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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.

The amount of β-1 structures in lignin is small; however, they are assumed to significantly affect the reactivity of lignin because they form dienone structures. A method employing thioacidolysis and subsequent desulfurization yields products that can be analyzed via gas chromatography-mass spectrometry (GC-MS) to quantify these β-1 structures. However, the retention times and response factors of the reaction products have not been accurately determined thus far. Here, 12 standard compounds combined with p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units were synthesized, and their retention times and response factors were determined through GC-MS, using selective ions. Based on these data, we also investigated the β-1 structures of lignocellulosic lignin samples. Our results clarified that the successful formation of the β-1 structure was dependent on the type of aromatic rings present; there were very few β-1 structures containing H units; and the amount of G-G type was higher and that of the heterotype, i.e., G-S type, was lower than the stochastic value.

Podocyte injury is an independent risk factor for the progression of renal diseases. Semaphorin3A (SEMA3A), expressed in podocytes and tubular cells in the mammalian adult kidneys, has been reported to regulate diverse biological functions and be associated with renal diseases. Here, we investigated pathological roles of SEMA3A signaling on podocyte injury using a doxorubicin (Dox)-induced mouse model and examined the therapeutic effect of SEMA3A-inhibitor (SEMA3A-I). We demonstrated that Dox caused massive albuminuria and podocyte apoptosis as well as an increase of SEMA3A expression in podocytes, all of which were ameliorated with SEMA3A-I treatment. In addition, c-Jun N-terminal kinase (JNK), known as a downstream of SEMA3A signaling, was activated in Dox-injected mouse podocytes while SEMA3A-I treatment partially blocked the activation. In vitro, SEMA3A-I protected against Dox-induced podocyte apoptosis and recombinant SEMA3A caused podocyte apoptosis with activation of JNK signaling. JNK inhibitor, SP600125, attenuated SEMA3A-induced podocyte apoptosis, indicating that the JNK pathway would be involved in SEMA3A-induced podocyte apoptosis. Furthermore, the analysis of human data revealed a positive correlation between levels of urinary SEMA3A and protein, suggesting that SEMA3A is associated with podocyte injury. In conclusion, SEMA3A has essential roles in podocyte injury and it would be the therapeutic target for protecting from podocyte injury.

Obesity is supposed to cause renal injury via autophagy deficiency. Recently, sodium glucose co-transporter 2 inhibitors (SGLT2i) were reported to protect renal injury. However, the mechanisms of SGLT2i for renal protection are unclear. Here, we investigated the effect of SGLT2i for autophagy in renal proximal tubular cells (PTCs) on obesity mice. We fed C57BL/6J mice with a normal diet (ND) or high-fat and -sugar diet (HFSD) for nine weeks, then administered SGLT2i, empagliflozin, or control compound for one week. Each group contained N = 5. The urinary N-acetyl-beta-d-glucosaminidase level in the HFSD group significantly increased compared to ND group. The tubular damage was suppressed in the SGLT2i&ndash, HFSD group. In electron microscopic analysis, multi lamellar bodies that increased in autophagy deficiency were increased in PTCs in the HFSD group but significantly suppressed in the SGLT2i group. The autophagosomes of damaged mitochondria in PTCs in the HFSD group frequently appeared in the SGLT2i group. p62 accumulations in PTCs were significantly increased in HFSD group but significantly suppressed by SGLT2i. In addition, the mammalian target of rapamycin was activated in the HFSD group but significantly suppressed in SGLT2i group. These data suggest that SGLT2i has renal protective effects against obesity via improving autophagy flux impairment in PTCs on a HFSD.

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.

