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Ginkgo biloba L. saplings were cultivated in an airtight growth chamber with 13CO2 for two months. The 13C ratio of the newly developed xylem region was ca. 85%, evaluated by high lateral resolution secondary ion mass spectrometry and thioacidolysis/GC-MS. Quantitative solid-state 13C direct polarisation/magic angle spinning (DP/MAS) NMR measurements with high-speed MAS of 70.0 kHz were conducted for cutting-milled wood (CMW), ball-milled wood (BMW), and enzymatically saccharified lignin (EL) samples. In addition, quantitative liquid-state 13C NMR measurements were carried out for EL in DMSO-d 6. Major lignin aromatic signals were classified into three groups of aromatic carbons of C–H, C–C, and C–O, and their area ratio was compared within these measurements. EL samples in solid- and liquid-state showed nearly the same results. However, the results for CMW and BMW in solid-state NMR suggest the structural alteration of lignin within the sample preparation procedure, including ball milling.

The physical, chemical and biological properties of wood depend on the supramolecular assembly of cellulose microfibrils (CMFs), hemicelluloses (HCs) and lignin in the growing cell walls. Based on the 13C-tracer studies of ginkgo xylem formation, a hypothetical scenario for the role of monolignol glucosides (MLGs) in the assembly is proposed as follows: (1) Both moieties, aglycone monolignols and glycone d-glucose (d-Glc), play essential roles in a cooperative manner in delivery of hydrophobic and highly reactive p-hydroxycinnamyl- (H), coniferyl- (G) alcohols to the hydrophilic site of lignin deposition. (2) The d-Glc liberated at lignification site is converted into essential HCs mainly via Golgi apparatus under the influence of diurnally changing turgor pressure, and partly converted in the apoplast. (3) At cell corner middle lamella, a pressure-resistant layer of HG-lignin-HCs-CMFs is formed, and allows expansion of new cells in cambium region by elevation of turgor pressure. The deformable G-lignin-HCs-CMFs layer at secondary wall shrinks by dehydration of the swollen gel of HCs-CMFs during differentiation, and contributes posture control of standing tree. On-demand quick supply of a large amount of monolignols and HCs can be achieved by the large storage and delivery of MLGs in the growing ginkgo xylem.

Various phenomena in living physiological systems are conducted on the hydrated conditions, and in many cases, they do not work in a dry state. Imaging mass spectrometry is one of the direct detection methods scanning the sample surface with some focused and pulsed energy and analysing the sputtered components. However, under the high vacuum conditions required for usual imaging mass spectrometry, the sample surface is rapidly dried. It is difficult for the target cell to survive, and the original situation are lost soon. Here, the combination of a freeze-fixation and a cryo sample stage is a promising method to do mass spectrometry while maintaining the original situation. By rapidly freezing the cells, the momentary situation as a living cell is fixed. The situation in a living cell can be captured as still images by cryo imaging mass spectrometry. This mini-review introduces the outline of imaging mass spectrometry especially for low molecular weight components and recent results for frozen-hydrated samples by cryo secondary ion mass spectrometry.

A simple method was developed to isolate the compound middle lamella (CML) in its natural state using wet-beating, sedimentation, and methanol dialysis. The isolated CML fraction was characterised. The CML was isolated in 2.0% yield from the mature wood of Ginkgo biloba. Scanning electron microscopy (SEM) images of the isolated CML fraction showed the presence of flake-like structures. However, some small amounts of secondary wall substance was observed. The lignin content of the CML fraction was approximately 45%. Thioacidolysis reactions with the isolated CML fraction revealed that the lignin β-O-4 structure was scarce in the isolated fraction and that numerous structures with two or more consecutive condensed-type bonds were present. Results from the solid-state carbon-13 nuclear magnetic resonance (13C NMR) experiments indicated that the CML fraction had low crystallinity, indicating low cellulose content. Liquid-state NMR analysis of the lignin from the CML fraction revealed the absence of p-hydroxyphenyl (H) units.

Lignin is thought to be covalently bound to carbohydrates like hemicellulose during biosynthesis to form a lignin-carbohydrate complex (LCC). However, successive polymerisation with monolignols after the formation of LCC has not yet been clarified. To investigate the reaction of LCC, its enzymatic dehydrogenative polymerisation was conducted using deuterium-labelled coniferyl alcohol and model compounds, i.e., a lignin model (β-O-4 dimer model) compound (LM) and an LCC model (benzyl ether type) compound (LCCM). The obtained polymers (DHPs) were methylated and subjected to thioacidolysis, and the degradation products were quantified by gas chromatography-mass spectrometry (GC-MS). The results showed that the amount of coniferyl alcohol connected to the LCCM via β-O-4 binding was almost the same as that bound to the LM. However, the amount of unreacted LCCM was larger than that of LM, suggesting that the LCCM is less likely to form condensed structures, i.e., 5-5, β-5, and 4-O-5 structures. This could be due to the steric hindrance of the carbohydrate at the benzylic position.

