Sally A. Ralph

FT-Raman Spectroscopy of Wood: Identifying Contributions of Lignin and Carbohydrate Polymers in the Spectrum of Black Spruce (Picea Mariana):

Good-quality Raman spectra of most wood species can now be obtained by using near-infrared Fourier transform Raman spectroscopy. To make effective use of such spectroscopic information, one needs to interpret the data in terms of contributions from various wood components and, for each component polymer, in terms of vibrational modes of its substructural units/groups. In the present work, Raman spectral features of black spruce (Picea mariana) wood were associated with lignin and/or carbohydrate polymers. Lignins spectral contributions were recognized in several ways. In addition to spectra of milled-wood and enzyme lignins, a spectrum of native lignin was obtained by subtracting the spectrum of acid chlorite delignified black spruce from the spectrum of an untreated wood sample. A comparison of lignin spectra indicated that the Raman features of the three lignins are very similar. Raman contributions of carbohydrate polymers, namely, those of cellulose and hemicellulose, were identified by using authentic and/or isolated samples and, in the case of cellulose, by using previously published spectra. Such an analysis showed that the hemicellulose present in black spruce did not give rise to any new, unique features that were not already present due to cellulose. Therefore, it was concluded that the hemicellulose contribution is broad and is hidden under the Raman contribution of cellulose. Also, peak positions of lignin contributions did not overlap with those of cellulose, and there were spectral regions where either lignin or cellulose contributed.

Effects of Coumarate 3-Hydroxylase Down-regulation on Lignin Structure

Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant guaiacyl (G) and syringyl (S) units. Stem levels of up to ∼65% P (from wild-type levels of ∼1%) resulting from down-regulation of C3H were measured by traditional degradative analyses as well as two-dimensional13C-1H correlative NMR methods. Such levels put these transgenics well beyond the P:G:S compositional bounds of normal plants; p-hydroxyphenyl levels are reported to reach a maximum of 30% in gymnosperm severe compression wood zones but are limited to a few percent in dicots. NMR also revealed structural differences in the interunit linkage distribution that characterizes a lignin polymer. Lower levels of key β-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of β-1 coupling products, highlighting the significant differences in the reaction course for p-coumaryl alcohol versus the two normally dominant monolignols, coniferyl and sinapyl alcohols. A larger range of dibenzodioxocin structures was evident in conjunction with an approximate doubling of their proportion. The nature of each of the structural units was revealed by long range13C-1H correlation experiments. For example, although β-ethers resulted from the coupling of all three monolignols with the growing polymer, phenylcoumarans were formed almost solely from coupling reactions involving p-coumaryl alcohol; they resulted from both coniferyl and sinapyl alcohol in the wild-type plants. Such structural differences form a basis for explaining differences in digestibility and pulping performance of C3H-deficient plants.

Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

Profiling of Oligolignols Reveals Monolignol Coupling Conditions in Lignifying Poplar Xylem

Lignin is an aromatic heteropolymer, abundantly present in the walls of secondary thickened cells. Although much research has been devoted to the structure and composition of the polymer to obtain insight into lignin polymerization, the low-molecular weight oligolignol fraction has escaped a detailed characterization. This fraction, in contrast to the rather inaccessible polymer, is a simple and accessible model that reveals details about the coupling of monolignols, an issue that has raised considerable controversy over the past years. We have profiled the methanol-soluble oligolignol fraction of poplar (Populus spp.) xylem, a tissue with extensive lignification. Using liquid chromatography-mass spectrometry, chemical synthesis, and nuclear magnetic resonance, we have elucidated the structures of 38 compounds, most of which were dimers, trimers, and tetramers derived from coniferyl alcohol, sinapyl alcohol, their aldehyde analogs, or vanillin. All structures support the recently challenged random chemical coupling hypothesis for lignin polymerization. Importantly, the structures of two oligomers, each containing a γ-p-hydroxybenzoylated syringyl unit, strongly suggest that sinapyl p-hydroxybenzoate is an authentic precursor for lignin polymerization in poplar.

