Synthesis of 4-O-glycosylated 1,5-anhydro-d-fructose and of 1,5-anhydro-d-tagatose from a common intermediate 2,3-O-isopropylidene-d-fructose

Four novel disaccharides of glycosylated 1,5-anhydro-d-ketoses have been prepared: 1,5-anhydro-4-O-β-d-glucopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-galactopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-glucopyranosyl-d-tagatose, and 1,5-anhydro-4-O-β-d-galactopyranosyl-d-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-β-d-fructopyranose, was prepared from d-fructose and was converted into the d-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

Stereoselective entry into the d-GalNAc series starting from the d-Gal one: a new access to N-acetyl-d-galactosamine and derivatives thereof

A new stereoselective preparation of N-aceyl-d-galactosamine (1b) starting from the known p-methoxyphenyl 3,4-O-isopropylidene-6-O-(1-methoxy-1-methylethyl)-β-d-galactopyranoside (10) is described using a simple strategy based on (a) epimerization at C-2 of 10 via oxidation-reduction to give the talo derivative 11, (b) amination with configurational inversion at C-2 of 11 via a S_N2-type reaction on its 2-imidazylate, (c) anomeric deprotection of the p-methoxyphenyl β-d-galactosamine glycoside 14, (d) complete deprotection. Applying the same protocol to 2,3:5,6:3′,4′-tri-O-isopropylidene-6′-O-(1-methoxy-1-methylethyl)-lactose dimethyl acetal (4), directly obtained through acetonation of lactose, the disaccharide β-d-GalNAcp-(1→4)-d-Glcp (1a) was obtained with complete stereoselectivity in good (40%) overall yield from lactose.     

Kinetically controlled Ferrier rearrangement of 3-O-mesyl-d-glycal derivatives

The Ferrier rearrangement, which is widely used in carbohydrate chemistry, is generally performed under acidic conditions to give an α anomer with high stereoselectivity. We have found that 3-O-mesyl-d-glycals 2-4 were smoothly reacted with alcohols in the presence of triethylamine. The present reaction was shown to proceed under kinetic control to give ∼1.3:1.0 mixture of α and β anomers, indicating that a kinetic anomeric effect does not operate.     

Synthesis and antitumor activity of new d-seco and d-homo androstane derivatives

Starting from 3β-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4-9 were synthesized. On the other hand, 3β-hydroxy-17-oxa-d-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, prostate cancer AR−, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC₅₀ values being 2 μM and 0.55 μM, respectively. Compounds 6 (10 μM) and 14 (9 μM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1-3, 5-8, 10 and 12-15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.     

Isolation and structure of d-xylans from pericarp seeds of Opuntia ficus-indica prickly pear fruits

Xylans were isolated from the pericarp of prickly pear seeds of Opuntia ficus-indica (OFI) by alkaline extraction, fractionated by precipitation and purified. Six fractions were obtained and characterized by sugar analysis and NMR spectroscopy. They were assumed to be (4-O-methyl-d-glucurono)-d-xylans, with 4-O-α-d-glucopyranosyluronic acid groups linked at C-2 of a (1→4)-β-d-xylan. The sugar composition and the ¹H and ¹³C NMR spectra showed that their chemical structures were very similar, but with different proportions of d-Xyl and 4-O-Me-d-GlcA. Our results showed that, on average, the water soluble xylans have one nonreducing terminal residue of 4-O-methyl-d-glucuronic acid for every 11 to 14 xylose units, whereas in the water non-soluble xylans, xylose units can varied from 18 to 65 residues for one nonreducing terminal residue of 4-O-methyl-d-glucuronic acid.     

Reaction kinetics and mechanism of acid-catalyzed anomerization of 1-O-acetyl-2,3,5-tri-O-benzoyl-l-ribofuranose

The mechanism of the acid-catalyzed anomerization of 1-O-acetyl-2,3,5-O-benzoyl-α- and -β-l-ribofuranoses in different acetic acid-acetic anhydride mixtures was investigated. The progress of the reactions was followed by NMR spectroscopy and the rate constants for the reactions were determined by the use of a kinetic model. The site of anomeric activation was clarified by the use of ¹³C-labeled acetic acid and acetic anhydride, respectively, proving that the anomerization takes place by exocyclic C-O cleavage, thus ruling out anomerization via acyclic intermediates. The role of the acetyl cation as the catalytically active species was further verified.     