To discuss the macromolecular structure and properties of lignin, the complementary use of solid- and liquid-state nuclear magnetic resonance (NMR) combined with a specific 13C-enrichment technique may provide useful information. The 13C-enriched lignin was prepared by administering [guaiacyl ring-5(G5)-13C]-coniferin to a growing Ginkgo biloba L. shoot. The 13C-enriched cellulolytic enzyme lignin (EL) and its acetate prepared from the ginkgo shoot were examined by solid- and liquid-state 13C NMR spectroscopy. The 13C NMR spectrum derived only from the G5 carbon was obtained as a difference spectrum based on the spectra of the G5-13C enriched and un-enriched (UE) samples. The condensed structures, including the enriched G5-carbon, were evaluated using difference spectra. The chemical shifts of the enriched G5 carbon in both the solid- and liquid-state 13C NMR spectra agreed with each other in principle. The quantitative ratio of the condensed and uncondensed structures at G5 was found to be larger by solid-state cross polarization/magic angle spinning (CP/MAS) NMR than by liquid-state NMR.

No clear picture has yet been elaborated concerning the mechanism of lignin growth, and thus this topic is the focus of the present paper. Namely, the enzymatic dehydrogenative polymerization (DHP formation) of coniferyl alcohol (CA, as a monolignol) and three dilignols and their reaction kinetics were investigated. The dilignols [guaiacylglycerol-β-coniferyl ether (IβO4 ), dehydrodiconiferyl alcohol (IIβ5 ), and pinoresinol (IIIββ )] and CA as a monolignol [(3-OCD3)-coniferyl alcohol (CAOCD3 )] were synthesized and subjected to enzymatic DHP formation. The dilignol derived from CAOCD3 could be identified by its higher molecular weight in comparison with the starting dilignols (IβO4 , IIβ5 , and IIIββ ). Based on the observed consumption rate of the CA and its dilignols, it was proposed that a radical transfer system exists between the dilignols, which is generated from the CA and the starting substrates.

Glycol lignin possessing thermoplastic and fusible properties can be a biorefinery platform material; however, glycol lignin is obtained as very small particles in suspension and the low dewatering efficiency is a major difficulty for the mass production. To improve the productivity of glycol lignin in the refinery plant, glycol lignin-based flocculants were prepared and their flocculation performance was examined. The flocculants (cationized glycol lignins) were synthesized by a reaction with glycidyltrimethylammonium chloride (GTA) in various reaction conditions. The prepared flocculants exhibited high flocculation performance and successfully economized the production cost of glycol lignin. Their flocculation efficiency was affected by the precipitation temperature, the length of the glycol chain, the molecular weight and the functional group of the glycol lignin. The flocculation behavior was interpreted by phase separation theory at a lower critical solution temperature (LCST).

An occluding substance (gum) was observed in the vessels of Phellodendron amurense Rupr. and analysed by spectroscopic and chemical methods. Following safranin-alcian blue staining, the gum in sapwood (sW) turned to blue and in heartwood (hW) to red. The gum was studied in situ by UV and Raman microscopies, time-of-flight secondary ion mass spectrometry (TOF-SIMS). The gum was isolated by laser microdissection (LMD) and it was alkali hydrolysed and the degradation products were analysed by GC-MS. The staining experiments, and the UV and Raman microscopies indicated that the major component of the sW gum is constituted of polysaccharides, while in the hW gum the aromatic character is dominating. TOF-SIMS measurements were interpreted as showing the aromatic substances in the hW gum did not contain lignin. The GC-MS analysis revealed the presence of vanillic acid in the degradation products of hW gum.

The stability and chemical reactions of lignans during alkaline cooking is not yet fully clarified although their degradation products may have a considerable effect on the pulp and its bleaching, such as spotting and color contamination. To fill this gap, a phenolic lignan (pinoresinol) and four non-phenolic furofuran lignans (gmelinol, paulownin, eudesmin and sesamin) were submitted to alkaline cooking at various conditions. The effect of cooking duration shows that non-phenolic lignans are more stable during a 3-h reaction time than pinoresinol. After a 24-h reaction, most of the lignans were decomposed or polymerized. The effect of cooking temperature shows that pinoresinol, eudesmin and sesamin are more susceptible to degradation with increasing temperature. The concentration of the alkaline liquors only affect significantly to pinoresinol because of its acidity. It was also observed that pinoresinol, gmelinol and eudesmin give rise to small amounts of vanillin and acetovanillone, while paulownin and sesamin were mainly polymerized or degraded. To confirm the reaction mechanism of the occurrence of acetovanillone in the reaction, 13C-labeled pinoresinol was also submitted to alkaline cooking. It was revealed that the cleavage at β-β′ position occurred during alkaline reaction. The stability of these compounds during alkaline cooking are not affected by the hydroxyl group in the C-β′ position.

