Synthesis and structural analyses of 3-acetamido-1,4-di-O-acetyl-2,3,5-trideoxy-5-C-(isopropylphosphinyl)-D-erythro-pentopyranoses

1H NMR spectroscopy of phosphorus containing hetero sugars (phospha sugars), revealed the alpha and beta configurations and chair conformations for 3-acetamido-1,4-di-O-acetyl-2,3,5-trideoxy-5-C-(isopropylphosphinyl)-alpha- and beta-D-erythro-pentopyranoses. The conformation of the title compounds was determined by 1H NMR as 1C4 in CDCl3 and the conformation was in accord with that in solid state determined by X-ray crystallographic analysis.     

Transformations of 4- and 17alpha-substituted testosterone analogues by Fusarium culmorum

A series of 4- and/or 17alpha-substituted testosterone analogues has been incubated with the hydroxylating fungus Fusarium culmorum AM282. It was found that 19-norandrostenedione, 19-nortestosterone, 4-methoxytestosterone, 4-methyltestosterone, and 4-chloro-17alpha-methyltestosterone were hydroxylated exclusively or mainly at the 6beta-position. The mixtures of 6beta-, 15alpha-, and 12beta- or 11alpha-monohydroxy derivatives were obtained from 17alpha-methyltestosterone and 17alpha-ethyl-19-nortestosterone--the substrates with alkyl group at C-17alpha. 4-Chlorotestosterone was predominantly hydroxylated at 15alpha-position, but the reaction was accompanied by the reduction of 4-en-3-one system, which proceeded in the sequence: reduction of ketone to 3beta-alcohol and then reduction of the double 4,5 bond. The results obtained indicate an influence of stereoelectronic and steric effects of substitutes on regioselectivity of the hydroxylation of 4-en-3-one steroids by F. culmorum.     

An alpha-glucan elicitor from the cell wall of a biocontrol binucleate Rhizoctonia isolate

Binucleate Rhizoctonia (BNR) isolate (232-C6) is an effective biocontrol agent for protection of potato from Rhizoctonia canker, a disease caused by Rhizoctonia solani. Production of hydrolytic enzymes is one of the best known inducible defense responses following microbial infection. We isolated and characterized a cell wall alpha-glucan from BNR, which induces beta-1,3 glucanase activities in potato sprouts, the primary site of infection by R. solani. An autoclaving method, previously reported for isolation of oligosaccharide elicitors was used, and the glucan purified by chromatographic techniques. Maximal induction of beta-1,3 glucanase activity in potato sprouts was obtained with 250 microg of the alpha-glucan elicitor after 6 days from inoculation time. Both, BNR mycelium and the alpha-glucan produced a similar kinetic response of beta-1,3 glucanase. However, the alpha-glucan did not induce phytoalexin accumulation, previously correlated with the defense response. Uronic acids (approximately 10% with respect to total neutral sugars) were determined and identified as glucuronic acid by high-pH anion-exchange chromatography. Methylation analysis showed that the glucan consists of (1-->3) and (1-->4)-linked glucose units with preponderance of the first ones. Some of the (1-->4) linkages were branched at position 6. The glucan was partially degraded with amyloglucosidase. This, together with the NMR spectra data and the high optical rotation of the original (+195 degrees ) and degraded glucans (+175 degrees ) proved the alpha configuration. Further methylation of the amyloglucosidase degraded glucans indicated that they consist of (1-->3)-linked glucoses. The present study is the first report on the isolation and characterization of an alpha-glucan from Rhizoctonia, that may be important as a biocontrol factor.     

Reaction on D-glucal by an inverting phosphorylase to synthesize derivatives of 2-deoxy-beta-D-arabino-hexopyranosyl-(1-->4)-D-glucose (2II-deoxycellobiose)

Four derivatives of 2(II)-deoxycellobiose were synthesized from d-glucal and acceptor sugars (d-glucose, d-xylose, d-mannose, and 2-deoxy-d-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the beta-(1-->4) linkage of 2-deoxy-d-arabino-hexopyranoside. The acceptor specificity for the d-glucal reaction was identical to that for the alpha-d-glucose 1-phosphate reaction, but the activity of d-glucal was approximately 500 times less than that of alpha-d-glucose 1-phosphate, using 10mM substrates.     

