An expedient synthesis of l-ribulose and derivatives

A significant improvement in the production of l-ribulose from inexpensive and commercially available starting materials, l-arabinose and sodium aluminate, is demonstrated. This has facilitated expeditious access to gram-scale quantities of l-ribulofuranoside derivatives.     

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD⁺)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD⁺-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.     

Intracellular l-arginine concentration does not determine NO production in endothelial cells: Implications on the “l-arginine paradox”

We examined the relative contributory roles of extracellular vs. intracellular l-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of ¹⁵N₄-ARG, ARG, or l-arginine ethyl ester (ARG-EE) for 2 h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, ¹⁵N₄-ARG, dimethylarginines, and l-citrulline by an LC–MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by¹⁵N-nitrite or estimated ¹⁵N₃-citrulline concentrations when ¹⁵N₄-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced ¹⁵N₄-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by ¹⁵N-nitrite, total nitrite and ¹⁵N₃-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the “l-arginine paradox” should not consider intracellular ARG concentration as a reference point.     

Determination of [d-Ala, d-Leu]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

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.     

A Metal Ion as a Cofactor Attenuates Substrate Inhibition in the Enzymatic Production of a High Concentration of <Emphasis Type="SmallCaps">D</Emphasis>-glutamate Using <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">D</Emphasis>-glutamate Amidohydrolase

N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL- glutamate was used as a substrate. The addition of 1 mM Co²⁺ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co²⁺ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn²⁺ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h.     

Development of a high-throughput method for the determination of pharmacological levels of plasma d-serine

d-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, d-serine must be administered at high doses to observe clinical effects. This is due in large part to d-serine undergoing oxidation by d-amino acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of d-serine with a DAAO inhibitor has been suggested as a way to lower the dose of d-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma d-serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure d-serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor d-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate d-serine from plasma followed by quantitation of d-serine using the DAAO-catalyzed reaction. Plasma d-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.     

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}).     

Determination of N-nitroso-l-arginine in rat brain extracts by capillary electrophoresis using photodiode-array detection

N-Nitroso-l-arginine was described as one of the products of l-arginine metabolism in biological media. A simple and rapid method to determine its concentration in rat brain was developed. Capillary electrophoresis with a photodiode-array detector was used at 254 nm, permitting the quantification of N-nitroso-l-arginine. The detection limit in biological solution was 1 μg/ml.     

N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Novel sugar bola-amphiphiles with a pseudo macrocyclic structure

Novel bola-amphiphilic compounds have been synthesised with d-glucose or d-galactose moieties as polar head groups. The sugar groups are coupled to the hydrophobic bridge, a chain of ten or twelve methylene groups, by an amide function at the C-2 site (2-alkylamido-2-deoxy-d-glucose derivative) or a glycuronamido function at the C-6 site. The former series is glycosylated with a cinnamyl group and the latter with octyl or decyl groups to afford pseudo macrocyclic compounds.     

Comparison of detection methods for liquid chromatographic determination of 3-nitro-l-tyrosine

A liquid chromatographic method has been developed for the determination of 3-nitro-l-tyrosine. Different detection methods, including UV, oxidative and redox electrochemistry, and postcolumn photolysis followed by electrochemical detection, have been optimized and compared in terms of analysis time, detection limit and dynamic range. It was demonstrated that liquid chromatography with postcolumn photolysis followed by electrochemical detection is the most effective method, with an analysis time of 5 min, detection limit of 0.01 pmol, and a linear dynamic range from 2 nM to 100 μM.     

Synthesis of disaccharide fragments of the AT-III binding domain of heparin and their sulfonatomethyl analogues

d-Glucuronate and l-iduronate-containing disaccharides related to the antithrombin-binding domain of heparin were prepared. The carboxylic function of the uronic acid unit was formed on a disaccharide level in the case of the glucuronate, while on a monosaccharide level in the case of the iduronate derivatives. Synthesis of their sulfonic acid analogues was carried out analoguosly applying sulfonatomethyl-containing acceptors in the form of either salts or methyl esters. Significant difference could be observed in the methyl ether formation reactions of the sulfonatomethyl-containing uronate disaccharides and the non-sulfonic acid uronates.     

