C-prolinylquercetins from the yellow cocoon shell of the silkworm, Bombyx mori

Two flavonoids containing the l-proline moiety, 6-C-[(2S,5S)-prolin-5-yl] quercetin (prolinalin A) and 6-C-[(2S,5R)-prolin-5-yl] quercetin (prolinalin B), were isolated from the cocoon shell of the silkworm, Bombyx mori. Their structural elucidation was achieved by application of acid hydrolysis and spectroscopic methods. These compounds were not found in the leaves of mulberry (Morus alba L.), the host plant of the silkworm, suggesting that the flavonoids are metabolites of the insect. This is the first time that flavonoids with an amino acid moiety have been found as naturally occurring compounds.     

Reexamination of phenoloxidase in larval circulating hemocytes of the silkworm, Bombyx mori

We have developed a modified method to detect phenoloxidase activity on hemocytes by using freshly prepared l-DOPA (1 mg/ml in 35% ethanol) to fix and incubate larval hemocytes. This method is more sensitive than the common method, in which hemocytes were fixed in 4% formaldehyde and then incubated with 2 mg/ml l-DOPA in water separately. Phenoloxidase assayed using this modified method can be inhibited by phenyltiourea (phenoloxidase inhibitor). After incubation with l-DOPA solution in ethanol, most prohemocytes, all plasmatocytes and young granulocytes are stained brown due to oxidation of l-DOPA into pigments, indicating that they have phenoloxidase. Oenocytoids are dimly stained because many of their cell inclusions have been released during the treatment. Large propidium-iodide-negative prohemocytes have strong phenoloxidase activity and are easily misunderstood as propidium-iodide-positive oenocytoids if the fluorescent method is not used for identification. Thus, in addition to oenocytoids and plasmatocytes, some prohemocytes and granulocytes in the silkworm also have phenoloxidase.     

Efficient synthesis of 5-thio-D-arabinopyranose and 5-thio-D-xylopyranose from the corresponding D-pentono-1,4-lactones

5-Thio-D-arabinopyranose (5) and 5-thio-D-xylopyranose (10) were synthesized from the corresponding D-pentono-1,4-lactones. After regioselective bromination at C-5, transformation into 5-S-acetyl-5-thio derivatives, reduction into lactols and deprotection afforded the title compounds in 49 and 42% overall yield, respectively.     

Aromatic compound glucosides,alkyl glucoside and glucide from the fruit of anise

From the polar portion of the methanolic extract of the fruit of anise (Pimpinella anisum L.), which has been used as a spice and medicine since antiquity, four aromatic compound glucosides, an alkyl glucoside and a glucide were isolated together with 24 known compounds. The structures of the new compounds were clarified as (E)-3-hydroxyanethole beta-D-glucopyranoside, (E)-1'-(2-hydroxy-5-methoxyphenyl)propane beta-D-glucopyranoside, 3-hydroxyestragole beta-D-glucopyranoside, methyl syringate 4-O-beta-D-glucopyranoside, hexane-1,5-diol 1-O-beta-D-glucopyranoside and 1-deoxy-L-erythritol 3-O-beta-D-glucopyranoside by spectral investigation.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

Sesquiterpene lactone glucosides and alkyl glycosides from the fruit of cumin

From the polar portion of the methanolic extract of cumin (fruit of Cuminum cyminum L.), two sesquiterpenoid glucosides, cuminoside A and B, and two alkyl glycosides were isolated together with five known compounds. Their structures were established as (1S,5S,6S,10S)-10-hydroxyguaia-3,7(11)-dien-12,6-olide beta-D-glucopyranoside, (1R,5R,6S,7S,9S,10R,11R)-1,9-dihydroxyeudesm-3-en-12,6-olide 9-O-beta-D-glucopyranoside, methyl beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside and ethane-1,2-diol 1-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, respectively.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Evaluation of carbohydrate molecular mechanical force fields by quantum mechanical calculations