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), one of the major biorefinery industry residues from the acid saccharification process on lignocellulosic materials for bio-fuel generation, is becoming a major problem for the sustainable development of the bio-refinery industry. Meanwhile, the recalcitrant and highly complex structure of SAL makes it insoluble in water and other organic solvents, resulting in a low reactivity. This study investigated the effects of hydrothermal reactions on SAL to enhance its solubility and reactivity. A hydrothermal reaction was first conducted on SAL from Cryptomeria japonica to obtain a completely water-soluble lignin. Fourier transform infrared spectroscopy, gel permeation chromatography, gas chromatography, and nuclear magnetic resonance (NMR) analyses revealed that the hydrothermal reaction of the SAL caused depolymerisation, demethylation, and the introduction of phenolic hydroxyls. Furthermore, another experiment was conducted on synthesizing a lignin polymer model compound (DHP) with 13C-labelled on the aromatic ring, followed by hydrothermal reaction and acetylation. Detailed structural information of DHP under hydrothermal reaction was obtained by solid-state cross polarization magic angle spinning NMR analysis. Reactivity of the hydrothermally treated SAL was enhanced by the hydrothermal reaction through the reversed increasing of the phenolic hydroxyl/aliphatic hydroxyl ratio in the condensed lignin units.

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.

Lignin is an essential component of higher plants, which is built by the enzymatic dehydrogenative polymerization of monolignols. First, monolignol is enzymatically oxidized to produce the phenoxy radical, which can form resonance hybrids. Two radical resonant hybrids are coupled with each other to yield dilignol with various linkage types, of which the main structures are β- O -4′ ( I ), β-5′ ( II ) and β-β′ ( III ). However, the reaction mechanism behind the addition lignol radicals to dilignol is not yet fully understood. Here, we show an unexpected reaction with structure II during enzymatic dehydrogenative polymerization, which involves cleavage of a covalent linkage and creation of a new radical coupling site. This implied that the β-5 dilignol diversifies the growing pattern of lignin. This discovery elucidates a novel mechanism in lignin polymerization.

To utilize the by-product lignin (SAL) that is generated by bioethanol production systems that use lignocellulosic materials with a sulfuric acid catalyst, the antioxidant activity, reducing power, and acetylcholinesterase inhibitory activity of SAL were investigated. Hydrothermal treatment effectively increased the total phenolic content (TPC) and had positive effects on the antioxidant activity and reducing power. By using model compounds, the reaction mechanism of the increase in TPC by hydrothermal treatment was determined. The antioxidant activity, reducing power, and acetylcholinesterase inhibitory activity of hydrothermalized SAL were insensitive to the methoxy group content, molecular weight, and differences in nucleus species (such as guaiacyl or syringyl). This suggests that the species of lignocellusic materials have little relationship to the potential utilization of SAL as a bioactive agent.

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.

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.

The antioxidant activities of two persimmon (Diospyros kaki T.) branches were evaluated. After preparing acetone, 70% acetone, and methanol extracts from Fuyu and Nishimurawase persimmon branches, DPPH radical scavenging activity (RSA), ABTS RSA, reducing power (RP), and total phenol contents (TPC) were determined. The acetone extracts of Fuyu and Nishimurawase showed higher antioxidant properties than the other extracts. The highest TPC (22.48 mg/g), DPPH RSA ($IC_{50}

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.

Several kinds of natural woods and isolated lignins with various syringyl to guaiacyl (S/G) ratios were subjected to thioacidolysis followed by Raney nickel desulfuration to elucidate the relationships between the S/G ratio and the interunit linkage types of lignin. Furthermore, enzymatic dehydrogenation polymers (DHP) were produced by the Zutropf (gradual monolignol addition) method from mixtures of various ratios of coniferyl alcohol and sinapyl alcohol. The analysis of DHPs and natural wood lignins exhibited basically a similar tendency. The existence of both syringyl and guaiacyl units is effective for producing higher amounts of beta-O-4 and 4-O-5 structures, but it lowers the total amount of cinnamyl alcohol and aldehyde end groups. The relative frequency of the beta-beta structure increased, whereas that of beta-5 and 5-5 structures decreased with increasing syringyl units.

