Thiolated Hemicellulose As a Versatile Platform for One-Pot Click

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Thiolated Hemicellulose As a Versatile Platform for One-Pot ClickType Hydrogel Synthesis Laleh Maleki, Ulrica Edlund, and Ann-Christine Albertsson* Fiber and Polymer Technology, Royal Institute of Technology (KTH), Teknikringen 56, SE-100 44 Stockholm, Sweden S Supporting Information *

ABSTRACT: A one-pot synthetic methodology for the thiolation of O-acetyl-galactoglucomannan (AcGGM) was developed to merge hemicellulose chemistry with “click” chemistry. This was realized by the AcGGM-mediated nucleophilic ring-opening of γ-thiobutyrolactone via the activation of the polysaccharide pendant hydroxyl groups. The incorporation of thiol functionalities onto the hemicellulose backbone was visualized by 1H and 13C NMR spectroscopy and was assessed by an Ellman’s reagent assay of the thiol groups. The versatility of the thiolated AcGGM was elaborated and demonstrated by conducting several postmodification reactions together with hydrogel formation utilizing thiol−ene and thiol-Michael addition “click” reactions. The one-pot synthesis of thiolated AcGGM is a straightforward approach that can expand the applications of hemicelluloses derived from biomass by employing “click” chemistry. ability, has attracted tremendous attention.14 In fact, the thiolation of natural polymers opens up new avenues toward a vast number of postmodification “click” reactions and, hence, the development of engineered renewable products such as hydrogels and micelles. Among the most abundant naturally occurring polymers, we have turned our attention toward hemicelluloses, such as O-acetyl-galactoglucomannan (AcGGM) extracted from spruce (Picea abies). Hemicelluloses are a family of heteropolysaccharides that, along with cellulose and lignin, are the main constituents of plant cell walls.15 In recent years, the interest in hemicelluloses as a plentiful biopolymer has been escalating. Consequently, various methodologies for the conversion of these renewable resources into value-added products, regardless of their origin or state of purity, have been developed.16−18 Because of their abundant hydroxyl groups, hemicelluloses can be the target of diverse chemical modifications.19−21 However, the majority of these chemical modifications are achieved through multistep procedures, which highlights the advantages of developing methodologies that can accomplish the functionalization of hemicelluloses in one step. We have previously demonstrated that functional monomers with a low tendency to homopolymerize are valuable building blocks for the synthesis of functionalized macromolecules.22 Among these functional monomers, γ-thiobutyrolactone is an interesting sulfur-bearing candidate because of its inability to selfpolymerize under anionic conditions.23 Nevertheless, the

1. INTRODUCTION Natural polymers are renewable resources that, although sophisticated and widely used in their natural state, can be developed into tailored materials with endless applications through chemical modifications. Within the realm of polymer science, the derivatization of natural polymers such as polysaccharides has now become an established practice. By the start of the new century, the introduction of “click” chemistry1 as a modular approach to construct complex architectures has led to a paradigm shift in polymer science. The impact of this rapidly expanding field of study on various disciplines of polymer science, including polysaccharide chemistry, is significant.2−4 Among the reactions with “click” characteristics, the thiolation of a CC double bond, although known for more than a century,5 has recently gained momentum in the field of polymer chemistry.6−8 Such thiolation reactions can proceed via either a radical pathway involving thiyl radicals6 or a nucleophilic mechanism associated with the formation of thiolate anions.9 These fast-yielding thiol“click” reactions are attractive and versatile tools for carbohydrate chemistry due to their tolerance to the presence of oxygen and moisture, their regioselectivity, and the possibility of performing such reactions in aqueous solutions.7,10,11 However, for this chemistry to be implemented in renewable material design, imparting thiol functionalities to natural macromolecules is indispensable. Polythiol polymers and their hydrophilic counterparts known as thiomers12 were recently introduced as a class of thiolbearing macromolecules.13 In line with a growing number of applications, the thiolation of naturally occurring polymers such as polysaccharides, with their biocompatibility and biodegrad© 2015 American Chemical Society

Received: December 19, 2014 Revised: January 8, 2015 Published: January 9, 2015 667