Bamboos are among the largest woody grasses and grow very rapidly. Although lignin is a crucial factor for the utilization of bamboo biomass, the lignification mechanism of bamboo shoots is poorly understood. We studied lignification in the bamboo Sinobambusa tootsik during culm development. Elongation growth began in May and ended in late-June, when the lignin content was approximately half that in mature culms. Thioacidolysis analysis indicated that p-hydroxyphenyl units in lignin formed even at late stages of lignification. The syringyl/guaiacyl ratio varied during culm development. Various lignin precursors were detected in developing culms by liquid chromatography–mass spectrometry. The ferulic acid content decreased from May to June, indicating that ferulic acid was utilized in early stages of cell wall formation. Monolignol glucosides were detected at early stages of lignification, whereas the contents of monolignols, coniferaldehyde, sinapaldehyde, p-coumaric acid, and ferulic acid peaked at later stages of lignification. Therefore, lignin precursors may be supplied differentially during the lignification process. In August, the rate of lignification decreased, although the contents of various lignin precursors peaked, implying that the rate-limiting step in the cessation of lignification in bamboo is transport or polymerization of lignin precursors, rather than their biosynthesis.

To investigate the possibility of decontaminating 137Cs-contaminated Cryptomeria japonica wood, kraft pulping was conducted and the Cs behavior in the reaction process was examined. 133Cs-treated or 137Cs-contaminated bark, sapwood, and heartwood chips of Cryptomeria japonica were digested using an aqueous solution of NaOH and Na2S. The pulp was washed with ultrapure water and filtered, after which the filtrate (black liquor) was collected. The black liquor was acidified to separate the supernatant and precipitation. The Cs (133Cs and 137Cs) concentrations in the chip and reaction products were measured. As for wood samples, the majority of Cs was present in black liquor, while only a minor amount of Cs was retained in the pulp (

To elucidate the effect of soil conditions on the in planta distribution of inorganic elements, an aluminium (Al)-tolerant plant, Hydrangea macrophylla, was cultivated with the addition of Al ion to soils. Freeze-dried stems from the plants were analysed by time-of-flight secondary ion mass spectrometry (dry-TOF-SIMS). Freeze-fixed stems of the plants were analysed by cryo-TOF-SIMS. The inorganic metal content was quantified by inductively coupled plasma atomic absorption spectrometry (ICP-AES). The dry- and cryo-TOF-SIMS mapping analyses showed that in the native sample, inorganic elements are mainly localised in the cortex and pith. Al-treatment [i.e. Al2(SO4)3 administration to the soil] altered the distribution and content of inorganic metals. The actual amount of inorganic elements quantified by ICP-AES showed that Al-treatment on the soil increased the amounts of Na, Mg, Al and Ca and decreased that of K in the stem. The secondary ion counts of inorganic elements in freeze-dried and -fixed samples, determined by dry-/cryo-TOF-SIMS measurements, showed similar variations as that observed with ICP-AES measurements. These results are interpreted as that Al-treatment altered the distribution and amount of inorganic elements in the stems of Al-tolerant H. macrophylla plants.

In order to understand the kraft pulp decolouring mechanism on using a nonionic detergent, the pulp washing process and the resulting pulp handsheets were investigated by examining the brightness, kappa number, thioacidolysis product yield, and dewatering efficiency in the pressing sheet making process. The pulp decolouring could be attributed to a decrease in the lignin content and an improvement in the dewatering efficiency. Furthermore, the detergent distribution in the aqueous pulp suspension obtained during the pulp washing process was visualised using cryo-time-of-flight secondary ion mass spectrometry/scanning electron microscopy (cryo-TOF-SIMS/SEM). The detergent was clearly observed at the transverse surface of the pulp fibre cell wall and was also detected in the lumen of the fibres, suggesting the permeation of the detergent into the pulp fibre cell wall. Based on these results, the pulp decolouring mechanism can be proposed as follows: the detergent permeates into the pulp fibre cell wall and promotes the solution-exchange between the inside and the outside parts of the fibre cell wall, finally washing away the chromophoric substances such as lignin and its degradation products owing to the enhanced dewatering efficiency.

The aim of the present study was to investigate the extractives of Gmelina arborea stem from the sapwood (sW) to heartwood (hW) including the transition zone (tZ) between them by means of quantitative GC-MS, while the spatial distribution of four typical compounds was analysed by TOF-SIMS. The focus was on gmelinol, paulownin, 7′-O-ethyl arboreol, and β-sitosterol, which were isolated and purified from hW. The four compounds revealed a characteristic distribution pattern corresponding to their key role in biosynthesis and depending on their susceptibility to secondary reactions in the hW.