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     

Bi- and trivalent glycopeptide mannopyranosides as inhibitors of type 1 fimbriae-mediated bacterial adhesion: variation of valency, aglycon and scaffolding

In order to test relevant structural parameters for effective inhibition of mannose-specific bacterial adhesion, bi- and trivalent glycopeptide α-d-mannopyranosides were synthesized that differ in their conformational properties as well as in the spatial arrangement of attached mannosyl residues. They were tested in an inhibition adhesion assay with fluorescent Escherichia coli bacteria and testing results were referenced to the inhibitory potency of methyl α-d-mannopyranoside. It was shown, that besides the nature of the mannoside aglycon moiety, scaffolding of α-d-mannopyranosides on a peptide backbone was important for the performance of the synthesized glycopeptides as inhibitors of bacterial adhesion.     

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.     

Is there a correlation between age and <Emphasis Type="SmallCaps">D</Emphasis>-aspartic acid in human knee cartilage?

Summary. L-Aspartic acid (L-Asp) is one of the fastest racemizing amino acids such that the abnormal D-form (D-Asp) has been found in stable biological human tissues such as dentin in teeth, eye lens and brain. Earlier reports showed that there was a linear correlation between age and D-Asp in teeth. We have previously reported that significant levels of D-Asp were found in normal and osteoarthritic knee cartilage. Since cartilage is a slow regenerating tissue, we hypothesized that D-Asp should accumulate in knee cartilage and that there might be a correlation between the age of the person and the amount of D-Asp found in cartilage. Our analysis of approximately 100 samples of normal knee cartilage showed that there are detectable amounts of D-Asp (2–4% of total Asp) in knee cartilage. However, there was only a slight correlation (r = 0.35) between the age of the person and the amount of D-Asp (nmoles/g). Surprisingly, there was a better correlation between age and the amount of D-Asp in the male subjects (r = 0.57) than in the female subjects (r = 0.21).     

The pathway ofD-cycloserine biosynthesis inStreptomyces garyphalus

Incubation experiments using washed cells and toluene treated cells ofStreptomyces garyphalus showed that O-acetyl-L-serine and hydroxyurea are intermediates in the biosynthesis ofD-cycloserine. The formation of [¹⁴C]O-ureidoserine from O-acetyl-L-serine and hydroxyurea was demonstrated by incubating an enzyme solution with¹⁴C-labelled substrates. Desalted cell-free extract catalyzed the conversion of O-ureido-D-serine toD-cycloserine in a reaction requiring ATP and Mg²⁺. The results suggested the following pathway forD-cycloserine biosynthesis.     

A novel mechanism involved in the metabolism of the tartaric acid stereoisomers in Rhodopseudomonas sphaeroides: enzymatic conversion of meso-tartaric acid to D(-)-glyceric acid and CO2

In cell extracts of Rhodopseudomonas sphaeroides grown on meso-tartrate the activities of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and 1.1.1.83, respectively) could be measured spectrophotometrically but not the activity of a meso-tartrate dehydrogenase or dehydratase. However, an enzyme activity was detected manometrically that catalyzed the stoichiometric release of CO₂ from mesotartrate in a molar ratio of 1:1. This reaction required catalytic amounts of NAD and the presence of both divalent (Mn²⁺ or Mg²⁺) and monovalent (NH ₄ ⁺ or K⁺) cations. Purification of the meso-tartrate decarboxylase showed that it was part of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating), which thus possessed a third catalytic function. The homogeneous enzyme catalyzed the stoichiometric conversion of incso-tartaric acid to D(-)-glyceric acid and CO₂. All interfering catalytic activities had been eliminated during the course of enzyme purification.     

n-carbamoyl-d-p-hydroxyphenylglycine production using immobilized d-hydantoinase from recombinant E. coli

An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl-d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The V_max and K_m of the immobilized d-hydantoinase at 50°C were 6.28 mm min⁻¹ g⁻¹ biocatalyst and 71.6 mm, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol⁻¹. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability.