Are lignins optically active?

The accepted derivation of lignins from non(enzymatically)-controlled radical coupling reactions has been recently challenged, and it is relevant to ascertain unequivocally whether lignins are or are not (as normally assumed) optically active. Two approaches were used. First, DFRC (derivatization followed by reductive cleavage) dimers derived from beta-5- and beta-beta-units in pine lignins, which certainly retain unaltered chiral centers (as well as beta-1- and beta-O-4-units where the intactness may be debated), were shown to be optically inactive by circular dichroism (CD) and chiral high-performance liquid chromatography. CD of beta-5-derived dimers following enantiomeric separation readily demonstrated the sensitivity of the method. Second, no optical activity could be detected (above 250 nm to avoid carbohydrate contributions) by CD of lignin isolates from pine, kenaf, maize, or a CAD-deficient pine mutant. Representative lignins are therefore not, within limits of detection by these methods, optically active.     

Structural characterization of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems

The structure of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems was studied both in situ and in isolated milled "wood" lignins by several analytical methods. The presence of p-coumarate and ferulate in the cortex and pith, as well as in their isolated lignins, was revealed by pyrolysis in the presence of tetramethylammonium hydroxide, and by 2D NMR, and indicated that ferulate acylates the carbohydrates while p-coumarate acylates the lignin polymer. 2D NMR showed a predominance of alkyl aryl ether (β-O-4') linkages (82% of total interunit linkages), with low amounts of "condensed" substructures, such as resinols (β-β'), phenylcoumarans (β-5'), and spirodienones (β-1'). Moreover, the NMR also indicated that these lignins are extensively acylated at the γ-carbon of the side chain. DFRC analyses confirmed that p-coumarate groups acylate the γ-OHs of these lignins, and predominantly on syringyl units.     

Comparative studies on the delignification of pine kraft-anthraquinone pulp with hydrogen peroxide by binucleus Mn(IV) complex catalysis

Pine kraft-anthraquinone (kraft-AQ) pulp was bleached in alkaline solution with hydrogen peroxide catalyzed by either [L(1)Mn(IV)(micro-O)(3)Mn(IV)L(1)](PF(6))(2)] (C1) or [LMn(IV)(2)(micro-O)(3)] (ClO(4))(2) (C2) at 60 and 80 degrees C for 120 min with a catalyst charge of 10 ppm on pulp. The resulting bleached pulp was hydrolyzed with cellulase to obtain insoluble and soluble residual lignins. The alkaline bleaching effluents were acidified to precipitate alkaline-soluble lignins. These lignin preparations were then characterized by 2D heteronuclear multiple-quantum coherence (HMQC) NMR spectroscopic techniques. The results showed that biphenyl (5-5) and stilbene structures of the residual lignin in the pulp are preferentially degraded in both the C1- and C2-catalyzed bleachings, whereas beta-O-4, beta-5, and beta-beta structures undergo degradation to a lesser extent. In both cases, the degradation of the residual lignin increased with the increase in reaction temperature from 60 to 80 degrees C. Thus, the result of C1-catalyzed delignification is not in agreement with the observed decrease in the disappearance rate for substrates in the C1-catalyzed oxidation of lignin model compounds with hydrogen peroxide when the reaction temperature is increased from 60 to 80 degrees C. In addition, the resulting residual lignins in the C2-catalyzed bleaching at 80 degrees C are less degraded than the corresponding lignins in the C1-catalyzed bleaching at both 60 and 80 degrees C. Thus, C1 is more effective than C2 as catalyst in the binucleus Mn(IV) complex-catalyzed bleaching of pine kraft-AQ pulp with hydrogen peroxide.     

Elucidation of the structures of residual and dissolved pine kraft lignins using an HMQC NMR technique

Comparative studies on the structures of residual and dissolved lignins isolated from pine kraft pulp and pulping liquor have been undertaken using the (1)H-(13)C HMQC NMR technique, GPC, and sugar analysis to elucidate the reaction mechanisms in kraft pulping and the lignin reactivity. A modified procedure for the isolation of enzymatic residual lignins has resulted in an appreciable decrease in protein contaminants in the residual lignin preparations (N content < 0.2%). The very high dispersion of HMQC spectra allows identification of different lignin moieties, which signals appear overlapped in 1D (13)C NMR spectra. Elucidation of the role of condensation reactions indicates that an increase in the degree of lignin condensation during pulping results from accumulation of original condensed lignin moieties rather than from the formation of new alkyl-aryl structures. Among aryl-vinyl type moieties, only stilbene structures are accumulated in lignin in appreciable amounts. Benzyl ether lignin-carbohydrate bonds involving primary hydroxyl groups of carbohydrates have been detected in residual and dissolved lignin preparations. Structures of the alpha-hydroxyacid type have been postulated to be among the important lignin degradation products in kraft pulping. The effect of the isolation method on the lignin structure and differences between the residual and dissolved lignins are discussed.     