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Biomacromolecules nucleophilic ring-opening of this thiolactone can be used as a convenient route to the formation of thiolated structures.24 To expand the applications of a biomass-derived polysaccharide, we aspired to functionalize hemicelluloses with suitable reactive sites that allow us to merge hemicellulose chemistry with “click” chemistry through a straightforward one-step process. Our aim was to develop and demonstrate a thiolation pathway of hemicellulose and the subsequent robust synthesis of various hemicellulose-based products through click-type reactions. For this purpose, the ring-opening of γ-thiobutyrolactone, a thiolactone monomer with low homoreactivity, achieved by the activation of the pendant hydroxyl groups of Oacetyl-galactoglucomannan (AcGGM) was developed. The versatility of the thiolated AcGGM was demonstrated by elaborating a number of click reactions leading to the synthesis of hemicellulose-based hydrogels and glycopolymers.

sodium phosphate buffer (0.02 M, pH 8) were prepared. A total of 250 μL of each solution was then added to a vial containing 50 μL of solution of Ellman’s reagent (4 mg/mL) mixed with 2.5 mL of phosphate buffer. After incubating the vials for 20 min in a dark place, their UV absorbance at 412 nm was measured on a UV-2401 UV−vis spectrophotometer. The amount of sulfohydryl groups in the samples was calculated using two different methods. In the first method, a standard curve was created by measuring the absorbance of N-acetyl cysteine (internal standard) solutions of varying concentrations. The thiol content per 100 mg of the sample was then calculated by fitting the UV absorbance of a sample solution into this standard curve. In the second approach, eq 1 (Lambert−Beer law) was used to calculate the number of moles of thiol per 100 mg AcGGM, where A is the absorbance, c is the concentration, b is the path length in cm, and E denotes the molar absorptivity of Ellman’s reagent.

2. EXPERIMENTAL SECTION

2.3. Thiol-Michael Addition Click Reactions Using AcGGMSH. Michael Addition to N-Phenyl Maleimide (AcGGM-S-Ph). AcGGM-SH0.5 (25 mg) was dissolved in 1 mL of DMSO. Approximately 2.3 mg N-phenyl maleimide (4 equiv according to the amount of thiol groups/100 mg AcGGM) was dissolved in 0.5 mL of DMSO. After complete dissolution, the two solutions were combined in a glass vial under constant stirring. The reaction was allowed to proceed for 1 h at room temperature. Finally, a solid product was obtained after precipitation into cold acetone followed by drying under reduced pressure. Michael Addition to N,N′-Methylenebis(acrylamide) (MBAA). AcGGM-SH0.75 (25 mg) was dissolved in 0.4 mL of sodium phosphate buffer solution (0.02 M, pH 8). MBAA (4 mg, 4 equiv according to the amount of thiol groups/100 mg AcGGM) was dissolved in 0.1 mL of the aforementioned buffer. The two solutions were then mixed by vortexing in a glass vial. Gelation was achieved after 15 min, and the reaction was continued for another 45 min. Hydrogels synthesized using this technique are herein denoted AcGGM-S-Gm and were retrieved by breaking the glass vials prior to purification. 2.4. Thiol−Ene Click Reactions Using AcGGM-SH. Thiol−Ene Reaction with N,N′-Methylenebis(acrylamide) (MBAA). AcGGMSH0.75 (25 mg) was dissolved in 0.5 mL of deionized water in a glass vial. To this solution, MBAA (4 mg, 4 equiv according to the amount of thiol groups on 100 mg AcGGM) was added under stirring. To initiate the reaction, 75 μL of a KPS solution (8 g L−1) in water and 75 μL of a sodium sulfite solution (8 g L−1) in water were added to the mixture. The glass vial was then homogenized by vortexing before being placed in a water bath at 70 °C. The gelation was achieved after 15 min, and the reaction was further continued for 1 h. Finally, cylindrical hydrogels were obtained after breaking the glass vials. Hydrogels synthesized via thiol−ene reaction with MBAA are denoted AcGGM-S-Gt Grafting of Polyethylene Glycol Monomethacrylate to AcGGMSH. AcGGM-SHx (50 mg) was dissolved in 2 mL of deionized water. PEG-MA (24.6 mg) equal to 4 equiv relative to the amount of thiol groups on the AcGGM backbone was dissolved in 2 mL of deionized water prior to mixing with the AcGGM-SHx. To this mixture, 150 μL of a KPS solution (8 g L−1) in water and 150 μL of a sodium sulfite solution (8 g L−1) in water were injected under stirring. After adding the initiator, the reaction mixture was placed in a water bath at 70 °C, and the reaction was carried out for 3 h. This mixture was then precipitated into 2-propanol at room temperature, and an off-white colored product was collected by centrifugation. The solid product was washed with 5 mL aliquots of chloroform and filtered before being vacuum-dried. The product of this synthesis was labeled AcGGM-SxPEG, where x is either 0.5 or 0.75, depending on the molar ratio of TBL used in the synthesis of its corresponding AcGGM-SHx. 2.5. Hydrogel Synthesis via Disulfide Bond Formation. AcGGM-S-Gs hydrogels were produced through radical-mediated disulfide bond formation. For this purpose, 25 mg AcGGM-SH0.75 was dissolved in 0.25 mL of deionized water in a glass vial. To this solution, 30 μL of a KPS solution (20 g L−1) and a sodium sulfite solution (20 g

C=

2.1. Materials. Sodium hydride, 60% dispersion in mineral oil, γthiobutyrolactone (TBL), 98%, N-phenyl maleimide, 97%, N,N′methylenebis(acrylamide) (MBAA), 99%, 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent), dimethyl sulfoxide anhydrous, 99.9%, ammonium chloride, 99.5%, N-acetyl-L-cysteine, 99%, sodium phosphate buffer (0.02 M, pH 8), potassium persulfate, ≥99% (KPS), sodium sulfite, ≥98% anhydrous, and polyethylene glycol methacrylate (PEG-MA, Mn 500) were all supplied by Sigma-Aldrich (Sweden) and used as received. O-Acetyl-galactoglucomannan (AcGGM) was extracted from thermomechanical pulping of spruce chips. The extracted fraction was then purified and concentrated by ultrafiltration (membrane cutoff 1 kDa) prior to freeze-drying. The carbohydrate composition of the AcGGM isolate was 17% glucose, 65% mannose, and 15% galactose.25 This AcGGM isolate had an average molecular weight of 7500 g mol−1 and a dispersity (Đ) of 1.3 obtained from size exclusion chromatography (SEC) calibrated with MALDI-TOF-MS according to a previously published protocol.26,27 A degree of acetylation (DSAC) of 0.30 has been determined for this AcGGM fraction using the aforementioned protocol. 2.2. One-Pot Synthesis of Thiol-Modified AcGGM (AcGGMSH). Thiol-functionalized AcGGM was synthesized by the ring opening of γ-thiobutyrolactone using the pendant hydroxyl groups of AcGGM as nucleophiles. In a typical procedure, 1 mmol (0.34 g) hexose units of AcGGM were dissolved in 20 mL of anhydrous DMSO. Subsequently, this solution was added to a three-neck roundbottom flask containing 1 mmol (0.024 mg = 1 equiv) NaH equipped with a magnetic stirrer under a N2 atmosphere. The addition was performed dropwise over 20 min at room temperature. After the complete addition, the mixture was stirred for another 20 min until a homogeneous solution was achieved. Thereafter, 0.5 or 0.75 mmol of TBL (43 or 65 μL) was added to the mixture dropwise, and the reaction was allowed to proceed for 2 h at room temperature. To terminate the reaction, 2 mL of saturated solution of NH4Cl was introduced into the reaction mixture. After 10 min of further stirring, the final mixture was precipitated into cold acetone, and a solid product was collected using centrifugation. The product was then washed repeatedly with ethanol preceding the second precipitation and centrifugation. The purification step was concluded by dissolving the product in 20 mL of deionized water and dialyzing (500 Da MWCO) against a constant flow of deionized water for 5 days followed by freeze-drying. Yield 60−70%. The products achieved after the thiolation of AcGGM are herein denoted AcGGM-SHx, where x refers to the amount of TBL used in their corresponding modification reactions (0.5 or 0.75 mmol). Determination of Thiol Group Content. Ellman’s reagent was used to assess the extent of the TBL ring-opening and the content of thiol groups. The procedure was performed according to an established protocol for the determination of sulfohydryl groups.28 Typically, 0.1, 0.2, 0.5, 2, and 5 mg/mL solutions of the modified AcGGM in 1 mL of 668

A bE

(1)

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Figure 1. 1H NMR spectra of AcGGM before and after thiolation (left) and 13C NMR spectra of AcGGM and AcGGM-SH0.5 (right) in D2O. L−1) was added. The mixture was vortexed for 2 min and then placed in a water bath at 70 °C until gelation was achieved (approximately 15 min). The hydrogels were retrieved by breaking the vials, followed by a leaching-out step performed in deionized water for 24 h to remove the excess initiator. 2.6. Characterization. Nuclear magnetic resonance (NMR) was performed on a Bruker Avance DMX-400 NMR operating at room temperature at 400 MHz. To determine the degree of modification of AcGGM, 40 mg of each AcGGM sample before and after any modification was dissolved in 0.6 mL of either DMSO-d6 or D2O. The solutions were transferred into NMR tubes with a 5 mm outer diameter. The approximate degree of thiol substitution (DSSH) was calculated by comparing the integral of C2 proton response at 2.51 ppm (Figure 1) to the acetyl response at 2.02 ppm based on eq 2:

DSSH =

ICH2 /2 ICH3/3

× DSAc

was measured at regular intervals after carefully drying the basket. The swelling ratio was calculated according to eq 3, in which m0 refers to the initial weight of the dry hydrogel and mt stands for the weight of the hydrogel after “t” hours: Q=

mt − m 0 m0

(3)

3. RESULTS AND DISCUSSION To expand the potential applications for hemicellulose as a renewable noncellulosic feedstock of great value, we derived a one-pot synthetic method for the thiolation of O-acetylgalactoglucomannan (AcGGM). The hypothesis was that γthiobutyrolactone (TBL), a five-membered thiolactone ring unlikely to homopolymerize under anionic conditions, can undergo ring-opening by nucleophilic attack of the activated pendant hydroxyl groups on the AcGGM backbone.23,29 The ring-opening of this thiolactone and thiolactones with similar structures via the nucleophilic attack of an amine has been reported elsewhere.24,30 NaH, a strong non-nucleophilic base conventionally used for the methylation of polysaccharides,31−33 was used to deprotonate the polyhydroxylated AcGGM. 3.1. Synthesis of Thiolated Acetyl Galactoglucomannan. Hitherto, the attempts to attach thiol groups on polysaccharide chains (i) have been isolated to polysaccharides with pendant amine or carboxylic acid moieties, (ii) have followed transesterification between pendant hydroxyl groups and thioglycolic acid, or (iii) have been achieved based on multistep procedures.34−36 However, we have developed a onepot procedure for thiol-functionalization of AcGGM, a hemicellulose with only hydroxyl functionality, Scheme 1. The pendant hydroxyl groups of AcGGM are powerful initiating sites for a number of chemical reactions. However, in the absence of a catalyst, these hydroxyl groups are not sufficiently strong to undergo a 1,2-nucleophilic addition leading to the ring-opening of a thiolactone. Deprotonating these inherent nucleophiles by using a non-nucleophile strong base such as NaH can enhance their nucleophilicity, in turn, making the ring-opening of a thiolactone viable. The ringopening of TBL leading to the covalent attachment of thiol groups to AcGGM was verified by NMR spectroscopy. The emergence of the chemical shifts stemming from the methylene protons and carbons in the 1H and 13C NMR spectra of AcGGM-SH0.5 and AcGGM-SH0.75 is shown in Figure 1. The

(2)

Fourier transform infrared spectroscopy (FTIR) measurements were recorded using a PerkinElmer Spectrum 2000 spectrometer equipped with an attenuated total reflectance (ATR) accessory. A mean of 32 scans with atmospheric water and carbon dioxide corrections were registered in the range of 4000 to 600 cm−1 at a 4 cm−1 resolution. Size exclusion chromatography (SEC) with 10 mM NaOH as the mobile phase was used to assess the molecular weight and molecular weight distribution of the pure and modified AcGGM. The analysis was conducted on a Dionex Ultimate-3000 HPLC system (Dionex Sunnyval, CA, U.S.A.), equipped with a WPS-3000SL autosampler, an LPG-4300 SD gradient pump, and a DAD-3000 UV/vis detector in combination with a Waters-410 refractive index (RI) detector. A combination of three PSS suprema columns (300 × 8 mm, 10 μm particle size) with 30, 1000, and 1000 Å connected in series together with a guard column (50 × 8 mm, 10 μm particle size) was used. A SEC calibration method based on pullulan standards with an Mp range of 342−708000 g/mol (PSS, Germany) were created. For each analysis, 250 μL of each sample solution was injected at 40 °C with a flow rate of 1 mL/min. A PSS WinGPC Unity software was used to analyze the data. Scanning electron microscopy (SEM) was used to study the topography and cross-section of AcGGM-S-Gt hydrogels before and after swelling. Each dry and swollen hydrogel sample was first immersed in liquid nitrogen and subsequently subjected to freezedrying for 24 h. The freeze-dried samples were then mounted on a carbon tape-coated stub and sputter coated with a 4 nm thick layer of gold/palladium using a Cressington 20HR Au/Pd sputter coater. Swelling ratio of AcGGM-S-Gt hydrogels was assessed gravimetrically by immersing a dry hydrogel weighing approximately 20 mg in 50 mL of deionized water. To facilitate the handling of the hydrogels, each sample was entrapped within a stainless steel basket (mesh size, 0.20 mm). The weight gain of the hydrogels in the initial stages of swelling 669

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color stemming from the nitromercaptobenzoate anion released into the reaction mixture upon mixing Ellman’s reagent with a solution of AcGGM-SH, Figure 2a,b.37 In the next step, the

Scheme 1. Schematic Outline of the Pathway for One-Pot Thiolation of AcGGM in DMSO

chemical shift at 2.51 ppm corresponds to the methylene protons adjacent to the thiol group (C2). The peak at approximately 1.85 ppm arises from the response of the protons of C3 methylene. The response of the protons corresponding to the methylene group neighboring the carbonyl is believed to be merged with the chemical shift assigned to the AcGGM acetyl CH3. The approximated degree of thiol substitution (DSSH) for AcGGM-SH0.5 and AcGGMSH0.75 was calculated by comparing the C2 proton response with the acetyl response at 2.01 ppm, assuming no variation in the degree of acetylation (DSAc) for AcGGM throughout the reaction (certified by 1H NMR), see Table 1. The DSSH calculated from 1H NMR peak integrals is not significantly influenced by the increase in the molar ratio of TBL from 0.5 to 0.75, which could be a consequence of assuming a constant DSAc before and after the modification reaction. In the 13C NMR spectrum of AcGGM-SH0.5, three additional peaks at 23, 28, and 32 ppm originating from C2, C3, and C4 of the opened ring of TBL are visible (Figure 1). The peak corresponding to the lactone carbonyl appears at 175 ppm, although it is difficult to distinguish from the baseline noise. The molecular weights of AcGGM before and after thiolation were assessed using size exclusion chromatography (SEC). The alkaline SEC traces of AcGGM, AcGGM-SH0.5, and AcGGMSH0.75 (Supporting Information, Figure S1) show that the reaction did not cause any concomitant hemicellulose degradation, verifying benign reaction conditions. The successful ring opening of TBL and the existence of thiol groups on AcGGM chains was corroborated by Ellmans’s reagent assay of the modified AcGGM. Ellman’s reagent has been used for decades as a sensitive tool to detect and calculate the sulfhydryl content in proteins and other macromolecules.37−39 First, the presence of sulfhydryl groups was confirmed by the immediate formation of an intense yellow

Figure 2. Solution of AcGGM-SH0.75 in sodium phosphate buffer (pH 8) (a) instantly turning yellow upon the addition of Ellman’s reagent (b).

content of thiols per 100 mg AcGGM was calculated using two methods based upon either the molar absorptivity of Ellman’s reagent at 412 nm or a standard curve created with N-acetyl cysteine as a reference. By comparing the results obtained from these two methods with the DSSH calculated from 1H NMR spectra of the modified AcGGM, a rough estimation of the number of thiol groups per AcGGM chain was made, Table 1. The means of these estimated values were used as the basis for postmodification and gel-formation reactions. 3.2. Hydrogel Synthesis Using AcGGM-SH. 3.2.1. Hydrogel Synthesis via Thiol−Ene Click Reaction. The radicalmediated addition of a thiol to an alkene known as thiol−ene chemistry is a rapid reaction with high efficiency and tolerance toward the reaction milieu.7,40 Because of its considerable merits, it has been recognized as one of the most promising reactions in the field of hydrogels and cross-linked networks.41,42 The synthesis of hemicellulose hydrogels via thiol− ene chemistry is a step forward in expanding the possible applications of this renewable resource. To accomplish this,

Table 1. Denotation and the Thiol Content of Thiolated AcGGM with Varying Degrees of Modification sample denotation

DSSH based on eq 2

free −SH groupsa (mmol/100 mg of sample)

free −SH groupsb (mmol/100 mg of sample)

free −SH groupsc (mmol/100 mg of sample)

AcGGM-SH0.5 AcGGM-SH0.75

0.15 ± 0.01 0.16 ± 0.02

0.08 ± 0.01 0.09 ± 0.01

0.012 ± 0.002 0.013 ± 0.002

0.025 ± 0.010 0.062 ± 0.023

Results calculated based on 1H NMR. bResults calculated using molar absorptivity of Ellman’s reagent at 412 nm. cResults calculated by fitting the absorbance of each sample into a standard curve made for N-acetyl cysteine. a

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Figure 3. FTIR spectra (wavenumber vs absorption) of (from bottom to top) unmodified AcGGM, thiol-modified AcGGM, disulfide hydrogel, Michael addition hydrogel, and thiol−ene hydrogel (left), and images (right) of (a) AcGGM-S-Gm, (b) AcGGM-S-Gs, and (c) AcGGM-S-Gt.

samples in dry and swollen states were examined using SEM. By comparing Figure 5a and c, topographical alterations as a consequence of the swelling of the hydrogel are observed. SEM micrographs of the hydrogel cross sections in Figure 5b and d clearly show the porous structure of AcGGM-S-Gt and the swelling-driven expansion of the pores. 3.2.2. Hydrogel Synthesis via Thiol-Michael Addition. Gel formation through thiol-Michael addition reactions has found extensive use in biomedical applications.43,44 Mild catalytic conditions and fast gelation under ambient conditions, along with the potential for obtaining in situ formation of hydrogels, are among the many favorable features of this chemistry.45 To explore the applicability of thiolated AcGGM for this method of hydrogel fabrication, AcGGM-S-Gm was synthesized via a Michael addition approach. AcGGM-SH0.75 was dissolved in sodium phosphate buffer (pH 8) to assist in the formation of thiolate anions and their corresponding nucleophilic attack on the double bond in MBAA. Gelation was achieved at ambient conditions within 1 h, Figure 3a. The FTIR spectrum of AcGGM-S-Gm is compared to AcGGM-S-Gt and AcGGMSH0.75 in Figure 3. A comparably weak peak at 1530 cm−1 in addition to the resemblance of the spectrum to the FTIR spectrum of AcGGM-SH0.75 indicates that only a small fraction of the gel consists of MBAA. In fact, the possible reactions of the thiolate anions in this system are not limited to their addition to an α,β-unsaturated system, but side reactions might also have occurred. A thiol−disulfide exchange reaction is another plausible pathway through which cross-links can be formed. Already existing intermolecular disulfide bonds may undergo cleavage upon the nucleophilic attack of a thiolate anion, creating an intramolecular disulfide bond and a new thiol derived from the intermolecular S−S.46 The competing effect of the thiol−disulfide exchange reaction can be suppressed by replacing MBAA with a more reactive Michael acceptor bearing maleimides or vinyl sulfones.47 3.2.3. Hydrogel Synthesis via Disulfide Bond Formation. Disulfide bond formation is nature’s robust mechanism for protein stabilization.48 Inspired by this natural phenomenon and taking the reversibility of disulfide bonds into account, the design of intelligent materials using this facile approach has been adopted by many researchers.49,50 Given the considerable merit in designing hemicellulose-based hydrogels fabricated by facile techniques, AcGGM-S-Gs hydrogels were prepared via the formation of intramolecular disulfide bonds between thiolated AcGGM chains, Figure 3b. A thermal radical initiation system was used to generate thiyl radicals in an aqueous solution of AcGGM-SH0.75, resulting in simultaneous thiyl−

