Research Article pubs.acs.org/journal/ascecg
Lignin Functionalization through Chemical Demethylation: Preparation and Tannin-Like Properties of Demethylated GuaiacylType Synthetic Lignins Kaori Sawamura,† Yuki Tobimatsu,†,‡ Hiroshi Kamitakahara,† and Toshiyuki Takano*,† †
Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
‡
S Supporting Information *
ABSTRACT: Demethylation of guaiacyl-type synthetic lignin (GDHP) with three different reagents, 1-dodecanethiol (DSH), hydroiodic acid (HI), and iodocyclohexane (ICH), was investigated as a basic study for lignin functionalization. Demethylation did not proceed efficiently in the reaction with DSH, although cleavage of the lignin side chains and nucleophilic substitution by DSH occurred. Demethylation proceeded efficiently in the reactions with HI and ICH, with significant cleavage of the lignin side chains. Furthermore, a recondensation reaction also occurred in the reaction with ICH. As a result, ICH was found to be the most effective demethylation reagent for increasing the phenolic-OH levels in the synthetic lignin. GDHP demethylated with ICH showed higher tannin-like properties (bovine serum albumin adsorption, 2,2-diphenyl-1-picrylhydrazyl radical scavenging, and iron(III) binding abilities) than nondemethylated GDHP. The latter two abilities especially were significantly improved. In addition, it was found from preliminary experiments that the demethylated GDHP was a useful precursor for lignin-based epoxy resin. These results suggested that chemical demethylation is an effective method for the functionalization of lignin. KEYWORDS: Demethylation, DHP, Lignin, Polyphenol, Softwood, Tannin
■
INTRODUCTION With the depletion of fossil resources and environmental concerns regarding their use, lignocellulosic biomass is becoming an important resource for the renewable production of fuels, chemicals, and energy. Lignin, a heterogeneous phenylpropanoid polymer, which encrusts cell wall polysaccharides (cellulose and hemicelluloses), composes 15−30% of lignocellulosic biomass. Despite its abundance, and the fact that it is currently the only feasible renewable feedstock for aromatic compounds, lignin has been underused in the current biomass utilization processes, and it is typically burned to generate the power needed to isolate polysaccharides from biomass. However, with an increasing demand for a cost-effective biorefinery process, the development of lignin valorization technologies, in particular, for the production of high-value aromatic materials and chemicals, is becoming an important research focus.1−3 Along with lignin, tannin is a common plant-derived polyphenol. Swain and Bate-Smith defined tannin as plantderived high-molecular weight phenolic compounds that precipitate alkaloids and proteins and display blue coloration in a solution containing iron(III) ions.4 Tannin is classically divided into hydrolyzable tannin and condensed tannin: the former includes commercially important extracts such as tannic © 2017 American Chemical Society
acid, and the latter includes Wattle tannin, quebracho tannin, persimmon tannin, and bark tannin. Tannin is well-known to exhibit several useful properties including protein precipitation, antioxidant, and heavy metal adsorption capabilities.5,6 Such properties of tannin and related polyphenols have been extensively investigated for the application to functional materials.7,8 Importantly, many of these unique functionalities of tannin and related polyphenols are considered to be primarily due to the abundantly present free phenolic-OH groups in the molecules. Lignin has been reported to exhibit tannin-like properties that might be leveraged for lignin varolization. Protein precipitation,9−11 antioxidant,12−14 and metal adsorption15 abilities of natural, synthetic, and technical lignins have been documented in the literature. However, such tannin-like abilities of lignin preparations are typically low, probably because the number of free phenolic-OH groups in lignin is low compared with tannin. Lignin contains substantial amounts of phenolic oxygens, but most of them are “capped” by etherification as aromatic methoxy groups and in the Received: March 10, 2017 Revised: April 17, 2017 Published: April 25, 2017 5424
DOI: 10.1021/acssuschemeng.7b00748 ACS Sustainable Chem. Eng. 2017, 5, 5424−5431
Research Article
ACS Sustainable Chemistry & Engineering
Figure 1. Representative structures of lignins (a) and tannins (b).
■
intermonomeric ether linkages (Figure 1). It is thereby conceivable that the tannin-like functionality of lignin could be enhanced by increasing the number of free phenolic-OH groups via chemical, and/or biochemical modification, such as demethylation. Lignin demethylation has mostly been studied for two purposes: (1) determination of methoxy group content in lignin; and (2) improvement of the reactivity of lignin preparations, in particular for applications to lignin-based thermosets, e.g., resins and adhesives. With respect to the former, hydroiodic acid has been widely used for quantification of methoxy groups in various lignin samples.16 With respect to the latter, chemical demethylation of technical lignins, e.g., Kraft and soda lignins, using various nucleophilic reagents such as thiols,17,18 or Lewis acids,19−21 as well as enzymatic demethylation using fungal enzymes22 have been investigated. Several reagents were found to be effective in increasing the concentration of free phenolic-OH groups in lignins. However, knowledge regarding the detailed chemical structures of the demethylated lignins prepared with various demethylation reagents is currently limited. In addition, to our knowledge, there have been no reports examining tannin-like functionalities of the demethylated lignins. Herein we explore the potential applicability of lignin demethylation for the production of lignin-based functional materials. In this study, we used a synthetic lignin (dehydrogenation polymer, DHP) as a pure lignin polymer model precursor for chemical demethylation to closely investigate the structure and properties of the demethylated lignin polymer products. Guaiacyl-type DHP (GDHP: softwood-type DHP) was demethylated using 1-dodecanethiol (DSH), hydroiodic acid (HI), and iodocyclohexane (ICH), and structures of the resultant demethylated GDHPs (deMeGDHPs) were characterized using 2D NMR in combination with chemical analysis and gel permeation chromatography (GPC). The functionality of the resultant deMeGDHPs was then evaluated in terms of the tannin-like properties as well as their usability as a precursor for lignin-based epoxy-resin materials.