In vitro hydroxylation of the norlignan agatharesinol to sequirin C and metasequirin C was demonstrated for the first time. After incubating agatharesinol with a microsomal preparation from the heartwood side of the intermediate wood of Cryptomeria japonica in the presence of cofactors, the aromatic ring-monohydroxylated derivatives of agatharesinol, sequirin C and metasequirin C, were formed. Although hydroxylation hardly occurred in the absence of cofactors, it could be initiated by adding NADPH or NADH, and was enhanced by further adding FAD or FMN. When microsomal preparations from the sapwood or from the sapwood side of the intermediate wood were used, hydroxylation did not occur. This in vitro conversion of the norlignans indicates that the hydroxylation of agatharesinol to sequirin C and metasequirin C is part of the in vivo biosynthetic pathway of norlignans. Another C. japonica norlignan, sugiresinol, which is a side chain-cyclized isomer of agatharesinol, does not seem to be accepted as a substrate, because hardly any hydroxysugireinol was formed after similar incubation with the enzyme.

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.

Dimeric lignin model compounds with non-phenolic and phenolic moieties and a β -O-4 type artificial lignin polymer were subjected to thioacidolysis to evaluate the reaction efficiency of thioacidolysis for cleavage of β -O-4 interunitary linkage. The obtained yields of thioethylated monomeric products from the dimeric lignin model compounds reached nearly 100% under the conventional condition, whereas that from the artificial lignin polymer was as low as 74%. Neither prolonged reaction time nor increased concentration of ethanethiol (the thioacidolysis reagent) enhanced the resulting monomer yields from the polymer (69–79%). Thioacidolysis of the lignin model compounds followed by HPSEC analysis also showed the dimeric model compounds were degraded almost quantitatively, but that the artificial lignin polymer was not. Thioacidolysis followed by desulfurization gave at least one dimeric product resulting from incomplete β -O-4 cleavage at significant yield. These results suggested the conventional...

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.

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.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was applied to the investigation of heartwood extractives in Sugi (Cryptomeria japonica). Sugi heartwood tissue generated secondary ions that were not produced from sapwood tissue by TOF-SIMS. Among the peculiar ions generated from heartwood, two positive ions of m/z 285 and 301 were remarkable due to their appearance in a larger mass range and with a high intensity. These two ions were not generated from heartwood tissue preextracted with n-hexane, and the n-hexane extract of Sugi heartwood produced both ions. Gas chromatography-mass spectrometry of the n-hexane extract demonstrated that ferruginol, a diterpene phenol, the molecular weight of which is 286, constituted one of the predominant constituents of the extract. Authentic ferruginol also generated both ions by TOF-SIMS. The molecular formula of the m/z 285 ion generated from Sugi heartwood tissue was estimated to be C(20)H(29)O, which corresponds well with that of ferruginol, i.e. C(20)H(30)O, by peak identification. All these results strongly suggest that the m/z 285 ion generated from Sugi heartwood tissue originated significantly from ferruginol in Sugi heartwood. By TOF-SIMS imaging, the m/z 285 ion was detected uniformly in the tracheid cell walls, in the cell walls of the axial parenchyma cells and ray parenchyma cells, and also inside these parenchyma cells. These results indicate that ferruginol was distributed almost evenly in Sugi heartwood tissue.

ロジングリセリンエステルを含む中性ロジンサイズ剤は, 中性条件下でも効果のあるサイズ剤として応用が広がりつつある。これまでの研究から, ロジングリセリンエステルにはアビエチン酸やマレイン化ロジンの溶出を防ぐ役割があることが示されている。今回はこのことを明らかにするため, いろいろな抄紙条件におけるロジングリセリンエステルの挙動をToF-SIMSでの可視化により解析することを試みた。ToF-SIMSを用いてイメージングをする場合, 物質特有のピークを見つけ出さなければならない。我々は, ロジングリセリンエステルのみを確実に検出するために, 重水素ラベルのロジングリセリンエステルを合成し, その重水素イオンを検出することとした。自然界には重水素はほとんど存在しないため, 目的とするロジングリセリンエステルのみを選択的に可視化することに成功した。ToF-SIMSのイメージングとサイズ度の測定結果から, ロジングリセリンエステルは中性条件のみならず, アルカリ条件においても可溶化せずにパルプ表面に留まっていることが明らかとなった。一方, ロジングリセリンエステルを含まないサイズ剤を用いた場合, 中性条件やアルカリ条件ではアビエチン酸やマレイン化ロジンは溶出し, サイズ効果は現れなかった。以上のことは, ロジングリセリンエステルはアビエチン酸やマレイン化ロジンの溶出を防ぐ役割を担っているという以前の研究結果を支持する。