Tricin, a Flavonoid Monomer in Monocot Lignification

Tricin, a flavonoid, is a lignification monomer in grasses where it functions as a nucleation site for lignin polymer formation. Tricin was recently discovered in lignin preparations from wheat (Triticum aestivum) straw and subsequently in all monocot samples examined. To provide proof that tricin is involved in lignification and establish the mechanism by which it incorporates into the lignin polymer, the 4′-O-β-coupling products of tricin with the monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) were synthesized along with the trimer that would result from its 4′-O-β-coupling with sinapyl alcohol and then coniferyl alcohol. Tricin was also found to cross couple with monolignols to form tricin-(4′-O-β)-linked dimers in biomimetic oxidations using peroxidase/hydrogen peroxide or silver (I) oxide. Nuclear magnetic resonance characterization of gel permeation chromatography-fractionated acetylated maize (Zea mays) lignin revealed that the tricin moieties are found in even the highest molecular weight fractions, ether linked to lignin units, demonstrating that tricin is indeed incorporated into the lignin polymer. These findings suggest that tricin is fully compatible with lignification reactions, is an authentic lignin monomer, and, because it can only start a lignin chain, functions as a nucleation site for lignification in monocots. This initiation role helps resolve a long-standing dilemma that monocot lignin chains do not appear to be initiated by monolignol homodehydrodimerization as they are in dicots that have similar syringyl-guaiacyl compositions. The term flavonolignin is recommended for the racemic oligomers and polymers of monolignols that start from tricin (or incorporate other flavonoids) in the cell wall, in analogy with the existing term flavonolignan that is used for the low-molecular mass compounds composed of flavonoid and lignan moieties.

NMR analysis of lignins in CAD-deficient plants. Part 1. Incorporation of hydroxycinnamaldehydes and hydroxybenzaldehydes into lignins

Peroxidase/H2O2-mediated radical coupling of 4-hydroxycinnamaldehydes produces 8-O-4-, 8-5-, and 8-8-coupled dehydrodimers as has been documented earlier, as well as the 5-5-coupled dehydrodimer. The 8-5-dehydrodimer is however produced kinetically in its cyclic phenylcoumaran form at neutral pH. Synthetic polymers produced from mixtures of hydroxycinnamaldehydes and normal monolignols provide the next level of complexity. Spectral data from dimers, oligomers, and synthetic polymers have allowed a more substantive assignment of aldehyde components in lignins isolated from a CAD-deficient pine mutant and an antisense-CAD-downregulated transgenic tobacco. CAD-deficient pine lignin shows enhanced levels of the typical benzaldehyde and cinnamaldehyde end-groups, along with evidence for two types of 8-O-4-coupled coniferaldehyde units. The CAD-downregulated tobacco also has higher levels of hydroxycinnamaldehyde and hydroxybenzaldehyde (mainly syringaldehyde) incorporation, but the analogous two types of 8-O-4-coupled products are the dominant features. 8-8-Coupled units are also clearly evident. There is clear evidence for coupling of hydroxycinnamaldehydes to each other and then incorporation into the lignin, as well as for the incorporation of hydroxycinnamaldehyde monomers into the growing lignin polymer. Coniferaldehyde and sinapaldehyde (as well as vanillin and syringaldehyde) co-polymerize with the traditional monolignols into lignins and do so at enhanced levels when CAD-deficiency has an impact on the normal monolignol production. The implication is that, particularly in angiosperms, the aldehydes behave like the traditional monolignols and should probably be regarded as authentic lignin monomers in normal and CAD-deficient plants.