X-ray structures of Bacillus pallidusd-arabinose isomerase and its complex with l-fucitol

d-Arabinose isomerase (d-AI), also known as l-fucose isomerase (l-FI), catalyzes the aldose–ketose isomerization of d-arabinose to d-ribulose, and l-fucose to l-fuculose. Bacillus pallidus (B. pallidus) d-AI can catalyze isomerization of d-altrose to d-psicose, as well as d-arabinose and l-fucose. Three X-ray structures of B. pallidusd-AI in complexes with 2-methyl-2,4-pentadiol, glycerol and an inhibitor, l-fucitol, were determined at resolutions of 1.77, 1.60 and 2.60 Å, respectively. B. pallidusd-AI forms a homo-hexamer, and one subunit has three domains of almost equal size; two Rossmann fold domains and a mimic of the (β/α) barrel fold domain. A catalytic metal ion (Mn²⁺) was found in the active site coordinated by Glu342, Asp366 and His532, and an additional metal ion was found at the channel for the passage of a substrate coordinated by Asp453. The X-ray structures basically supported the ene-diol mechanism for the aldose–ketose isomerization by B. pallidusd-AI, as well as Escherichia coli (E. coli) l-FI, in which Glu342 and Asp366 facing each other at the catalytic metal ion transfer a proton from C2 to C1 and O1 to O2, acting as acid/base catalysts, respectively. However, considering the ionized state of Asp366, the catalytic reaction also possibly occurs through the negatively charged ene-diolate intermediate stabilized by the catalytic metal ion. A structural comparison with E. colil-FI showed that B. pallidusd-AI possibly interconverts between “open” and “closed” forms, and that the additional metal ion found in B. pallidusd-AI may help to stabilize the channel region.     

Prebiotic feeding elevates central brain derived neurotrophic factor,N-methyl-d-aspartate receptor subunits and d-serine

The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action.     

Direct labelling of peptides with 2-[F]fluoro-2-deoxy-d-glucose ([F]FDG)

The study describes the use of [¹⁸F]FDG as ¹⁸F building block for the direct labelling of various aminooxy-functionalised peptides via chemoselective oxime formation.     

Oral supplementation with a combination of l-citrulline and l-arginine rapidly increases plasma l-arginine concentration and enhances NO bioavailability

Background

Chronic supplementation with l-citrulline plus l-arginine has been shown to exhibit anti-atherosclerotic effects. However, the short-term action of this combination on the nitric oxide (NO)–cGMP pathway remains to be elucidated. The objective of the present study was to investigate the acute effects of a combination of oral l-citrulline and l-arginine on plasma l-arginine and NO levels, as well as on blood circulation.

Methods

Rats or New Zealand white rabbits were treated orally with l-citrulline, or l-arginine, or a combination of each at half dosage. Following supplementation, plasma levels of l-arginine, NO_x, cGMP and changes in blood circulation were determined sequentially.

Results

l-Citrulline plus l-arginine supplementation caused a more rapid increase in plasma l-arginine levels and marked enhancement of NO bioavailability, including plasma cGMP concentrations, than with dosage with the single amino acids. Blood flow in the central ear artery in rabbits was also significantly increased by l-citrulline plus l-arginine administration as compared with the control.

Conclusion

Our data show for the first time that a combination of oral l-citrulline and l-arginine effectively and rapidly augments NO-dependent responses at the acute stage. This approach may have clinical utility for the regulation of cardiovascular function in humans.     

An approach to stereoselective preparation of 3-C-glycosylated d- and l-glucals

An approach to stereoselective synthesis of α- or β-3-C-glycosylated l- or d-1,2-glucals starting from the corresponding α- or β-glycopyranosylethanals is described. The key step of the approach is the stereoselective cycloaddition of chiral vinyl ethers derived from both enantiomers of mandelic acid. The preparation of 1,5-anhydro-4,6-di-O-benzyl-2,3-dideoxy-3-C-[(2,3,4,6-tetra-O-benzyl-β-d-glucopyranosyl)methyl]-l-arabino-hex-1-enitol, 1,5-anhydro-4,6-di-O-benzyl-2,3-dideoxy-3-C-[(2,3,4,6-tetra-O-benzyl-β-d-glucopyranosyl)methyl]-d-arabino-hex-1-enitol, and 1,5-anhydro-4,6-di-O-benzyl-2,3-dideoxy-3-C-[(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)methyl]-d-arabino-hex-1-enitol serves as an example of this approach.     

Formation of l-quebrachitol from d-bornesitol in leaves of Acer pseudoplatanus

d-Bornesitol and l-quebrachitol have been found in the leaves of Acer pseudoplatanus L. The results of incorporation studies using labeled myo-inositol-¹⁴C, l-inositol-¹⁴C and d-bornesitol-¹⁴C indicate that l-quebrachitol is produced by epimerization of d-bornesitol. In Artemisia vulgaris, however, the precursor of l-quebrachitol is l-inositol.     