Fluorinated acyclo-C-nucleoside analogues from glycals in two steps

A convenient two-step strategy is reported for the synthesis of fluorinated optically pure acyclo-C-nucleoside analogues starting from simple glycals. In the first step, benzyl- or p-methoxybenzyl-protected glycals are treated with trifluoroacetic anhydride, bromodifluoroacetyl chloride, trichloroacetyl chloride, and perfluorooctanoyl chloride, respectively, in the presence of Et3N. This one-pot procedure yields 1,2-unsaturated sugars (1,5-anhydro-3,4,6-tri-O-benzyl (or p-methoxybenzyl) 2-deoxy-2-perhalogenoacyl-D-arabino / lyxo-hex-1-enitols 4-9) acylated at C-2. In the second step, a selective ring transformation is induced by treatment of the C-acylated glycals with bis-nucleophiles (hydrazine, phenylhydrazine, o-phenylenediamine, hydroxylamine). In particular, 1,5-anhydro-3,4,6-tri-O-benzyl-2-deoxy-2-trifluoroacetyl-D-arabino-hex-1-enitol (4) and 1,5-anhydro-2-deoxy-2-trifluoroacetyl-3,4,6-tri-O-(p-methoxybenzyl)-D-arabino-hex-1-enitol (8) were reacted with these nucleophiles generating the final C-nucleoside analogues of pyrazole (10, 11, and 12), diazepine (13), and isoxazole (15), respectively, containing a carbohydrate side chain linked to the heterocyclic ring.     

Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Structural features of a pectic arabinogalactan with immunological activity from the leaves of Diospyros kaki

A water-soluble acidic heteroglycan, DL-3Bb, isolated from the leaves of Diospyros kaki, had [alpha](D)(20) -19.9 degrees (c 0.30, water), and contained rhamnose, arabinose, xylose, galactose and galacturonic acid in the molar ratio of 1.0:4.5:0.7:1.5:1.0. About 44% of the galacturonic acid existed as its methyl ester, and O-acetyl groups (approx 5.7%) were also identified. Its molecular weight was determined to be 9.0x10(5) Da by high-performance gel-permeation chromatography. Its structural features were elucidated by a combination of methylation analysis, periodate oxidation, two steps of partial acid hydrolysis, and 1H and 13C NMR spectroscopy and ESI mass spectrometry. The data obtained indicated that DL-3Bb possessed a backbone of a disaccharide of [-->4)-alpha-GalAp-(1-->2)-alpha-Rhap-(1-->], with approx 58.7% substitution at O-4 of the rhamnopyranosyl residues by beta-(1-->4)-linked xylopyranosyl residues, and by beta-(1-->3) and beta-(1-->6)-linked galactopyranosyl (galactan) residues. The side chains were further substituted by arabinofuranosyl residues at O-2 by beta-(1-->4)-linked xylopyranosyl residues and at O-3 by beta-(1-->6)-linked galactopyranosyl residues. Preliminary tests in vitro revealed that it could stimulate LPS-induced B lymphocyte proliferation, but not for ConA-induced T lymphocyte proliferation. It was proposed that the acid-labile arabinofuranosyl residues in the side chains would not be needed for the expression of the enhancement of the immunological activity, and that the presence of GalAp in the backbone has an important, but not crucial effect on the expression of the activity.     

Triterpenoids from Sanguisorba officinalis

Seven triterpenoids, i.e., 3beta-[(alpha-L-arabinopyranosyl)oxy]-19beta-hydroxyurs-12,20(30)-dien-28-oic acid (1), 3beta-[(alpha-L-arabinopyranosyl)oxy]-urs-11,13(18)-dien-28-oic acid beta-D-glucopyranosyl ester (2), 2alpha,3alpha,23-trihydroxyurs-12-en-24,28-dioic acid 28-beta-D-glucopyranosyl ester (3), 3beta-[(alpha-L-arabinopyranosyl)oxy]-urs-12,19(20)-dien-28-oic acid (4), 3beta-[(alpha-L-arabinopyranosyl)oxy]-urs-12,19(29)-dien-28-oic acid (5), 3beta-[(alpha-L-arabinopyranosyl)oxy]-19alpha-hydroxyolean-12-en-28-oic acid (6), 2alpha,3beta-dihydroxy-28-norurs-12,17,19(20),21-tetraen-23-oic cid (7), together with three known ones (8-10), were isolated from the roots of Sanguisorba officinalis. Their structures were determined by spectroscopic and chemical methods. Compounds 7 and 10 showed marginal inhibition activity against the growth of tumor cell lines.     

