Sequential synthesis and 13C-N.m.r. spectra of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides

The reaction of 2,3-di-O-acetyl-4-O-benzyl-α,β-d-xylopyranosyl bromide (2) with methyl 2,3-di-O-acetyl-β-d-xylopyranoside gave methyl O-(2,3-di-O-acetyl-4-O-benzyl-β-d-xylopyranosyl)-(1→4)-2,3-di-O-acetyl-β-d-xylopyranoside (22). Catalytic hydrogenolysis of 22 exposed HO-4′ which was then condensed with 2. This sequence of reactions was repeated three more times to afford, after complete removal of protecting groups, a homologous series of methyl β-glycosides of (1→4)-β-d-xylo-oligosaccharides. ¹³C-N.m.r. spectra of the synthetic methyl β-glycosides (di- to hexa-saccharide) are presented together with data for six other, variously substituted, homologous series of (1→4)-d-xylo-oligosaccharides.     

Interactions glycanne-protéine. Synthése des 4-méthylombelliféryl-(2-acétamido-2-désoxy-β-D-glucopyranoside), -DI-N-acétyl-β-chitobioside et -tri-N-acétyl-β-chitotrioside. Interaction de ces osides avec le lysozyme

4-Methylumbelliferyl 2-acetamido-2-deoxy-β-D-glucopyranoside, 2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-β-D-glucopyranoside (di-N-acetyl-β-chitobioside), and O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (tri-N-acetyl-β-chitotrioside) were obtained in good yield from the corresponding peracetylated glycosyl chlorides by condensation with the sodium salt of 4-methylumbelliferone in N,N-dimethylformamide. The trisaccharide glycoside is hydrolyzed by lysozyme and is, therefore, a convenient substrate for this enzyme; the 4-methylumbelliferone produced can be determined by the increase of the fluorescence intensity at 442 nm. The intensity of the fluorescence of 4-methylumbelliferyl tri-N-acetyl-β-chitotrioside is enhanced upon binding with lysozyme without modification of the position of the absorption maximum. The binding constant and the rate of hydrolysis of the trisaccharide glycoside by lysozyme are higher than those obtained with p-nitrophenyl tri-N-acetyl-β-chitotrioside.     

Uniformity and distinctness of phyllocladus as evidenced by flavonoid accumulation

The conifer genus Phyllocladus is shown by comparative flavonoid chemistry to be remarkably homogeneous and quite distinct from other studied genera in the Podocarpaceae. It is characterized by the accumulation (in the foliage) of a predominance of flavone O-glycosides, and in particular, luteolin 7- and 3′-O-glycosides. Lower levels of flavonol O-glycosides are also evident. Two flavone glycosides are reported for the first time, luteolin 3′-O-α-L-rhamnopyranoside and luteolin 7-O-α-L-rhamnoside.     

4-Methylumbelliferyl esters as fluorogenic substrates for proteases

4-Methylumbelliferyl esters of amino acid derivatives have been synthesized using the carbodiimide, disulphite and carbonate methods. Of these, the first was shown capable of preparing 2-naphthyl and 4-methylumbelliferyl esters of benzoylglycine, benzyloxycarbonyl glycine and benzyloxycarbonyl-citrulline but not of benzoyl-N^G-nitroarginine. 2-Naphthyl benzoyl-N^G-nitroargininate was prepared successfully using di(2-naphthyl)sulphite. Bis(4-methylumbelliferyl)sulphite could not be prepared but 4-methylumbelliferyl benzoyl-N^G-nitroargininate was obtained by the use of an equilibrium method using diphenyl sulphite in the presence of 4-methylumbelliferone. A new reagent, phenyl 4-methylumbelliferyl carbonate, was synthesized and used for the preparation of the 4-methylumbelliferyl esters of benzoylglycine, benzyloxycarbonylglycine and benzoyl-N^G-nitroarginine. The 4-methylumbelliferyl esters of benzyloxycarbonylglycine and benzyloxycarbonylcitrulline were shown to be good substrates for the assay of proteases, including chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4). Disadvantages of 4-methylumbelliferyl esters are discussed.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

Depolymerization of heparin with diazomethane. Structure of N,O-methylated,even-numbered oligosaccharides produced by β-eliminative cleavage of the 2-amino-2-deoxyglycosylic linkage

The long-period reaction of heparin with excess diazomethane at 20° resulted in cleavage at the β-position of the uronic acid carboxyl group to give a mixture of methyl α- and β-glycosides of N,O-methylated di-, tetra-, and hexa-saccharides having a 4,5-unsaturated uronic acid, nonreducing end-group. The major disaccharides obtained were methyl O-(4-deoxy-3-O-methyl-α-l-threo-hex-4-enopyranosyluronic acid 2-sulfate)-(1→4)-2-deoxy-3-O-methyl-2-(N-methylsulfoamino)-α- and -β-d-glucopyranoside. The reaction of heparin at 4° yielded a mixture of methylated, higher-molecular-weight oligosaccharides, which retained some affinity for antithrombin III-Sepharose.     

