Lignans of Larix leptolepis

Five lignans have been isolated from wood of Larix leptolepis. They are identified as 1-(4-hydroxy-3-methoxyphenyl)-2-4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy-propane- 1,3-diol, 1-(4-hydroxy-3-methoxyphenyl)-2-2-methoxy-4-[1-(E)-propen-3-ol]-phenoxy- propane-1,3-diol, 1-(4-hydroxy-3-methoxyphenyl)-2-(4-formyl-2-methoxyphenoxy)-propane-1,3-diol, 1,2-bis-(4-hydroxy-3-methoxyphenyl)-propane-1,3-diol and a trilignol, leptolepisol C.     

Metabolism of non-phenolic β-O-4 lignin model compounds by the white-rot fungus Phlebia radiata

Summary The degradation of three non-phenolic -O-4 diarylpropane lignin model compounds was studied in cultures of the white-rot fungus Phlebia radiata. The degradation pattern of the model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) was also compared with that of Phanerochaete chrysosporium under conditions where both fungi were cultivated without agitation in an oxygen atmosphere. Compound I was readily degraded by both fungi, and qualitatively the degradation patterns were quite similar. The product, after C-C bond cleavage, was veratraldehyde (IV) which was almost stoichiometrically reduced to veratryl alcohol (V). However, large amounts of V were detected only in P. chrysosporium cultures. Experiments with the model compound 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (II) showed that in the presence of II, the total amount of veratryl compounds accounted for 15–33 m in standing cultures of Phlebia radiata. The model compound 1-(3,4-dimethoxyphenyl)-2-(4-methoxyphenoxy) propane-1,3-diol (III) was more readily degraded than I and II. The results indicated that, in P. radiata cultures, the acting enzymes were lignin peroxidases and IV reducing enzyme, while laccase was less important. Offprint requests to: A. Hatakka     

Characterization of an extracellular lignin peroxidase of the lignocellulolytic actinomycete Streptomyces viridosporus

Previously we reported production of an extracellular lignin-inducible peroxidase by Streptomyces viridosporus (M. Ramachandra, D.L. Crawford, and A.L. Pometto III, Appl. Environ. Microbiol. 53:2754-2760, 1987). This peroxidase was shown to oxidize 3,4-dihydroxyphenylalanine, 2,4-dichlorophenol, homoprotocatechuic acid, caffeic acid, and N,N,N',N'-tetramethylphenylenediamine and was found in higher than normal levels in strains enhanced for lignocellulose degradation. In the present study, we used a pure extracellular enzyme preparation with high peroxidase isoform P3 activity to oxidize lignin substructure model compounds of both the 1,2-diaryl propane and arylglycerol-beta-aryl ether types and containing C alpha-carbonyl and C alpha-hydroxyl groups. The reactions were monitored by gas chromatography-mass spectrometry and high-pressure liquid chromatography techniques. In the presence, but not the absence, of hydrogen peroxide, the enzyme preparation catalyzed C alpha-C beta bond cleavage in the side chains of the diaryl ethers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propan-1-one (II) and the diaryl ethane 1-(4-methoxyphenyl)-2-(phenyl)ethan-1-one (III). Rapid hydrogen peroxide consumption was observed when the enzyme preparation was added to either milled corn lignin or lignocellulose. Additional characterizations showed that this enzyme is a heme protein (Soret band, 408 nm) and a major component of the ligninolytic system of S. viridosporus T7A. This is the first report of a lignin peroxidase in a bacterium. We have designated this new lignin peroxidase as ALiP-P3.     

Characterization of an extracellular lignin peroxidase of the lignocellulolytic actinomycete Streptomyces viridosporus.

Previously we reported production of an extracellular lignin-inducible peroxidase by Streptomyces viridosporus (M. Ramachandra, D.L. Crawford, and A.L. Pometto III, Appl. Environ. Microbiol. 53:2754-2760, 1987). This peroxidase was shown to oxidize 3,4-dihydroxyphenylalanine, 2,4-dichlorophenol, homoprotocatechuic acid, caffeic acid, and N,N,N',N'-tetramethylphenylenediamine and was found in higher than normal levels in strains enhanced for lignocellulose degradation. In the present study, we used a pure extracellular enzyme preparation with high peroxidase isoform P3 activity to oxidize lignin substructure model compounds of both the 1,2-diaryl propane and arylglycerol-beta-aryl ether types and containing C alpha-carbonyl and C alpha-hydroxyl groups. The reactions were monitored by gas chromatography-mass spectrometry and high-pressure liquid chromatography techniques. In the presence, but not the absence, of hydrogen peroxide, the enzyme preparation catalyzed C alpha-C beta bond cleavage in the side chains of the diaryl ethers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propan-1-one (II) and the diaryl ethane 1-(4-methoxyphenyl)-2-(phenyl)ethan-1-one (III). Rapid hydrogen peroxide consumption was observed when the enzyme preparation was added to either milled corn lignin or lignocellulose. Additional characterizations showed that this enzyme is a heme protein (Soret band, 408 nm) and a major component of the ligninolytic system of S. viridosporus T7A. This is the first report of a lignin peroxidase in a bacterium. We have designated this new lignin peroxidase as ALiP-P3.     

Degradation mechanisms of phenolic beta-1 lignin substructure model compounds by laccase of Coriolus versicolor

Phenolic beta-1 lignin substructure model compounds, 1-(3,5-dimethoxy-4-hydroxy-phenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)propa ne-1, 3-diol (I) and 1-(3,5-dimethoxy-4-ethoxyphenyl)-2-(3, 5-dimethoxy-4-hydroxyphenyl)propane-1,3-diol (II) were degraded by laccase of Coriolus versicolor. Substrate I was converted to 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)-3- hydroxypropanone (III), 1-(3,5-dimethoxy-4-ethoxyphenyl)-2-hydroxyethanone (IV), syringaldehyde (V), 1-(3,5-dimethoxy-4-ethoxyphenyl)-3-hydroxypropanal (VI), 2,6-dimethoxy-p-hydroquinone (VII), and 2,6-dimethoxy-p-benzoquinone (VIII). Furthermore, incorporations of 18O of 18O2 into ethanone (IV) and 18O of H218O into hydroquinone (VII) and benzoquinone (VIII) were confirmed. Substrate II gave 1-(3,5-dimethoxy-4-hydroxyphenyl)ethane-1, 2-diol (IX), 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-hydroxyethanone (X), and 3,5-dimethoxy-4-ethoxybenzaldehyde (XI). Also 18O of H218O was incorporated into glycol (IX) and ethanone (X). Based on the structures of the degradation products and the isotopic experiments, it was established that three types of reactions occurred via phenoxy radicals of substrates caused by laccase: (i) C alpha-C beta cleavage (between C1 and C2 carbons); (ii) alkyl-aryl cleavage (between C1 carbon and aryl group); and (iii) C alpha (C1) oxidation.     

Incorporation of 18O2 and absence of stereospecificity in primary product formation during fungal metabolism of a lignin model compound

The metabolism of a lignin substructure model compound, 1,2-bis(3-methoxy-4-ethoxyphenyl)propane-1,3-diol (Ia) in ligninolytic cultures of Phanerochaete chrysosporium was studied to help elucidate the biochemical mechanism of lignin degradation. The primary reaction was cleavage of the model compound between C₁ and C₂ of the propane moiety to produce 1-(3-methoxy-4-ethoxyphenyl)ethane-1,2-diol and a C₆-C₁ product (probably 3-methoxy-4-ethoxybenzaldehyde). Other identified products arose secondarily; all were further metabolized. Even though the model compound was a mixture of four stereoisomers, no stereoselectivity was observed in its metabolism. In cultures under ¹⁸O₂, the initial cleavage produced the diol product with ≈70% enrichment by ¹⁸O in the benzyl alcohol group. The diol was a mixture of the two possible enantiomers, and the O₂-derived hydroxyl was incorporated at the asymmetric (benzyl) carbon. (Limited optical activity in the diol was traced to selective further metabolism of the D form.) These results show that the primary cleavage reaction lacked stereospecificity and was primarily oxygenative, implicating a nonspecific oxygenase or a nonenzymatic reaction involving activated oxygen. Preliminary experiments demonstrated no cell homogenate activity against Ia.     

Design and synthesis of novel chloramphenicol amine derivatives as potent aminopeptidase N (APN/CD13) inhibitors

Herein we report a series of novel chloramphenicol amine derivatives as aminopeptidase N (APN)/CD13 inhibitors. All compounds were synthesized starting from commercially available (1S,2S)-2-amino-1-(4-nitrophenyl) propane-1,3-diol. The preliminary biological screening showed that some compounds exhibited potent inhibitory activity against APN. It should be noted that one compound, 13b (IC₅₀ = 7.1 μM), possess similar APN inhibitory activity compared with Bestatin (IC₅₀ = 3.0 μM).     