A branched beta-D-(1-->3,1-->6)-glucan from the marine diatom Chaetoceros debilis (Bacillariophyceae) characterized by NMR

The chrysolaminaran from the marine diatom Chaetoceros debilis was isolated and characterized by NMR spectroscopy. Cells were harvested in the stationary phase of growth after the medium had been depleted of nitrate when the chrysolaminaran content was expected to be at its highest. The chrysolaminaran was isolated with an yield of 17.5 mg/L, which corresponds to 15.8 pg/cell. 1H NMR indicated that the structure was similar to that of a beta-(1-->3) main chain with beta-(1-->6)-linked side chains. The degree of polymerization was found to be 30, corresponding to a molecular weight of approximately 4900. Thirty-three percent of the residues were found to be beta-(1-->6)-linked branches. The characteristics of the beta-(1-->6) branching were examined by NOESY NMR, which suggested pustulan-like branches, being beta-(1-->6) linked chains connected to the main chain with few branch points. Confirmation of the 1H NMR data was done by 13C-DEPT, TOCSY, COSY and HMQC NMR spectroscopy. The assignment of the resonances of the main beta-(1-->3) and beta-(1-->6) chains is presented. The structure proposed from our analyses is compared against other chrysolaminaran structures.     

Steroidal glycosides from the rhizomes of Ruscus hypophyllum

Seven steroidal glycosides, along with one known glycoside, were isolated from the rhizomes of Ruscus hypophyllum (Liliaceae). Comprehensive spectroscopic analysis, including 2D NMR spectroscopy, and the results of acid hydrolysis allowed the chemical structures of the compounds to be assigned as (23S,25R)-23-hydroxyspirost-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (1), 1beta-hydroxyspirosta-5,25(27)-dien-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (2), (22S)-16beta,22-dihydroxycholest-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (3), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (4), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3beta-yl beta-d-glucopyranoside (5), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-3beta,22-dihydroxycholest-5-en-1beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-(3,4-di-O-acetyl-beta-d-xylopyranoside) (6), and (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-3beta,22-dihydroxycholest-5-en-1beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-O-[beta-d-xylopyranosyl-(1-->3)]-beta-d-xylopyranoside (7), respectively. This is the first isolation of a series of cholestane glycosides from a Ruscus species.     

The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk)

The physiologically active, gel-forming fraction of the alkali-extractable polysaccharides of Plantago ovata Forsk seed husk (psyllium seed) and some derived partial hydrolysis products were studied by compositional and methylation analysis and NMR spectroscopy. Resolving the conflicting claims of previous investigators, the material was found to be a neutral arabinoxylan (arabinose 22.6%, xylose 74.6%, molar basis; only traces of other sugars). With about 35% of nonreducing terminal residues, the polysaccharide is highly branched. The data are compatible with a structure consisting of a densely substituted main chain of beta-(1-->4)-linked D-xylopyranosyl residues, some carrying single xylopyranosyl side chains at position 2, others bearing, at position 3, trisaccharide branches having the sequence L-Araf-alpha-(1-->3)-D-Xylp-beta-(1-->3)-l-Araf. The presence of this sequence is supported by methylation and NMR data, and by the isolation of the disaccharide 3-O-beta-D-xylopyranosyl-L-arabinose as a product of partial acid hydrolysis of the polysaccharide.     

Production of a new sucrose derivative by transglycosylation of recombinant Sulfolobus shibatae beta-glycosidase

The gene encoding beta-glycosidase of the hyperthermophilic archaea Sulfolobus shibatae (SSG) was expressed in Escherichia coli. Recombinant SSG (referred to as rSSG hereafter) was efficiently purified, and its transglycosylation activity was tested with lactose as a donor and various sugars as acceptors. When sucrose was used as an acceptor, we found a distinct intermolecular transglycosylation product and confirmed its presence by TLC and high performance anion exchange chromatography (HPAEC). The sucrose transglycosylation product was isolated by paper chromatography, and its chemical structure was determined by 1H and 13C NMR. The sucrose transfer product was determined to be beta-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-beta-d-fructofuranoside with a galactose molecule linked to sucrose via a beta-(1-->6)-glycosidic bond.     

