Β-ether Cleavage Research Articles

Influences of 2-ethylanthraquinone on lignin degradation in alkaline cooking of Eucalyptus pellita wood

The impact of 2-ethylanthraquinone (2-EAQ) on alkaline cooking of Eucalyptus pellita wood was examined by analyzing pulp yield, kappa number, the structure and weight-averaged molecular weight (Mw) of dissolved lignin in black liquor and residual lignin in pulp (RL). During soda cooking under 23% of active alkaline (AA) condition, adding 0.06% 2-EAQ reduced the kappa number by 12 points compared to cooking without it. The addition of 0.06% 2-EAQ to kraft cooking increased pulp yield by about 4% at about 19 of kappa number compared to the no addition. The analysis of lignin structure by using semiquantitative heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR) showed that the addition of 2-EAQ promoted β-ether cleavage during alkaline cooking and reduced peeling reaction of polysaccharides and prevention of the elimination of γ-OH of lignin to form enol ethers and stilbenes. Overall, the addition of 2-EAQ promoted the degradation of lignin to lower molecular weight compounds, with a more pronounced effect observed in soda cooking than in kraft cooking. Under optimal conditions, the delignification efficiency and pulp yield of soda-EAQ pulping were comparable to kraft cooking.

In Vitro Enzymatic Depolymerization of Lignin with Release of Syringyl, Guaiacyl, and Tricin Units.

ABSTRACTNew environmentally sound technologies are needed to derive valuable compounds from renewable resources. Lignin, an abundant polymer in terrestrial plants comprised predominantly of guaiacyl and syringyl monoaromatic phenylpropanoid units, is a potential natural source of aromatic compounds. In addition, the plant secondary metabolite tricin is a recently discovered and moderately abundant flavonoid in grasses. The most prevalent interunit linkage between guaiacyl, syringyl, and tricin units is the β-ether linkage. Previous studies have shown that bacterial β-etherase pathway enzymes catalyze glutathione-dependent cleavage of β-ether bonds in dimeric β-ether lignin model compounds. To date, however, it remains unclear whether the known β-etherase enzymes are active on lignin polymers. Here we report on enzymes that catalyze β-ether cleavage from bona fide lignin, under conditions that recycle the cosubstrates NAD+ and glutathione. Guaiacyl, syringyl, and tricin derivatives were identified as reaction products when different model compounds or lignin fractions were used as substrates. These results demonstrate an in vitro enzymatic system that can recycle cosubstrates while releasing aromatic monomers from model compounds as well as natural and engineered lignin oligomers. These findings can improve the ability to produce valuable aromatic compounds from a renewable resource like lignin.IMPORTANCE Many bacteria are predicted to contain enzymes that could convert renewable carbon sources into substitutes for compounds that are derived from petroleum. The β-etherase pathway present in sphingomonad bacteria could cleave the abundant β–O–4-aryl ether bonds in plant lignin, releasing a biobased source of aromatic compounds for the chemical industry. However, the activity of these enzymes on the complex aromatic oligomers found in plant lignin is unknown. Here we demonstrate biodegradation of lignin polymers using a minimal set of β-etherase pathway enzymes, the ability to recycle needed cofactors (glutathione and NAD+) in vitro, and the release of guaiacyl, syringyl, and tricin as depolymerized products from lignin. These observations provide critical evidence for the use and future optimization of these bacterial β-etherase pathway enzymes for industrial-level biotechnological applications designed to derive high-value monomeric aromatic compounds from lignin.

Pyrolytic reactivities of deuterated β-ether-type lignin model dimers

To achieve a better understanding of the radical chain reactions in lignin pyrolysis, the reactivities of three phenolic and nonphenolic deuterated lignin model dimers, i.e., 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-propan-1-d-ol, 1-(4-hydroxy-d-3-methoxyphenyl)-1-hydroxy-d-2-(2-methoxyphenoxy)-propane, and 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1-propan-1-d-ol, were studied in closed ampoule reactors under N2 at 250–400°C, the temperature at which the major primary pyrolysis reactions of lignin normally occur, including cleavage of the β-ether linkages. Deuteration at Cα and hydroxyl groups decreased the β-ether cleavage reactivities of the dimers, which indicated that hydrogen abstractions from Cα and hydroxyl groups play direct roles in pyrolytic β-ether cleavage reactions. The influences of deuteration on the yields of the cleavage products indicated the reaction pathways. Significant changes in the reactivity of a mixture of pyrolysis dimers supported the involvement of radical chain reactions. The phenolic Cα-deuterated dimer had complex effects on the reactivity, probably because of the secondary isotope effects of Cα-d on the conformation.

Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate

Lignin is a major component of plant cell walls that is essential to their function. However, the strong bonds that bind the various subunits of lignin, and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of monolignols including p-coumaryl, coniferyl, and sinapyl alcohols to produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin, and these monolignol conjugates can also be "monomer" precursors of lignin. Although monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate monolignol transferase activity and determined its kinetic parameters.

Different pyrolytic cleavage mechanisms of β-ether bond depending on the side-chain structure of lignin dimers

Influence of C γ–OH on pyrolytic cleavage mechanism of β-ether-linkage in lignin dimer was studied in N 2 with C γ-deoxy-type dimers, which have various p-substituents (–H, –Cl, –OCH 3) at their C β-phenoxy groups. Reactivities at 400 °C and the influence of radical scavenger (tetralin) indicated that C γ-deoxy types, especially in phenolic forms, are degraded through radical chain mechanism. Furthermore, substituent effects on their reactivities under excess amount of tetralin as discussed with Hammett's substituent constant ( σ p) and bond dissociation energy (BDE)-reducing parameter (ΔBDE) revealed that this chain reaction is initiated with the radical species formed through C β–O bond homolysis. These chain reactions were only effective in a closed-type reactor and not effective in an open-type reactor. Contrary to this, C γ–OH type in phenolic form was degraded at 250 °C, which is much lower than that (400 °C) of the C γ-deoxy types, and the reactivity was not influenced by tetralin. These results indicate that introducing OH at C γ changes the β-ether cleavage mechanism in phenolic form from radical chain to quinone methide mechanism.

Effects of side-chain hydroxyl groups on pyrolytic β-ether cleavage of phenolic lignin model dimer

Effects of side chain hydroxyl groups on pyrolytic β-ether cleavage of phenolic model dimers were studied with various deoxygenated dimers under pyrolysis conditions of N2/400°C/1 min. Although phenolic dimer with hydroxyl groups at the C α − and C γ −positions was much more reactive than the corresponding nonphenolic type, deoxygenation at the C γ -position substantially reduced the reactivity up to the level of the nonphenolic type. These results are discussed with the cleavage mechanism via quinone methide intermediate formation, which is activated through intramolecular hydrogen bonds between C α − and C γ − hydroxyl groups.

An alternative β-ether cleavage pathway for a non-phenolic β- O-4 lignin model dimer catalyzed by a laccase-mediator system

A non-phenolic β- O-4 lignin substructure model dimer, 1-(4-ethoxy-3-methoxyphenyl)-2-(2,6-dimethoxyphenoxy)ethanol ( I) was oxidized by Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole (1-HBT) to give many degradation products ( II– VII). When the degradation was performed under 18 O 2 , one oxygen atom ( 18 O ) was incorporated into the C2(β)-OH of 1-(4-ethoxy-3-methoxyphenyl)-2-hydroxyethanone ( III) formed via a β-ether cleavage reaction. When the C2(β)- 2 H 2 labeled substrate I- 2 H was synthesized and oxidized by Laccase/1-HBT couple, deuterium atoms were retained in the product ethanone III. It is clearly indicated that ethanone III might be formed from substrate I without the abstraction of C2(β)-protons during the oxidation. We conclude that ethanone III was formed via a peroxy radical intermediate produced from the reaction between the benzylic radical of substrate I catalyzed by the laccase/1-HBT couple and molecular oxygen.

Delignification Mechanism During High-Boiling Solvent Pulping. IV. Effect of a Reducing Sugar on the Degradation of Guaiacylglycerol-β-guaiacyl Ether

A phenolic β-O-4 type lignin model compound, guaiacylglycerol-β-guaiacyl ether was treated with 70 wt% aqueous 1,3-butanediol solution in the presence of glucose at 160–200°C to investigate the effect of reducing sugars on the degradation of lignin during high-boiling solvent (HBS) pulping. Addition of glucose increased the formation of guaiacol, coniferyl alcohol, and its γ-ethers, and decreased the formation of radical coupling compounds dramatically. These results suggest that reducing sugars may stabilize phenoxy radicals formed by homolysis of phenolic β-ethers. The kinetic studies also revealed that the disappearance of the β-ether model compound was enhanced substantially by the presence of glucose, which suggests that in addition to homolysis of the β-ether, a reducing sugar-assisted β-ether cleavage may be involved under the conditions used.