Oxidation and reduction of lignin-related aromatic compounds by Aureobasidium pullulans

Summary A yeast-like fungus, identified as Aureobasidium pullulans, was isolated from a kraft mill settling pond by enrichment culture on 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-propane-1,3-diol (I). The fungus was also able to use the following aromatic acids as sole carbon source: Benzoic, p-hydroxybenzoic, vanillic, syringic, ferulic and protocatechuic acids. Various aromatic alcohols were oxidised to their corresponding aldehydes and acids during aerobic culture, while aromatic aldehydes were both oxidised and reduced. However, the aromatic acids were not reduced, but were slowly metabolised. Dimer I was cleaved at the alkyl-phenyl linkage to give glycerol-2-guaiacyl ether in high yield. The identity of the latter was determined by mass spectrometry and proton nmr. The dimers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-propane-1,3-diol (II), 3,4-dimethoxy--(2,6-dimethoxy-4-carboxyphenoxy)-acetophenone (III) and 5-carboxy-7-methoxy-2-(4-hydroxy-3-methoxyphenyl)-3-methyl-2,3-dihydrobenzo-[b]-furan (IV) were not metabolised. It is concluded that the fungus resembles Fusarium in many of its metabolic properties, and could be considered as a potential lignin degrader.     

Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system

Phenolic and nonphenolic (permethylated) synthetic [14C]lignins were depolymerized by Trametes villosa laccase in the presence of a radical mediator, 1-hydroxybenzotriazole (HOBT). Gel permeation chromatography of the treated lignins showed that approximately 10% of their substructures were cleaved. The system also cleaved a beta-O-4-linked model compound, 1-(4-ethoxy-3-methoxy-ring-[14C]phenyl)-2-(2-methoxyphenoxy)-propane- 1,3-diol, and a beta-1-linked model, 1, 2-bis-(3-methoxy-4-[14C]methoxyphenyl)-propane-1,3-diol, that represent nonphenolic substructures in lignin. High performance liquid chromatography of products from the oxidized models showed that they were produced in sufficient yields to account for the ability of laccase/HOBT to depolymerize nonphenolic lignin.     

Novel aminopeptidase N (APN/CD13) inhibitors derived from chloramphenicol amine

Aminopeptidase N (APN) is involved in different physiological and pathological processes of tumor cells, including proliferation, invasion, apoptosis and metastasis. Herein one series of compounds derived from commercially available (1S,2S)-2-amino-1-(4-nitrophenyl) propane-1,3-diol have been designed and synthesized. Furthermore, preliminary activity evaluation showed that some compounds elicited moderate inhibitory activity against APN with compounds 10e (IC(50)=6.1±0.5 μM) possessing the best efficacy, which could be used as the lead compound in the future for anticancer agents research.     

The C-C bond cleavage of a lignin model compound, 1,2-diarylpropane-1,3-diol, with a heme-enzyme model catalyst tetraphenylporphyrinatoiron(III)chloride in the presence of tert-butylhydroperoxide

The catalytic C-C bond cleavage of a lignin model compound was investigated by use of tetraphenylporphyrinatoiron(III)chloride as a model for enzymic degradation of lignin. The C-C bond of the lignin model compound 1,2-bis(4-ethoxy-3-methoxyphenyl) propane-1,3-diol was oxidatively cleaved by catalysis of iron-porphyrins in the presence of tert-butylhydroperoxide or iodosylbenzene at a room temperature. The products formed after complete oxidation of the substrate were identified as 4-O-ethylvanillin, alpha-hydroxy-4-ethoxy-3-methoxyacetophenone, 4-O-ethylvanillic acid, 4-ethoxy-3-methoxyphenylglycol, 4-ethoxy-3-methoxy-alpha-(4-ethoxy-3-methoxyphenyl)-beta-hydroxypropi ophenone and formaldehyde.     

Ferrocene Compounds XXXVI. Lipase-catalyzed Enantioselective Acetylation of Prochiral 2-(ω-Ferrocenylalkyl)propane-1,3-diols

Enantioselective acetylation (desymmetrization) of prochiral 2-(ferrocenylmethyl)propane-1,2-diol (1), 2-(2-ferrocenylethyl)propane-1,2-diol (2) and 2-(3-ferrocenylpropyl)propane-1,2-diol (3) into chiral monoacetates [(+)-4-(+)-6], with a series of microbial lipases in benzene at 27°C, revealed the lipase from Pseudomonas sp (PSL) as the most selective. Acetylation was fastest and most enantioselective for conversion 1→(+)-4 by PSL (97% e.e.). By comparison of the compounds (+)-4-(+)-6 with their benzene analogues of the known (R) absolute configuration, on the basis of their elution orders on Chiracel OD, and the same direction of their optical rotations, an R-configuration is proposed for (+)-monoacetates 4-6.     