EXPERIMENTAL SECTION
Preparation of GDHP. Coniferyl alcohol23 was synthesized and subjected to horseradish peroxidase (HRP)-catalyzed polymerization using the “end-wise” polymerization method according to literature.24 Briefly, solutions A [HRP (100 U/mg, 5.4 mg, Wako, Osaka, Japan) in 0.01 M phosphate buffer (pH 6.5, 267 mL)], B [coniferyl alcohol (800 mg) in distilled water (1065 mL)], and C [30% H2O2 (0.465 mL) in distilled water (1065 mL)] were prepared. Solutions B and C were simultaneously added to solution A using a peristaltic pump over a period of 24 h at ambient temperature. The reaction mixture was then treated with additional HRP (5.4 mg) and allowed to stand at ambient temperature for 24 h. The resulting precipitate was collected by centrifugation, washed with distilled water, and lyophilized to afford GDHP (typically 80%−90% weight yield). Demethylation of GDHP with DSH. A solution of DSH (0.2 mL) in DMF (0.5 mL) was treated with a solution of 28% NaOMe/ MeOH (0.29 mL) at 5 °C. After the solution had warmed to 25 °C, a solution of GDHP (100 mg) in DMF (0.5 mL) was added, and the resultant mixture was heated to reflux for 1 h (for deMeGDHP-DSH1) or 12 h (for deMeGDHP-DSH2). The reaction mixture was then cooled to 5 °C and acidified with 1 M HCl. The resultant precipitates were filtered, washed with distilled water and n-hexane, and dried in vacuo to afford deMeGDHP-DSH1 or deMeGDHP-DSH2. Demethylation of GDHP with HI. A solution of GDHP (50 mg) in DMF (2.5 mL) was treated with a 55% HI solution (0.21 mL) in a pressure-proof test tube at ambient temperature and then heated at 160 °C for 1 h (for deMeGDHP-HI1) or 12 h (for deMeGDHP-HI2). The reaction mixture was then cooled to ambient temperature, washed with n-hexane, and poured into a saturated Na2S2O5 solution. The resulting precipitate was collected by filtration, washed with distilled water, and lyophilized to afford deMeGDHP-HI1 or deMeGDHPHI2. Demethylation of GDHP with ICH. A solution of GDHP (100 mg) in DMF (2.5 mL) was treated with ICH (0.715 mL) and heated to reflux for 5 min (for deMeGDHP-ICH1), 1 h (deMeGDHP-ICH2), or 12 h (deMeGDHP-ICH3). The reaction mixture was then cooled to ambient temperature, washed with n-hexane, and poured into a saturated Na2S2O5 solution. The resulting precipitate was collected by filtration, washed with distilled water, and lyophilized to afford deMeGDHP-ICH1, deMeGDHP-ICH2, or deMeGDHP-ICH3. Acetylation of DHPs. A sample of DHP (∼20 mg) was dissolved in acetic anhydride/pyridine (1:1, v/v, 2 mL). After stirring at ambient temperature overnight, the mixture was poured into distilled water (200 mL). The resultant precipitate was collected by filtration, washed 5425
DOI: 10.1021/acssuschemeng.7b00748 ACS Sustainable Chem. Eng. 2017, 5, 5424−5431
Research Article
ACS Sustainable Chemistry & Engineering Table 1. Summary of Structural Characterization of DHPs NMR signal intensity ratioa entry reagent 1 2 3 4 5 6 7 8
− DSH DSH HI HI ICH ICH ICH
reaction time yield (wt %) − 1h 12 h 1h 12 h 5 min 1h 12 h
− 82 92 65 62 57 76 65
GPC datac
product
Ph-OAc/AliphOAc
Ph-OMe/PhOAc
phenolic-OH (mmol/g)
GDHP deMeGDHP-DSH1 deMeGDHP-DSH2 deMeGDHP-HI1 deMeGDHP-HI2 deMeGDHP-ICH1 deMeGDHP-ICH2 deMeGDHP-ICH3
0.12 0.39 0.46 0.49 2.0 0.83 4.0 15.0
10.3 4.9 3.6 4.0 0.73 3.8 1.0 0.03
2.1 2.2 2.1 3.0 5.3 3.2 5.3 7.8
b
Mw (× 103)
Mw/ Mn
6.4 4.8 4.0 3.8 3.4 4.8 5.6 5.6
1.9 1.6 1.6 1.5 1.5 1.8 2.0 1.9
a
Determined in HSQC NMR spectra of acetlyated DHPs. Ph-OAc, methyl signals attached to phenolic acetoxy groups. Aliph-OAc, methyl signals attached to aliphatic acetoxy groups. Ph-OMe, methyl signals attached to aromatic methoxyl groups. bDetermined by Folin−Ciocalteu assay. c Determined after acetylation using polystyrene standards. Protein Adsorption Test. The protein adsorption test was performed according to the method described by Kawamoto et al.9 Briefly, deMeGDHP samples (1 mg) were added to a solution of bovine serum albumin (BSA) in 50 mM acetate buffer (pH 4.5, 0.5 mL). The mixture was sonicated for 3 min, stirred at ambient temperature for 1 h, and then centrifuged. The supernatant was filtered through cotton wool and then treated with Bradford reagent32 (3 mL). The solution was kept at ambient temperature for 10 min and then subjected to UV−vis measurement at 595 nm. The calibration curve was constructed using BSA solutions in the acetate buffer. The test was performed three times, and the data reported here are mean ± standard deviations. DPPH Free Radical Scavenging Ability Test. The test was performed according to the method described by Aquino et al. with a modification.33 Briefly, 2,2-diphenyl-1-picrylhydrazyl (DPPH) (2.5 mg) was dissolved in 90% dioxane/water (v/v, 100 mL). A solution of deMeGDHP samples in 90% dioxane/water (200 μg/mL, 75 μL) was mixed with the DPPH solution (2 mL). The mixture was stirred at ambient temperature for 36 h and then subjected to UV−vis measurement at 515 nm. The calibration curve was constructed using (+)-catechin solutions in 90% dioxane/water. The test was performed three times, and the data reported here are mean ± standard deviations. Iron(III) Binding