Degradation mechanisms of a nonphenolic β-O-4 lignin model dimer by Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole

Past studies have shown that Trametes versicolor laccase catalyzed Cα-Cβ cleavage, Cα-oxidation, β-ether cleavage and aromatic ring cleavage of a nonphenolic β-O-4 lignin model dimer, 1,3-dihydroxy-2-(2,6-dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I), in the presence of 1-hydroxybenzotriazole. In this study, we investigated the detailed degradation mechanism for these pathways of substrate I by laccase/1-hydroxybenzotriazole couple, based on incorporation experiments from H218O and 18O2. Oxidation of substrate I in H218O resulted in the incorporation of 18O into three aromatic ring cleavage products and a β-ether cleavage product. These results were identical with those for lignin peroxidase. We inferred that these cleavages of substrate I proceed via the β-aryl cation radical intermediate of I. However, the Cα-Cβ cleavage degradation product of I by the laccase/1-hydroxybenzotriazole couple was 4-ethoxy-3-methoxybenzoic acid, whereas it was 4-ethoxy-3-methoxybenzaldehyde for lignin peroxidase. It is clear that one atom of 18O from 18O2 was incorporated into 4-ethoxy-3-methoxybenzoic acid during the oxidation of substrate I. To confirm the reaction intermediate, we carried out the chemical reaction of 2-(2,6-dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)- 3-hydroxypropanone with alkaline hydrogen peroxide, since the expected hydroperoxide intermediate is quite similar to that formed from the reaction between the benzylic radical of I and O2. The result showed that the main degradation product was 4-ethoxy-3-methoxybenzoic acid. Thus, we propose a novel Cα-Cβ cleavage via a kind of Baeyer-Villiger reaction for nonphenolic β-O-4 lignin model dimer by laccase/1-hydroxybenzotriazole couple.

Aromatic ring cleavage of a non-phenolic β- O-4 lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole

The novel cleavage products, 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)-1-formyloxypropane (II) and 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-2,3-cyclic carbonate (III) were identified as products of a non-phenolic β- O-4 lignin model dimer, 1,3-dihydroxy-2-(2,6-dimethoxylphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I), by a Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole (1-HBT). An isotopic experiment with a 13C-labeled lignin model dimer, 1,3-dihydroxy-2-(2,6-[U-ring- 13C]dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I- 13C) indicated that the formyl and carbonate carbons of products II and III were derived from the β-phenoxy group of β- O-4 lignin model dimer I as aromatic ring cleavage fragments. These results show that the laccase-1-HBT couple could catalyze the aromatic ring cleavage of non-phenolic β- O-4 lignin model dimer in addition to the β-ether cleavage, Cα-Cβ cleavage, and Cα-oxidation.

Degradation of a non-phenolic β- O-4 substructure and of polymeric lignin model compounds by laccase of Coriolus versicolor in the presence of 1-hydroxybenzotriazole

A β- O-4 lignin substructure model compound, 2-(2,6-dimethoxy-4-formylphenoxy)-1,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)propane, was oxidized by Coriolus versicolor laccase in the presence of 1-hydroxybenzotriazole to form four products: 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)propanone, 1-(4-ethoxy-3-methoxyphenyl)-3-hydroxypropanone, 4-ethoxy-3-methoxybenzoic acid, and 2,6-dimethoxy- p-benzoquinone. The results show that three types of the reactions, β-ether cleavage, Cα-Cβ cleavage, and Cα-oxidation are catalyzed by laccase–1-hydroxybenzotriazole couple. Furthermore, depolymerization of a polymeric guaiacyl lignin model compound prepared from [β- 14C]coniferyl alcohol was also caused by this system.

Degradation of a non-phenolic β-O-4 substructure and of polymeric lignin model compounds by laccase ofCoriolus versicolorin the presence of 1-hydroxybenzotriazole

A β-O-4 lignin substructure model compound, 2-(2,6-dimethoxy-4-formylphenoxy)-1,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)propane, was oxidized by Coriolus versicolor laccase in the presence of 1-hydroxybenzotriazole to form four products: 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)propanone, 1-(4-ethoxy-3-methoxyphenyl)-3-hydroxypropanone, 4-ethoxy-3-methoxybenzoic acid, and 2,6-dimethoxy-p-benzoquinone. The results show that three types of the reactions, β-ether cleavage, Cα-Cβ cleavage, and Cα-oxidation are catalyzed by laccase–1-hydroxybenzotriazole couple. Furthermore, depolymerization of a polymeric guaiacyl lignin model compound prepared from [β-14C]coniferyl alcohol was also caused by this system.