Reaction mechanisms in delignification of pine Kraft-AQ pulp with hydrogen peroxide using Mn(IV)-Me4DTNE as catalyst

Pine Kraft-AQ pulp was bleached with hydrogen peroxide catalyzed by [LMn(IV)(2) (mu-O)(3)](ClO(4))(2) at 80 degrees C for 120 min under optimum reaction conditions. The resulting bleached pulp was hydrolyzed with cellulase to obtain insoluble and soluble residual lignins. The alkaline effluent from the bleaching was acidified to precipitate alkaline soluble lignin. These lignin preparations were purified, and then analyzed by 2D HMQC NMR spectroscopic techniques. The results showed that biphenyl (5-5) and stilbene structures are preferentially degraded in the bleaching process, while beta-O-4, beta-5, and beta-beta structures undergo degradation only to a lesser extent. This implies that hydrogen peroxide bleaching using the catalyst is more effective in delignification of softwood pulps than hardwood pulps. The possible reaction mechanisms for the delignification of residual lignin in the pine Kraft-AQ pulp in the bleaching process are discussed on the basis of the 2D HMQC NMR spectroscopic data and the model compound experiments.     

Analysis of technical lignins by two- and three-dimensional NMR spectroscopy

Modern multidimensional NMR spectroscopic methods were applied to investigate the effects of kraft pulping and oxygen delignification on lignin side-chain structures. In addition to the two-dimensional HSQC measurements, the three-dimensional HSQC-TOCSY technique was utilized to elucidate the (1)H-(1)H and (1)H-(13)C correlations of individual spin systems and thus indicate a certain lignin side-chain structure. Unlike earlier, nonlabeled samples were used for 3D measurements. According to 2D and 3D NMR spectra, most of the structures identified in milled wood lignin (MWL) are still present in technical lignins after kraft pulping and oxygen delignification. Although the main reaction during kraft pulping is the cleavage of beta-O-4 linkages, these structures are still left in spent liquor lignin as well as in residual lignin. The amount of coniferyl alcohol and dihydroconiferyl alcohol end groups, as well as some unidentified saturated end groups, is higher in technical lignins than in MWL. Contrary to our earlier observations, no diphenylmethane structures were observed in any technical lignins. Vinyl aryl ether structures could not be detected in technical lignins either.     

Effects of increased nitrogen supply on the lignification of poplar wood

The short-term influence of adequate and high nitrogen fertilization on poplar lignification was investigated. The high nitrogen supply decreased lignin staining in the newly formed secondary xylem, indicating that lignin deposition was affected. Acetyl bromide determinations gave a 9-10% decrease in lignin content; however, Klason lignin content was unchanged. Thioacidolysis showed that elevated N supply affected lignin structure such that there was a reduced frequency of lignin units involved in beta-O-4 bonds, a reduced syringyl/guaiacyl ratio, an increased frequency of p-hydroxyphenyl lignin units, more guaiacyl units with free phenolic groups, and more p-hydroxybenzoic acid ester-linked to poplar lignins. These features suggest that lignins from poplars grown under high N bear structural similarities to lignins formed during early stages of wood development. The findings also indicate that a gravitational stimulus inducing the formation of tension wood and high N availability lead to similar and additive effects on lignin content and structure.     