Ferrocene Compounds XXXVI. Lipase-catalyzed Enantioselective Acetylation of Prochiral 2-(ω-Ferrocenylalkyl)propane-1,3-diols

Enantioselective acetylation (desymmetrization) of prochiral 2-(ferrocenylmethyl)propane-1,2-diol (1), 2-(2-ferrocenylethyl)propane-1,2-diol (2) and 2-(3-ferrocenylpropyl)propane-1,2-diol (3) into chiral monoacetates [(+)-4-(+)-6], with a series of microbial lipases in benzene at 27°C, revealed the lipase from Pseudomonas sp (PSL) as the most selective. Acetylation was fastest and most enantioselective for conversion 1→(+)-4 by PSL (97% e.e.). By comparison of the compounds (+)-4-(+)-6 with their benzene analogues of the known (R) absolute configuration, on the basis of their elution orders on Chiracel OD, and the same direction of their optical rotations, an R-configuration is proposed for (+)-monoacetates 4–6.     

Antioxidative phenylpropanoids from berries of Pimenta dioica

A phenylpropanoid, threo-3-chloro-1-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol, was isolated from the berries of Pimenta dioica together with five known compounds, eugenol, 4-hydroxy-3-methoxycinnamaldehyde, 3,4-dimethoxycinnamaldehyde, vanillin and 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol. In addition, the stereochemistry of 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol was determined. The phenylpropanoids inhibited autoxidation of linoleic acid in a water-alcohol system.     

Preparation of antibodies against a novel immunosuppressant, FTY720, and development of an enzyme immunoassay for FTY720

FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin). To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA) was developed. Two kinds of haptens, 3 and 4, for 1 were synthesized and coupled to ovalbumin (OVA). Rabbits were immunized with 3-OVA or 4-OVA, and corresponding antibodies were obtained. Both antibodies recognized the 2-amino-2-(2-phenylethyl)propane-1,3-diol moiety in 1. Using the anti-3-OVA antibody, a competitive EIA for 1 was developed and evaluated. The range of quantification by the EIA was 0.06-10 ng/mL. The application of the EIA has enabled us to measure the FTY720 concentration in serum after oral administration of 1 (1mg/kg) to rats.     

An access to various sulfation patterns in dermatan sulfate: chemical syntheses of sulfoforms of trisaccharide methyl glycosides

The syntheses are reported for the first time of alpha-L-IdopA2SO(3)-(1-->3)-beta-D-GalpNAc4SO(3)-(1-->4)-alpha-L-IdopA2SO(3)-(1-->OMe), its disulfated analogue alpha-L-IdopA2SO(3)-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-L-IdopA2SO(3)-(1-->OMe), and of beta-D-GalpNAc4SO(3)-(1-->4)-alpha-L-IdopA2SO(3)-(1-->3)-beta-D-GalpNAc4SO(3)-(1-->OMe), which represent structural fragments of dermatan sulfate, unavailable directly by chemical or enzymatic degradation of the glycosaminoglycan polymer. These molecules were readily obtained from a pair of key disaccharide intermediates, in which the relative difference of stability of the D-GalNAc 4-hydroxy protecting groups (acetate or pivalate) toward saponification conditions allowed access to various sulfoforms from a common precursor. For the preparation of these blocks, the 4-O-pivaloyl-D-galacto moiety was readily obtained through a one-pot stereospecific intramolecular nucleophilic displacement on an easily available 3-O-pivaloyl-D-gluco precursor, and the L-IdoA moiety through selective radical oxidation at C-6 of a L-ido 4,6-diol derivative with oxoammonium salts.     

Structural analysis of a new glycosphingolipid from the lipopolysaccharide-lacking bacterium Sphingomonas adhaesiva.

A new glycosphingolipid, GSL-4B, was isolated from Sphingomonas adhaesiva and found to share the ceramide moiety with GSL-1 and GSL-3 from Sphingomonas capsulata studied earlier [Kawahara, K.; Moll, H.; Knirel, Y. A.; Seydel, U.; Z?hringer, U. Eur. J. Biochem. 2000, 267, 1837-1846]. It is heterogeneous with respect to the long-chain bases erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol, and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol which in GSL-4B are present in the ratios of 1.1:1.0:1.1, and all bearing amide-linked (S)-2-hydroxymyristic acid. Methylation analysis and MALDI-TOF-MS along with 1H and 13C NMR spectroscopy showed that the carbohydrate part of GSL-4B has the structure of alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-GlcpA-(1-->1)-Cer     

