hemicellulose from lignin in white pine sawdust using boron trihalide reagents

Accepted Manuscript Separation of cellulose/hemicellulose from lignin in white pine sawdust using boron trihalide reagents M. Zain H. Kazmi, Abhoy Karmakar, Vladimir K. Michaelis, Florence J. Williams PII:

S0040-4020(19)30147-4

DOI:

https://doi.org/10.1016/j.tet.2019.02.009

Reference:

TET 30132

To appear in:

Tetrahedron

Received Date: 1 November 2018 Revised Date:

24 January 2019

Accepted Date: 1 February 2019

Please cite this article as: Kazmi MZH, Karmakar A, Michaelis VK, Williams FJ, Separation of cellulose/ hemicellulose from lignin in white pine sawdust using boron trihalide reagents, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Separation of Cellulose/Hemicellulose from Lignin in White Pine Sawdust Using Boron Trihalide Reagents M. Zain H. Kazmi, Abhoy Karmakar, Vladimir K. Michaelis, and Florence J. Williams*

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Department of Chemistry, University of Alberta, Edmonton, AB, Canada * corresponding author. Email address: [email protected]

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Abstract: A mild method for the separation of cellulose/hemicellulose from extractive free sawdust is described. Sequential treatments with an equimolar mixture of BCl3 and BBr3 remove polysaccharide components from a white pine sawdust sample. Spectroscopic analyses, including solution and solid state NMR spectroscopy, confirm a reduction in the amount of aliphatic sugars in solid samples and show that extracted components consist only of polymeric sugars and are free of aromatics. Staining with fluorescent and colorimetric dyes confirm that the sawdust sample after boron trihalide treatment is primarily lignin, with no detectable polysaccharides.

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1. Introduction

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Boron Lewis acids have great potential as reagents in sustainable chemistry. Boric acid byproducts are environmentally benign and non-toxic,1 yet the electronic configuration and metallic character of boron results in strong Lewis acidic and catalytic capabilities. Moreover, the high bond dissociation energy of boron–oxygen bonds provides a thermodynamic driving force for the activation of oxygenated species.2 Of particular interest in the development of sustainable chemical feedstocks is the degradation of lignocellulose and lignin, which are biopolymers defined by ether and acetal linkages of phenolic and sugar monomers (Figure 1).3

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Lignocellulose is the structural component of plant cell walls. It consists of tubular bundles of cellulose surrounded and interconnected by hemicellulose and ultimately by lignin. The degradation of lignocellulose from common feedstocks such as wood shavings and switchgrass has been the subject of significant academic interest and commercial development.3-11 An ideal method would separate cellulose from lignin while avoiding the introduction of new C–C bonds in the lignin polymer structure, commonly referred to as lignin condensation. Classic methods to separate lignin from lignocellulose employ acid, base, and/or oxidants at elevated temperatures, but these methods have the unfortunate effect of increasing aromatic carbon–carbon connections.12-16 Since C–C bonds complicate the degradation of lignin into monomeric, high value chemicals, yields of lignin valorization are currently limited to 20-45%.12

Figure 1.17 Representation of lignin, cellulose, and hemicellulose chemical structures. Quantum chemical calculations by Sturgeon and coworkers indicate that under Brønsted acid conditions, lignin condensation has a lower activation energy than lignin hydrolysis.18 Such predictions have led to increased exploration of Lewis acid-mediated hydrolysis in lignin separation and valorization strategies, with reasonable success.19-22 These same calculations predict that lower temperature processes are favorable to minimize condensation.

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Having previously developed a chemo- and regioselective method of ether cleavage at room temperature using a combination of boron trihalide reagents which exhibited a superior reactivity profile as compared to boron tribromide alone,23 we sought to explore whether the same reaction conditions could be applied to a “lignin first” cleavage of aromatic lignin monomers from lignocellulose directly.16 Selective degradation of lignin would allow its solubilization and subsequent extraction from the lignocellulose material. Indeed, the ability of boron tribromide to degrade lignin has already been demonstrated by the Zhang lab.21 Alternatively, the boron Lewis acid reagents could preferentially degrade polysaccharide acetal linkages, which would result in the degradation and extraction of cellulosic material. In either case, if there is sufficient chemoselectivity, a boron Lewis acid strategy would provide a new method to separate the fundamental components of lignocellulose under ambient conditions.

