Crystal structures of rare disaccharides, α-d-glucopyranosyl β-d-psicofuranoside, and α-d-galactopyranosyl β-d-psicofuranoside

The crystal structures of α-d-glucopyranosyl β-d-psicofuranoside and α-d-galactopyranosyl β-d-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R₁ = 0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between ⁴E and ⁴T₃. Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

Physicochemical and biological properties of 2-O-α-d-galactosyl-cyclomaltohexaose (α-cyclodexterin) and -cyclomaltoheptaose (β-cyclodextrin)

The physicochemical and biological properties of the new branched cyclomaltooligosaccharides (cyclodextrins; CDs), 2-O-α-d-galactosyl-cyclomaltohexaose (2-O-α-d-galactosyl-α-cyclodextrin, 2-Gal-αCD) and 2-O-α-d-galactosyl-cyclomaltoheptaose (2-O-α-d-galactosyl-β-cyclodextrin, 2-Gal-βCD), were investigated. The formation of inclusion complexes of 2-Gal-CDs with various kinds of guest compounds (clofibrate, cholesterol, cholecalciferol, digitoxin, digitoxigenin, and prostaglandin A₁) was examined by a solubility method, and the results were compared with those of non-branched CDs and other 6-O-glycosyl-CDs such as 6-O-α-d-galactosyl-CDs, 6-O-α-d-glucosyl-CDs, and 6-O-α-maltosyl-CDs. The inclusion abilities of 2-Gal-αCD for clofibrate and prostaglandin A₁, and 2-Gal-βCD for clofibrate, cholecalciferol, cholesterol, and digitoxigenin were markedly weaker than those of non-branched CD and other 6-O-glycosyl-CDs in each series, probably because of a steric hindrance caused by the α-(1→2)-galactoside linkage. The hemolytic activities of 2-Gal-CDs on human erythrocytes were the lowest among each CD series, and the compounds showed negligible cytotoxicity towards Caco-2 cells up to at least 100 mM.     

A facile synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol

A facile and short synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol (1) has been achieved in high yield starting from 4,5-epoxycyclohex-1-ene by using a catalytic asymmetric allylic oxidation reaction.     

Studies on the stereoselective synthesis of a protected α-d-Gal-(1→2)-d-Glc fragment

A novel 1,2-cis stereoselective synthesis of protected α-d-Gal-(1→2)-d-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-d-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-d-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-d-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-d-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH₃ at C-1 and benzoyl groups at C-4 and C-6.     

Characterisation of oligosaccharides from the chondroitin/dermatan sulphates: H and C NMR studies of oligosaccharides generated by nitrous acid depolymerisation

The polymers chondroitin sulphate and dermatan sulphate have been fragmented by an anhydrous hydrazine/nitrous acid procedure. The resulting disaccharides from the polymer repeat sequences were reduced with NaBH₄ and purified by ion exchange chromatography. Whereas enzymatic depolymerisation leads to the loss of the distinction between glucuronic and iduronic acids of CS and DS in the resultant disaccharides, nitrous acid depolymerisation retains these structures. Complete ¹H and ¹³C NMR data have been derived for the major components which were shown to have the structures: GlcA-(β1→3)-anTal6S-ol (I) and l-IdoA-(α1→3)-anTal4S-ol (II), where anTal-ol represents (2,5)anhydro-d-talitol and 6S/4S represent O-ester sulphate groups at C-6/C-4 sites.     

Solvent effect on enzyme-catalyzed synthesis of β-d-glucosides using the reverse hydrolysis method: Application to the preparative-scale synthesis of 2-hydroxybenzyl and octyl β-d-glucopyranosides

Almond β-d-glucosidase was used to catalyze alkyl-β-d-glucoside synthesis by reacting glucose and the alcohol in organic media. The influence of five different solvents and the thermodynamic water activity on the reaction have been studied. The best yields were obtained in 80 or 90% (v/v) tert-butanol, acetone, or acetonitrile where the enzyme is very stable. In this enzymatic synthesis under thermodynamic control, the yield increases as the water activity of the reaction medium decreases. Enzymatic preparative-scale syntheses were performed in a tert-butanol-water mixture which was found to be the most appropriate medium. 2-Hydroxybenzyl β-d-glucopyranoside was obtained in 17% yield using a 90:10 (v/v) tert-butanol-water mixture. Octyl-β-glucopyranoside was obtained in 8% yield using a 60:30:10 (v/v) tert-butanol-octanol-water mixture.     