Lignin is a biopolymer consisted of monolignols but the growth mechanism of lignin has not yet been elucidated. In this study, enzymatic dehydrogenative polymerization of dilignols, guaiacylglycerol-β-coniferyl ether (I), dehydrodiconiferyl alcohol (II), and pinoresinol (III), was conducted using crude enzyme obtained from Japanese cypress (Chamaecyparis obtusa) in order to investigate the step following the initial radical coupling between monolignols. The results suggested the occurrence of radical transfer during the reaction, similar to the reaction using horseradish peroxidase (HRP). The behavior of erythro and threo of I during the reaction was analyzed, and it was found that the crude enzyme has a substrate specificity toward erythro rather than threo forms, but HRP shows negligible specificity. And that erythro of I shows a slightly greater tendency to form a radical by radical transfer from II or III than the threo form.

Sulfuric acid lignin (SAL) is a byproduct of the acid saccharification of lignocellulosic materials for bio-fuel generation. The recalcitrant and highly complex structure of SAL significantly limits its industrial use, making it a useless industrial waste. In a previous study, we proposed a hydrothermal reaction for SAL that can convert it into water-soluble compounds (HSALs), which considerably expanded its industrial use scenario. In this study, we converted this water-soluble HSAL into two ionic modifications with applications as flocculants and dispersants. Firstly, the introduction of a carboxy group was carried out with bromoacetic acid. In the carboxymethylated HSAL (C-HSAL), the carboxymethyl groups content was as high as 3.66 mmol/g. Dispersibility tests using gypsum paste showed that C-HSAL exhibited a dispersibility almost equivalent to that of commercial lignosulfonate. Furthermore, another investigation was conducted synthesizing cationized HSAL derivatives by the reaction with glycidyl trimethylammonium chloride (GTA). Dye removal tests were performed with the obtained lignin-based flocculants using three organic dyes. The results showed ideal dye removal ratios for the GTA-HSALs (higher than 90% for all dyes tested), thereby suggesting that they have a huge potential as core molecules to develop high-efficient dye flocculants.

Lignification of ray parenchyma cells (RPCs) in the sapwood (sW), transition zone (TZ), and heartwood (hW) of Phellodendron amurense Rupr. has been investigated by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and thioacidolysis. The results of TOF-SIMS indicate that the relative ion intensity of lignin in RPCs increased from sW to hW, while there was almost no difference in the case of axial wood fibers. The ratio of syringyl (S) to guaiacyl (G) lignin units (S/G value) in RPCs was higher than that in wood fibers. Samples containing more RPCs were prepared by laser microdissection (LMD) and analyzed by thioacidolysis, including also the subsequent Raney nickel desulfurization, and the resulting monomers and dimers were quantitatively analyzed by GC/MS. The monomer analysis shows that lignin content and S/G values in RPCs rich samples increased from sW towards hW. The dimer analysis reveals that the S-S dimers are dominant in all types of samples. The results of TOF-SIMS and thioacidolysis of LMD samples are interpreted that the lignification of RPCs progresses from the sW towards the hW and that the chemical structure of lignin in RPCs is different from that in axial elements.

The structure and biosynthesis of lignin are not yet fully understood, especially the step following the initial dimerization of monolignol. Liquid chromatograph mass spectrometer (LC–MS) was used to analyze the consumption rates of monolignol dimers formed by β-O-4, β-5, and β-β couplings between coniferyl alcohols in efforts to understand the activity of monolignol dimers in enzymatic dehydrogenative polymerization. We investigated the reaction kinetics in single-component and mixed-component reaction systems containing one and two species of the dimers, respectively. A difference was observed between the consumption rates of the three dimers we tested, and the consumption rate of one dimer in the single-component reaction was different from that in a mixed-component reaction. In qualitative LC–MS analyses, coniferyl alcohol oligomers were detected in the reaction products. Some monolignol tetramers were formed by 5-5 and 5-O-4 coupling between the dimers. The results of this work suggested that monolignol dimers with β-5 and β-β linkages could function as radical mediators in enzyme-catalyzed polymerization.

The lignification of ray parenchyma cells in the sapwood (sW) and heartwood (hW) of Pinus densiflora was investigated by thioacidolysis and the subsequent Raney nickel desulfuration. The samples rich and less rich in ray parenchyma cells were prepared by laser microdissection (LMD). The whole sections burned randomly by the laser served as the controls. Guaiacyl (G) monomers were detected in all the samples, and p-hydroxyphenyl (H) monomers were detectable only in trace amounts, while syringyl (S) units were absent, as expected in softwood. In sW samples rich in ray parenchyma cells, the yields of G monomers are significantly lower than in the other samples. The various types of G-G and one G-H dimers were detected, and the β-1′, β-5′, and 5-5′ dimers were dominant. The relative distributions of lignin interunit linkages were very similar in all the samples regardless of the abundance of the ray parenchyma cells in the sW or hW tissues.

