 1650 and 1270 cm−1 characteristic for amides (Figure S2). See Table S1 for elemental analysis results. CMC Adsorption Experiments. CMC (25 mg), CMC-propargyl (25 mg), and CMC-EO-azide (25 mg) were dissolved in 50 mL of 25 mM CaCl2 solution in separate flasks. These solutions were then introduced to the QCM-D chamber (0.1 mL flow) containing the relevant cellulose model film deposited on a quartz crystal. When the adsorption plateaus were reached the films were rinsed with 25 mM CaCl2 solution and Milli-Q-water (Figure S3). Click Reaction between Azide-Modified Cellulose Model Surface and Alkyne-Modified BSA. CMC-EO-azide (25 mg) was dissolved in 50 mL of 25 mM CaCl2 solution. This solution was then introduced to the QCM-D chamber (0.1 mL flow) containing the relevant cellulose model film deposited on a quartz crystal. When the adsorption plateau of CMC-EO-azide was reached quartz crystal was rinsed with 25 mM CaCl2 solution, Milli-Q water and PBS buffer in prior of introducing the alkyne-modified BSA. Two sets of solutions of alkyne-modified BSA (100 μg/mL in PBS-buffer) was prepared, one containing CuSO4 × 5H2O and L-ascorbic acid (Cu1 catalyst) and one without the Cu1 catalyst (control). After the reaction cellulose model surface was rinsed with PBS and Milli-Q water and the resulting films on quartz crystals were characterized with AFM imaging. Fluorescent Labeling of Cellulose Filter Paper. Approximately 50 mg of filter paper was immersed (30 min) in a 25 mM CaCl2 solution (20 mL) that contained 10 mg of CMC-EO-azide. Sample was extensively washed with D.I. water to remove unbound CMC-EOazide. Next, activated filter paper was immersed (30 min) in a solution (50:50 D.I. H2O/acetone) that contained 5-(dimethylamino)-N-(2propyl)-1-naphthalenesulfonamide (dansyl probe, 5 mg), CuSO4 × 5H2O (75 mg) and L-ascorbic acid (176 mg). Finally, the sample was subjected to extensive washings with saturated EDTA-solution (3 × 30 mL), acetone (3 × 30 mL), and D.I. water (3 × 30 mL) to remove the copper and unbound dansyl probe. After drying the samples overnight at RT, the fluorescence response was measured. The elemental composition of the sample surface was measured by XPS (Table S2). Identical experiment without the Cu1 catalyst was conducted as a control reference. Click Reaction between Alkyne-Modified Cellulose Surface and OMe-PEG-N3. CMC-propargyl (25 mg) was dissolved in 50 mL of 25 mM CaCl2 solution. This solution was then introduced to the QCM-D chamber (0.1 mL flow) containing the relevant cellulose model film deposited on a quartz crystal. When the adsorption plateau of CMC-propargyl was reached quartz crystal was rinsed with 25 mM CaCl2 solution and Milli-Q water in prior of introducing the OMePEG-N3 (500 μg/mL). Two sets of solutions of OMe-PEG-N3 were prepared, one containing CuSO4 × 5H2O and L-ascorbic acid (Cu1 catalyst) and one without the Cu1 catalyst (control). After the reaction cellulose model surface was rinsed with Milli-Q water. Click Reaction between CMC-EO-azide and Propargylamine. CMC-EO-azide (65 mg) was dissolved in D.I. water (50 mL). Next, propargylamine (50 μL) was added, closely followed by the addition of CuSO4 × 5H2O (10 mg) and L-ascorbic acid (35 mg) that were predissolved in 2 mL of D.I. water. Reaction mixture was kept shaking for 6 h and then dialyzed against saturated ethylenediaminetetraacetic acid (EDTA) solution (1 day) to remove the remaining copper. Finally, the solution was dialyzed against D.I. water for 2 days and collected by lyophilization (60 mg). 738


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Figure 1. QCM-D curves and AFM images of CMC-EO-azide/BSA-alkyne/Cu1 (blue line, lower image) and CMC-EO-azide/BSA-alkyne (red line, upper image). Decreased frequency denotes increased mass on the substrate.