The preparation of dispersants with carboxyl groups from acid lignin (a byproduct in the saccharification process of woody materials that are renewable resources) was investigated, and their dispersion efficiency was evaluated. In this study, sulfuric acid lignin (SAL) was selected as a representative acid lignin. To convert SAL to water-soluble polymers, it was first subjected to phenolation with sulfuric acid to enhance its chemical reactivity. The introduction of a carboxy group was carried out with two methods. One was carboxymethylation with bromoacetic acid, and the other was the Mannich reaction with glycine, sarcosine, and iminodiacetic acid. The carboxymethylation product [carboxymethylated phenolized sulfuric acid lignin (CP-SAL)] with 1.4 carboxymethyl groups per phenolated phenylpropane lignin base unit was soluble in water. The Mannich reaction products were also soluble in water with a lower addition of carboxyl groups than that for CP-SAL. Surprisingly, all the prepared water-soluble polymers possessed a higher dispersibility for gypsum paste than commercial lignosulfate. This may be due to the higher molecular weights of acid lignin derivatives. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 2508–2513, 2005

To examine the behavior of cinnamaldehyde and cinnamyl alcohol glucosides in lignifying tissues, angiosperms were subjected to tracer experiments using radioisotope-labeled and stable isotope-labeled glucosides. The aglycone from coniferaldehyde glucoside was efficiently incorporated into guaiacyl and syringyl lignin as a cinnamyl alcohol unit. The aglycone from coniferin was also incorporated into guaiacyl and syringyl lignin. However, some of the coniferin-derived aglycone that was incorporated into lignin passed through a cinnamaldehyde form prior to dehydrogenative polymerization. When coniferin was administered together with coniferaldehyde glucoside, syringyl units were rarely synthesized from coniferin via the cinnamyl alcohol stage, whereas numerous syringyl units were synthesized from coniferaldehyde glucoside. These observations suggest that the coniferaldehyde form is crucial for the biosynthesis of syringyl lignin in angiosperms.

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.

Aglycons derived from 4-O-beta-D-glucosides of both caffeyl and 5-hydroxyconiferyl alcohols were incorporated into guaiacyl (G) and syringyl (S) units in the lignin of newly formed xylem of several angiosperms. It is likely that these aglycons enter the cinnamyl alcohol pathway as intermediates in the introduction of methoxyl groups onto aromatic rings, and serve as precursors for the biosynthesis of lignin. The S/G ratio in this pathway was coincident with the ratio in the cell wall lignin of each tree. Our results indicate that the cinnamyl alcohol pathway involves the same mechanisms as the cinnamic acid and cinnamyl CoA pathways and they suggest that this novel pathway might be part of a metabolic grid in the biosynthesis 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 develop more effective oxidative hair coloring products, it is important to understand the localization of colored chromophores, which are formed from oxidative dyes, in the fine structure of hair. However, the dyeing regions of oxidative hair dyes in the fine structure of hair have not been extensively examined. In this study, we investigated the distribution and localization of colored chromophores formed by an oxidative hair coloring product in the fine structure of human hair by using a stable isotope-labeled oxidative dye with nanoscale secondary ion mass spectrometry (NanoSIMS). First, formation of the colored chromophore from a deuterium-labeled oxidative dye was examined by visible spectra similarly to a study of its formation using nonlabeled oxidative dye. Furthermore, the formation of binuclear indo dye containing deuterium in its chemical structure was confirmed using time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis. As a result of the NanoSIMS image on a cross-sectional dyed hair, although deuterium ions were detected in whole hair cross-section, quite a few of them were detected at particulate regions. These particulate regions of the dyed black hair in which deuterium ions were intensely detected were identified as melanin granules, by comparing the dyeing behaviors of black and white hair. NanoSIMS analysis revealed that melanin granules of black human hair are important dyeing regions in oxidative hair coloring.