DFRC Method for Lignin Analysis. 1. New Method for β-Aryl Ether Cleavage: Lignin Model Studies

A new method for selective and efficient cleavage of arylglycerol-β-aryl (β-O-4) ether linkages in lignins is described and applied to several lignin β-ether models. The term “DFRC” was coined for derivatization followed by reductive cleavage. Derivatization, accompanied by cell wall solubilization, is accomplished with acetyl bromide (AcBr); reductive cleavage of resulting β-bromo ethers utilizes zinc in acetic acid. Degradation monomers, 4-acetoxycinnamyl acetates, from β-ether cleavage by the DFRC method were identified by NMR, GC−MS, and comparison of GC retention times with authentic compounds. Under the conditions used in this study, the β-ether linkage of all models was cleaved in very high (>92%) yield. The DFRC method produces simpler mixtures of monomers with higher yields than alternative hydrolytic methods. Because of its relative simplicity, mild conditions, and exceptional selectivity, this method should become a powerful analytical method for lignin characterization. Keywords: Acetyl bromid...

P-coumaroylated syringyl units in maize lignin: Implications for β-ether cleavage by thioacidolysis

Recent NMR studies of lignin isolated from maize demonstrated that p-coumarate esters are attached exclusively to the γ-position of phenylpropane side chains. Thioacidolysis/desulphuration experiments have revealed that p-coumarate units are attached primarily ( ca 90%) to syringly moieties in maize lignin. In model studies with guaiacylglycerol and syringylglycerol-β-guaiacyl ethers, cleavage of β-ether linkages by thioacidolysis was reduced 40% by γ-acylation of phenylpropane side chains with p-coumarate. Our results indicate that γ- p-coumarate esters significantly reduce the yields of syringyl products recovered after thioacidolysis of grass lignins.

Dimeric ß-Ether Thioacidolysis Products Resulting from Incomplete Ether Cleavage

Four β-ether dimers which result from incomplete β-ether cleavage of syringyl-guaiacyl lignins following non-optimized thioacidolysis/desulfurization of plant materials were identified. The compounds were ; the guaiacyl-guaiacyl dimer 3-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxy-4-propylphenoxy)propan-1-ol, the guaiacyl-syringyl dimer 2-(2,6-dimethoxy-4-propylphenoxy)-3-(4-hydroxy-3-methoxyphenyl)-propan-1-ol, the syringyl-guaiacyl dimer 3-(3,5-dimethoxy-4-hydroxyphenyl)-2-(2-methoxy-4-propylphenoxy)-propan-1-ol, and the syringyl-syringyl dimer 3-(3,5-dimethoxy-4-hydroxyphenyl)-2-(2,6-dimethoxy-4-propylphenoxy)-propan-1-ol. Synthesis of each dimer, by extending dimeric lignin model β-ether strategies, provided authentication of the structures.

Lignin peroxidase-type activity of soybean peroxidase

Soybean peroxidase (SBP), an acidic peroxidase isolated from the hulls of the bean, catalyzes the efficient oxidation of veratryl alcohol to veratraldehyde in the presence of H 2O 2. The reaction is optimal at pH 2.4 in the presence of 0.2 m CaCl 2. Soybean peroxidase is highly thermostable at pH 2.4, with half-lives of 210 and 2.5 h at 30 and 50°C, respectively. This compares favorably to the thermostability of lignin peroxidase (LiP) from Phanerochaete chrysosporium under equally acidic conditions. In fact, SBP is at least 150-fold more stable than LiP at 30°C and the latter is completely labile at 50°C. Soybean peroxidase follows a ping-pong, bi-bi catalytic reaction mechanism with a k cat K m (veratryl alcohol) of2.47 · 10 2 m −1s −1, ca. 1500-fold lower than a similar value for lignin peroxidase. This lower value of catalytic efficiency is due both to a higher K m (veratryl alcohol) and lower k cat for SBP as compared to LiP. Oxidation of methoxybenzenes suggests that the approximate oxidation potential of SBP is 1.42 V, yet this is high enough to effect the oxidation (and eventual β-ether cleavage) of 1-(3,4-dimethoxyphenyl)-2-(phenoxy)propan-1,3-diol, a lignin model dimer. In addition to SBP, horseradish peroxidase (HRP) is also capable of oxidizing veratryl alcohol as well as methoxybenzenes (the latter up to an oxidation potential of 1.34 V). Horseradish peroxidase, however, is extremely labile at pH 2.4 and is inactivated within minutes under such acidic conditions. The oxidization bySBP, and to a lesser extent by HRP, of veratryl alcohol directly in the presence of H 2O 2 is the first reported case of plant peroxidases catalyzing the efficient oxidation of high oxidation potential nonphenolics and demonstrates that SBP may be an effective alternative to lignin peroxidase.