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2. Results and Discussion

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In order to test the chemoselectivity and reactivity of boron trihalides, we performed test reactions using model lignin compounds 1 and 4, and commercial nanocellulose (Eqs. 1-3). While an equimolar mixture of boron trichloride and boron tribromide resulted in complete consumption of starting model compounds, with high conversion to the expected catechol product 2, the same reagents and conditions had no observable effect on nanocellulose (Eq. 3).

Next, we sought to test the reactivity on lignocellulose directly. In order to obtain reasonably clean lignocellulose, we performed a standardized series of solvent treatments to remove extractives from white pine sawdust.24 This extractive free sawdust was then treated with an equimolar boron trihalide mixture in dichloromethane. After 18 hours, the reaction was quenched with water, filtered, and the aqueous and organic layers were examined for cleaved byproducts (Table 1, entry 1). Surprisingly, the organic layer contained minimal extracted material (~15 mg), while the aqueous layer was evaporated to provide 870 mg of material. 1H NMR analysis of the aqueous extract showed significant peaks around 3.5-5 ppm, but only trace aromatic protons, indicative of 3

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oligomeric or polymeric sugars rather than lignin oligomers. We observed in the first and all subsequent reactions (vide infra), significant levels of cellulosic polymer in the aqueous layer, as confirmed by solution nuclear magnetic resonance (NMR) spectroscopy and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) (see supplemental information (SI)). MALDI-MS analysis of the material obtained from the aqueous extract is highly similar to a MALDI-MS spectrum obtained from commercial nanocellulose mixed with boric acid.

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These observations indicate that boron trihalide reagents facilitate the release of cellulose and/or hemicellulose. This release could be achieved either through hydrolysis of acetal linkages in polysaccharide polymers, generating oligomers that are solubilized, or by cleavage of ethers in the lignin structure, providing gaps that allow the liberation of cellulose. In the latter case, degradation of the lignin structure must occur to such a minimal degree that it is insufficient to liberate lignin oligomers.

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Table 1. Exposure of sawdust to boron trihalides for degradation of polymer structure.

Intrigued by the potential of this mild room temperature method to separate cellulose from lignin, we exposed the solid residue of the first reaction to further boron trihalide treatment until there was a minimal decrease in mass (Table 1, entries 2-4). The retention of 16% mass at the end of four reaction cycles is consistent with expectations, since softwood lignocellulose is ~25% lignin by mass.25 Throughout each of the four reactions, no evidence of aromatic material was observed in solution. Dialysis (0.5-1 kDa membrane) of a portion of the aqueous extract from each reaction cycle (entries 1-4) was performed to remove boric acid. Quantification of the polysaccharide remaining after dialysis provided an estimate of the total cellulosic extract over all four cycles to be 0.30 g.

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At the conclusion of this sequential treatment, a new reaction with extractive free sawdust was performed with boron trihalide addition amounts equivalent to that used in the initial four entries combined (Table 1, entry 5, 19.2 mL for 300 mg starting material as compared to 8 mLx4 for 500 mg starting material). This reaction showed increased degradation of sawdust, but not to the same extent as the batch-wise treatment performed by entries 1-4.

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Seeking to verify that the solid residue obtained at the conclusion of four sequential boron trihalide treatments was lignin stripped of cellulose and hemicellulose, we performed a series of analytical assays and spectroscopic characterizations. Solid-state 13C cross-polarization magicangle spinning (CPMAS) NMR was used to verify a relative increase in aromatic resonances (120-160 ppm) as compared to the starting sawdust samples (Figure 2). While solid-state NMR avoids the ambiguity of solution NMR resulting from soluble versus insoluble (and therefore silent) components of lignocellulose, the differences in spectra can be subtle.26 Lignin contains aliphatic oxygenated functionality with chemical shifts similar to polysaccharides. Moreover, due to the abundance of hydrogen atoms, aliphatic carbons often appear stronger in intensity than aromatic carbons in 13C{1H} CPMAS NMR spectra. Nevertheless, a single source of lignocellulose before treatment and after treatment can be compared for changes (Figure 2, b and c). The 13C{1H} CPMAS NMR spectrum of nanocellulose (a) is consistent with previous literature and informs the analysis of differences observed between spectra b and c.26-28 Spectrum c shows a decrease in intensity at 65 ppm, and increases at 100-160 ppm (Figure 2d). These changes are indicative of a higher concentration of aromatics in the sample due to a loss in polysaccharides. Analysis using infrared spectroscopy (IR) also confirmed a relative decrease in the abundance of hydroxyl functionality in the treated sawdust sample as compared to untreated sawdust (see Appendix A, Supplemental Information (SI)).