The distribution of aluminum (Al) and other inorganic elements has been mapped at the cellular level in the mature xylem of maple by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging. This method permits to image inorganic elements along with organic molecular species with high spatial resolution, and it was demonstrated that Na, K, Ca, and Mg are almost uniformly distributed in several contiguous growth rings of the air-dried wood sample. In contrast, Al was accumulated heterogeneously and intensely in the cell walls of the secondary xylem, including several growth rings, despite the long-term air-drying during storage. These results are interpreted that Al is immobilized within the cell wall, or it is closely interacted with the components of the secondary xylem. TOF-SIMS mapping also shows that Al has a tendency to be localized around vessels in the inner rings.

The experiments with model compounds revealed that time-of-flight secondary ion mass spectrometry (ToF-SIMS) is able to split the common interunit linkages of lignin, except the 5-5 linkage. In a previous study, ToF-SIMS produced characteristic secondary ions with m/z 137 and 151 (C6-C1 fragments) from the phenylcoumaran-type lignin model compound, the benzofuran ring of which has a β-5 linkage and an α-O-4 linkage. However, it is still unclear whether the fragments are from ring A with the free phenolic OH as a result of the sole cleavage of the α-O-4 link by opening the furan ring or from ring B as a result of the β-5 cleavage. In this study, the phenylcoumaran-type lignin model compound and its deuterium- and/or 13C-labeled analogues were synthesized and analyzed by ToF-SIMS. It could be clarified that the β-5 linkage is not cleaved by ToF-SIMS.

The structure of lignin in the compound middle lamella (CML) of softwood tracheids differs from that in the secondary wall (SW) in regard to the content of condensed structures (5-5′-biphenyl, dibenzodioxocin and 4-O-5′-diphenyl ether). In an early stage of cell wall formation, random coarse networks composed of thin cellulose microfibrils (CMFs), hemicelluloses, and pectin are formed in the CML, then globular p-hydroxyphenyl/guaiacyl lignin (HG-lignin) is deposited quickly into the network. The globular lignin is assumed to be a micellar aggregate of oligolignols folded at the β-O-4 bond with their phenolic ends on the outer part of the aggregate. When 3D clusters of the globules are deposited on the preformed network of polysaccharides, further growth of the oligolignols by endwise addition of new monolignols is spatially limited, so frequent condensation occurs between growing aromatic ends of adjacent HG-oligolignols within the globule and between the wide contact boundaries of the 3D clustered globules to produce a highly condensed supramolecule in CML. In SW, the folded G-oligolignols are deposited slowly in the narrow tubular space surrounding thick CMFs coated with hemicelluloses. Condensation occurs mostly between adjacent growing ends of the oligolignols within the tubular aggregates. Spatial regulation of condensation of folded polylignols is one of the factors producing a different supramolecular structure for CML lignin than for SW lignin.

The distribution of ethyl acetate extracts and 4,8-dihydroxy-5-methoxy-2-naphthaldehyde (compound I), which is a major constituent of the extracts obtained from the blackened heartwood of Diospyros kaki, was analyzed via gas chromatography-mass spectrometry (GC-MS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). According to GC-MS, the extracts and compound I are high in concentration at the pith and at the edges of the blackened heartwood. ToF-SIMS analysis revealed a peak at a mass-to-charge ratio of (m/z) 218, which is characteristic of the ionic form of compound I. The ToF-SIMS imaging of compound I in the blackened heartwood based on m/z 218 shows that compound I is located in parenchyma cells and their neighboring axial elements.

In this study, polyesters were prepared from sulfuric acid lignin (SAL) that was generated as a by-product during the production of bioethanol from lignocellulosic materials using ɛ-caprolactone (CL). Carboxylic groups were introduced into SAL by a hydrothermal reaction under alkali conditions. The acid form (-COOH) of the obtained polymer (HSAL) was dissolved into ɛ-caprolactone and the mixture was heated at 150°C for 12 h with ZnCl2 as a catalyst. The structure of the obtained polymer (PCL-HSAL) was proposed based on results of differential scanning calorimetric analysis, swelling tests, and dynamic mechanical thermal analysis. The hydroxyl group in HSAL participated in the ring-opening polymerization of CL. PCL-HSAL with a high HSAL content formed a three-dimensional network that did not dissolve in organic solvents and did not melt at high temperature. PCL-HSAL showed a high swelling property for a suitable ratio of CL to HSAL.

4,8-Dihydroxy-5-methoxy-2-naphthaldehyde (Compound I) was isolated from blackened heartwood of Diospyros kaki and was methylated with diazomethane. Antioxidant and cytotoxic activities of Compound I and two methylated derivatives [4-hydroxy-5,8-dimethoxy-2-(2-oxopropyl)-naphthalene (D-1) and 2-glycidyl-4-hydroxy-5,8-dimethoxy naphthalene (D-2)] were evaluated. Compound I showed higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and reducing power than D-1 and D-2. However, D-1 and D-2 exhibited slightly stronger 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)+ (ABTS+) radical scavenging activity than Compound I. Compound I also exhibited stronger cytotoxic activity than D-1 and D-2 against the growth of HT-29 colon cancer cells. The results supported the hypothesis that methylation of naphthalene derivatives slightly increased ABTS+ radical scavenging activity, but significantly decreased DPPH radical scavenging activity, reducing power, and cytotoxic activity.