Ability Test. The test was performed according to the method described by Nishida with a modification.34 Briefly, deMeGDHP samples (2 mg) were added to a solution of 0.5 mM FeCl3 in 50 mM acetate buffer (pH 5.5, 9 mL). The mixture was sonicated for 3 min, stirred at ambient temperature for 12 h, and then centrifuged. The supernatant was treated with 1.5 mL of 2.5 mM Chrome Azurol S (CAS) aqueous solution (Nacalai Tesque Inc.), kept at ambient temperature for 10 min, and then subjected to UV−vis measurement at 630 nm. The calibration curve was constructed using a series of FeCl3 solutions in the acetate buffer. The test was performed three times, and the data reported here are mean ± standard deviations. Preliminary Evaluation of deMeGDHP as a Hardener for Epoxy Resin. In a silicon cup, deMeGDP-ICH3 (40 mg), diglycidyl ether bisphenol A (DGEBA) (14.6 mg), and 2-ethyl-4-methyl imidazole (2E4MZ) (0.55 mg) were dissolved in DMF (2 mL).35 The solution was stirred for 30 min at ambient temperature and dried in vacuo at 60 °C to remove the DMF solvent. The residue (noncuring resin mixture, deMeGDHP-ICH3-EP) was then cured by heating successively at 110 °C for 30 min, 150 °C for 2 h, and 180 °C for 3 h, to afford deMeGDHP-ICH3-EP-cured. The control samples (deMeGDHP-EP and deMeGDHP-EP-cured) were prepared from GDHP in the same manner. FTIR spectroscopy was performed using a typical KBr method with a Shimadzu 8600PCs FT-IR spectrophotometer (Shimadzu Co., Kyoto, Japan; resolution mode, 4.0, 100 scans). Thermogravimetric analysis (TGA) was conducted under nitrogen (flow rate: 50 mL/min) with a Shimadzu TGA-50
thoroughly with distilled water, and lyophilized to yield acetylated DHP. NMR Spectroscopy. Acetylated DHPs were dissolved in CDCl3 (∼30 mg/mL) and subjected to NMR analysis on a Varian FT-NMR (500 MHz) spectrometer (Agilent Technologies, Santa Clara, CA, USA) operated with Varian VnmrJ 3.2 software. The central chloroform peaks were used as internal reference (δC/δH: 77.0/7.26 ppm). Adiabatic HSQC experiments were performed using the Varian standard implementation (“HSQCAD”), and experiment parameters were set according to the literature.25,26 Data processing was performed using Bruker Topspin 3.1 (Mac) software and used typical matched Gaussian apodization in F2 (LB = −0.5, GB = 0.001) and squared cosine-bell and one level of linear prediction (32 coefficients) in F1. For volume integration, linear prediction was turned off, and no correction factors were used; therefore, the reported data represent volume integrals only. GPC Analysis. Acetylated DHPs were dissolved in CHCl3 (1 mg/ mL) and subjected to GPC analysis for molecular weight determination on a Shimadzu LC-10 system (Shimadzu Co., Kyoto, Japan) equipped with a Shimadzu UV−vis detector (SPD-10Avp) under the following conditions: columns: Shodex columns K-802, K802.5, and K-805 connected in series (Showa Denko K. K., Tokyo, Japan); column temperature: 40 °C; eluent: CHCl3; flow rate: 1.0 mL/min; and sample detection: UV absorbance at 280 nm. The molecular mass calibration used polystyrene standards (Shodex, Showa Denko K. K.). Folin−Ciocalteu Assay. The total free phenolic-OH content in DHPs was determined using a Folin−Ciocalteu assay following a modified literature procedure.27−29 Briefly, 5 mL of Folin−Ciocalteu phenol reagent (Nacalai Tesque Inc., Kyoto, Japan) was mixed with 90% dioxane/water (v/v) (45 mL). The mixture was kept at ambient temperature for 15 min and then filtered to remove precipitates. The solution (2 mL) was then mixed with the solution containing DHP samples (200 μg/mL in 90% dioxane/water, 200 μL). The mixture was stirred at ambient temperature for 3 min, and then a 7.5% Na2CO3 aqueous solution (1.6 mL) was added. The mixture was stirred at ambient temperature for a further 54 h and then subjected to UV−vis measurement at 765 nm. UV−vis spectra were recorded with a JASCO V-560 UV−vis spectrometer (Jasco, Hachioji, Japan). The calibration curve was constructed using solutions of (+)-catechin (Nacalai Tesque Inc.) in 90% dioxane/water. The data reported in Table 1 are means of triplicates (SD < 7%). Nitroso Assay. A nitroso assay for the qualitative analysis of catechol residues in DHP samples was performed according to the literature.30,31 A solution of DHP in DMF (0.5 mg/mL, 1.2 mL) was treated successively with 10% NaNO2 aqueous solution (600 μL), 20% urea aqueous solution (w/v) (600 μL), and 10% CH3COOH aqueous solution (600 μL). The mixture was stirred at ambient temperature for 3 min, then 2 M NaOH (1.2 mL) was added, and the solution color was visually observed. 5426
DOI: 10.1021/acssuschemeng.7b00748 ACS Sustainable Chem. Eng. 2017, 5, 5424−5431
Research Article
ACS Sustainable Chemistry & Engineering
Figure 2. Short range 1H−13C correlation (HSQC) NMR spectra of acetylated samples of guaiacyl synthetic lignin, GDHP-Ac (a) and demethylated GDHPs prepared with (b) 1-dodecanethiol, DSH (deMeGDHP-DSH2-Ac), (c) hydroiodic acid, HI (deMeGDHP−HI2-Ac), and (d) iodocyclohexane, ICH (deMeGDHP-ICH3-Ac). thermal analyzer (Shimadzu Co.). The samples were heated from 25 to 700 °C at a rate of 10 °C/min.