Electron Transfer Reactions in Pulping Systems (VII): Degradation Reactions of β-Methoxy Lignin Models

Abstract Two β-methoxy lignin model compounds were heated at 150°C in aqueous sodium hydroxide and in mixtures of NaOH/glucose and NaOH/glucose/AQ. Practically no β-ether cleavage was observed under these simulated pulping conditions. The principal products were vinyl ethers and Cα-reduction products; the former dominating in NaOH, the latter in the anthraquinone case. The observation of Cα-reduction products indicates that electron transfer reactions had occurred. Attempts to prepare β-(3-butenyl)-β-methoxy lignin models failed.

Some mechanistic aspects of the light-induced yellowing of lignin: A further study of the photochemical reactions of α-guaiacoxy-β-hydroxy-proprio-veratrone, and some substituted methoxybenzenes

The primary photoreduction of a model lignin system α-guaiacoxy-β-hydroxypropioveratrone occurs via a triplet mechanism leading to polarized ketyl radicals which undergo β-ether cleavage similarly to the photochemical reactions of α-guaiacoxyacetoveratrone. Comparison of the photoyellowing rates of solutions of the two lignin models showed a slightly greater rate for the derivative with the βhydroxymethyl function, with some subtle differences in behavior between the two compounds probably due to the intramolecular hydrogen bonding in the former. In addition it was found that photooxidation of several methoxybenzenes was considerably slower than that of corresponding methoxyphenols. This is reassuring that the ubiquitous presence of methoxybenzene groups in lignin matrices is unlikely to contribute significantly on the timescale relevant to the photoyellowing of CTMP pulps.

Dissolution of Wood with Acetyl Bromide Solutions-Reactions of Lignin Model Compounds

Abstract A Pinus radiata refiner mechanical pulp (RMP) sample and a range of lignin model compounds were treated with a solution of 25% w/w acetyl bromide (AcBr) in acetic acid to assess the reactions which take place during the dissolution of wood with the AcBr solution. The lignin model reactions were also carried out in solutions to which 4% w/w perchloric acid was added. The RMP sample gave a product which contained 9.2% bromine, and was severely degraded to low molecular weight products, the polysaccharides more so than the lignin fraction. The reactions of the lignin model compounds with AcBr included O-acetylation of phenolic and aliphatic hydroxyl groups, cleavage of β-ether bonds, C-acetylation of aromatic rings, particularly in positions para to methoxyl groups, demethylation of aromatic methoxyl groups and probable replacement of benzyl alcohol groups by bromine functions. The reactions were accelerated by perchloric acid, and the rates appeared to decrease in the order O-acetylation π bromine substitution >> β-ether cleavage π C-acetylation > demethylation. It is concluded that dissolution of wood in the AcBr solution is a consequence of depolymerisation of the polysaccharides and lignin, and acetylation to give products which would be soluble in the reaction medium.

The mechanism of the M�ule colour reaction introduction of methylated syringyl nuclei into softwood lignin

Ezo spruce (Picea jezoensis) wood meal and milled wood lignin were successively reduced with sodium borohydride, methylated with methanol-HCl, oxidized with Fremy's salt, reduced with sodium dithionite, and methylated with diazomethane. Permanganate oxidation of the treated milled wood lignin showed that 0.08–0.1/C9 units of 3,4,5-trimethoxyphenyl groups were introduced into the softwood lignin. Although hardwood meal (beech, Fagus crenata) methylated with diazomethane gave a purple-red colour with the Maule test, the treated softwood meal gave only a dark brown colour. The aromatic nuclei of lignin were broken down by the Maule treatment. The consumption of permanganate by treated softwood lignin was higher than by hardwood lignin, which suggests that the guaiacyl nuclei were broken down severely. It is proposed that the purple-red colour obtained from methylated hardwood lignin with the Maule colour test is generated by reaction of syringyl groups which were liberated by β-ether cleavage under the permanganate oxidation conditions.