Figure 2. Solid-state 13C{1H} CPMAS NMR spectra of commercial nanocrystalline cellulose (a), extractive free sawdust (b), solid residue after four cycles of borohalide treatment (c) and 5

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enlarged overlay of regions of interest (d). Spectra (a-c) were plotted so that the most intense resonance (~75 ppm) had identical intensity between spectra.

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We then employed Calcofluor White staining to visualize the presence of cellulose and hemicellulose in our samples (Figure 3).29,30 Differential interference contrast (DIC) microscopy was used to provide images of the samples to compare to fluorescence images. Comparison of extract free sawdust (a/b), cotton (e/f) (cellulose positive control), commercial lignin (g/h, negative control for cellulose/hemicellulose), and the residual solid from boron trihalide treatment of sawdust (c/d), showed a clear absence of fluorescence in both commercial lignin and our residual solid (d and h). Since Calcofluor White is a selective stain for polysaccharide, the absence of fluorescence can be ascribed to the absence of polymeric sugars. These results therefore indicate that boron trihalide treatments result in an efficient removal of cellulose and hemicellulose from sawdust.

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Figure 3. Calcofluor White staining. Individual samples were stained with Calcofluor White and mounted on glass slides. Slides were imaged on an epifluorescence microscope (Nikon eclipse Ti) using a DAPI filter at 20X magnification. The exposure time was set to 150 ms for all fluorescence images. DIC images of the same field of view were captured at identical magnification. Scale bars were set using ImageJ. (a) Sawdust DIC image (b) Sawdust fluorescence image (c) Solid residue DIC image (d) Solid residue fluorescence image (e) Cotton DIC image (f) Cotton fluorescence image (g) Lignin DIC image (h) Lignin fluorescence image. To verify the identity of the remaining solid, all samples were stained with Toluidine Blue O (Figure 4). Toluidine Blue O is a colorimetric stain which is known to produce a blue-green color when interacting with lignin, and a purple-pink color when interacting with cellulose.30,31 In samples containing both, such as wood samples and plant stems, a purple-blue color is typically observed. When the residual solid from our reaction is treated with Toluidine Blue O, a characteristic green-blue color is present in thick particulates (b), which is distinct from the purple-blue hue of untreated sawdust (a). Commercial lignin presents as a dark green color (c), while the nanocellulose control shows only pink and purple (d). 6

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Toluidine Blue O and Calcofluor White have previously been applied to the molecular composition analysis of whole plant slices,30,31 rather than particulate sawdust and lignin particles. The results of both Calcofluor White and Toluidine Blue O treatment of our samples demonstrate that such stains are also effective for the analysis of lignocellulose fractionation.

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Figure 4. Toluidine Blue O staining. Individual samples were stained with Toluidine Blue O and mounted on a 6 well plate. Slides were imaged using a bright field color channel at 20X magnification on a Cytation 5 microscope (BioTek). Scale bars were set using ImageJ. (a) Sawdust (b) Solid residue (c) Commercial lignin (d) Nanocellulose. 3. Conclusion

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Boron trihalide treatment successfully and efficiently separated cellulosic material from a white pine sawdust sample. The procedure does not require elevated temperature, which compares favorably to commonly employed methods (Organosolv, Kraft, Klason, etc) in which high temperatures may contribute to the undesirable condensation of lignin.13,32 The exact mechanism of this process remains elusive and the reactivity is seemingly inconsistent with model reactions that predict chemoselective lignin degradation. This inconsistency may be indicative of a controlled release of protic acids from boron halide reagents being operative rather than boronmediated hydrolysis.34

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This work represents the first investigation using boron trihalides alone to process lignocellulose into constituent components. Future work will pursue characterization of the structural features of the lignin remaining after polysaccharide extraction, including quantifying the level of lignin condensation. Such studies should not only provide insight into the operative mechanism in boron trihalide treatment of lignocellulose, but also determine the potential for valorization of the remaining solids. 4. Experimental Details

All reactions were carried out under air and water free conditions. Dichloromethane (CH2Cl2) was passed through a column of activated molecular sieves (4Å, LC technologies). Deuterated chloroform (CDCl3) was treated with oven-dried molecular sieves (4 Å), whereas deuterated water (D2O) was used without further treatment. Both boron reagents, BBr3 (1.0 M in CH2Cl2) and BCl3 (1.0 M in CH2Cl2), Toluidine Blue O stain and Calcofluor White stain were purchased from Sigma Aldrich. Commercial lignin (dealkaline lignin) was purchased from TCI Chemicals. Pine sawdust was obtained by sanding a sample of white pine wood. Solution NMR spectra (1H NMR) were recorded at 400, 500 and 600 MHz. Fourier transform infrared (FT-IR) spectra were 7

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recorded by incorporating the analyte within a KBr pellet, and Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectra were recorded using a dihydroxybenzoic acid (DHB) matrix. MALDI-MS analysis was performed on an ultraflexXtreme™ MALDITOF/TOF (Bruker Daltonics) mass spectrometer in negative MS mode. Dialysis was done using a micro float-a-lyzer (0.5-1 kDa, cellulose membrane) purchased from spectrum labs. Reverse phase HPLC was performed on an Agilent Technologies 1260 Infinity instrument (C18, 150x4.6 mm, 3mL/min). Refer to Appendix A (Supplemental Information) for experimental data from FT-IR, MALDI, and solution NMR analyses. Reaction of lignin model compounds with BBr3:BCl3 reagent system

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Model lignin monomer 1 (25.0 mg, 0.117 mmol, 1.00 equiv.) was dissolved in CH2Cl2 (2 mL) and added to a flame-dried 5 mL round bottom flask. The solution was cooled to 0 oC, and BCl3 (120 µL, 1.0 M in CH2Cl2) was added, followed by BBr3 (120 µL, 1.0 M in CH2Cl2). The reaction mixture was allowed to come to room temperature and was stirred for 18 h. Solvent was then removed in vacuo and the resulting residue was dissolved in CDCl3 for NMR analysis. The solution was concentrated in vacuo again and α,α,α-trifluorotoluene (0.100 mmol) was added as an internal standard for HPLC. The reaction mixture was then diluted in 3 mL acetonitrile and 2 mL water to produce a homogeneous solution. Reverse phase HPLC was used to provide yields of catechol 2 (75%) and benzyl bromide 3 (38%).

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Model lignin monomer 4 (25.0 mg, 0.102 mmol, 1.00 equiv.) was dissolved in CH2Cl2 (2 mL) and added to a flame-dried 5 mL round bottom flask. The solution was cooled to 0 oC, and BCl3 (160 µL, 1.0 M in CH2Cl2) was added, followed by BBr3 (160 µL, 1.0 M in CH2Cl2). The reaction mixture was allowed to come to room temperature and was stirred for 18 h. Solvent was then removed in vacuo and the resulting residue was dissolved in CDCl3 for NMR analysis. The solution was concentrated in vacuo again α,α,α-trifluorotoluene (0.100 mmol) was added as an internal standard for HPLC. The reaction mixture was then diluted in 3 mL acetonitrile and 2 mL water to produce a homogeneous solution. Reverse phase HPLC was used to provide yields of catechol 2 (80%) and brominated compound 5 (quant.).

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HPLC conditions (C18, 3 mL/min; solvent A = acetonitrile, solvent B = water): 0-5 min: 10-30% A gradient; 5-10 min: 30-50% A gradient; 10-20 min: 50-60% A gradient. Two standard mixtures of starting materials, expected products, and α,α,α-trifluorotoluene (1:2:3:trifluorotoluene = 1:1:1:1; 4:2:5:trifluorotoluene = 1:1:1:1) were analyzed to establish the UV absorption ratios of all components relative to α,α,α-trifluorotoluene. 0.4 mL of reaction solution with α,α,α-trifluorotoluene internal standard was then analyzed by HPLC, and UV absorption intensities at 254 nm were used to quantify yields. Catechol 2 Rt = 0.85-0.87 min; benzyl bromide 3 Rt = 13.34-13.36 min; brominated compound 5 Rt = 15.64-15.72 min. Benzyl bromide loss was presumed to have occurred during the removal of solvent in vacuo. 4.2

Reaction of commercial nanocellulose with BBr3:BCl3 reagent system

To a sample of nanocellulose (0.05 g) in CH2Cl2 (3 mL) under vigorous stirring was added BCl3 (0.8 mL, 1.0 M in CH2Cl2), followed by BBr3 (0.8 mL, 1.0 M in CH2Cl2). The reaction mixture was stirred for 18 h, filtered through 2 glass microfibre filter papers, and then rinsed with 5 mL 8

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of hot distilled water. The aqueous and organic layers were separated. The organic layer was dried over Na2SO4 and both layers were then concentrated under reduced pressure. Solution NMR was performed on materials extracted into the aqueous layer.

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Similarly, nanocellulose was subjected to the same reaction conditions using just BBr3 (1.6 mL, 1.0 M in CH2Cl2). Solution NMR was performed on materials extracted in the aqueous layer. Removal of extractives from pine sawdust24

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Pine sawdust (4.0 g) was heated at reflux in 50 mL of ethanol-benzene (1:2) for 6 h. The reaction mixture was filtered and washed with 20 mL ethanol. After drying in vacuo, the residual solid was added to 50 mL of ethanol and the mixture was heated at reflux for 4 h. The reaction mixture was filtered and washed with 50 mL water. After drying in vacuo, the residual solid was transferred to a beaker with 120 mL of distilled water, and the mixture was brought to a boil for 1 h. Finally, the mixture was filtered and washed with 150 mL distilled boiling water. The final solid residue obtained after the extract was dried under vacuum (0.1 torr) overnight to yield 3.3 g. Removal of polysaccharides from extractive-free sawdust

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To a mixture of extractives-free pine sawdust (0.50 g) in CH2Cl2 (30 mL) under vigorous stirring was added BCl3 (8.0 mL, 1.0 M in CH2Cl2), followed by BBr3 (8.0 mL, 1.0 M in CH2Cl2). The reaction mixture was stirred for 18 h, filtered through 2 glass microfibre filter papers, and then rinsed with 50 mL of hot distilled water. The aqueous and organic layers were separated. The organic layer was dried over Na2SO4 and both layers were then concentrated under reduced pressure. The solid material remaining after filtration (solid residue) was dried under high vacuum overnight to give 320 mg solid residue. The reaction sequence was then repeated using this solid residue an additional 3 times to ultimately provide 79 mg of residual solid material for analytical assays (see Table 1). Solution NMR as well as MALDI mass spectrometry and FT-IR spectroscopy was performed on material extracted into the aqueous layers (see SI). FT-IR, solidstate NMR (section 4.7) and microscopic analysis with staining (sections 4.8, 4.9) were performed on the residual solid. One-pot boron trihalide treatment of extractive-free sawdust

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To a mixture of extractives-free pine sawdust (0.30 g) in CH2CL2 (20 mL) under vigorous stirring was added BCl3 (19.2 mL, 1.0 M in CH2Cl2), followed by BBr3 (19.2 mL, 1.0 M in CH2Cl2). The reaction mixture was stirred for 18 h, filtered through 2 glass microfibre filter papers, and then rinsed with 30 mL of hot distilled water. The aqueous and organic layers were separated. The organic layer was dried over Na2SO4 and both layers were then concentrated under reduced pressure. The solid material remaining after filtration (solid residue) was dried under high vacuum overnight to provide 97 mg of residual solid material (see Table 1, entry 4). 4.6

Dialysis of aqueous extractives obtained in lignocellulose BCl3:BBr3 treatment

Lyophilized aqueous extract from the reaction in Table 1, entry 1 (10.7 mg) was dissolved in water (0.50 mL) and dialysed in water (1.0 L, 12 h x 2), resulting in 1.40 mg of material 9

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4.7

Solid-state 13C NMR spectroscopy

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remaining after drying in vacuo. Similarly, lyophilized aqueous extracts from entries 2-4 (12.5 mg, 25.0 mg and 25.0 mg, respectively) were dialysed in the same manner to provide 2.00 mg, 1.60 mg, and 0.800 mg, respectively. By extrapolating these dialysis yields, the total mass of extracted cellulosic material from the lignocellulose sawdust sample is calculated to be 0.30 g dry weight. MALDI analysis on dialyzed material remains consistent with polymeric and oligomeric cellulose.

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Toluidine Blue O staining30

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Magic-angle spinning (MAS) NMR experiments for cellulose nanocrystals and sawdust treated samples were acquired at 7.05 T (νL(13C) = 75.53 MHz) on a Bruker Avance 300 NMR spectrometer equipped with a 4 mm double resonance MAS NMR Bruker probe. The samples were packed in 4 mm outer diameter ZrO2 rotors and sealed with Kel-F top caps. Carbon-13 cross-polarization (CP)2 MAS NMR spectra were acquired with a MAS frequency, ωr/2π = 14 kHz, a 4.0 µs π/2 pulse (ɣB1/2π = 62.5 kHz), a contact time of 3.0 ms, 1,000 to 30,000 co-added transients and a recycle delay time of 3 s. The nanocrystalline cellulose sample was acquired under slightly different conditions: a contact time of 5.0 ms, a recycle delay time of 10 s and ωr/2π = 13 kHz. All spectra were acquired with high power TPPM3 1H decoupling (ɣB1/2π = 62.5 kHz). NMR spectra were referenced to TMS (δ(13C) = 0.00 ppm) by setting the high frequency 13C peak of solid adamantane to 38.56 ppm.33

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Toluidine blue O (20.0 mg) was dissolved in 100 mL Millipore water to make a stock staining solution (0.0200%). Solid compounds (sawdust, solid residue, commercial lignin (TCI), nanocellulose) (2.0 mg each) were added to microcentrifuge tubes followed by stain solution (500 µL). After five minutes, the tubes were centrifuged. Excess stain was removed (480 µL) and water was added (480 µL) before centrifuging again. This process was repeated twice. The remaining supernatant (480 µL) was removed. 10.0 µL of suspension was placed in a 6 well plate. Stained solids were observed using bright-field microscopy on a Cytation 5 microscope at 20X magnification. Calcofluor White staining30

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Calcofluor white (2.00 mL, 0.1% solution) was dissolved in 98 mL Millipore water to make a stock staining solution (0.00200%). Solid compounds (sawdust, residue, lignin, cotton) (2.0 mg each) were added microcentrifuge tubes (2.00 mL) separately followed by stain solution (500 µL). After ten minutes, the tubes were centrifuged. Excess stain was removed (480 µL) and water was added (480 µL) before centrifuging again. The remaining supernatant (480 µL) was removed. 10.0 µL of suspension was placed on the glass slide, followed by the addition of 20.0 µL of water before addition of the coverslip, which was fixed to the glass slide. Stained solids were observed through a DAPI filter on an epifluorescence microscope (Nikon eclipse Ti) at 20X magnification. 10

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Acknowledgements

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We would like to acknowledge Khyati Gohil for her assistance in microscopic imaging, as well as the Stryker laboratory at University of Alberta for providing model lignin compounds 1 and 4.

Appendix A. Supplemental Data

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Supplementary data to this article can be found online. References

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This work was supported by the American Chemical Society (ACS) PRF Grant 59191-ND-1, the Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery Grant Program (NSERC DG), Grant RGPIN-2016-04843 and RGPIN-2016-05447, as well as the NSERC Research Tools and Instruments Grant Program RTI-2016-00639, and Alberta Innovates CNC Program.

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