Lignin modification during Eucalyptus globulus kraft pulping followed by totally chlorine-free bleaching: a two-dimensional nuclear magnetic resonance, Fourier transform infrared, and pyrolysis-gas chromatography/mass spectrometry study

Chemical modification of eucalypt lignin was investigated during kraft pulping and chlorine-free bleaching by comparing milled wood lignin, kraft lignin, and pulp enzymatic residual lignins. The syringyl-to-guaiacyl ratio (S/G) from analytical pyrolysis slightly changed during pulping and bleaching (S/G, 3-4) but was higher in the kraft lignin. Semiquantitative heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance (NMR) showed that the relative amount of beta-O-4' (around 80% side chains) and resinol type substructures (15%) was slightly modified during pulping and oxygen delignification. However, a decrease of resinol substructures (to only 6%) was found after alkaline peroxide bleaching. The relative amount of surviving linkages in the highly phenolic kraft lignin was dramatically modified; resinols were predominant. Oxygen delignification did not change interunit linkages, but a relative increase of oxidized units was found in the HSQC aromatic region, in agreement with the small increase of pyrolysis markers with oxidized side chains. NMR heteronuclear multiple bond correlations showed that the oxidized units after oxygen delignification bore conjugated ketone groups.     

Controlled release of a herbicide from matrix granules based on solvent-fractionated organosolv lignins

An organosolv (Alcell) lignin was fractionated with both sequential solvent-extraction and solvent-mixture precipitation using various organic solvent systems. The resulting fractionated lignins were used to prepare matrix granules with bromacil as a model compound using a melt process. The release rates of bromacil in vitro from the granular matrix system were influenced by both the lignin used as carrier and the solution fractionation. The variations in release rates were related to the high proportion of low-molecular-weight fraction and its associated water-soluble lignin in the fractionated lignins. Fickian diffusion was a predominant factor in controlling the release of bromacil from the matrix granules based on the low-molecular-weight fraction (L(fr1)). For the matrix granules based on the other fractionated lignins, the release of bromacil was of super-case II transport. The fine structure of the matrix granules before and after release was also studied. An unique release pattern of bromacil was observed for the matrix granules based on the low-molecular-weight fraction (L(fr1)), showing a fast release followed by zero-order release, which was a result of remarkable changes in the structure of the matrix granules during the course of release.     

Trapping of p-Coumaryl and Coniferyl Alcohol during Soda-Anthraquinone Treatment: A Means of Estimating Uncondensed β-O-4 Structures in Native Lignin

In most native lignins, at least 50% of the phenylpropane (C(9)) units are involved in β-O-4 linkages. It was recently observed that ethylguaiacol (EG) was efficient at trapping coniferyl alcohol generated from the cleavage of uncondensed β-O-4 dimeric structures during soda-anthraquinone (AQ) or SAQ delignification of sugar maple wood meal. Some of the coniferyl alcohol was transformed to vinylguaiacol and isoeugenol, and the α-carbon atom in all three monomers formed C-C bonds with the C-5 position of EG. In the present research, eucalyptus and sugar cane bagasse meals were also investigated, and the yields of uncondensed β-O-4 structures in the nonsyringyl fraction were quantitated. The estimates of the uncondensed fraction of the lignin in the three samples (assuming S units are 90-95% uncondensed) were in close agreement with results from traditional but more tedious methods such as permanganate oxidation or spectroscopic methods requiring a sample representative of native lignin.     

Extraction and characterization of original lignin and hemicelluloses from wheat straw

Original lignin and hemicelluloses were sequentially extracted with high yield/purity, using acidic dioxane/water solution and dimethyl sulfoxide, from ball-milled wheat straw. The acidic dioxane lignin fraction is distinguished by high beta-O-4' structures and by low amounts of condensed units (beta-5', 5-5', and beta-1'). Hemicelluloses contain arabinoxylans as the major polysaccharides, which are substituted by alpha-l-arabinofuranose, 4-O-methylglucuronic acid, acetyl group (DS = 0.1), and xylose at O-3 and/or O-2 of xylans. It was found that arabinoxylans form cross-links with lignins through ferulates via ether bonds, glucuronic acid via ester bonds, and arbinose/xylose via both ether and glycosidic bonds, respectively, in the cell walls of wheat straw. Diferulates are also incorporated into cross-links between lignin and hemicelluloses as well as lignification of wheat straw cell walls. The guaiacyl unit is considered to be a significant condensed structural constructor in extracted lignin and a connector between lignin and carbohydrates.     