A quantitative evaluation of 20 second-generation carbohydrate force fields was carried out using ab initio and density functional methods. Geometry-optimized structures (B3LYP/6-31G(d)) and relative energies using augmented correlation consistent basis sets were calculated in gas phase for monosaccharide carbohydrate benchmark systems. Selected results are: (i). The interaction energy of the alpha-d-glucopyranose.H(2)O heterodimer is estimated to be 4.9 kcal/mol, using a composite method including terms at highly correlated (CCSD(T)) level. Most molecular mechanics force fields are in error in this respect; (ii). The (3)E envelope (south) pseudorotational conformer of methyl 5-deoxy-beta-d-xylofuranoside is 0.66 kcal/mol more stable than the (3)E envelope (north) conformer and the alpha-anomer of methyl d-glucopyranoside is 0.82 kcal/mol more stable than the beta-anomer; (iii). The relative energies of the (gg, gt and tg) rotamers of methyl alpha-d-glucopyranoside and methyl alpha-d-galactopyranoside are (0.13, 0.00, 0.15) and (0.64, 0.00, 0.77) kcal/mol, respectively. The results of the quantum mechanical calculations are compared with the results of calculations using the 20 second-generation carbohydrate force fields. No single force field is consistently better than the others for all the test cases. A statistical assessment of the performance of the force fields indicates that CHEAT(95), CFF, certain versions of Amber and of MM3 have the best overall performance, for these gas phase monosaccharide systems.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

An improved synthesis of 5-thio-D-ribose from D-ribono-1,4-lactone

5-Thio-D-ribopyranose was synthesized from D-ribono-1,4-lactone (1) by two approaches: (i) 5-bromo-5-deoxy-D-ribono-1,4-lactone (2) was successively transformed into 5-bromo-5-deoxy, 5-S-acetyl-5-thio or 5-thiocyanato-D-ribofuranose derivatives; appropriate treatment then lead to 5-thio-D-ribopyranose (7) in 46-48% overall yield and; (ii) 2 was transformed into the 5-S-acetyl-5-thio-D-ribono-1,4-lactone derivative (11). Reduction and deprotection of 11 afforded 5-thio-D-ribopyranose (7) in 57% overall yield.     

Optimized synthesis of specific sizes of maltodextrin glycosides by the coupling reactions of Bacillus macerans cyclomaltodextrin glucanyltransferase

Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.19), in reaction with cyclomaltohexaose and methyl alpha-D-glucopyranoside, methyl beta-D-glucopyranoside, phenyl alpha-D-glucopyranoside, and phenyl beta-D-glucopyranoside gave four kinds of maltodextrin glycosides. The reactions were optimized by using different ratios of the individual d-glucopyranosides to cyclomaltohexaose, from 0.5 to 5.0, to obtain the maximum molar percent yields of products, which were from 68.3% to 78.6%, depending on the particular D-glucopyranoside, and also to obtain different maltodextrin chain lengths. The lower ratios of 0.5-1.0 gave a wide range of sizes from d.p. 2-17 and higher. As the molar ratio was increased from 1.0 to 3.0, the larger sizes, d.p. 9-17, decreased, and the small and intermediate sizes, d.p. 2-8, increased; as the molar ratios were increased further from 3.0 to 5.0, the large sizes completely disappeared, the intermediate sizes, d.p. 4-8, decreased, and the small sizes, d.p. 2 and 3 became predominant. A comparison is made with the synthesis of maltodextrins by the reaction of CGTase with different molar ratios of d-glucose to cyclomaltohexaose.     

Glycosylation of l,4:3,6-dianhydro-D-glucitol (isosorbide)

Condensation of 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl-, 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl- and of 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromides with l,4:3,6-dianhydro-D-glucitol under Koenigs-Knorr conditions, and using the Helferich modification of the reaction showed regioselectivity in glysosylation at C-5 of isosorbide.     

The Fermentative Formation of CDP-Choline by Haploid Cells of Yeasts and the Change of the Temperature-sensitivity of a Mutant of a Yeast,Saccharomyces rouxii

Phosphorylation of CMP and formation of CDP-choline were tested with various haploid cells of yeasts. Most of them had more or less the ability, but a mutant (Lys–M7, alpha type) of Saccharomyces rouxii was found to lack the ability. Further study revealed the change of the temperature-sensitivity of the mutant, which could not produce CDP-choline when treated at 37°C, whereas it could at 16°C. The growth of the mutant was more sensitive to temperatures than that of the wild strain. The former did not grow at 36.3°C, while the latter grew.     