Rhamnogalacturonan I in Solanum tuberosum tubers contains complex arabinogalactan structures

A rhamnogalacturonan I polysaccharide was isolated from potato (Solanum tuberosum cv. Posmo) tuber cell walls and characterised by enzymatic digestion with an endo-beta-1 --> 4-galactanase and an endo-alpha-1 --> 5-arabinanase, individually or in combination. The reaction products were separated using size-exclusion chromatography and further analysed for monosaccharide composition and presence of epitopes using the LM5 anti-beta-1 --> 4-galactan and LM6 anti-alpha-1 --> 5-arabinan monoclonal antibodies. The analyses point to distinct structural features of potato tuber rhamnogalacturonan I, such as the abundance of beta-1 --> 4-galactan side chains that are poorly substituted with short arabinose-containing side chains, the presence of alpha-1 --> 5-arabinan side chains substituted with beta-1 --> 4-galactan oligomers (degree of polymerisation > 4), and the presence of alpha-1 --> 5-arabinans that resist enzymatic degradation. A synergy between the enzymes was observed towards the degradation of arabinans but not towards the degradation of galactans. The effect of the enzymes on isolated RG I is discussed in relation to documented effects of enzymes heterologously expressed in potato tubers. In addition, a novel and rapid method for the determination of the monosaccharide and uronic acid composition of cell wall polysaccharides using high-performance anion exchange chromatography with pulsed amperometric detection is described.     

Cholinesterase inhibitory pregnane-type steroidal alkaloids from Sarcococca hookeriana

The bioassay-guided phytochemical investigation on Sarcococca hookeriana have resulted in the isolation of four new pregnane-type steriodal alkaloids: hookerianamide-D [(2'E,20S)-20-(N,N-formyl(methyl)amino)-3beta-(3',4'-dimethyl-2'-pentenamido)-5alpha-pregnane] (1), hookerianamide-E [(2'E,20S)-20-(N,N-dimethylamino)-3beta-(senecioylamino)-5alpha-pregn-14-en-2beta-O-acetate] (2), hookerianamide-F [(2'E,20S)-20-(N-methylamino)-3beta-(tigloylamino)-5alpha-pregn-2,14-dien-4-one] (3), and hookerianamide-G [(20S)-20-(N,N-dimethylamino)-3beta-(N-methylbenzamido)-5alpha-pregn-4beta-O-acetate] (4), along with five known alkaloids 5-9. Their structures were determined by spectroscopic analysis. These steroidal alkaloids and chemically derived derivatives of compound 5 have displayed varying degree of inhibitory activities against acetylcholinesterase and butyrylcholinesterase enzymes in a concentration-dependent fashion, with the IC(50) values ranging from 1.5 to 148.2 and 0.6 to 100.2 microM, respectively.     

Structures of polysaccharides and oligosaccharides of some Gram-negative marine Proteobacteria

The chemical structures of polysaccharides and LPS core oligosaccharides, isolated from various Gram-negative marine bacteria from the genera Pseudoalteromonas and Shewanella belonging to the Alteromonadaceae family and gamma-subclass of Proteobacteria, are reviewed. The polysaccharides are distinguished by the acidic character (e.g., due to the presence of hexuronic and aldulosonic acids and their derivatives) and the occurrence of unusual sugars, including N-acyl derivatives of 6-deoxyamino sugars, such as N-acetyl-D-quinovosamine, N-acetyl-L-fucosamine and N-acetyl-6-deoxy-L-talosamine, and higher sugars like 2,6-dideoxy-2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-D-galactopyranose (shewanellose). Many constituent sugars have various uncommon non-sugar substituents, such as alanine, formic, lactic and hydroxybutyric acids, sulfate, phosphate, and 2-aminopropane-1,3-diol.     

Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A beta-glucosidase

The nonnucleophilic mutant E383A beta-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of alpha-glucosyl and alpha-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 degrees C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pKa of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, beta-(1-->3) disaccharides are obtained in good to excellent yields, thus expanding the synthetic products available with current exo-glycosynthases. With xylopyranosyl acceptor, regioselectivity is poorer and results in the formation of a mixture of beta-(1-->3) and beta-(1-->4) linkages. In contrast, disaccharide acceptors produce exclusively beta-(1-->4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from beta-(1-->3) to beta-(1-->4). To improve operational performance, the E383A mutant was immobilized on a Ni2+-chelating Sepharose resin. Immobilization did not increase stability to pH and organic solvents, but the operational stability and storage stability were clearly enhanced for recycling and scaling-up.     

Purification and characterization of an endo-beta-(1-->6)-galactanase from Trichoderma viride

An endo-beta-(1-->6)-galactanase from Onozuka R-10, a commercial cellulase preparation from Trichoderma viride, was purified 57-fold. Apparent Mr values of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were 47,000 and 17,000, respectively. The enzyme was assayed with a galactan from Prototheca zopfii, which has a high proportion of beta-(1-->6)-linked galactosyl residues. It exhibited maximal activity toward the galactan at pH 4.3. The enzyme hydrolyzed specifically beta-(1-->6)-galactooligosaccharides with a degree of polymerization higher than 3 and their acidic derivatives with 4-O-methyl-glucosyluronic or glucosyluronic groups at the nonreducing terminals. The methyl beta-glycoside of beta-(1-->6)-galactohexaose was degraded to reducing galactooligomers with a degree of polymerization 2-5 as the products at the initial stage of hydrolysis, and galactose and galactobiose at the final stage, indicating that the enzyme can be classified as an endo-galactanase. The extent of hydrolysis of the carbohydrate portion of a radish root arabinogalactan-protein (AGP) increased when alpha-L-arabinofuranosyl residues attached to beta-(1-->6)-linked galactosyl side chains of the AGP were removed in advance. The enzyme released galactose, beta-(1-->6)-galactobiose, and 4-O-methyl-beta-glucuronosyl-(1-->6)-galactose as major hydrolysis products when allowed to act exhaustively on the modified AGP.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.     

Synthesis of C-glycopyranosylphloroacetophenone derivatives and their anomerization facilitated by 1,3-diaxial interactions

The reaction of 2,3,4-tri-O-benzyl-6-deoxy-alpha-D-glucopyranosyl fluoride, 2,3,4,6-tetra-O-benzyl-alpha-D-allopyranosyl fluoride, and 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl fluoride with 2,4-di-O-benzylphloroacetophenone, in the presence of boron trifluoride diethyl etherate, afforded, respectively, the corresponding 3-C-beta-D-glycopyranosylphloroacetophenone derivatives exclusively in anomerically pure form. Alternatively, the reaction of 2,3,4,6-tetra-O-benzyl-alpha-D-gulopyranosyl fluoride with 2,4-di-O-benzylphloroacetophenone afforded both the 3-C-beta-D-gulopyranosylphloroacetophenone derivative (4C(1) conformation) as the major product and the 3-C-alpha-D-gulopyranosylphloroacetophenone derivative (1C(4) conformation) as the minor product under identical conditions. Including the previously prepared C-glycosylphloroacetophenone derivatives that contain 3-C-beta-D-glucosyl, 3-C-beta-D-xylosyl, 3-C-beta-2-deoxy-D-arabino-hexosyl, 3-C-beta-D-galactosyl, 3-C-beta-L-arabinosyl, and 3-C-alpha-L-arabinosyl moieties, the conformation is dictated primarily by the preference of the bulky aromatic aglycon to orient equatorially, due to the strong repulsion of the aglycon. The anomerization is directed secondarily by the presence of 1,3-diaxial interactions in the sugar moiety.     

An acetylated galactoglucomannan from Picea abies L. Karst

A water-soluble galactoglucomannan composed of D-galactose, D-glucose, and D-mannose in 1:3:17 mole proportion has been isolated from the secondary cell walls of Picea abies L. Karst. About 33% of the polysaccharide units were substituted by acetyl groups. Structural studies of the polymer indicated a beta-(1-->4)-linked glucomannopyranosyl backbone with a low content of branch points at O-6 of mannosyl and glucosyl residues. A preference for mannosyl groups indicates the presence of a single D-galactosyl unit side-chain. About half of the mannose residues were O-acetylated at C-2 and C-3 in 1.7:1 mole proportion.