Microwave-assisted lignin isolation using the enzymatic mild acidolysis (EMAL) protocol

The use of microwaves is explored in an effort to further improve the recently developed lignin isolation protocol termed EMAL (enzymatic mild acidolysis lignin). Because the presence of the lignin-carbohydrate linkages seems to be rather pronounced within wood, a microwave reactor was used to replace traditional refluxing during the mild acidolysis step. This was done in an attempt to augment the selectivity of this step toward cleaving lignin-carbohydrate bonds as well as reducing the overall intensity of this step toward inducing changes in the lignin structure, thus affording lignin in greater yields and purities. Consequently, in this study the yields, purities, and structures of lignins isolated from spruce (softwood) by the EMAL protocol under various microwave conditions were examined. The variables studied included microwave power, microwave heating time, hydrochloric acid concentration and water content of the reaction medium. Microwave heating afforded EMAL samples of high purity (90%, comparable to the conventional protocol) but in significantly greater gravimetric yields. Quantitative (31)P NMR and SEC data confirmed that the structure of lignin was similar to that obtained by traditional EMALs, with comparable contents of beta-aryl ether bonds, phenolic hydroxyls (condensed and uncondensed), and carboxylic acids.     

Elucidation of lignin structure through degradative methods: comparison of modified DFRC and thioacidolysis

Milled wood and milled wood lignin (MWL) samples were subjected to DFRC and thioacidolysis. Despite the fact that both methods selectively cleave aryl ether bonds, substantial differences in results were obtained. Lignin thioacidolysis gave total molar yields of degradation monomer products in the range of 3.5-7 mol % higher than DFRC. GPC analysis showed that the thioacidolysis-treated lignin was degraded to a lower average molecular weight than that treated by DFRC. Contrary to results reported for lignin model compounds, these results indicate that the DFRC method does not completely or efficiently degrade the lignin polymer. In fact, the DFRC-degraded lignin retained much of the characteristics of the original MWL. Elemental analysis revealed the presence of bromine in the DFRC-treated lignin, and two-dimensional (1)H-(13)C HMQC NMR spectroscopy showed the presence of beta-O-4 linkages in the DFRC-treated lignin. No beta-O-4 interunit linkages were detected in the thioacidolysis-treated lignin. These results are consistent with the lower monomer yields and the higher average molecular weight of the DFRC-treated lignin and indicate inefficiency in the chemistry of the method, probably due to steric constraints of the polymeric nature of lignin.     

Lignins and ferulate-coniferyl alcohol cross-coupling products in cereal grains

Plant cell walls containing suberin or lignin in the human diet are conjectured to protect against colon cancer. To confirm the existence of authentic lignin in cereal grain dietary fibers, the DFRC (derivatization followed by reductive cleavage) method was applied to different cereal grain dietary fibers. By cleavage of diagnostic arylglycerol-beta-aryl (beta-O-4) ether linkages and identification of the liberated monolignols, it was ascertained that lignins are truly present in cereal grains. From the ratios of the liberated monolignols coniferyl alcohol and sinapyl alcohol, it is suggested that lignin compositions vary among cereals. Furthermore, dimeric cross-coupling products, comprising ferulate and coniferyl alcohol, were identified in most cereal fibers investigated. These ferulate 4-O-beta- and 8-beta-coniferyl alcohol cross-coupled structures indicate radical cross-coupling of polysaccharides to lignin precursors via ferulate.     

Highly acylated (acetylated and/or p-coumaroylated) native lignins from diverse herbaceous plants

The structure of lignins isolated from the herbaceous plants sisal ( Agave sisalana), kenaf ( Hibiscus cannabinus), abaca ( Musa textilis) and curaua ( Ananas erectifolius) has been studied upon spectroscopic (2D-NMR) and chemical degradative (derivatization followed by reductive cleavage) methods. The analyses demonstrate that the structure of the lignins from these plants is highly remarkable, being extensively acylated at the gamma-carbon of the lignin side chain (up to 80% acylation) with acetate and/or p-coumarate groups and preferentially over syringyl units. Whereas the lignins from sisal and kenaf are gamma-acylated exclusively with acetate groups, the lignins from abaca and curaua are esterified with acetate and p-coumarate groups. The structures of all these highly acylated lignins are characterized by a very high syringyl/guaiacyl ratio, a large predominance of beta- O-4' linkages (up to 94% of all linkages), and a strikingly low proportion of traditional beta-beta' linkages, which indeed are completely absent in the lignins from abaca and curaua. The occurrence of beta-beta' homocoupling and cross-coupling products of sinapyl acetate in the lignins from sisal and kenaf indicates that sinapyl alcohol is acetylated at the monomer stage and that, therefore, sinapyl acetate should be considered as a real monolignol involved in the lignification reactions.     

