Initial Stage of Syringyl Lignin Formation from Sinapyl Alcohol.

Elucidating the detailed structure and formation mechanism of lignin, especially understudied syringyl (S) lignin, advances our knowledge of lignocellulosic biomass. To examine the early stages of S-lignin formation from sinapyl alcohol (SA), the FMR (flow microreactor) method and the Zutropf (gradual addition of SA) method with limited amounts of H2O2 were employed for the peroxidase-catalyzed dehydrogenative polymerization of SA. Only ß-ß dimers and not ß-O-4 dimers were obtained as initial dimerization products. Six new oligoligognols up to pentamers with ß-ß and ß-O-4 structures were identified. The erythro isomer was preferentially formed over the threo isomer in the ß-O-4 structures, similar to that found in naturally occurring S-rich hardwood lignin. Although minor substructures, the α-oxidized ß-ß and ß-O-4 structures and spirodienone (ß-1) structure identified in this study demonstrate the characteristic features of S-rich lignin. Based on the identified products, the initial formation mechanism of S-lignin from SA was proposed.

Effect of pH on the Dehydrogenative Polymerization of Monolignols by Laccases from Trametes versicolor and Rhus vernicifera.

Dehydrogenative polymerization of coniferyl alcohol (CA) and sinapyl alcohol (SA) was conducted using commercial laccases, fungal laccase from Trametes versicolor (LacT) and plant laccase from Rhus vernicifera (LacR), at pH 4-7 to investigate how the enzymatic polymerization of monolignols differs between these two laccase systems. The enzyme activity of LacT was the highest at pH 4, whereas that of LacR was the highest at pH 7. A dehydrogenation polymer (DHP) was obtained only from CA in both laccase systems, although the consumption rate of SA was higher than that of CA. 1H-13C HSQC NMR analysis showed that DHPs obtained using LacT and LacR contained lignin substructures, including ß-O-4, ß-O-4/α-O-4, ß-ß, and ß-5 structures. At pH 4.5, the ß-O-4 structure was preferentially formed over the ß-O-4/α-O-4 structure, whereas at pH 6.5, the ß-O-4/α-O-4 structure was preferred. The pH of the reaction solution was more vital to affect the chemical structure of DHP than the origin of laccases.

Characterization of Immature Bamboo (Phyllostachys nigra) Component Changes with Its Growth via Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance Spectroscopy.

A 6.2 m high immature bamboo (Phyllostachys nigra) was divided into seven fractions. The bamboo cell walls and lignin samples from young to old were characterized by 1H-13C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy both qualitatively and semiquantitatively. Mature bamboo and bamboo shoot samples were used as comparison references. HSQC-NMR analysis proved that cellulose and arabinoxylan have already deposited in bamboo shoot, and cellulose amount increased during growth. Lignin side chain linkage formation started from ß-ether (ß-O-4), then phenylcoumaran (ß-5), and finally resinol (ß-ß). Ferulic acid and p-coumaric acid (pCA) were formed at the earlier stages in the immature bamboo, and the pCA proportion decreased throughout the lignification process. We propose that the bamboo lignification process is distinct from both woody and other herbaceous plants, where syringyl units deposited at the early stage and polymerized with the ß-O-4 linkage. Then guaiacyl units formed gradually, and finally, p-hydroxyphenyl units formed.

Biomimetic Oxidation of Monolignol Acetate and p-Coumarate by Silver Oxide in 1,4-Dioxane.

Lignin acylated with acetate and/or p-coumarate is common in many herbaceous plants. Herein, the biomimetic oxidation of γ-acylated monolignols with Ag2O was studied to understand the effect of γ-acyl groups on monolignol polymerization. The oxidation of sinapyl acetate gave γ-acylated and α-acylated ß-O-4 dimers in 71 and 9.5% yields, respectively. The oxidation of sinapyl p-coumarate produced γ-acylated ß-O-4 and γ-acylated tetralin ß-ß dimers in 53 and 16% yields, respectively. Only the sinapyl alcohol moiety in sinapyl p-coumarate reacted, and the p-coumarate moiety remained unchanged, suggesting that p-coumaric acid is not incorporated into the lignin backbone in the acylated lignins. All of the γ-acylated monolignols used in this study produced the γ-acylated ß-O-4 dimers, which suggests that the γ-acylated monolignols act as lignin monomers. The relatively high yields of the ß-O-4 dimers indicate that Ag2O oxidation of the monolignols can be used as an easy method for synthesizing the ß-O-4 dimer model compounds.