phenolic-OH and aliphatic-OH groups and from aromatic methoxy groups (OMe) in the lignin polymers. The structural changes of the lignin polymer could be assessed based on the integral ratios of the OAc and OMe contour signals. It was clear that the signal ratio of phenolic-OAc and aliphatic-OAc groups (Ph-OAc/Aliph-OAc) increased, whereas the ratio of aromatic methoxy and phenolic-OAc signals (Ph-OMe/Ph-OAc) decreased by treatment with DSH (Table 1). This suggests that the anticipated release of free phenolic-OH groups in the lignin polymer takes place in the reaction with DSH. However, the Ph-OAc/Aliph-OAc and Ph-OMe/Ph-OAc signal ratios did not change much with an increase of reaction time after 1 h. In addition, both the aromatic methoxy groups (OMe) and nondemethylated guiacyl aromatic unit (G) signals were still clearly visible even after 12 h (Figure 2B). These data suggest that only partial aromatic demethylation of GDHP occurs with DSH at least under the present conditions. The signals from the β−O−4 linkages (I and IV) markedly decreased in the spectra of deMeGDHP-DSH1-Ac and deMeGDHP-DSH2-Ac (Figures 2 and S1 in the Supporting Information). This suggests cleavage of the β-aryl ether linkages, which, along with aromatic demethylation, also contributed to the increase of free phenolic-OH groups in the lignin polymer. The decreased molecular weights of the deMeGDHP-DSHs were also supportive of such partial cleavage of intermonomeric linkages in the polymer (Table 1). In addition, the deMeGDHP-DSH spectra displayed intense signals from alkyl methylene and methyl groups of DSH (Figures 2B and S1 in the Supporting Information), which suggested incorporation of DSH into the polymer during the reaction. Incorporation of the DSH reagent into demethylated kraft lignin was also reported in the literature.17 Taken together, it is most likely that de-etherification of β−O−4 linkages took
■
RESULTS AND DISCUSSION Demethylation of GDHP. Three different demethylation reagents (DSH, HI, and ICH) were used for the preparation of demethylated synthetic lignins (deMeGDHPs). HI efficiently converted the aromatic methoxy groups in lignin into the corresponding free phenolic-OHs and stoichiometrically generated MeI, which could be quantified for the estimation of the methoxy group content in the original lignin polymer.16,36,37 Although the reagent has long been used for methoxy content analysis of various lignin samples, very little is known regarding the structure and properties of the remaining demethylated lignin polymer fractions. DSH38 and ICH39 are relatively recently developed reagents for the demethylation of aryl methyl ethers in small-molecule organic chemistry. Although both reagents have been recently applied to the demethylation of technical lignins,17,21 the reaction details are unclear. In the present study, we used a modern 2D NMR technique, as well as chemical methods and GPC, to comparatively characterize the deMeGDHPs prepared with the above three demethylation reagents with different reaction times. Demethylation with DSH. Demethylation of GDHP with DSH in refluxing DMF17 for 1 and 12 h afforded deMeGDHPDSH1 and deMeGDHP-DSH2, respectively, in good yields (Table 1). The HSQC NMRs of acetylated GDHP (GDHPAc) and acetylated deMeGDHP-DSHs (deMeGDHP-DSH1Ac and deMeGDHP-DSH2-Ac) are shown in Figures 2 and S1 in the Supporting Information. All the DHP spectra displayed typical lignin aromatic (G) and side-chain (I−IV) signals as well as those from acetoxy groups (OAc) attached to the 5427
DOI: 10.1021/acssuschemeng.7b00748 ACS Sustainable Chem. Eng. 2017, 5, 5424−5431
Research Article
ACS Sustainable Chemistry & Engineering place mainly by nucleophilic displacement with DSH at the βcarbons, although further investigation is required. Given that β−5 (II) and β−β (III) linkage signals mostly remained in the deMeGDHP-DSH spectrum (Figure 2B), the cleavage of β-aryl ethers in the β−O−4 linkages exceeds the cleavage of the stabilized cyclic α-ethers in β−5 (II) and β−β (III) linkages. The phenolic-OH content of deMeGDHP-DSHs was also evaluated using the Folin−Ciocalteu method (Table 1). There was almost no change in the phenolic-OH content between GDHP and deMeGDHP-DSHs. This is apparently contradictory to our NMR data, which showed a relative increase of phenolic-OH signals over aliphatic-OH and aromatic methoxyl signals in the deMeGDHP-DSH spectra. It can, however, be explained by the fact that incorporation of the DSH alkyl chains in the polymer decreased the concentration of aromatic units in the polymer, which offset the increase of phenolic-OH content per sample weight. We also checked the presence of catechol units in deMeGDHP-DSH2 using a nitroso assay.30,31 The polymer did not clearly exhibit positive red coloration (Figure 3), suggesting that only a small amount of catechols were