NMR characterization of lignins isolated from fruit and vegetable insoluble dietary fiber

Compositional information for lignins in food is rare and concentrated on cereal grains and brans. As lignins are suspected to have important health roles in the dietary fiber complex, the confusing current information derived from nonspecific lignin determination methods needs to be augmented by diagnostic structural studies. For this study, lignin fractions were isolated from kiwi, pear, rhubarb, and, for comparison, wheat bran insoluble dietary fiber. Clean pear and kiwi lignin isolates allowed for substantive structural profiling, but it is suggested that the significance of lignin in wheat has been overestimated by reliance on nonspecific analytical methods. Volume integration of NMR contours in two-dimensional (13)C-(1)H correlation spectra shows that pear and wheat lignins have comparable guaiacyl and syringyl contributions and that kiwi lignins are particularly guaiacyl-rich (approximately 94% guaiacyl) and suggest that rhubarb lignins, which could not be isolated from contaminating materials, are as syringyl-rich (approximately 96% syringyl) as lignins from any known natural or transgenic fiber source. Typical lignin structures, including those newly NMR-validated (glycerols, spirodienones, and dibenzodioxocins), and resinols implicated as possible mammalian lignan precursors in the gut are demonstrated via their NMR correlation spectra in the fruit and vegetable samples. A novel putative benzodioxane structure appears to be associated with the kiwi lignin. It is concluded that the fruits and vegetables examined contain authentic lignins and that the detailed structural analysis exposes limitations of currently accepted analytical methods.     

Using transgenic poplars to elucidate the relationship between the structure and the thermal properties of lignins

In an attempt to draw relationships between the molecular structure and the thermal behavior of lignins, thermomechanical analyses were run on six milled wood and enzyme poplar lignin fractions prepared from genetically modified and control woods. All the lignin samples displayed similar thermal profiles with a clear inflection point assigned to the glass transition point. The temperature (T(g)) at which this transition occurs showed large variations from 170 to 190 degrees C, depending both on the genetic modification and on the age of the tree. These variations were found to be closely related to the condensation degree of lignins evaluated by thioacidolysis.     

NMR evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into Lignins of O-methyltransferase-deficient poplars

Benzodioxane structures are produced in lignins of transgenic poplar plants deficient in COMT, anO-methyltransferase required to produce lignin syringyl units. They result from incorporation of 5-hydroxyconiferyl alcohol into the monomer supply and confirm that phenols other than the three traditional monolignols can be integrated into plant lignins.     

Characterization of milled wood lignin (MWL) in Loblolly pine stem wood, residue, and bark

Milled wood lignin samples from Loblolly pine stem wood, forest residue, and bark were isolated and characterized by quantitative (13)C and (31)P nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC) for molecular weight determination. Results from (13)C NMR show the stem wood and forest residue samples have similar functional group contents. However, the bark has fewer methoxyl groups, β-O-4 structures, dibenzodioxocin, and side chains than the other two lignins. The bark lignin has the highest amounts of p-hydroxyphenyl (h) and C-5 condensed lignin, stem wood has the lowest, and the residue lies between. (31)P NMR analysis indicates that bark lignin contains more C-5 substituted phenolics and fewer aliphatic hydroxyl groups than the lignin isolated from stem wood or residue. The molecular weight distribution analysis indicates the bark lignin has higher weight-average molecular weight (M(w)) and polydispersity index than the lignin recovered from stem wood or residue.     

Structural characterization of guaiacyl-rich lignins in flax (Linum usitatissimum) fibers and shives

The structural characteristics of the lignins from flax (Linum usitatissimum) fibers and shives were studied. Significant differences in the content and composition of the lignin from both parts were observed. The lignin contents were 3.8% in the fibers and 29.0% in the shives. Analysis by Py-GC/MS indicated a H:G:S molar ratio of 13:72:15 in the milled wood lignin (MWL) isolated from flax fibers and a molar ratio of 5:87:8 in the MWL isolated from flax shives. In addition, 2D-NMR showed a predominance of β-O-4' aryl ether linkages, followed by β-5' phenylcoumaran and β-β' resinol-type linkages in both MWLs, with a higher content of condensed linkages in flax shives. Thioacidolysis (followed by Raney nickel desulfurization) gave further information on the lignin units involved in the different linkages and confirmed the enrichment of G units. The thioacidolysis dimers released were similar from both lignins, with a predominance of the β-5' followed by β-1' and 5-5' structures.