Biomimetic oxidative coupling of sinapyl acetate by silver oxide: preferential formation of ß-O-4 type structures.

Biomimetic oxidations of sinapyl alcohol and sinapyl acetate were carried out with Ag2O to better understand the high frequency of ß-O-4 structures in highly acylated natural lignins. The major products from the Ag2O oxidation of sinapyl alcohol were sinapyl aldehyde (14% yield), ß-O-4-coupled dimer (32% yield), and ß-ß-coupled dimer (3% yield). In contrast, the Ag2O oxidation of sinapyl acetate produced ß-O-4-coupled dimer in 66% yield. Oligomeric products with predominantly ß-O-4 structures were also obtained in 18% yield. The yield of the ß-O-4-coupled products from sinapyl acetate was much higher than that from sinapyl alcohol. Computational calculations based on density functional theory showed that the negative charge at Cß was significantly reduced by the γ-acetyl group. The computational calculations suggest that the Coulombic repulsion between Cß and O4 in sinapyl acetate radicals was significantly reduced by the γ-acetyl group, contributing to the preferential formation of ß-O-4 structures from sinapyl acetate.

Targeting the GPIbα binding site of thrombin to simultaneously induce dual anticoagulant and antiplatelet effects.

Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis-Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin.

A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo.

BACKGROUND: Some prominent cultured plant cell lines, such as the BY-2 cell line of tobacco (Nicotiana tabacum cv. 'Bright Yellow 2') and the T87 cell line of Arabidopsis (Arabidopsis thaliana L. Heynh., ecotype Columbia) are used as model plant cells. These suspension cell culture systems are highly applicable for investigating various aspects of plant cell biology. However, no such prominent cultured cell lines exist in bamboo species. RESULTS: We standardized a novel xylogenic suspension culture model in order to unveil the process of lignification in living bamboo cells. Initial signs of lignin deposition were able to be observed by a positive phloroglucinol-HCl reaction at day 3 to 5 under lignification conditions (LG), i.e., modified half-strength Murashige and Skoog medium (m1/2MS) containing 10 µM 6-benzyladenine (BA) and 3% sucrose. Two types of xylogenic differentiation, both fiber-like elements (FLEs) with cell wall thickening and tracheary elements (TEs) with formation of perforations in the cell wall, were observed under these conditions. The suspension cells rapidly formed secondary cell wall components that were highly lignified, making up approximately 25% of the cells on a dry weight basis within 2 weeks. Detailed features involved in cell growth, differentiation and death during lignification were characterized by laser scanning microscopic imaging. Changes in transcript levels of xylogenesis-related genes were assessed by RT-PCR, which showed that the transcription of key genes like PAL1, C4H, CCoAOMT, and CCR was induced at day 4 under LG conditions. Furthermore, interunit linkage of lignins was compared between mature bamboo culms and xylogenic suspension cells by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The presence of the most common interunit linkages, including ß-aryl ether (ß-O-4), phenylcoumaran (ß-5) and resinol (ß-ß) structures was identified in the bamboo cultured cell lignin (BCCL) by HSQC NMR. In addition to these common features of lignin, several differences in lignin substructures were also found between the BCCL and the bamboo milled wood lignin (BMWL). CONCLUSIONS: Our xylogenic suspension culture model could be used for detailed characterization of physiological and molecular biological events in living bamboo cells.

Thermal mobility of ß-O-4-type artificial lignin.