positive red coloration, suggesting that some catechol units were present in the polymer (Figure 3). The lignin side-chain signals, especially those from the β− O−4 linkages (I and IV), were considerably depleted in the spectrum of deMeGDHP-HI1-Ac, while the methoxy (OMe) and guiacyl aromatic (G) signals were still clearly present (Figure S2 in the Supporting Information). The data suggested that cleavage of β-aryl ethers prevailed prior to aromatic demethylation at the initial stage of the HI reaction. On the other hand, all the major side-chain signals, including β−5 (II) and β−β (III) linkage signals, as well as the methoxy (OMe) and guiacyl aromatic (G) signals were considerably depleted in the spectrum of deMeGDHP-HI2-Ac (Figure 2C). In addition, new broad aromatic signals appeared at δC/δH = 122/7.2 (colored in red, Figure 2). It has been reported that HSQC spectra of acetylated samples of caffeyl alcohol-derived ligninlike polymers, which contain substantial amounts of free catechol units, displayed acetylated catechol ring signals likewise in a region at δC/δH = 122−126/7.2.40 Hence, although further studies are needed for a conclusive assignment, we presumably attributed the new aromatic signals appearing in the spectra of deMeGDHP-HI2 to catechol-like aromatic systems generated via demetylation of guaiacyl aromatic rings at the later stage of the HI reaction. Taken together, our NMR data suggested that the cleavage of the lignin side-chains, presumably by HI-induced de-etherification and hydrolysis, is dominant at the initial stage and the anticipated aromatic demethylation occurs at the later stage of the HI reaction. GPC analysis showed that a substantial decrease of DHP molecular weight had already occurred after 1 h of the HI reaction (Table 1), which further supports cleavage of the lignin side chains prior to the aromatic demethylation. Demethylation with ICH. Demethylation of GDHP with ICH was performed under similar conditions as in the reactions with DSH and HI described above. The 5 min, 1 and 12 h reactions afforded deMeGDHP-ICH1, deMeGDHP-ICH2, and deMeGDHP-ICH3, respectively (Table 1). NMR analysis of the acetylated deMeGDHP-ICHs (deMeGDHP-ICH1-Ac, deMeGDHP-ICH2-Ac, and deMeGDHP-ICH3-Ac) showed that the Ph-OAc/Aliph-OAc signal ratio in the HSQC spectra increased markedly with an increase of reaction time and reached 15.0 after 12 h, 125-fold higher than that determined for the original GDHP, and concurrently, the Ph-OMe/Ph-OAc signal ratio decreased from 10.3 to 0.03 after 12 h reaction. The Folin−Ciocalteu assay determined the phenolic-OH contents of deMeGDHP-ICH1, deMeGDHP-ICH2, and deMeGDHPICH3 to be 1.5-, 2.5-, and 3.7-fold higher than that of the original GDHP (Table 1). In addition, the nitroso assay for deMeGDHP-ICH3 gave a positive deep red coloration and suggested that the catechol level of the polymer was substantially higher than those of the other deMeGDHPs prepared with DSH or HI (Figure 3). These data collectively suggest that ICH increases free phenolic-OHs via demethylation more efficiently than DSH and HI. The reaction of ICH with GDHP most likely proceeds in a similar manner to the HI reaction, as it is known that ICH produces HI in situ via an elimination process.39 As observed in the HI reaction, all the lignin side chain signals were markedly depleted in the spectrum of deMeGDHP-ICHs (Figure 2D). Based on the signals from the major lignin linkages (I−IV), the order of the reactivity of these linkages was β−O−4 (I and IV, completely disappeared at 5 min reaction) > β−β (III, disappeared at 1 h reaction) > β−5 (II, disappeared at 12 h
Figure 3. Solutions of guaiacyl synthetic lignin (GDHP) and demethylated GDHPs (deMeGDHPs) treated with a nitroso reagent for the detection of catechol units.
formed in the DSH reaction. Collectively, our data suggest that, at least under the present reaction conditions, DSH treatment only partially increases free phenolic-OH groups in the lignin polymer, which is most likely mainly via cleavage of β-aryl ethers rather than aromatic demethylation. Demethylation with HI. Demethylation of GDHP with HI was carried out under similar conditions to the reaction with DSH. Reaction times of 1 and 12 h afforded deMeGDHP-HI1 and deMeGDHP-HI2, respectively (Table 1). HSQC NMR spectra of the acetylated deMeGDHP-HIs (deMeGDHP-HI-1Ac and deMeGDHP−HI2-Ac) are shown in Figures 2C and S2 in the Supporting Information, and the Ph-OAc/Aliph-OAc and Ph-OMe/Ph-OAc NMR signal ratios are listed in Table 1. The Ph-OAc/Aliph-OAc signal ratio dramatically increased by 4-fold and 17-fold in the spectra of deMeGDHP-HI1-Ac and deMeGDHP-HI2-Ac, respectively, compared to that in the spectrum of GDHP-Ac. At the same time, the Ph-OMe/PhOAc signal ratio markedly decreased as the reaction time increased. These NMR data collectively suggest an efficient release of free phenolic-OHs via aromatic demethylation in the HI reaction. In line with this, the Folin−Ciocalteu assay determined the phenolic-OH contents of deMeGDHP-HI1 and deMeGDHP-HI2 to be 1.4-fold and 2.5-fold higher than that of the original GDHP (Table 1). In addition, treatment of deMeGDHP-HI2 solution with the nitroso reagent gave a 5428
DOI: 10.1021/acssuschemeng.7b00748 ACS Sustainable Chem. Eng. 2017, 5, 5424−5431
Research Article
ACS Sustainable Chemistry & Engineering
Figure 4. Tannin-like property characterization of guaiacyl synthetic lignin (GDHP) and demethylated GDHPs prepared with iodocyclohexane (ICH) with 5 min (deMeGDHP-ICH1), 1 h (deMeGDHP-ICH2), and 12 h (deMeGDHP-ICH3) reaction times. (a) BSA adsorption capacity. (b) DPPH scavenging capacity. (c) Fe3+ ion adsorption capacity. Values are means ± standard deviation, SD (n = 3), and asterisks indicate significant differences between GDHP and deMeGDHP-ICHs (Student’s t-test, *: p < 0.05; **: p < 0.01).