A detailed chemical investigation of the extractives of the blackened heartwood of Diospyros kaki was carried out to understand their chemical characteristics and to obtain chemotaxonomic information. Three novel naphthalene derivatives were isolated, i.e., 4-hydroxy-5,6-dimethoxy-2-naphthaldehyde (1), 5,6,8-trimethoxy-3-methyl-1-naphthol (2), and 4,8-dihydroxy-5-methoxy-2-naphthaldehyde (3), in addition to two previously reported 2-naphthaldehydes: 4-hydroxy-5,8-dimethoxy-2-naphthaldehyde (4) and 4-hydroxy-5-methoxy-2-naphthaldehyde (5) Their structures were identified by 1D and 2D nuclear magnetic resonance spectroscopy, as well as by high-resolution mass spectrometry.

Electropolymerization of coniferyl alcohol was carried out in an aqueous system (0.2 M NaOH) and in an organic solvent system [CH2Cl2/methanol (4:1 v/v) in the presence of 0.2 M LiClO4] to produce a dehydrogenation polymer (DHP) - artificial lignin. In both systems, the polymerization of coniferyl alcohol was visually confi rmed. In the aqueous system, no dimer was detected in the reaction medium after electropolymerization, suggesting that endwise polymerization occurred on the electrode surface. Thioacidolysis degradation revealed that the obtained polymers had numerous 8-O-4′ linkages. The electropolymerization products obtained in the organic solvent system also had numerous 8-O-4′ linkages; in particular, the polymers obtained in the initial polymerization stage. This was probably because of the limited area available for reaction and the orientation of coniferyl alcohol on the electrode surface controlled the polymerization.

Heartwood extracts from Amazonian trees cumaru-ferro (Dipteryx odorata), jatoba (Hymenaea courbaril), and guarita (Astronium lecointei) exhibit antioxidant activities comparable with that of α-tocopherol, a well-known antioxidant. This article reports the characterization of the antioxidant compounds in the extracts of the three heartwoods. Silica gel column chromatography of the cumaru-ferro EtOAc extract yielded (−)-(3R)-7,2′,3′-trihydroxy-4′-methoxyisoflavan and (+)-(3R)-8,2′,3′-trihydroxy-7,4′-dimethoxyisoflavan. Silica gel column chromatography followed by preparative high-performance liquid chromatography of the jatoba EtOAc extract yielded (−)-fisetinidol and (+)-trans-taxifolin. Chemical structures were assigned using electron-ionization mass spectrometry, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy including nuclear Overhauser effect spectroscopy (NOESY), as well as optical rotation and circular dichroism. Gas chromatography-mass spectrometry demonstrated that the isolated compounds were predominant in the EtOAc extracts. In the guarita EtOAc extract, catechin and gallic acid were identified by comparing their retention times and mass fragmentation patterns with those of authentic samples. Antioxidant activity determined by the 1,1-diphenyl-2-picrylhydrazyl assay demonstrated that all these compounds had activities comparable with that of α-tocopherol.

The heartwood and sapwood characteristics of 11 Amazonian trees were investigated. Whereas 7 of the specimens had densities greater than 0.7 g/cm3, the heartwood density of ipe amarelo (Tabebuia serratifolia), macaranduba (Manilkara huberi), cumaru-ferro (Dipteryx odorata), and guarita (Astronium lecointei) exceeded 1.0 g/cm3. Jatoba contained small amounts of Klason lignin and α-cellulose, and large amounts of holocellulose and alkali extract, suggesting that it has a high polysaccharide content that can be dissolved in an alkaline medium. The difference in the syringyl/guaiacyl (S/G) ratios of the samples before and after alkali extraction suggests that alkali extracts contain syringyl-type polyphenols. In all of the samples, the heart-wood methanol extracts were larger in volume than the sapwood methanol extracts, and the sapwood alkali extracts were larger in volume than the heartwood alkali extracts. The antioxidant activities of the methanol and alkali extracts were assayed by measuring the levels of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and super oxide dismutase (SOD)-like activity, respectively. The heart-wood methanol extract of jatoba (Hymenaea courbaril) exhibited the highest level of activity (EC50 = 44 mg/l), which exceeded that of α-tocopherol (EC50 = 48 mg/l), and the heartwood alkali extracts of jatoba and ipe amarelo had high SOD-like activity comparable with red wine.