AcGGM-S-Gt hydrogels were synthesized by reacting AcGGMSHx with a difunctional acrylamide cross-linker, N,N′methylenebis(acrylamide) (MBAA), under thermal initiation using KPS and sodium sulfite. The thiol−ene click reaction between AcGGM-SH and MBAA led to a rapid gelation within 15 min, Figure 3c. To validate that MBAA is covalently incorporated into the network of AcGGM-S-Gt, this hydrogel together with its precursor (AcGGM-SH0.75) and pristine AcGGM were analyzed by FTIR spectroscopy. In the FTIR spectrum of AcGGM-S-Gt, a band at approximately 1530 cm−1 arising from the amide N−H bending vibrations in the MBAA structure is evidence for the covalent cross-linking of AcGGM-SH, Figure 3. The presence of MBAA in the hydrogel structure is further confirmed by an increase in the intensity of the band at 1650 cm−1, which is attributed to the CO stretching vibrations in MBAA molecules, Figure 3. The swelling behavior of this hydrogel formulation was evaluated to measure the achieved hydrogel-like properties. The swelling kinetics of AcGGM-S-Gt were assessed by placing each hydrogel in excess deionized water and following the weight gain gravimetrically over 54 h, Figure 4. An equilibrium swelling

Figure 4. Swelling kinetics of AcGGM-S-Gt in deionized water together with visual variation of AcGGM-S-Gt upon swelling.

ratio of 11 was achieved for this hydrogel formulation within the test period, attributed to the hydrophilic interactions of AcGGM and water. AcGGM-S-Gt does not dissolve upon prolonged immersion in water, further suggesting the successful formation of a covalently bonded three-dimensional network. To better explore the network structure of AcGGM-S-Gt and the effect of swelling on its morphology, lyophilized hydrogel 671

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Figure 5. SEM images taken at ×250 magnification of (a) dry AcGGM-S-Gt surface, (b) dry AcGGM-S-Gt cross-section, (c) swollen AcGGM-S-Gt surface, and (d) swollen AcGGM-S-Gt cross-section.

thiyl radical coupling that afforded a hydrogel network. The FTIR spectrum of AcGGM-S-Gs is shown in Figure 3. The close resemblance between the FTIR spectra of AcGGM-S-Gs and AcGGM-SH indicates that AcGGM-S-Gs is made solely of thiolated AcGGM. However, the IR vibrations corresponding to S−H and S−S expected at 2600−2550 and 540−500 cm−1, respectively, are very weak and not detectible. 3.3. Postmodification of AcGGM-SH. In order to establish a synthetic platform for the further exploitation of hemicelluloses, two methods of AcGGM-SH postmodifications are exemplified. 3.3.1. Phenyl-Functionalization of AcGGM. Maleimides are known to be reactive Michael acceptors because of their ring strain and the cis-conformation of their two carbonyl groups.45 Postmodification of AcGGM-SH with N-phenylmaleimide was herein designed as a model reaction to demonstrate the feasibility of hemicellulose functionalization by virtue of a Michael addition click reaction. N-phenylmaleimide was selected because the position at which the 1H NMR response of the phenyl ring appears (7−8 ppm) does not overlay the sugar ring peaks in the 3−5 ppm region, allowing a more accurate analysis of the reaction product by means of NMR spectroscopy. Due to the insolubility of N-phenylmaleimide in water, this Michael addition reaction was conducted in DMSO at room temperature. 1H NMR spectra of AcGGM-SH0.5 before and after phenyl-functionalization are shown in Figure 6. In the 1 H NMR spectrum of AcGGM-S-Ph, three distinct peaks at 7.29, 7.44, and 7.52 ppm, respectively, arising from the C1, C3, and C2 protons of the phenyl ring in N-phenylmaleimide, indicate the presence of phenyl groups on AcGGM. The absence of a chemical shift arising from the CC bond in the maleimide ring (expected at 6−7 ppm) also verifies a completed click reaction. It is noteworthy that by increasing the degree of phenyl substitution, this reaction may serve as a promising means of reducing the hydrophilicity of hemicelluloses. 3.3.2. Grafting of PEG to AcGGM. The versatility of thiolated hemicellulose as a precursor for tailored materials was