scavenging capacity of deMeGDHP-ICHs was up to 3.9-fold higher than that of nondemethylated GDHP (Figure 4B). Thus, demethylation is effective in increasing the antioxidant properties of lignin and might be useful for applications such as the production of lignin-based biomedical, cosmetic, and dietary products. Metal binding/chelating is an important feature of tannin and related polyphenols and can be exploited for the development of functional materials such as heavy metal sorbents to be used in wastewater treatment or in the collection of precious metals. Iron(III) binding of tannin is also important for understanding tannin antioxidant ability in relation to the Fenton reaction.48 As for lignin, there is one report concerning the absorption of lignin with iron(III), which is one of the most abundant transition-metal ions in soil.15 Our iron(III) binding ability test using a CAS reagent34,49 determined that the binding capacity of deMeGDHP-ICHs was significantly enhanced (up to 2.6-fold higher) compared to the nondemethylated GDHP control. Like we observed for the DPPH radical scavenging ability described above, deMeGDHP-ICH exhibited a good correlation between iron-binding ability and the phenolic-OH content (Figure 4C). We also tested the association of deMeGDHP-ICH3 with the iron(III) cation in a solution-state. A buffer-dioxane solution containing deMeGDHP-ICH3 displayed a deep blue coloration upon the addition of the iron(III) cation, a typical coloration observed for a tannin solution (Figure 5). It is most likely that the
reaction) (Figure S3 in Supporting Information). The nondemethylated aromatic ring (G) signals almost completely disappeared, and, as observed in the HI reaction, the new broad aromatic signals, presumably from catechol units (at δC/δH = 122/7.2, colored in red), were visible in the spectrum of deMeGDHP-ICH3-Ac after a 12 h reaction (Figure 2D). The molecular weight of GDHP decreased at 5 min, but had increased at the 1 and 12 h reactions (Table 1). This implies that an acid-induced recondensation reaction also occurred in the later stage of the ICH reaction. Overall, ICH was found to be the most effective demethylation reagent among the three reagents examined in this study. Evaluation of the Tannin-Like Properties of deMeGDHP-ICHs and GDHP. Next, the tannin-like properties of deMeGDHP-ICHs with different phenolic-OH levels were evaluated. BSA adsorption, DPPH free radical scavenging, and iron binding tests were selected to assess the major tannin properties, i.e., protein precipitation, antioxidant, and metal-ion adsorption abilities, respectively. The BSA adsorption test is the most popular method for evaluating the protein precipitation ability of tannins41,42 and has also been used for the characterization of lignins.9−11 The BSA adsorption abilities of the deMeGDHP-ICHs were significantly higher than that of GDHP (Figure 4A). However, there was no clear correlation between the BSA adsorption ability and the phenolic-OH content. The nature and extent of the associations between tannins and proteins depend on chemical structure and related physical properties of the tannin molecules.43,44 Kawamoto et al. (1992) previously reported that various isolated lignins displayed relatively weak BSA adsorption capacities with no clear correlation with their phenolic-OH content.9 Our data suggest that not only the phenolic-OH content but also some other factors, such as the distribution of phenolic-OHs in the polymer, might affect the protein adsorption abilities of the demethylated lignins. The DPPH free radical-scavenging ability test is a convenient antioxidant test for various polyphenols including tannin45−47 and lignins.12−14 The conventional method was modified for our assay for deMeGDHP-ICHs and GDHP using a 90% dioxane/water solvent system. It was clear that the DPPH free radical-scavenging ability of deMeGDHP-ICHs significantly increased with an increase in phenolic-OH levels. The
Figure 5. Solutions of guaiacyl synthetic lignin (GDHP) and demethylated GDHP prepared with 12 h ICH reaction (deMeGDHP-ICH3) untreated and treated with ferric (III) ion for chelate formation. 5429
DOI: 10.1021/acssuschemeng.7b00748 ACS Sustainable Chem. Eng. 2017, 5, 5424−5431
Research Article
ACS Sustainable Chemistry & Engineering
content in the lignin polymer under the present conditions. The deMeGDHPs showed significantly higher BSA adsorption, DPPH free radical-scavenging, and iron(III)-binding abilities than those of the nondemethylated GDHP control. The latter two abilities were significantly higher than those of the control and correlated well with the phenolic-OH content in the lignin polymer. Our results demonstrate that the tannin-like functionality of lignin can be substantially enhanced by demethylation, as expected, i.e., lignin can be converted to a tannin-like polymer by demethylation; while we used a guaiacyl DHP as a model lignin precursor in this study, it is highly conceivable that the approach can be applied to various types of lignin materials derived from biomass, which will be further investigated in our future studies. Overall, we envision that chemical demethylation could be an alternative strategy for improving lignin varolization for future biorefinery.
catechol units newly formed in deMeGDHP via demethylation can chelate with the iron(III) cations.48 Overall, demethylation was found to be effective in enhancing the iron(III) binding of lignin. Preliminary Evaluation of deMeGDHP-ICH3 as a Hardener for Epoxy Resin. Lignin-based epoxy resin is one of the targets for new lignin utilization.1 We evaluated, as a preliminary study, the usability of deMeGDHP prepared with ICH as a hardener for epoxy resin. The curing reaction between deMeGDHP-ICH3 and DGEBA (phenol group:epoxy group = 1:1) in the presence of 2E4MZ35 afforded the cured epoxy resin, deMeGDHP-ICH3-EP-cured. A control sample (GDHPEP-cured) was also prepared from GDHP. The characteristic epoxy vibration bands at 910 cm−1, visible both in the FT-IR spectra of deMeGDHP-ICH3-EP and GDHP-EP, completely disappeared in the spectra of deMeGDHP-ICH3-EP-cured and GDHP-EP-cured (Figure S4 in Supporting Information), suggesting that the curing reaction had proceeded successfully. TGA curves of deMeGDHP-ICH3-cured and GDHP-EP-cured are shown in Figure 6. The 5% and 10% weight loss
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00748. Figures S1−S3: HSQC NMR spectra of GDHP and deMeGDHPs; Figure S4: FT-IR spectra of epoxyGDHPs and epoxy-deMeGDHP-ICHs (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Toshiyuki Takano: 0000-0001-8244-223X Notes
The authors declare no competing financial interest. Figure 6. TGA curves for cured epoxy resins from guaiacyl synthetic lignin, GDHP (GDHP-EP-cured), and demethylated GDHP prepared with 12 h ICH reaction (deMeGDHP-ICH3-EP-cured).
■
ABBREVIATIONS
■
REFERENCES
BSA, bovine serum albumin; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DSH, 1-dodecanethiol; HI, hydroiodic acid; ICH, iodocyclohexane; GDHP, guaiacyl-type dehydrogenation polymer
temperature (Td5 and Td10) of deMeGDHP-ICH3-EP-cured (366 and 399 °C) were substantially higher than those of GDHP-EP-cured (327 and 372 °C), clearly indicating an enhanced thermal durability through demethylation. Taken together, our results suggest that demethylation using ICH could be an effective pretreatment for lignin-based epoxy resin.
(1) Upton, B. M.; Kasko, A. M. Strategies for the conversion of lignin to high-value polymeric materials: review and perspective. Chem. Rev. 2016, 116, 2275−2306. (2) Galkin, M. V.; Samec, J. S. Lignin valorization through catalytic lignocellulose fractionation: a fundamental platform for the future biorefinery. ChemSusChem 2016, 9 (13), 1544−1558. (3) Rinaldi, R.; Jastrzebski, R.; Clough, M. T.; Ralph, J.; Kennema, M.; Bruijnincx, P. C.; Weckhuysen, B. M. Paving the way for lignin valorisation: recent advances in bioengineering, biorefining and catalysis. Angew. Chem., Int. Ed. 2016, 55 (29), 8164−8215. (4) Swain, T.; Bate-Smith, C. Flavonoid compounds. In Comparative Biochemistry: A Comprehensive Treatise, Constituent of life − Part A; Florkin, M., Mason, H. S., Eds.; Academic Press: New York, 1962; Vol. III (5) Santos-Buelga, C.; Scalbert, A. Proanthocyanidins and tannin-like compounds − nature, occurrence, dietary intake and effects on nutrition and health. J. Sci. Food Agric. 2000, 80, 1094−1117. (6) Quideau, S.; Deffieux, D.; Douat-Casassus, C.; Pouysegu, L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew. Chem., Int. Ed. 2011, 50 (3), 586−621.
■
CONCLUSIONS The reactions of GDHP with three different demethylation reagents, DSH, HI, and ICH, were investigated. With all three reagents, demethylation proceeded along with the cleavage of lignin side chains. In the reaction with DSH, while the cleavage of β−O−4 linkages proceeded efficiently, most likely via nucleophilic substitutions by DSH, the aromatic demethylation was not so good. Efficient aromatic demethylations and substantial increases in the phenolic-OH levels in the lignin polymer were observed for the reactions with HI and ICH. In both cases, cleavage of the major lignin linkages, including β− O−4, β−5, and β−β linkages, proceeded smoothly. Furthermore, a recondensation reaction was suggested in the reaction with ICH. Consequently, ICH was found to be the most reactive demethylation reagent for increasing the phenolic-OH 5430
DOI: 10.1021/acssuschemeng.7b00748 ACS Sustainable Chem. Eng. 2017, 5, 5424−5431
Research Article
ACS Sustainable Chemistry & Engineering (7) Arbenz, A.; Avérous, L. Chemical modification of tannins to elaborate aromatic biobased macromolecular architectures. Green Chem. 2015, 17, 2626−2646. (8) Bacelo, H. A. M.; Santos, S. C. R.; Botelho, C. M. S. Tanninbased biosorbents for environmental applications−A review. Chem. Eng. J. 2016, 303, 575−587. (9) Kawamoto, H.; Nakatsubo, F.; Murakami, K. Protein-adsorbing capacities of lignin samples. Mokuzai Gakkaishi 1992, 38 (1), 81−84. (10) Zahedifar, M.; Castro, F. B.; Ørskov, E. R. Effect of hydrolytic lignin on formation of protein−lignin complexes and protein degradation by rumen microbes. Anim. Feed Sci. Technol. 2002, 95 (1), 83−92. (11) Yoshida, T.; Lu, R.; Han, S.; Hattori, K.; Katsuta, T.; Takeda, K.; Sugimoto, K.; Funaoka, M. Laccase-catalyzed polymerization of lignocatechol and affinity on proteins of resulting polymers. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 824−832. (12) Dizhbite, T.; Telysheva, G.; Jurkjane, V.; Viesturs, U. Characterization of the radical scavenging activity of lignins − natural antioxidants. Bioresour. Technol. 2004, 95, 309−317. (13) Nsimba, R. Y.; West, N.; Boateng, A. A. Structure and radical scavenging activity relationships of pyrolytic lignins. J. Agric. Food Chem. 2012, 60, 12525−12530. (14) Zhang, S.; Zhang, Y.; Liu, L.; Fang, G. Antioxidant activity of organosolv lignin degraded using SO42‑/ ZrO2 as catalyst. BioResources 2015, 10 (4), 6819−6829. (15) Guillon, E.; Merdy, P.; Aplincourt, M.; Dumonceau, J.; Vezin, H. Structural characterization and iron (III) binding ability of dimeric and polymeric lignin models. J. Colloid Interface Sci. 2001, 239, 39−48. (16) Chen, C. L. Determination of methoxyl groups. In Methods in Lignin Chemistry ; Lin, Y. S., Dence, C. W., Eds; Springer: Berlin, Germany, 1992. (17) Hu, L.; Pan, H.; Zhou, Y.; Hse, C.-Y.; Liu, C.; Zhang, B.; Xu, B. Chemical groups and structural characterization of lignin via thiolmediated demethylation. J. Wood Chem. Technol. 2014, 34, 122−134. (18) Li, J.; Wang, W.; Zhang, S.; Gao, Q.; Zhang, W.; Li, J. Preparation and characterization of lignin demethylated at atmospheric pressure and its application in fast curing biobased phenolic resins. RSC Adv. 2016, 6, 67435−67443. (19) Chung, H.; Washburn, N. R. Improved lignin polyurethane properties with lewis acid treatment. ACS Appl. Mater. Interfaces 2012, 4 (6), 2840−2846. (20) Ferhan, M.; Yan, N.; Sain, M. A new method for demethylation of lignin from woody biomass using biophysical methods. J. Chem. Eng. Process Technol. 2013, 4, 1000160. (21) Song, Y.; Wang, Z.; Yan, N.; Zhang, R.; Li, J. Demethylation of wheat straw alkali lignin for application in phenol formaldehyde adhesives. Polymers 2016, 8, 209. (22) Zou, L.; Ross, B. M.; Hutchison, L. J.; Christopher, L. P.; Dekker, R. F. H.; Malek, L. Fungal demethylation of Kraft lignin. Enzyme Microb. Technol. 2015, 73−74, 44−50. (23) Quideau, S.; Ralph, J. Facile large-scale synthesis of coniferyl, sinapyl, and p-coumaryl alcohol. J. Agric. Food Chem. 1992, 40 (7), 1108−1110. (24) Tobimatsu, Y.; Takano, T.; Kamitakahara, H.; Nakatsubo, F. Studies on the dehydrogenative polymerizations of monolignol βglycosides. Part 2: Horseradish peroxidase-catalyzed dehydrogenative polymerization of isoconiferin. Holzforschung 2006, 60, 513−518. (25) Tobimatsu, Y.; Davidson, C. L.; Grabber, J. H.; Ralph, J. Fluorescence-tagged monolignols: Synthesis, and application to studying in vitro lignification. Biomacromolecules 2011, 12, 1752−1761. (26) Mansfield, S. D.; Kim, H.; Lu, F.; Ralph, J. Whole plant cell wall characterization using solution-state 2D NMR. Nat. Protoc. 2012, 7, 1579−1589. (27) Bond, T. J. Analysis and purification of catechins and their transformation products. In Methods in polyphenol analysis; SantosBuekga, C., Williamson, G., Eds.; Athenaeum Press: Gateshead, U.K., 2003.
(28) Georgé, S.; Brat, P.; Alter, P.; Amiot, M. J. Rapid determination of polyphenols and vitamin C in plant-derived products. J. Agric. Food Chem. 2005, 53, 1370−1373. (29) dos Santos, P. S. B.; Erdocia, X.; Gatto, D. A.; Labidi, J. Characterisation of kraft lignin separated by gradient acid. Ind. Crops Prod. 2014, 55, 149−154. (30) Reeve, R. M. Histological and histochemical changes in developing and ripening peaches. I. The catechol tannins. Am. J. Bot. 1959, 46, 210−217. (31) Tobimatsu, Y.; Chen, F.; Nakashima, J.; Escamilla-Treviño, L. L.; Jackson, L.; Dixon, R. A.; Ralph, J. Coexistence but independent biosynthesis of catechyl and guaiacyl/syringyl lignin polymers in seed coats. Plant Cell 2013, 25, 2587−2600. (32) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilization the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (33) Aquino, R.; Cáceres, A.; Morelli, S.; Rastrelli, L. An extract of Tagetes lucida and its phenolic constituents as antioxidants. J. Nat. Prod. 2002, 65, 1773−1776. (34) Nishida, H. A study on the spectrophotometric determination of iron (III) with Chromazurol S. Bunseki Kagaku 1970, 19, 221−226. (35) Tsuda, S.; Nakagawa, K.; Oyama, T.; Takahashi, A.; Okabe, Y.; Kagawa, H.; Yamada, S.; Okabe, Y. Study on lignophenol-cured epoxy resins. J. Network Polym. 2010, 31, 75−83. (36) Baker, S. M. Rapid methoxyl analysis of lignins using gas chromatography. Holzforshung 1996, 50, 573−574. (37) Li, H.; Chai, X.-S.; Liu, M.; Deng, Y. Novel method for the determination of the methoxyl content in lignin by headspace gas chromatography. J. Agric. Food Chem. 2012, 60, 5307−5310. (38) Chae, J. Practical demethylation of aryl methyl ethers using an odorless thiol reagent. Arch. Pharmacal Res. 2008, 31 (3), 305−309. (39) Zuo, L.; Yao, S.; Wang, W.; Duan, W. An efficient method for demethylation of aryl methyl ethers. Tetrahedron Lett. 2008, 49, 4054− 4056. (40) Chen, F.; Tobimatsu, Y.; Havkin-Frenkel, D.; Dixon, R. A.; Ralph, J. A polymer of caffeyl alcohol in plant seeds. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 1772−1777. (41) Kawamoto, H.; Nakatsubo, F.; Murakami, K. Stoichiometric studies of tannin-protein co-precipitation. Phytochemistry 1996, 41 (5), 1427−1431. (42) Naczk, M.; Oickle, D.; Pink, D.; Shahidi, F. Protein precipitating capacity of crude canola tannins: effect of pH, tannin, and protein concentrations. J. Agric. Food Chem. 1996, 44, 2144−2148. (43) Frazier, R. A.; Papadopoulou, A.; Mueller-Harvey, I.; Kissoon, D.; Green, R. J. Probing protein-tannin interactions by isothermal titration microcalorimetry. J. Agric. Food Chem. 2003, 51, 5189−5195. (44) Quideau, S.; Deffieux, D.; Douat-Casassus, C.; Pouysegu, L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew. Chem., Int. Ed. 2011, 50, 586−621. (45) Diouf, P.-F.; Merlin, A.; Perrin, D. Antioxidant properties of wood extracts and colour stability of woods. Ann. For. Sci. 2006, 63, 525−534. (46) Makino, R.; Ohara, S.; Hashida, K. Radical scavenging characteristics of condensed tannins from bark of various tree species compared with quebracho wood tannin. Holzforschung 2011, 65, 651− 657. (47) Chai, W.-M.; Shi, Y.; Feng, H.-L.; Qiu, L.; Zhou, H.-C.; Deng, Z.-W.; Yan, C.-L.; Chen, Q.-X. NMR, HPLC-ESI-MS and MALDITOF MS analysis of condensed tannins from Delonix regia (Bojer ex Hook.) Raf. And their bioactivities. J. Agric. Food Chem. 2012, 60, 5013−5020. (48) Perron, N. R.; Brumaghim, J. L. A review of the antioxidant mechanisms of polyphenol compounds related to iron binding. Cell Biochem. Biophys. 2009, 53, 75−100. (49) Engels, C.; Gänzle, M. G.; Schieber, A. Fractionation of gallotannins from mango (Mangifera indica L.) kernels by high-speed counter-current chromatography and determination of their antibacterial activity. J. Agric. Food Chem. 2010, 58, 775−780.
5431
DOI: 10.1021/acssuschemeng.7b00748 ACS Sustainable Chem. Eng. 2017, 5, 5424−5431