Apigenin and amentoflavone glycosides in the psilotaceae and their phylogenetic significance

Documentation of amentoflavone O-glucosides as the predominant flavonoid glycosides in both genera of the Psilotaceae clearly distinguishes this family from all other families of vascular plants. Psilotum and Tmesipteris also possess apigenin C- and O-glycosides as common flavonoid types. Apigenin 7-O-rhamnoglucoside occurs in both genera and the previously undocumented apigenin 7-O-rhamnoglucoside-4′-O-glucoside, although identified only in Tmesipteris, may also be present in Psilotum. The existence of flavone C-glycosides in both genera may provide a phytochemical relationship between the Psilotaceae and some ferns. The phylogenetic significance of these results is discussed.     

Anthocyanins and flavonols from the blue flowers of six Meconopsis species in Bhutan

Blue flowers of six Bhutani Meconopsis species, M. bhutanica, M. bella, M. horridula, M. simplicifolia, M. primulina and M. polygonoides, were surveyed for anthocyanins and other flavonoids. Four anthocyanins were isolated and identified as cyanidin 3-O-sambubioside-7-O-glucoside (1), cyanidin 3-O-[xylosyl-(1 → 2)-(6″-malonylglucoside)]-7-O-glucoside (2), cyanidin 3-O-sambubioside (4) and cyanidin 3-O-[xylosyl-(1 → 2)-(6″-malonylglucoside)] (5). On the other hand, 12 flavonols were isolated from their Meconopsis species with various combination and characterized as kaempferol 3-O-glycosides (8–12), kaempferol 3,7-O-glycosides (13–16), quercetin 3-O-glycosides (17 and 18) and isorhamnetin 3-O-glycoside (19). Of six Meconopsis species which were surveyed in this experiment, anthocyanin and flavonol composition of five species except for M. horridula was clarified for the first time. Their Meconopsis species showed the different flavonoid profiles, respectively, and flavonoid diversity within the glycosylation level of Meconopsis flowers were indicated.     

Preparation of (aryl α-l-idopyranosid)uronic acids

The earlier preparation of cyclohexylammonium (phenyl α-l-idopyranosid)-uronate has been improved, and (4-methylumbelliferyl α-l-idopyranosid)uronic acid (14), a more sensitive substrate for α-l-iduronidase, has been synthesized by an analogous route. Zinc chloride-catalyzed condensation of 4-methylumbelliferone with 1,2,3,4,6-penta-O-acetyl-α-l-idopyranose (4) in 1,2-ethanediol diacetate gave crystalline 4-methylumbelliferyl 2,3,4,6-tetra-O-acetyl-α-l-idopyranoside (7). O-Deacetylation and catalytic oxidation gave 14, characterized as a cyclohexylammonium salt. The starting material 4 was prepared, in 21 % yield from l-glucose, by conversion of the intermediate 1,2,3,4,6-penta-O-acetyl-β-l-glucopyranose to 2,3,4,6-tetra-O-acetyl-β-l-glucopyranosyl chloride and acetoxonium ion rearrangement, as described for the D-series.     

O-GLYCOSIDES OF N-HYDROXYINDOLES[1]

First O-glycosides of N-hydroxyindole were synthesized by the interaction of the indoles containing electron withdrowing substituents with acyl halogenoses in the presence of alkaline reagents. 1-O-β-D-Glucopyranosides of 1-hydroxy-5-(or 6)-nitroindoles, 1-O-β-D-ribofuranoside of 1-hydroxy-5-nitroindole and also 1-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]-2-methoxycarbonylindole were obtained. 1-[(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)-oxy]-6-nitro-indole was transformed into 1-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-oxy]indole.     

Conversion of amylose into a (1-4)-2-amino-2-deoxy-alpha-D-glucopyranuronan and its 2-N-sulfo derivative, a heparin analog

By a modification of a previously established reaction-sequence involving successive oxidation with methyl sulfoxide-acetic anhydride, oximation, and reduction with lithium aluminum hydride, 6-O-tritylamylose (1) was converted into a 6-O-tritylated (1→4)-α-D-linked glucan (3) containing 2-amino-2-deoxy-D-glucose residues and some O-(methylthio)methyl groups. Removal of the ether groups from this product gave a 2-aminated amylose (4) of degree of substitution (d.s.) by amine of 0.54 that underwent cleavage by fungal alpha-amylase to give oligosaccharides containing amino sugar residues. N-Trifluoroacetylation of 3 followed by removal of the ether groups, oxidation at C-6 with oxygen-platinum, and removal of the N-substituent, gave a (1 →4)-2-amino-2-deoxy-α-D-glucopyranuronan 7 having d.s. by amine of up to 0.65, and by carboxyl, of 0.46. Sulfation of this product with sulfur trioxide-pyridine and then with chlorosulfonic acid-pyridine gave a (1→4)-2-deoxy-2-sulfoamino-α-D-glucopyranuronan, isolated as its sodium salt 8, which showed appreciable blood-anticoagulant activity.     