Several lignin model polymers and their derivatives comprised exclusively of ß-O-4 or 8-O-4' interunitary linkages were synthesized to better understand the relation between the thermal mobility of lignin, in particular, thermal fusibility and its chemical structure; an area of critical importance with respect to the biorefining of woody biomass and the future forest products industry. The phenylethane (C6-C2)-type lignin model (polymer 1) exhibited thermal fusibility, transforming into the rubbery/liquid phase upon exposure to increasing temperature, whereas the phenylpropane (C6-C3)-type model (polymer 2) did not, forming a char at higher temperature. However, modifying the Cγ or 9-carbon in polymer 2 to the corresponding ethyl ester or acetate derivative imparted thermal fusibility into this previously infusible polymer. FT-IR analyses confirmed differences in hydrogen bonding between the two model lignins. Both polymers had weak intramolecular hydrogen bonds, but polymer 2 exhibited stronger intermolecular hydrogen bonding involving the Cγ-hydroxyl group. This intermolecular interaction is responsible for suppressing the thermal mobility of the C6-C3-type model, resulting in the observed infusibility and charring at high temperatures. In fact, the Cγ-hydroxyl group and the corresponding intermolecular hydrogen bonding interactions likely play a dominant role in the infusibility of most native lignins.

Heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR) characterization of acetylated fir (Abies sachallnensis MAST) wood regenerated from ionic liquid.

An ionic liquid, 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), was used to dissolve Japanese fir (Abies sachallnensis MAST) wood. Milled woods prepared by planetary ball-milling for 8 h dissolved completely in [Bmim]Cl at 100 °C in 2 h. The dissolved woods were then subjected to in situ acetylation, and the fully acetylated woods were regenerated from [Bmim]Cl. (1)H-(13)C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments were successfully conducted with the acetylated woods in dimethyl sulfoxide (DMSO)-d(6). The acetylated lignin and polysaccharide signals dispersed reasonably well on the 2D spectra. Characterization of the NMR signals for the whole cell-wall components, including lignin, cellulose, and hemicelluloses, was achieved by comparison with isolated lignin and commercial cellulose and hemicelluloses (arabinoxylan, galactomannan, and glucomannan). The procedure used here is applicable for the characterization of cell-wall components in various plant biomasses.

Influence of syringyl to guaiacyl ratio on the structure of natural and synthetic lignins.

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.

Synthesis of beta-O-4-type artificial lignin polymers and their analysis by NMR spectroscopy.

We describe the synthesis and NMR spectroscopic analysis of three artificial lignin polymers containing only the beta-O-4 substructure: syringyl-type homopolymer, p-hydroxyphenyl-type homopolymer and guaiacyl/syringyl-type heteropolymer. Using gel permeation chromatography, the weight-average degree of polymerization (DP(w)) of the three polymers was determined as 19.2, 38.6, and 13.9, respectively. The polymers were prepared based on the synthetic methodology of guaiacyl-type homopolymer, and were fully characterized using (1)H-, (13)C-, and (1)H-(13)C NMR spectroscopy of the acetylated and non-acetylated forms. The spectra of guaiacyl/syringyl-type heteropolymers were in good agreement with those of the beta-O-4 substructure of milled wood lignin obtained from the hardwood of Japanese white birch.

Low molecular weight lignin suppresses activation of NF-kappaB and HIV-1 promoter.

Human immunodeficiency virus type 1 (HIV-1) is a cytopathic retrovirus and the primary etiological agent of acquired immunodeficiency syndrome (AIDS) and related disorders. In cells chronically infected with HIV-1, nuclear factor-kappaB (NF-kappaB) activation by external stimuli such as tumor necrosis factor alpha (TNFalpha) augments replication of HIV-1. NF-kappaB involves in many diseases such as inflammation, cancer, and Crohn's disease. In this paper, we exhibit that (i) HIV-1gene expression was inhibited by lignin, (ii) fraction of small molecular mass in HBS lignin (less than 0.5kDa) had stronger inhibitory effects than large molecular mass (more than 1.3kDa), (iii) lignin also inhibited activation of NF-kappaB induced by TNFalpha, (iv) among six lignin dimer-like compounds, compound 6 containing beta-5 bond has more potent inhibitory activity than compounds 1, 2, 3, 4 and 5, which contain beta-1, beta-O-4, 5-5, or beta-beta structural units. These results suggested that small molecules of lignin inhibit HIV-1 replication through suppression of HIV-1 transcription from LTR including activation via NF-kappaB. Low molecular lignin may be a beneficial material or drug leads as a new class for AIDS and NF-kappaB-related diseases.