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Interestingly, the experiment with Cu1 as a catalyst showed significant negative frequency shift due to the successful click reaction between the cellulose surface containing azideactivated CMC and alkyne-modified BSA (Figure 1, blue line). Although rinsing appears to remove the unbound BSA from the cellulose surface, a significant amount of BSA remained intact even after extensive rinsing sequences. This indicates successful click reaction and, thus, covalent linking of the alkyne-modified BSA to the azide-modified cellulosic surface. It should be noted here that by using ethylenediaminetetraacetic acid (EDTA) as a metal ion scavenger the technique can be expanded for the biological applications that do not tolerate copper.33 Alternatively, one can employ copper-free click chemistry, that is, bio-orthogonal ligations.34 AFM image of the BSA-modified cellulose model surface revealed globular structures deriving from the covalently bound BSA (Figure 1, bottom right). Topography of the control sample, on the other hand, remained unchanged, and no BSA was found on the cellulose model surface (Figure 1, above right). Identical modifications were also carried out with the filter paper and NFC model surfaces (Supporting Information, Table S2, Figure S4). Elemental Composition of Modified Cellulose Surfaces. The subsequent modified model surfaces were analyzed with XPS, and the results are listed in Table 1. As expected, the

RESULTS AND DISCUSSION Synthesis of Functionalized CMC. The syntheses of the CMCs containing either alkyne or azide functionalities were carried out via carbodiimide-mediated formation of an amide linkage between precursors carrying an amine functionality and the carboxylic acid groups of CMC.29−32 FTIR and elemental analysis confirmed the successful grafting reactions (Supporting Information, Figures S1 and S2 and Table S1). The grafting densities of the modified CMCs (CMC-propargyl, CMC-EOazide) were calculated based on the degree of substitution of the starting CMC (DS = 0.7) and they were found to be 85− 90%. The initial adsorption of alkyne- or azide-modified CMC and the subsequent click reaction in situ on the modified cellulose surface were followed by quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Adsorption of Functionalized CMC and Consequent Click Reaction on Cellulose Surface. First, the adsorption of the azide- and alkyne-modified CMCs onto cellulose was explored to elucidate the possible effects triggered by the derivatization reaction. QCM-D analysis displayed negative frequency shifts corresponding to the increased mass on the cellulose surface, that is, the adsorption of modified CMC onto the cellulose model surface. In fact, the adsorbed amounts of modified CMCs were on the same level with the unmodified CMC (Supporting Information, Figure S3). Furthermore, the adsorbed CMC layer was not removed even upon extensive washing of the model cellulose surface, which concurs with the previous reports on CMC adsorption on cellulose.21−23 Also, the adsorbed amount of CMC was equivalent to reported results in a similar system where the thickness of the waterswollen CMC layer was calculated to be 10 nm.23 The subsequent click reaction on the CMC as adsorbed in the cellulose model surface employed a high molecular weight substance carrying an alkyne functionality to react with the azide groups on cellulose surface. To demonstrate the functionalities, alkyne-modified bovine serum albumin (BSA) was chosen to provide a weight marker that is easily detectable in QCM-D monitoring (Supporting Information, Scheme S-3). As expected, the control experiment did not reveal negative frequency shifts since the reaction between the BSA weight marker and azide-modified cellulose surface do not proceed without the Cu1 catalyst (Figure 1, red line).

Table 1. XPS Data of Cellulose Model Surfaces sample CMC-EO-azide/BSA-alkyne/Cu1 CMC-EO-azide/BSA-alkyne (control) CMC-EO-azide/BSA/Cu1 (control)

O 1s

C 1s

N 1s

S 2p

O/C

22.8 42.6

64.2 56.5

12.6 0.9

0.4 0

0.36 0.75

40.9

57.5

1.6

0

0.71

amount of nitrogen in the sample that underwent the click reaction (CMC-EO-azide/BSA-alkyne/Cu1) was found to be significantly higher when compared to those of the two control samples, CMC-EO-azide/BSA-alkyne and CMC-EO-azide/ BSA/Cu1, respectively. In addition, the oxygen/carbon ratio was drastically altered after decorating the surface with BSA due to its high carbon content. Almost identical XPS results were achieved by using a cotton fiber based filter paper as a cellulosic substrate (Supporting Information, Table S2). When the 739