To study the behavior of hardwood sulfuric acid lignin (SAL) during phenolization, we compared the product yield, average molecular weight, methoxy content, and reactions of simple model compounds with those of softwood SAL, focusing on the difference between syringyl and guaiacyl units. The beech SAL reacted with phenol more readily than red pine SAL and yielded a larger soluble fraction of phenolized SAL. To investigate the difference in the phenolization activity of the syringyl and guaiacyl units in beech lignin, we prepared syringyl-nucleus-rich sulfuric acid lignin (S-rich-SAL) and guaiacyl-nucleus-rich sulfuric acid lignin (G-rich-SAL) from beech, which were subjected to phenolization. The results suggest that the syringyl unit in SAL had greater phenolization activity and its phenolized products were more soluble in acidic aqueous medium and introduced less phenol than the guaiacyl unit. Using model compounds, the study also showed that the syringyl unit had higher phenolization reactivity than the guaiacyl unit.

To examine the effect of gravity on lignin content and deposition in plant cells, we used ultraviolet (UV) microspectrophotometry and chemical methods to investigate the secondary xylem of Prunus jamasakura grown on a three-dimensional (3D) clinostat, which simulates microgravity. The stem of the 3D-clinostat specimens elongated with bending and the width of their secondary phloem increased. The UV absorbance of the 3D-clinostat specimens at 278 nm was higher than that of the control specimens, which were grown on the ground, in the wood fiber cell corner middle lamella, compound middle lamella, and fiber secondary wall; the UV absorbance in the vessel secondary wall did not differ between the specimens. The lignin content in the stem, including the bark, of the 3D-clinostat specimens, as determined using an acetyl bromide method, was less than that of the control specimens. In the specimens that differentiated on a 3D clinostat, the amount of lignin in the wood fibers increased, while the proportion of the lignified xylem in the stem decreased relative to control values.

Lignin in plant cell walls is a complex, irregular polymer built from phenylpropanoid C6-C3 units that are connected via various C-C and C-O linkages. A recent study using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with Ga primary ion bombardment showed that lignin polymers can be characterized by specific positive ions possessing a substituted aromatic ring (so-called guaiacyl or syringyl rings), which are the basic building units of lignin. To study the relationship between the characteristic ions of lignin and the common interunit linkages, various lignin dimer model compounds were investigated using ToF-SIMS. The resulting dimer spectra showed that the characteristic ions with a guaiacyl ring at m/z 137 and 151 result from rupture of most common interunit linkages, not only 8-O-4' linkages, which are the most abundant in lignin, but also 8-1', 8-5', and 8-8'. There was no evidence of rupture of 5-5' linkages. These results show that ToF-SIMS offers a new tool for the direct analysis of the depolymerized fragments of lignin polymers. The mechanisms for the fragmentation of lignin dimer models in ToF-SIMS were proposed that allow ToF-SIMS fragmentation rules to be deduced. Adduct ions such as [M + 13]+ ([M + CH]+) were also produced in fragmentation of the dimers and are thought to arise from the combination of the molecules with their stable fragments.

The lignification process and lignin distribution at different stages of cell wall differentiation in the secondary xylem of compression and normal woods of Pinus thunbergii were investigated by thioacidolysis and subsequent desulfuration. We prepared 50-µm-thick, contiguous tangential sections of pine shoots, cut from the cambial zone through to mature xylem. In compression wood, uncondensed guaiacyl (G) and p-hydroxyphenyl (H) lignins were deposited simultaneously from early to late stages of lignification. The various types of G-G, G-H, and H-H dimers were detected in compression wood, and the ratio of G-H and H-H dimers to total dimers increased as lignification proceeded. In contrast, uncondensed and condensed H units were detected in trace amounts in normal wood. Significant differences in the relative distributions of lignin interunit linkages were not observed between compression and normal woods or between differentiating and mature xylems in either compression or normal woods.

The chemical structure of lignin, a complex, irregular polymer of phenylpropane units that occurs in plant cell walls, was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The positive ToF-SIMS spectra of lignin isolated from pine and beech wood showed prominent secondary ions possessing guaiacyl (at m/z 137 and 151) or syringyl (at m/z 167 and 181) rings, which are the basic building units of lignin polymer. This shows that ToF-SIMS is a useful tool for lignin structural analysis. The peaks at m/z 137 and 167 were assigned as the C6-C1 ion, and the peaks at m/z 151 and 181 may be double-component, the C6-C1 ion and the C6-C2 ion. We confirmed the characteristic guaiacyl ions using a synthetic lignin model compound, dehydrogenation polymer (DHP), which was formed by polymerizing of unlabeled and deuterium-labeled coniferyl alcohols. The formation mechanism of the main secondary ions was deduced by labeling specific positions of coniferyl alcohols with a stable isotope to study the relationship between chemical structure and secondary ion formation in ToF-SIMS.