Chemical synthesis of beta-O-4 type artificial lignin.

An artificial lignin polymer containing only the beta-O-4 substructure was synthesized. The procedure consists of two key steps: 1) polycondensation of a brominated monomer by aromatic Williamson reaction; and 2) subsequent reduction of the carbonyl polymer. 13C-NMR and HMQC spectra of the polymer were consistent with beta-O-4 substructures in milled wood lignin isolated from Japanese fir wood. The weight average degree of polymerization (DP(w)) ranged from 19.5 to 30.6, which is comparable to enzymatically synthesized artificial lignin from p-hydroxycinnamyl alcohols (dehydrogenation polymer, DHP) and some isolated lignins. Using this new lignin model polymer, it will now be possible to reinvestigate the properties and reactivity of the main lignin structure in terms of its polymeric character.

A new analysis of the depolymerized fragments of lignin polymer using ToF-SIMS.

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.

Production of mesoscopically patterned cellulose film.

Honeycomb and stripe patterned films were prepared from cellulose triacetate (CTA)/chloroform solution, as a result of the self-organization of the polymer during evaporation of the solvent. The honeycomb patterned CTA films were prepared by two methods, a direct pattern formation method and a transcription method. The latter method gave a well-organized microporous honeycomb pattern. Both types of patterned CTA films were saponified to yield the corresponding patterned cellulose films.

Easy synthesis of beta-O-4 type lignin related polymers.

The beta-O-4 structure is the most abundant substructure in lignin. Lignin related polymers composed of only the beta-O-4 structure were prepared using simple aromatic compounds as starting materials. Acetophenone derivatives were brominated, polymerized in the presence of K2CO3 and reduced with NaBH4 to give the lignin related polymers. These are linear polymers which resemble natural lignins in their structures, although they do not have a gamma-hydroxymethyl group. The number average degree of polymerization (DPn) was determined with peracetate of the polymers by gel permeation chromatography. The DPn of guaiacyl type polymers ranged from 15.2-21.4, where the value for the syringyl type was 11.3 and for the p-hydroxyphenyl type 16.9. The Guaiacyl type polymer was very soluble in usual lignin solvents such as 1, 4-dioxane-water (96 : 4, v/v) and DMSO, but only slightly soluble in acetone-water (9 : 1, v/v).

Synthesis of some phenylpropanoid monoglycerides via the Mitsunobu protocol.

Natural monoglycerides of cinnamic, ferulic and p-coumaric acids were synthesized in good to high overall yields from isopropylidene glycerol via the Mitsunobu reaction and further deprotection of the corresponding acetonides with Amberlyst 15. The method avoids the need of protection of the phenolic hydroxyls. During the Mitsunobu esterifications a strong influence of the acid strength on the efficacy and outcome of the reaction was observed.

Biosynthesis of indocarbazostatin B, incorporation of D-[U-13C] glucose and L-[2-13C] tryptophan.

High incorporation of D-[U-13C] glucose and L-[indole-2-13C] tryptophan into indocarbazostatin B (2) was observed in a biosynthetic study using a mutant strain, Streptomyces sp. MUV-7-8. The original strain, Streptomyces sp. TA-0403 produced a small amount of indocarbazostatin (1) and indocarbazostatin B (2), which displayed potent biological activities. To facilitate biosynthetic studies, we selected high indocarbazostatin producing mutant strains. The first mutants, Streptomyces sp. MUV-6-83 and MUV-6-17, produced indocarbazostatins C (3) and D (4) as well as 1 and 2. When the production medium was supplemented with D-tryptophan, the MUV-6-17 mutant produced K252c (5), whereas when L-tryptophan was added, it produced K252d (6). On further UV treatment of the mutant strain MUV-6-83, we finally obtained a new mutant producer, Streptomyces sp. MUV-7-8, that produced 2 as a major metabolite with higher productivity. This mutant producer enabled us to do a feeding experiment of the envisioned precursors, glucose and tryptophan.