Sulfuric acid lignin (SAL), which is formed as a by-product during the production of bioethanol from woody biomass, was solubilized and functionalized by hydrothermal reaction. SAL could be easily dissolved in an alkaline medium, especially sodium hydroxide solution, by this reaction. The soluble part of the reaction products (S-HSAL) could be dissolved at neutral pH. IR spectrometric analysis of SAL revealed that hydrophilic groups were introduced in it during the reaction. The dispersibility of S-HSAL was increased by sulfonation (SS-HSAL), and it was found to be an effective dispersant for gypsum paste.

In this study, water-soluble anionic and cationic polymers were prepared from sulfuric acid lignin (SAL), an acid hydrolysis lignin, and the relationship between the surface tension of these polymers and industrial performance was examined. The SAL was phenolized (P-SAL) to enhance its solubility and reactivity. Sulfonation and the Mannich reaction with aminocarboxylic acids produced water-soluble anionic polymers and high-dispersibility gypsum paste. The dispersing efficiency increased as the surface tension decreased, suggesting that the fluidity of the gypsum paste increased with the polymer adsorption on the gypsum particle surface. Water-soluble cationic polymers were prepared using the Mannich reaction with dimethylamine. The cationic polymers showed high sizing efficiency under neutral papermaking conditions; the sizing efficiency increased with the surface tension. This suggests that the polymer with high hydrophilicity spread in the water and readily adhered to the pulp surface and the rosin, showing good retention.

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.

A tracer experiment using synthesized labeled lignin precursors was designed to confirm the actual biosynthetic pathway for syringyl lignin. Tetradeuteroferulic acid-[8-D, 3-OCD3] and heptadeuterosinapic acid-[8-D, 3,5-OCD3] were synthesized and fed to shoots of robinia (Robinia pseudoacacia) and oleander (Nerium indicum) trees. The incorporation of each labeled precursor into lignin was traced by gas chromatography−mass spectrometry. The synthesized sinapic acid, in which both methoxy groups were labeled, was useful in monitoring the conversion of sinapic acid into syringyl lignin. When heptadeuterosinapic acid was fed, syringyl units containing seven deuterium labels were detected. The results of this study support the traditionally accepted pathway that sinapic acid is converted to sinapyl alcohol via sinapoyl-CoA in robinia and oleander. Keywords: Lignin biosynthesis; heptadeutero sinapic acid-[8-D, 3,5-OCD3]; tracer experiments; sinapyl alcohol; robinia; oleander; 4-coumarate-CoA ligase (4CL)

For many angiosperms, a 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) isozyme that exhibits activity with a sinapic acid substrate has not been detected in vitro . Therefore, the possible role of sinapic acid as a precursor of syringyl lignin has been questioned. To further examine this issue, heptadeutero sinapic acid [β-D, (-OCD 3 ) 2 ] were synthesized and fed to shoots of oleander ( Nerium indicum Mill.) and Robinia ( Robinia pseudoacacia L.) trees, and the incorporation of these labeled precursors into lignin was traced. Lignin in the newly formed xylem of the treated stems was analyzed by derivatization followed by reductive cleavage (DFRC). The two labeled methoxyl groups of sinapic acid were useful in monitoring the conversion of sinapic acid into syringyl lignin. When heptadeutero sinapic acid was fed, syringyl units containing seven deuterium labels were detected. These results suggest that sinapic acid is converted to sinapoyl-CoA during lignin biosynthesis in oleander and Robinia trees in vivo .