Synthesis and structural analysis of a series of d-glucose derivatives as low molecular weight gelators

Low molecular weight gelators are an interesting new type of compounds that are important in supramolecular chemistry and advanced materials. Previously, we had synthesized several acyl derivatives of methyl 4,6-O-benzylidene-α-d-glucopyranoside and found that a number of terminal acetylene-containing esters are good gelators. To understand the structure requirement of the acyl chains, we synthesized a series of analogs containing different functional groups including aryl, alkenyl, and halogen derivatives. X-ray crystal structures of a monoester and a diester derivative were also obtained to help understand the relationship between structure and gelation. For good gelation properties, the carboxyl derivatives should possess alkyl groups containing a terminal acetylene group and aryl derivatives.     

Transport and metabolism of d-lactate in Jerusalem artichoke mitochondria

We report here initial studies on d-lactate metabolism in Jerusalem artichoke. It was found that: 1) d-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-d-lactoyl-glutathione. 2) Externally added d-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) d-lactate was metabolized inside mitochondria by a flavoprotein, a putative d-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added d-lactate by means of a d-lactate/H⁺ symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial d-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.     

α,β-dicarbonyl reduction by Saccharomycesd-arabinose dehydrogenase

An α,β-dicarbonyl reductase activity was purified from Saccharomyces cerevisiae and identified as the cytosolic enzyme d-Arabinose dehydrogenase (ARA1) by MALDI-TOF/TOF. Size exclusion chromatography analysis of recombinant Ara1p revealed that this protein formed a homodimer. Ara1p catalyzed the reduction of the reactive α,β-dicarbonyl compounds methylglyoxal, diacetyl, and pentanedione in a NADPH dependant manner. Ara1p had apparent K_m values of ∼ 14 mM, 7 mM and 4 mM for methylglyoxal, diacetyl and pentanedione respectively, with corresponding turnover rates of 4.4, 6.9 and 5.9 s^− 1 at pH 7.0. pH profiling showed that Ara1p had a pH optimum of 4.5 for the diacetyl reduction reaction. Ara1p also catalyzed the NADP⁺ dependant oxidation of acetoin; however this back reaction only occurred at alkaline pH values. That Ara1p was important for degradation of α,β-dicarbonyl substrates was further supported by the observation that ara1-Δ knockout yeast mutants exhibited a decreased growth rate phenotype in media containing diacetyl.     

Effects of 2-deoxy-d-glucose on the glucose metabolism in Saccharomyces cerevisiae studied by multinuclear-NMR spectroscopy and biochemical methods

The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30°C in a standard pyrophosphate medium containing 4.5 10⁷ cells/ml. ³¹P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates. The production of soluble metabolites of glucose was evaluated by ¹³C- and ¹H-NMR and biochemical techniques. The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-d-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites. The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM). The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration. The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG. The glucose consumption and the production of soluble metabolites of [1-¹³C]glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG. The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate. Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 ± 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0). Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose. However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated. DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG. Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible. By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation. Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG. The reasons why DG6P cannot accumulate indefinitely in cells are discussed, together with the reasons why the consumption of DG, but not glucose, becomes negligible after 30 min of incubation. In the absence of DG, the amount of polyphosphates (polyP) increased regularly with time as long as glucose was sufficiently present (≥ 5 mM) in the suspension. When glucose was exhausted, long chain polyphosphates disappeared to give rise, at first, to polyP with shorter chains and finally to inorganic phosphate. In the presence of 5 mM DG, the reduction in quantity of polyP can be explained by the fact that ATP, normally used for the polyP synthesis, is now diverted to phosphorylation of DG to DG6P. The presence of 5 mM DG also had significant effects on the glutamate C2, C3 and C4 signal intensity and the production of all aminoacids. The results seem to indicate that the enzymes involved in the Krebs cycle are also affected by the presence of DG.     

Occurrence of phosphatidyl-d-serine in the rat cerebrum

Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-l-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-d-serine (d-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of d-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence of d-PS. Free d-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the occurrence of a divalent cation-dependent enzyme that synthesizes d-PS by a base-exchange reaction.     

Lactose as an inexpensive starting material for the preparation of aldohexos-5-uloses: synthesis of l-ribo and d-lyxo derivatives

Partially protected derivatives of l-ribo- and d-lyxo-aldohexos-5-ulose have been prepared starting from triacetonlactose dimethyl acetal derivatives. Key steps of the synthetic sequences are (a) the synthesis of 4′-deoxy-4′-eno- and 6′-deoxy-5′-eno lactose derivatives, and (b) the epoxidation-methanolysis of the above-mentioned enol ethers to give 1,5-bis-glycopyranosides, masked form of the target 1,5-dicarbonyl hexoses.     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.