New preparations of lignin polymer models under conditions that approximate cell wall lignification. II. Structural characterization of the models by thioacidolysis

Summary Paris-Grignon, Lignin polymer model Dehydrogenation polymer The structures of novel lignin polymer models prepared from coniferin or coniferyl alcohol under conditions that approximate cell wal1 Signification were characterized by thioacidolysi s. The linkageof monolignol (DHP) type analysis by thioacidolysi s indicates that the structure of these polylignols approximated that of -. Monolignol glucoside native lignin more closely than did the structure of polylignols prepared by the conventional method Coniferyl ulcohol from coniferyl alcohol. Among v:arious possible factors affecting the structure of polylignols during Thioacidolysis polymerization under the present experimental conditions, the important ones appears to be: pH of the medium in which dehydrogenativc polymerization of coniferyl alcohol takes place; relative concentrations of monomer and oligomer radicals; and the carbohydrate matrix in which polymerization occurs.

FT–Raman Investigation of Milled-Wood Lignins: Softwood, Hardwood, and Chemically Modified Black Spruce Lignins

Abstract Raman spectroscopy is being increasingly applied to study wood and other lignin-containing biomass/biomaterials. Lignins contribution to the Raman spectra of such materials needs to be understood in the context of various lignin structures, substructures, and functional groups so that lignin-specific features could be identified and the spectral information could be interpreted usefully. Additionally, to enhance the utility of Raman as a characterization tool, an understanding of chemical-treatment-induced changes to the lignin spectrum is important. In the present work, Raman spectra of four milled-wood lignins (MWLs)—black spruce, loblolly pine, aspen, and sweetgum—were compared, and using black spruce MWL, spectral changes brought about by alkaline hydrogen peroxide bleaching, hydrogenation, acetylation, and methylation reactions were analyzed. The band intensity changes depended upon the nature of the chemical treatments.

New Preparations of Lignin Polymer Models under Conditions that Approximate Cell Wall Lignification. I. Synthesis of Novel Lignin Polymer Models and their Structural Characterization by 13 C NMR

Guaiacyl-type lignin polymer models were prepared from coniferin by the action of β-glucosidase and peroxidase, with hydrogen peroxide generated in situ through the action of oxygen and glucose oxidase on the glucose liberated from the coniferin. Polylignols were also prepared from coniferyl alcohol using procedures modified to more closely correspond to conditions prevailing in the cell wall environment. The structure of these novel polylignols approximated that of native lignin more closely than did the structure of polylignols prepared by the conventional method from coniferyl alcohol.

Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase

Many litter-decay fungi secrete heme-thiolate peroxygenases that oxidize various organic chemicals, but little is known about the role or mechanism of these enzymes. We found that the extracellular peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent cleavage of environmentally significant ethers, including methyl t-butyl ether, tetrahydrofuran, and 1,4-dioxane. Experiments with tetrahydrofuran showed the reaction was a two-electron oxidation that generated one aldehyde group and one alcohol group, yielding the ring-opened product 4-hydroxybutanal. Investigations with several model substrates provided information about the route for ether cleavage: (a) steady-state kinetics results with methyl 3,4-dimethoxybenzyl ether, which was oxidized to 3,4-dimethoxybenzaldehyde, gave parallel double reciprocal plots suggestive of a ping-pong mechanism (Km(peroxide), 1.99 ± 0.25 mm; Km(ether), 1.43 ± 0.23 mm; kcat, 720 ± 87 s−1), (b) the cleavage of methyl 4-nitrobenzyl ether in the presence of H218O2 resulted in incorporation of 18O into the carbonyl group of the resulting 4-nitrobenzaldehyde, and (c) the demethylation of 1-methoxy-4-trideuteromethoxybenzene showed an observed intramolecular deuterium isotope effect [(kH/kD)obs] of 11.9 ± 0.4. These results suggest a hydrogen abstraction and oxygen rebound mechanism that oxidizes ethers to hemiacetals, which subsequently hydrolyze. The peroxygenase appeared to lack activity on macromolecular ethers, but otherwise exhibited a broad substrate range. It may accordingly have a role in the biodegradation of natural and anthropogenic low molecular weight ethers in soils and plant litter.