Ferulic acid (FA), tetradeuteroferulic acid (DFA), sinapic acid (SA), or heptadeuterosinapic acid (DSA) was exogenously supplied to poplar (Populus alba L.) callus. Administration of FA or SA increased the lignin content of the callus to about twice that of the control callus. Gas chromatographic analysis of the alkali hydrolysate of the cell wall residue revealed that only a trace amount of SA was bound to the cell wall, and the amount of FA was less than 2% of the total callus lignin. Thioacidolysis of the DFA-treated callus indicated that DFA is effectively converted to both coniferyl and sinapyl alcohols and then incorporated into the corresponding lignin. Incorporation of DSA into syringyl lignin or guaiacyl lignin was not observed, but yields of syringyl lignin thioacidolysis products were markedly increased by DSA treatment of the callus. These results suggest that SA may not be a precursor of sinapyl alcohol and syringyl lignin per se, but it may induce or enhance the biosynthesis of syringyl lignin in poplar callus.

To elucidate the formation mechanism of acidsoluble lignin (ASL) formed in the Klason lignin determination, beech wood meals were treated with sulfuric acid (SA) under various conditions, and the ASL solution was extracted with CHC13. The results indicated the following: (1) wood components yielding ASL are dissolved in 72% SA during the initial stage; (2) the quantity of ASL is highest during the initial stage, then decreases with prolonged time of 72% SA treatment and finally reaches a constant value; (3) soluble lignin prepared by 72% SA treatment and subsequent standing in 3% SA again yield insoluble Klason lignin and ASL after boiling in 3% SA; and (4) about half the amount of ASL is dissolved in CHC13. The foregoing suggest that wood components yielding ASL are dissolved in 72% SA at the beginning and finally change to ASL after being subjected to depolymerization, hydrolysis, and other reactions. ASL may thus be composed of low-molecular-weight degradation products and hydrophilic derivatives of lignin.

The lignin biosynthetic pathway in Eucalyptus camaldulensis was investigated by feeding stems with deuterium-labeled precursor. Pentadeutero[γ,γ-D2 OCD3] coniferyl alcohol was synthesized and supplied to shoots of E. camaldulensis, and incorporation of the labeled precursor into lignin was traced by gas chromatography-mass spectrometry. In addition to the direct incorporation of labeled precursor into the guaiacyl unit, a pentadeuterium-labeled syringyl unit was detected. This finding indicates that the γ-deuterium atoms in the hydroxymethyl group of labeled coniferyl alcohol remain intact during modification of the aromatic ring. The relative level of trideuterium-labeled syringyl monomer (the result of conversion via the cinnamic acid pathway) was negligible, suggesting that the pathway at the monolignol stage is used for conversion of exogenously supplied precursor. Our results provide conclusive evidence of a novel alternative pathway for generation of lignin subunits at the monolignol stage even in plants that do not accumulate coniferin in lignifying tissues.

To examine the possible in vivo conversion of coniferin to syringin in the biosynthetic pathway of lignin, variously radio-labeled coniferin was fed to a shoot of Magnolia kobus. When coniferin-[side chain γ- 3 H, Glc-U- 14 C] was administered, only 3 H-activity was detected in syringin isolated from the shoot. The lack of 14 C-activity in isolated syringin suggests that the conversion from guaiacyl to syringyl proceeded at the stage of monolignols, and the glucose moiety is temporally removed during the process of methoxylation. These results suggest the possible role of monolignols in the control of monomer composition of lignin.

Selective labelling of p-hydroxyphenyl-, guaiacyl- and syringyl-propane moieties in protolignin was achieved by administration of corresponding 3 H-labeled monolignol glucosides to differentiating xylem of ginkgo. The growing process of the protolignin macromolecule in the specific morphological region was visualized by application of high resolution microautoradiography to the selectively labeled wood tissue. (...)

The dehydrogenative polymerization of coniferyl alcohol by horseradish peroxidase was performed in 0.10 M phosphate buffer at 27 degrees C. Dehydrogenative polymer (DHP) from coniferyl alcohol was characterized by size exclusion chromatography (SEC) and nuclear magnetic resonance (NMR) spectroscopy. The ratio of 8-O-4':8-5':8-8' linkages was determined by the 1H NMR spectrum of DHP acetate which had good solubility. In "end-wise like" polymerization (the slow addition of hydrogen peroxide), addition of alpha-cyclodextrin to the medium led to DHP with increased 8-O-4' content and a decrease in 8-5' linkages. Under higher pH conditions, DHP with higher 8-O-4' and 8-5' content was obtained in the presence of alpha-cyclodextrin. In the end-wise polymerization (the slow additions of coniferyl alcohol and hydrogen peroxide), using alpha-cyclodextrin also gave DHP with a 8-O-4' richer structure than that prepared in no additive system. The analysis of thioacidolysis products from DHP supported the results of the alpha-cyclodextrin effects on the 8-O-4'-rich structure of DHP. The 8-O-4' structure in DHP prepared in the presence of alpha-cyclodextrin had racemic form as shown by ozonation.