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atomic ratios in BSA and cellulose molecules are considered (and subtracting the contribution from control reference), the apparent coverage of BSA can be calculated from the XPS data, corresponding to about 75% surface coverage of BSA on cellulose. Despite the reservations involved in the quantification of XPS data, the result indicates that the degree of surface modification with the presented method is substantial. Furthermore, the surface modification appears to be quantitatively similar for very different cellulose substrates, namely an amorphous cellulose film (Table 1) and fibrous filter paper (Table S2). The real amount of BSA depends on the actual surface area of the cellulose substrate, and this depends on the porosity of the substrate, that is, its swelling and accessibility in water. The swelling, in turn, depends on the solution properties, for example, the electrolyte concentration, and it must be probed separately for each system in question. Fluorescent Labeling of Functionalized Cellulose Surface. The adaptability of the modification technique on generic cellulose, that is, beyond NFC was further explored by the fluorescence labeling of filter paper. Azide-modified CMC was adsorbed to the filter paper followed by the subsequent click reaction with the fluorescent probe 5-(dimethylamino)-N(2-propyl)-1-naphthalenesulfonamide (dansyl probe, Supporting Information). The modified filter paper is intensely bluegreen fluorescent, as shown by a photograph (Figure 2c). It is

Figure 3. QCM-D curves of CMC-propargyl/OMe-PEG-N3/Cu1 (blue line) and CMC-propargyl/OMe-PEG-N3 (red line). Decreased frequency denotes increased mass on the substrate.

activated cellulosic substrates increases the number of potential modification routes. Moreover, the scope of the method can be expanded with the aid of other click chemistry reactions, such as thiol−ene17−20 and copper-free click reactions.34 In comparison, the only present method for generic surface modification of cellulose is the so-called XET-technology, based on the strong adsorptive interaction of xyloglucans with cellulose.35,36 However, XET-method requires detailed knowledge on enzymatic reactions and the modifications are limited to the reducing end groups of xyloglucans, which lowers the final grafting density of the functionalized cellulose. Within our method, large, commercially available libraries of alkyne- and azide-modified functionalities are available for any nonspecialist as well as a wide selection of straightforward synthetic methods for alkyne and azide modifications for various molecules. Recently, increased utilization of cellulosic substrates for more sophisticated functional materials, in particular, in the realm of nanosized cellulose,37−47 has raised novel requirements for their surface modification. 13 C−CPMAS-NMR Analysis of Modified CMC. To verify a successful click reaction (formation of a triazole moiety), azide-modified CMC was reacted with propargylamine and the product was analyzed by 13C−CPMAS-NMR. Spectra of the unmodified CMC and click chemistry modified CMC are shown in Figure 4. The signals arising from the CMC moiety are clearly identified as well as the signals of the triazole ring carbons generated by the click reaction, at 140.6 and 126.4 ppm, respectively. The broad resonance in the click chemistry modified CMC at 172.4 ppm is due to the amide carbons of azide-modified CMC (Figure 4, bottom). Aliphatic carbon signals deriving from the ethyleneoxide chain and covalently bound propargylamine between 33.2, 38.6, and 49.9 ppm, respectively, are also present in the spectrum.

Figure 2. (a) Photograph of unmodified filter paper under UV light (wavelengths 254 and 366 nm), (b) photograph of the filter paper after the reaction with dansyl probe without Cu1 (negative control), and (c) photograph of the filter paper after Cu1-catalyzed reaction with dansyl probe.

noteworthy that both succinimide-assisted amidation and Cu1catalyzed azide−alkyne cycloaddition are relatively mild reaction conditions for cellulose modification. Moreover, the click reaction confirms that filter paper has been grafted with the fluorescent dansyl probe, because the copper-free reaction between azide-modified filter paper and dansyl probe does not produce a fluorescent filter paper (Figure 2b). In fact, it appears as nonfluorescent as the unmodified filter paper (Figure 2a). Furthermore, XPS analysis of modified filter paper revealed slightly elevated amount of nitrogen and sulfur when compared to those of the control sample (Supporting Information, Table S2). PEGylation of Functionalized Cellulose Surface. The versatility of the developed method was demonstrated starting from the alkyne-modified cellulose surface. Azide containing methoxy-PEG (Mw 20000 g mol−1) was reacted with the alkyne-modified cellulose model surface and the reaction was monitored by QCM-D. As expected, the introduction of azide containing PEG accompanied with Cu1 as a catalyst resulted in the mass increase observed in negative frequency shifts (Figure 3, blue line). Again, the control experiment in the absence of Cu1 did not suggest a surface reaction (Figure 3, red line). This illustrates the generic nature of the technique as one can start from either of the precursors (alkyne- and azide-modified CMC). The possibility to utilize either azide- or alkyne-