The total synthesis of ganglioside GM3

Previous syntheses of ganglioside GM3 (NeuAc alpha3Gal beta4Glc beta1Cer) are reviewed, and both chemoenzymatic and chemical total synthetic approaches were investigated. In a chemoenzymatic approach, (2S,3R,4E)-5'-acetyl-alpha-neuraminyl-(2' --> 3')-beta-galactopyranosyl-(1' --> 4')-beta-glucopyranosyl-(1' <--> 1)-2-azido-4-octadecene-1,3-diol (azidoGM3) was readily prepared utilizing recombinant beta-Gal-(1' --> 3'/4')-GlcNAc alpha-(2' --> 3')-sialyltransferase enzyme, and was evaluated as a synthetic intermediate to ganglioside GM3. The chemical total synthesis of ganglioside GM3 was performed on one of the largest scales yet reported. The highlights of this synthesis include minimizing the steps necessary to prepare the lactosyl acceptor as a useful anomeric mixture, which was present in excess for the highly regioselective and fairly stereoselective sialylation with a known neuraminyl donor to give the protected GM3 trisaccharide. The synthetic methodology maximized convergence by a subsequent glycosidic coupling of the well-characterized GM3 trisaccharide trichloroacetimidate derivative with protected ceramide. The ganglioside GM3 was nearly homogeneous as the two glycosidic couplings utilized preparative HLPC purifications, and variations in the sphingosine base and fatty acyl group were under 0.1 and 0.2%, respectively.     

Two New Norlignans and a New Lignanamide from Peperomia tetraphylla

Two new cyclobutane-type norlignans, methyl rel-(1R,2S,3S)-2-(7-methoxy-1,3-benzodioxol-5-yl)-3-(2,4,5-trimethoxyphenyl)cyclobutanecarboxylate (1), and methyl rel-(1R,2R,3S)-2-(7-methoxy-1,3-benzodioxol-5-yl)-3-(2,4,5-trimethoxyphenyl)cyclobutanecarboxylate (2), and a new lignanamide, 3-hydroxy-N-[2-(4-hydroxyphenyl)ethyl]-α-[4-(2-{N-[2-(4-hydroxyphenyl)ethyl]carbamoyl}ethenyl)-3-methoxyphenoxy]-4-methoxycinnamamide 4,8″-ether (3), along with five known amides, 4-8, were obtained from the whole plant of Peperomia tetraphylla. Their structures were elucidated mainly by the analysis of NMR and MS data. The new compounds 1-3 and the known compound 4 were tested for their cytotoxic activities against the HepG2 (human hepatocarcinoma), A549 (human lung cancer), and HeLa (human cervical cancer) cell lines. Compound 4 showed significant cytotoxicity against HepG2 cell lines with an IC(50) value of 9.4 ± 1.0?μM.     

The effect of propane-diols on the intestinal uptake of nutrients and brush border membrane enzymes in the rat

The effect on rats of oral doses (38.66 mM/kg body wt) of propane-1,2-diol (PD) administered daily for 10 (Group 1), 20 (Group 2), and 30 days (Group 3) was investigated. Weight gain was initially retarded (P less than 0.05) in Group 1, but was later reversed and elevated significantly (P less than 0.05) in Groups 2 and 3 as compared with their respective controls receiving an equal volume of saline. PD showed a tendency toward enhancing the activities of various enzymes involved in terminal digestion, with the significant effect exerted in few groups on sucrase (P less than 0.05), lactase (P less than 0.05), and gamma-glutamyl transpeptidase (P less than 0.05) when compared with the respective controls. Absorption of D-glucose, glycine, L-aspartic acid, L-lysine, and calcium was elevated and was especially significant in Groups 2 and 3 (P less than 0.001). The structural integrity of the jejunal surface was retained for the most part. A similar examination of the effects of PD was also carried out in vitro to ascertain whether PD itself or its metabolites are involved in its action. The in vitro effects of propane-1,2-diol were compared with those of the more toxic compound propane-1,3-diol. The former exerted greater inhibitory action on the activities of the disaccharidases. The degree of inhibition was in the order sucrase much greater than lactase greater than maltase. The kinetic data revealed that inhibition by 1,2-diol in native and detergent solubilized sucrase is noncompetitive, with Ki values in the range of 0.35-0.41 M. The two diols did not alter the nutrient transport in the brush border membrane vesicles. The present work on rats indicates that PD may influence the intestinal digestive and absorptive functions in vivo and that this in vivo effect of PD is different from that observed in vitro suggesting that the nutritional and toxicological effect of PD may be mediated by different mechanisms.