A direct enzymatic synthesis of β-d-galactopyranosyl-d-xylopyranosides and their use to evaluate rat intestinal lactase activity in vivo

By enzymatic β-d-galactosylation of d-xylose a mixture of 4-, 3-, and (1, 4, and 7, respectively) was obtained in 50% isolated yield. Disaccharides 1, 4, and 7 are substrates of intestinal lactase isolated from lamb small intestine with K_m values of 250.0, 4.5, and 14.0 mM, respectively. The mixture was used to monitor the normal decline in lactase activity in rats that takes place after weaning. The data obtained by this method correlated with the levels of intestinal lactase activity in the same animals after being sacrificed.     

New microbial mannan catabolic pathway that involves a novel mannosylglucose phosphorylase

The consecutive genes BF0771–BF0774 in the genome of Bacteroidesfragilis NCTC 9343 were found to constitute an operon. The functional analysis of BF0772 showed that the gene encoded a novel enzyme, mannosylglucose phosphorylase that catalyzes the reaction, 4-O-β-d-mannopyranosyl-d-glucose + Pi → mannose-1-phosphate + glucose. Here we propose a new mannan catabolic pathway in the anaerobe, which involves 1,4-β-mannanase (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772), finally progressing to glycolysis. This pathway is distributed in microbes such as Bacteroides, Parabacteroides, Flavobacterium, and Cellvibrio.     

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-l-glycero-d-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP⁺, instead of NAD⁺, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Facile syntheses of the disaccharide repeating unit of the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b and its dimer and trimer

A highly efficient strategy for the preparation of a disaccharide-repeating unit of the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b, and its dimer and trimer, has been developed through a regio- and stereoselective manner using p-methoxylphenyl 2,4,6-tri-O-benzoyl-α-d-glucopyranoside and 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-6-deoxy-α-l-talopyranosyl trichloroacetimidate as the key synthons. The target molecules were equipped with a p-methoxylphenyl handle at the reducing terminus to allow for their further functionalization and attachment to a carrier protein.     

Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage

Hydroxytyrosol (2-(3′,4′-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3′-O-β-d-glucuronide and 4′-O-β-d-glucuronide derivatives and 2-(3′,4′-dihydroxyphenyl)ethanol-1-O-β-d-glucuronide, in comparison with the parent compound, to inhibit H₂O₂ induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H₂O₂ treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.     

An improved synthetic approach to 7-[3-amino-4-O-(α-l-mycarosyl)-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone and its interaction with human serum albumin

An improved synthetic approach to 7-[3-amino-4-O-(α-l-mycarosyl)-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone (α1) with high stereoselectivity and good yield was developed. The feature of its binding to human serum albumin (HSA) was also investigated under simulative physiological conditions via fluorescence and UV–vis absorption spectroscopy and molecular modeling methods. The results revealed that α1 caused the fluorescence quenching of HSA by the formation of α1–HSA complexes. Hydrophobic interactions played a major role in stabilizing the complex, which was in good agreement with the results of the molecular modeling study. In addition, the effect of common ions on the binding constants of α1–HSA complexes at room temperature was also discussed. All the experimental results and theoretical data indicated that α1 bound to HSA and was effectively transported and eliminated in the body. Such findings may provide useful guidelines for further drug design.     

Structural studies of the O-specific polysaccharide from the lipopolysaccharide of Mesorhizobium huakuii strain S-52, the symbiotic partner of Astragalus sinicus

The O-specific polysaccharide (OPS) obtained by mild-acid degradation of the lipopolysaccharide isolated from Mesorhizobium huakuii strain S-52 was studied by sugar and ethylation analyses along with ¹H and ¹³C NMR spectroscopy. It was concluded that the OPS was composed of trisaccharide repeating units containing two residues of 6-deoxy-l-talose (6dTal) and one l-rhamnose (Rha), whose sequence in the OPS was determined by NOESY and HMBC experiments. The minor 3-O-acetylation (about 10%) of 6-deoxytalose glycosidically substituted at position-2 was judged by relative signal intensities of corresponding O-acetylated and non-acetylated 6dTal residues. Moreover, it was found that the non-reducing end of the OPS repeating unit was occupied by 3-O-methyl-d-fucose, which terminated the O-chain as a cap-residue. These data defined the structure of the OPS as:α-3-OMe-d-Fucp-(1→[2)-α-l-6dTalp-(1→3)-α-l-6dTalp-(1→2)-α-l-Rhap-(1→]_n     

Vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-d-glucopyranoside from the leaves of Diospyros Kaki stimulates the glucose uptake in HepG2 and 3T3-L1 cells

A novel α-glucosidase inhibitor, vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-d-glucopyranoside, was isolated for the first time from leaves of Diospyros Kaki and its bioactivity analyzed. This inhibitor exhibited strong anti-α-glucosidase activity with an IC₅₀ value of 170.62 nM and stimulated a dose-dependent increase in the uptake of a fluorescent d-glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), in HepG2 cells at a rate higher than that of insulin controls. It was also found to be associated with adipocyte differentiation and moderate increases in 2-NBDG uptake by 3T3-L1 cells. These findings suggest that vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-d-glucopyranoside could augment peripheral glucose as an insulin-sensitizing agent against Type 2 diabetes mellitus.     

A novel plastidial lipoxygenase of maize (Zea mays) ZmLOX6 encodes for a fatty acid hydroperoxide lyase and is uniquely regulated by phytohormones and pathogen infection

Lipoxygenases (LOXs) are members of a large enzyme family that catalyze oxygenation of free polyunsaturated fatty acids into diverse hydroperoxide compounds, collectively called oxylipins. Although LOXs have been well studied in dicot species, reports of the genes encoding these enzymes are scarce for monocots, especially maize. Herein, we reported the cloning, characterization and molecular functional analysis of a novel maize LOX gene, ZmLOX6. The ZmLOX6 nucleotide sequence encodes a deduced translation product of 892 amino acids. Phylogenetic analysis showed that ZmLOX6 is distantly related to previously reported 9- or 13-LOXs from maize and other plant species, including rice and Arabidopsis. Although sequence prediction suggested cytoplasmic localization of this protein, ZmLOX6 protein has been reportedly isolated from mesophyll cell chloroplasts, emphasizing the unique features of this protein. Plastidial localization was confirmed by chloroplast uptake experiments with the in vitro translated protein. Analysis of recombinant protein revealed that ZmLOX6 has lost fatty acid hydroperoxide forming activity but 13-LOX-derived fatty acid hydroperoxides were cleaved into odd-chain ω-oxo fatty acids and as yet not identified C5-compound. In line with its reported abundance in mesophyll cells, ZmLOX6 was predominantly expressed in leaf tissue. Northern blot analysis demonstrated that ZmLOX6 was induced by jasmonic acid, but repressed by abscisic acid, salicylic acid and ethylene and was not responsive to wounding or insects. Further, this gene was strongly induced by the fungal pathogen Cochliobolus carbonum during compatible interactions, suggesting that ZmLOX6 may contribute to susceptibility to this pathogen. The potential involvement of ZmLOX6 in maize interactions with pathogens is discussed.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

A facile synthesis of 5-thio-l-fucose and 5-thio-d-arabinose from d-arabinose

5-Thio-l-fucopyranose tetraacetate was synthesized in 11 steps from or d-arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a derivative was achieved to give the corresponding 6-deoxy-β-d-altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy-d-altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio-l-fucopyranose. A derivative was also prepared from 6-deoxy-β-d-altrofuranose derivatives. 5-Thio-d-arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio-l-fucose, was also synthesized from in 5 steps. 5-Thio-d-arabinose showed weak inhibitory activity against α-l-fucosidase from bovine kidney (K_{i = 0.77 mM}).     

Focused directed evolution of β-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening

Here we present an optimized procedure to generate amino acid variations at specific site(s) of proteins, followed by a simple one-step screen for mutants with the desired β-glucosidase activity.The procedure was evaluated by introducing sequence variation into a codon specifying a non-functional variant of the catalytic nucleophile (E401) of the maize β-glucosidase Zm-p60.1. Observed and theoretically expected frequencies of the four possible variants of the codon and the two possible phenotypes (functional and non-functional) were investigated. Deviations in codon and phenotype frequencies were expressed as a coefficient. This coefficient was then used to estimate the extent of oversampling, of the mutant library, which would be necessary to compensate for the underrepresentation of some sequences. This evaluation of the overall performance of the method allows experimentally derived parameters to be incorporated into mutant library design. This method combines the application of a well-defined distribution of variability with a reliable screening process. Thus, it facilitates the production of novel functional variants of β-glucosidases for either fundamental studies or potential biotechnological applications.