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CONCLUSIONS We have demonstrated that cellulose can be modified in a sequential and modular manner by exploiting the natural tendency of CMC to physically adsorb on cellulose in water medium, even after azide or alkyne functionalization. This property combined with a subsequent click chemistry reaction allowed for the modification of the cellulosic surfaces. Several cellulosic substrates (amorphous and nanofibrillar cellulose films, filter paper) as well as versatile modifications (protein 740


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Figure 4. 13C−CPMAS-NMR spectra of the unmodified CMC (top) and modified CMC that has undergone a click reaction with propargylamine (bottom). Label C7 refers to methylene carbons in the carboxymethyl moieties.

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decoration, fluorescent labeling, and PEGylation) were performed to demonstrate the generic nature of this method. We foresee that the universal and robust nature of this method, coupled with its mild reaction conditions in aqueous solutions, has a potential to set an altogether alternative trend for heterogeneous modification of cellulose.

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(1) Klemm, D.; Heublein, B.; Fink, H. P.; Bohn, A. Angew. Chem., Int. Ed. 2005, 44, 3358−3393. (2) Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors, M.; Gray, D.; Dorris, A. Angew. Chem., Int. Ed. 2011, 50, 5438−5466. (3) Bandyopadhyay-Ghosh, S.; Ghosh, S. B.; Sain, M. Ind. Appl. Nat. Fib. 2010, 459−480. (4) Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J. Chem. Soc. Rev. 2011, 40, 3941−3994. (5) Montañez, M. I.; Hed, Y.; Utsel, S.; Ropponen, J.; Malmström, E.; Wågberg, L.; Hult, A.; Malkoch, M. Biomacromolecules 2011, 12, 2114−2125. (6) Korhonen, J. T.; Kettunen, M.; Ras, R. H. A.; Ikkala, O. ACS Appl. Mater. Interface 2011, 3, 1813−1816. (7) Ali, M. M.; Aguirre, S. D.; Xu, Y.; Filipe, C. D. M.; Pelton, R.; Li, Y. Chem. Commun. 2009, 6640−6642. (8) Cheng, C.-M.; Martinez, A. W.; Gong, J.; Mace, C. R.; Phillips, S. T.; Carrilho, E.; Mirica, K. A.; Whitesides, G. M. Angew. Chem., Int. Ed. 2010, 49, 4771−4774. (9) Klemm, D.; Heinze, T.; Philipp, B.; Wagenknecht, W. Acta Polym. 1997, 48, 277−297. (10) Carter Fox, S.; Li, B.; Xu, D.; Edgar, K. J. Biomacromolecules 2011, 12, 1956−1972. (11) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004−2021. (12) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596−2599. (13) Tornøe, C. V.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057−3064. (14) Iha, R. K.; Wooley, K. L.; Nyström, A. M.; Burke, D. J.; Kade, M. J.; Hawker, C. J. Chem. Rev. 2009, 109, 5620−5686. (15) Click Chemistry for Biotechnology and Materials Science; Lahann, J., Ed.; John Wiley and Sons Ltd: United Kingdom, 2009. (16) Themed issue: Applications of click chemistry. Chem. Soc. Rev. 2010, 39 (4), 1221−1408.

ASSOCIATED CONTENT

S Supporting Information *

The characterization of CMC derivatives (propargyl and azide), the synthesis of alkyne-modified BSA and alkyne-modified dansyl probe. Adsorption experiments with modified CMCs and click reactions on nanofibrillated cellulose (NFC) surface and filter paper. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]; janne. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank M.Sc. Esa Haapaniemi for helping in 13C−CPMAS experiments and M.Sc. Nikolaos Pahimanolis for providing the fluorescent probe 5-(dimethylamino)-N-(2-propyl)-1-naphthalenesulfonamide. The work was partially funded by the Finnish Centre for Technology and Innovation (Tekes). E.K. acknowledges the Academy of Finland for financial support (No. 129068). 741


Biomacromolecules

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