Figure 6. 1H NMR spectrum of phenyl-functionalized AcGGM.

further demonstrated through the synthesis of PEG-grafted AcGGM. Our group has previously elaborated two “grafting from” processes performed by applying single-electron-transfer living radical polymerization (SET-LRP) and ring-opening polymerization (ROP).51,52 Pendant thiol groups of AcGGMSH may not be suitable sites to propagate a controlled polymerization of polymerizable species evolving along a chaingrowth pathway.7,53 However, they can perfectly fulfill the requirements for adopting a “grafting to” approach through their clickability. Polyethylene glycol monomethacrylate (PEG-MA) was grafted to AcGGM-SH by a thermally induced thiol−ene click reaction in water, using KPS and sodium sulfite as redox initiators. The grafted AcGGM was then characterized by NMR in order to assess the successful grafting process. The 1H NMR 672

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SEM and swelling properties. Phenyl-modification of AcGGM was accomplished through a thiol-maleimide Michael addition reaction using N-phenyl maleimide. PEG-grafted AcGGM was obtained after grafting PEG-MA to AcGGM-SH as a result of a thermally induced thiol−ene reaction. The grafted copolymer was assessed by 1H NMR spectroscopy to follow the extent of grafting. Thiol-modification of AcGGM is a powerful tool for the design of hemicellulose-based hydrogels and hybrid glycopolymers through a straightforward and highly efficient click-type approach.

spectra of the AcGGM-S-PEG copolymers are illustrated in Figure 7. The repeating unit of PEG is characterized by a

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ASSOCIATED CONTENT

* Supporting Information S

Alkaline SEC traces of pure and thiolated AcGGM, 1H NMR spectrum of AcGGM-S-Ph, and 1H NMR spectrum of AcGGM-S-PEG. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +46-8-7908274. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank Formas (Project Number 2011-1542) for financial support.

Figure 7. 1H NMR spectra of PEG-grafted AcGGM.

resonance at 3.62 ppm assigned to the CH2 protons in PEG main chain. However, no trace of any chemical shift stemming from the vinyl protons in the methacrylate end-groups expected between 5.5 and 6.5 ppm is observable. The grafting of PEG chains to AcGGM rather than accompanying it as homopolymer chains was evidenced by the absence of these vinyl peaks. The possibility to postmodify thiolated hemicellulose is here exemplified via grafting with PEG and phenylation but has many other potential uses. By “grafting-to” the hemicellulose backbone, a range of hybrid glycopolymers can be efficiently prepared. A related thio−bromo “click” reaction was proven a valuable tool to postmodify polymers synthesized via bromoinitiated controlled radical polymerization to facilitate accurate structural analysis and to determine the chain-end functionality.54−56

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4. CONCLUSIONS The synthesis of thiolated O-acetyl galactoglucomannan (AcGGM-SH) to a degree of modification of 0.16 yields a new, effective, and renewable platform enabling a wide array of click-type reactions. AcGGM was successfully thiol-functionalized by a one-pot procedure adopting AcGGM-mediated ringopening of γ-thiobutyrolactone at room temperature. The extent of thiol-modification was evaluated by 1H NMR spectroscopy together with a photometrical assay of the free thiol groups utilizing Ellman’s reagent. Thiol−ene and thiolMichael addition click reactions were performed as the basic paradigm of the valuable postmodification reactions applicable to such hemicellulose species. Three AcGGM-hydrogel formulations were synthesized using thiol−ene click reaction with MBAA, thiol-Michael addition to MBAA, and disulfide bond formation. Gel formation under these conditions is thus another confirmation of the successful thiolation of AcGGM. The hydrogels obtained from the thiol−ene reaction with MBAA were characterized by means of FTIR spectroscopy, 673

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