Characterization of a Novel Phenol Hydroxylase in Indoles Biotranformation from a Strain Arthrobacter sp. W1

Background

Indigoids, as popular dyes, can be produced by microbial strains or enzymes catalysis. However, the new valuable products with their transformation mechanisms, especially inter-conversion among the intermediates and products have not been clearly identified yet. Therefore, it is necessary to investigate novel microbial catalytic processes for indigoids production systematically.

Findings

A phenol hydroxylase gene cluster (4,606 bp) from Arthrobacter sp. W1 (PH_w1) was obtained. This cluster contains six components in the order of KLMNOP, which exhibit relatively low sequence identities (37–72%) with known genes. It was suggested that indole and all the tested indole derivatives except for 3-methylindole were transformed to various substituted indigoid pigments, and the predominant color products derived from indoles were identified by spectrum analysis. One new purple product from indole, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one, should be proposed as the dimerization of isatin and 7-hydroxylindole at the C-2 and C-6 positions. Tunnel entrance and docking studies were used to predict the important amino acids for indoles biotransformation, which were further proved by site-directed mutagenesis.

Conclusions/Significance

We showed that the phenol hydroxylase from genus Arthrobacter could transform indoles to indigoids with new chemical compounds being produced. Our work should show high insights into understanding the mechanism of indigoids bio-production.     

Flavonol glycosides acylated with 3-hydroxy-3-methylglutaric acid as systematic characters in Rosa

LC–UV–MS/MS analysis of leaf extracts from 146 accessions of 71 species of Rosa revealed that some taxa accumulated flavonol O-glycosides acylated with 3-hydroxy-3-methylglutaric acid, which are relatively uncommon in plants. The structures of two previously unrecorded examples isolated from Rosa spinosissima L. (syn. Rosa pimpinellifolia L.) were elucidated using spectroscopic and chemical methods as the 3-O-α-l-rhamnopyranosyl-(1 → 2)-[6-O-(3-hydroxy-3-methylglutaryl)-β-d-galactopyranosides] of kaempferol (3,5,7,4′-tetrahydroxyflavone) and quercetin (3,5,7,3′,4′-pentahydroxyflavone). The corresponding 3-O-[6-O-(3-hydroxy-3-methylglutaryl)-β-d-galactopyranoside] of quercetin was also present in R. spinosissima, but at lower levels, together with 17 other flavonol O-glycosides for which structures were assigned using LC–UV–MS/MS. The distribution of flavonol 3-hydroxy-3-methylglutarylgalactosides in Rosa was limited to some species of subgenus Rosa section Pimpinellifoliae and Rosa roxburghii Sw. of the monotypic subgenus Platyrhodon, indicating that this character could be of value in phylogenetic analyses of the genus.     

Effects of different mineral supplements on fertilization of phenol-contaminated soils by Corynebacterium glutamicum

This study focused on the effects of different mineral supplements on the ability of Corynebacterium glutamicum to degrade phenol in contaminated soil and convert the phenol into useful amino acids. Three types of minerals including FeSO₄, MgSO₄, and MnSO₄ were added at several concentrations to C. glutamicum culture media containing 1% yeast extract prior to treating the soil samples with 4.24 mM phenol. The reactor was incubated at 30°C and 150 rpm for 3 days, and the treated soil was sampled daily and analyzed using gas chromatography for residual phenol and the amino acids produced. Additionally, a plant toxicity assay was employed to examine the fertilization of the phenol-contaminated soil after C. glutamicum treatment supplemented with the three minerals. Our results suggested that among various tested concentrations, 72 μM of iron showed a significant effect on the utilization of phenol by C. glutamicum for conversion to amino acids, therefore enhancing fertilization of the phenol-contaminated soil.     

Further studies of flavonols of Clibadium (compositae)

The flavonoids of an additional eight species of Clibadium have been determined. The compounds are derivatives of kaempferol, quercetin and quercetagetin. O-Methylated quercetagetin derivatives were found in several taxa with the possibility that 6-methoxykaempferol may also exist in one collection. Kaempferol and quercetin exist as 3-O-glucosides, galactosides, rhamnosides, rutinosides and diglucosides although not all glycosides occur in each taxon. Quercetagetin derivatives occur as 7-O-glucosides. Observations on these newly investigated species confirm previous work in the genus that three types of flavonoid profiles exist: (1) kaempferol and quercetin 3-glycosides; (2) kaempferol and quercetin 3-glycosides plus quercetagetin 7-glucoside; and (3) kaempferol and quercetin 3-glycosides plus quercetagetin 7-glucoside and O-methylated derivatives of quercetagetin.     