Iridoid glycosides from Gardeniae Fructus for treatment of ankle sprain

The iridoid glycosides, genipin 1-O-β-d-isomaltoside (1) and genipin 1,10-di-O-β-d-glucopyranoside (2), together with six known iridoid glycosides, genipin 1-O-β-d-gentiobioside (3), geniposide (4), scandoside methyl ester (5), deacetylasperulosidic acid methyl ester (6), 6-O-methyldeacetylasperulosidic acid methyl ester (7), and gardenoside (8) were isolated from an EtOH extract of Gardeniae Fructus. The structures and relative stereochemistries of the metabolites were elucidated on the basis of 1D- and 2D-NMR spectroscopic techniques, high-resolution mass spectrometry, and chemical evidence. Geniposide (4), one of the main compounds of Gardeniae Fructus, was tested for treatment of ankle sprain using an ankle sprain model in rats. From the second to fifth day, the geniposide (4) (100 mg/ml) treated group exhibited significant differences (p < 0.01) with ∼21-34% reduction in swelling ratio compared with those of the vehicle treated control group. This indicated the potential effect of geniposide (4) for the treatment of disorders such as ankle sprain.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Facile synthesis of benzyl beta-D-galactofuranoside. A convenient intermediate for the synthesis of D-galactofuranose-containing molecules

Benzyl beta-D-galactofuranoside was efficiently obtained from 1,2,3,5,6-penta-O-benzoyl-alpha,beta-D-galactofuranose, via benzyl 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranoside. Conditions for the O-debenzylation were investigated in order to evaluate the synthetic application of the benzyl group as an anomeric protector of a galactofuranose moiety in synthetic strategies involving galactofuranose.     

Melting behaviour of D-sucrose, D-glucose and D-fructose

The melting behaviour of d-sucrose, d-glucose and d-fructose was studied. The melting peaks were determined with DSC and the start of decomposition was studied with TG at different rates of heating. In addition, melting points were determined with a melting point apparatus. The samples were identified as d-sucrose, alpha-d-glucopyranose and beta-d-fructopyranose by powder diffraction measurements. There were differences in melting between the different samples of the same sugar and the rate of heating had a remarkable effect on the melting behaviour. For example, T(o), DeltaH(f) and T(i) (initial temperature of decomposition) at a 1 degrees Cmin(-1) rate of heating were 184.5 degrees C, 126.6Jg(-1) and 171.3 degrees C for d-sucrose, 146.5 degrees C, 185.4Jg(-1) and 152.0 degrees C for d-glucose and 112.7 degrees C, 154.1Jg(-1) and 113.9 degrees C for d-fructose. The same parameters at 10 degrees Cmin(-1) rate of heating were 188.9 degrees C, 134.4Jg(-1) and 189.2 degrees C for d-sucrose, 155.2 degrees C, 194.3Jg(-1) and 170.3 degrees C for d-glucose and 125.7 degrees C, 176.7Jg(-1) and 136.8 degrees C d-fructose. At slow rates of heating, there were substantial differences between the different samples of the same sugar. The melting point determination is a sensitive method for the characterization of crystal quality but it cannot be used alone for the identification of sugar samples in all cases. Therefore, the melting point method should be validated for different sugars.     

Isolation and structure elucidation of 5′-O-β-d-glucopyranosyl-dihydroascorbigen from Cardamine diphylla rhizome

From the methanol extract of Cardamine diphylla rhizome, 5′-O-β-d-glucopyranosyl-dihydroascorbigen (1) and 6-hydroxyindole-3-carboxylic acid 6-O-β-d-glucopyranoside (2) were isolated. The structures of the compounds were elucidated using spectroscopic methods. This is the second report on the presence of a glucosylated indole ascorbigen in plants.     

Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thermatoga maritima

Fe-only hydrogenases contain a di-iron active site complex, in which the two Fe atoms have carbon monoxide and cyanide ligands and are linked together by a putative di(thiomethyl)amine molecule. We have cloned, purified and characterized the HydE and HydG proteins, thought to be involved in the biosynthesis of this peculiar metal site, from the thermophilic organism Thermotoga maritima. The HydE protein anaerobically reconstituted with iron and sulfide binds two [4Fe-4S] clusters, as characterized by UV and EPR spectroscopy. The HydG protein binds one [4Fe-4S] cluster, and probably an additional one. Both enzymes are able to reductively cleave S-adenosylmethionine (SAM) when reduced by dithionite, confirming that they are Radical-SAM enzymes.