Synthesis of 3-(2-aminoethylthio)propyl glycosides

Anomeric pairs of 3-(2-aminoethylthio)propyl d-galactopyranoside (4, 4a), d-glucopyranoside (5, 5a), and 2-acetamido-2-deoxy-d-glucopyranoside (6, 6a) were prepared by addition of 2-aminoethanethiol to the corresponding, anomeric, allyl glycosides. The allyl α-glycosides were prepared by refluxing the sugars with allyl alcohol in the presence of an acid catalyst; the allyl β-glycosides were prepared by the reaction of acetylated glycosyl bromides with allyl alcohol in the presence of mercuric cyanide, followed by O-deacetylation. The rate of thiol addition to the allylic group was found to be different for each glycoside.     

Flavonoid diversification in Achillea ptarmica and allied taxa

More than 50 collections of 12 species forming the A. ptarmica group have been analysed for their leaf flavonoids. C-Glycosylflavones (iso-orientin and derivatives, vicenins and lucenins) were found to be the main components, whereas flavonol 3-O-glycosides (based on quercetin and kaempferol) and flavone 7-O-glycosides (based on luteolin and diosmetin) were of restricted distribution. Infraspecific variability regarding C-glycosylflavones was observed in most of the taxa investigated. By contrast, flavonol 3-O-glycosides appeared to be stable characters and were sometimes accumulated instead of C-glycosylflavones. In addition to the flavonoids, the geographical distribution patterns and the possible origin of the A. sibirica in Eastern Asia are briefly discussed.     

Cloning and characterization of genes encoding an enzyme which oxidizes dimethyl sulfide in Acinetobacter sp. strain 20B

Acinetobacter sp. strain 20B was isolated based on the ability to utilize dimethyl sulfide as the sole sulfur source. Since strain 20B oxidized indole as well as dimethyl sulfide, indigo production by recombinant Escherichia coli clones carrying Acinetobacter DNA was used as a selection for cloning genes encoding dimethyl sulfide oxidation genes. The gene encoding an indole-oxidizing enzyme was also found to oxidize dimethyl sulfide. The dimethyl sulfide-oxidizing enzyme genes consisted of six open reading flames designated dsoABCDEF. The deduced amino acid sequences of dsoABCDEF were homologous with those of the multicomponent phenol hydroxylases. DsoABCDEF oxidized dimethyl sulfide to dimethyl sulfoxide, and dimethyl sulfoxide to dimethyl sulfone.     

Characteristics of transxylosylation by β-xylosidase from Aspergillus awamori K4

The Aspergillus awamori K4 β-xylosidase gene (Xaw1) sequence was deduced by sequencing RT-PCR and PCR products. The ORF was 2,412 bp and the predicted peptide was 804 amino acids long, corresponding to a molecular weight of 87,156 Da. The mature protein was 778 amino acids long with a molecular weight of 84,632 Da. A homology search of the amino acid sequence revealed that it was very similar to the Aspergillus niger β-xylosidase gene with only five amino acid differences. K4 β-xylosidase had the same catalytic mechanism as family 3 β-glucosidases, involving Asp in region A. At an early stage in the reaction with xylobiose and xylotriose, the hydrolysis rate was much lower than the transxylosylation rate, decreasing gradually as the substrate concentration increased, whereas the transxylosylation rate increased greatly. Aspergillus awamori K4 β-xylosidase had broad acceptor specificity toward alcohols, hydroxybenzenealcohols, sugar alcohols and disaccharides. A consensus portion involving the hydroxymethyl group of the acceptor was confirmed in the major transfer products 1(4)-O-β-d-xylosyl erythritol, (2-hydroxyl)-phenyl-methyl-β-d-xylopyranoside, 6S-O-β-d-xylosyl maltitol (S: sorbitol residue) and 6G-O-β-d-xylosyl palatinose (G: glucosyl residue). This might suggest that the methylene in the hydroxymethyl group facilitates base-catalyzed hydroxyl group attack of the anomeric center of the xylosyl–enzyme intermediate.     

The attachment of poly(ribitol phosphate) to lipoteichoic acid carrier

2-Acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(L-leucyl-L-threonyl-N²-tosyl-L-lysine p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (21) and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(L-leucyl-L-threonyl-N²-tosyl-L-lysine p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine (22), 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(glycine ethyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine, and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-1-oyl-(phenylalanine methyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine were synthesized by condensation of 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(benzyloxycarbonyl)-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine with the appropriate protected amino acids and tri- and tetra-peptides. The amino acid sequences of 21 and 22 correspond to the protected amino acid sequences 34–37 and 34–38 of ribonuclease B that are adjacent to the carbohydrate-protein linkage.