Direct analysis of nitrocatechol-type glucuronides in urine by capillary electrophoresis–electrospray ionisation mass spectrometry and tandem mass spectrometry

Direct, quantitative capillary electrophoresis–electrospray ionisation mass spectrometric (CE–ESI-MS) and tandem mass spectrometric (CE–ESI-MS–MS) methods are described for the quantitation of 3-O-glucuronides of E- and Z-entacapone isomers (EEG and EZG) and tolcapone (TG) in urine. 3-O-Glucuronide of nitecapone was used as internal standard. Good separation of glucuronides was achieved with 20 mM ammonium acetate as separation solution at pH 6.84. Stacking was used to increase the sensitivity of the method by introducing samples in 5 mM ammonium acetate. CE–ESI-MS and CE–ESI-MS–MS methods are linear with correlation coefficients better than 0.9983 and 0.9982, and repeatable with relative standard deviations below 9 and 14%, respectively. The limit of detection (LOD) in CE–ESI-MS at signal-to-noise ratio 3 is 100 ng/ml for EEG and EZG and 250 ng/ml for TG. The CE–ESI-MS–MS method was the more sensitive; LOD was 7 ng/ml for all compounds, without any concentration of the sample.     

Synthetic studies on amino-α-glycosides of aminocyclitols. Synthesis of kanamycin analogues

A diastereoisomer of Kanamycin C has been synthesized by a modified Koenigs—Knorr reaction of 3,4,6-tri-O-acetyl-2-(2,4-dinitroanilino)-2-deoxy-α-D-glucopyranosyl bromide with 4-O-(3-acetamido-2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl)-N,N′-di[(benzyloxy)carbonyl]-2-deoxystreptamine. Several Kanamycin analogues were synthesized by a similar condensation reaction. Each of the condensed products was isolated as its crystalline tetra-N-acetyl derivative and was proved by n.m.r. spectroscopy in D₂O to have the α-configuration.     

Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

Chemotaxonomic consideration of flavonoids from the leaves of Chrysanthemum arcticum subsp. arcticum and yezoense,and related species

We examined the foliar flavonoids of Chrysanthemum arcticum subsp. arcticum and yezoense, and related Chrysanthemum species. Five flavonoid glycosides (luteolin 7-O-glucoside and 7-O-glucuronides of luteolin, apigenin, eriodictyol and naringenin) were isolated from these taxa. Luteolin 7-O-xylosylglucoside, luteolin, apigenin and quercetin 3-methyl ether were found in subsp. yezoense as very minor compounds that were not recognised by high-performance liquid chromatography/photodiode array (HPLC/PDA). The related species C. yezoense contained acacetin 7-O-rutinoside and some methoxylated flavone aglycones as major compounds. Thus, C. arcticum was distinguished from C. yezoense according to their flavonoid profiles.     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulphate ester. I. Methods based upon the selective periodate oxidation of sialic acids

Summary Five new methods, based upon the selective oxidation of sialic acid residues with 0.4mm periodic acid in approximately 1m hydrochloric acid at 4°C for 1 h (PA^*), have been devised for the simultaneous visualization of neutral sugars and either sialic acid and its side chainO-acyl variants orO-sulphate ester. In the first of these, the selective periodate oxidation—borohydride reduction—saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (PA^*—Bh—KOH—PA^*—T—KOH—Bh—PAS) technique, sialic acids withO-acyl substituents at C7, C8 or C9 (or which have two of three side chainO-acyl substituents) stain blue while neutral sugars with periodate-sensitivevicinal diols (hexose, 6-deoxyhexose, andN-acetylhexosamine) stain magenta. The second method, the saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (KOH—PA^*—T—KOH—Bh—PAS), stains all sialic acids blue and neutral sugars magenta. In the third procedure, the selective periodate oxidation—Thionin Schiff—borohydride reduction—periodic acid—Schiff—saponification (PA^*—T—Bh—PAS—KOH) method, sialic acids without side chain substituents (or which have anO-acyl substituent at C7) stain blue and neutral sugars stain magenta. In the fourth method, the saponification-selective periodate oxidation—borohydride reduction—Alcian Blue pH 1.0—periodic acid—Schiff (KOH—PA^*—Bh—AB1.0—PAS) technique,O-sulphate esters stain aquamarine blue and neutral sugars stain magenta. In all of these techniques mixtures of the components stain in various shades of purple. Performance of the KOH—PA^*—Bh—AB1.0—PAS technique without the Alcian Blue pH 1.0 step provides a method for the selective identification of neutral sugars in macromolecules that also contain sialic acids.     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulphate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction

Summary Four methods based upon the periodic acid—phenylhydrazine—Schiff reaction have been developed for the simultaneous visualization of neutral sugars with periodate oxidizablevicinal diols (hexose, 6-deoxyhexose,N-acetylhexosamine) and either sialic acids or side chainO-acyl sialic acids. In the first of these procedures, the saponification—periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification (KOH—PA—DNPH—Az—KOH) method, all sialic acids stain Azure blue, neutral sugars with oxidizablevicinal diols stain yellow and mixtures of such components stain in various shades of green. In the second technique, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A Schiff—saponification (PA—DNPH—Az—KOH), Azure Blue staining is confined to sialic acids without side chain substituents or which have anO-acyl substituent at position C7, while in the third method, the selective periodate oxidation—borohydride reduction—saponification—periodic acid oxidation—2,4-dinitrophenyl hydrazine—Azure A—Schiff—saponification (PA^*—Bh—KOH—PA—DNPH—Az—KOH) technique, only sialic acids withO-acyl substituents at positions C7, C8 or C9 (or which have two or threeO-acyl side chain substituents) stain Azure blue. Finally in the fourth procedure, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification—borohydride reduction—periodic acid oxidation—Schiff (PA—DNPH—Az—KOH—Bh—PAS), sialic acids without side chain substituents or which haveO-acyl substituents at C7 stain Azure blue, sialic acids substituted at position C8 or C9 (or which are di- or tri-substituted) stain magenta and neutral sugars stain yellow. Where mixtures of these components are present, a wide range of colours is obtained.     

Effects of aluminum,iron, oxygen and nitrate additions on phosphorus release from the sediment of a Danish softwater lake

The effects of oxygen, aluminum, iron and nitrate additions on phosphate release from the sediment were evaluated in the softwater Lake Vedsted, Denmark, by a 34-day experiment with undisturbed sediment cores. Six treatments were applied: (1) Control - O₂ (0–20% saturation), (2) O₂ (100% saturation) (3) Al³⁺ – O₂, (4) Fe³⁺ + O₂, (5) Fe³⁺ – O₂, and (6) NO₃ ^– – O₂. Al₂(SO₄)₃*18 H₂O and FeCl₃*4H₂O were added in amounts that theoretically should immobilize the exchangeable P-pool in the top 5 cm of the sediment, while sodium nitrate concentrations were increased to 5 mg N l^–1. The four treatments with metals or NO₃ ^– reduced the P efflux from the sediment significantly as compared to the suboxic control treatment. Mean accumulated P-release rates for suboxic treatments with Al³⁺, Fe³⁺, and NO₃ ^– were: –0.27 mmol m^–2 (st. dev = 0.02 mmol m^–2, N = 5), 0.58 mmol m^–2 (st. dev = 0.30 mmol m^–2, N = 5) and 1.40 mmol m^–2 (st. dev = 0.14 mmol m^–2, N = 5), respectively. The oxic treatment with Fe³⁺ had a P efflux of 0.36 mmol m^–2 (st. dev = 0.08 mmol m^–2, N = 5). The two highest P-release rates were observed in the control treatment and the treatment with O₂ (14.50 mmol m^–2 (st. dev = 3.90 mmol m^–2, N = 5) and 2.31 mmol m^–2 (st. dev = 0.80 mmol m^–2, N = 5), respectively). In order to identify changes in the P and Fe binding sites in the sediment as caused by the treatments, a sequential P extraction procedure was applied on the sediment before and after the efflux experiment. Addition of O₂, Fe³⁺ and NO₃ ^– to the sediment increased the amounts of oxidized Fe³⁺ and P_BD. Al³⁺ addition resulted in a lower fraction of P_BD but a correspondingly higher fraction of Al-bound P. Addition of Al³⁺ decreased the Fe-efflux from the suboxic sediment as well as the amount of oxidized Fe³⁺ in the sediment. This questions the use of Al compounds that contain sulfate because of the possible formation of FeS, which will restrict upward migration of Fe²⁺ and the formation of new Fe-oxides in the surface sediment. Instead, we suggest the use of AlCl₃ for lake restoration purposes.     

Synthesis and Biological Activity of O,O-Dialkyl S-(5-Aryl-l,3,4-oxadiazol-2(3H)-on-3-yl)methyl Phosphorothioates and Phosphorodithioates

Some phosphorus derivatives of oxadiazoles were synthesized to seek insecticidal lead compounds. The l,3,4-oxadiazol-2-ones were converted via the N-methylol derivatives to the corresponding N-chloromethyl derivatives. From these derivatives a variety of O,O-dimethyl phosphorodithioates 4, O,O-dimethyl phosphorothioates 5 and O,O-di-i-propyl phosphorothioates 6 were prepared.

These phosphorus derivatives were examined for insecticidal activity towards houseflies and for anti-acetylcholinesterase (anti-AChE) activity using the housefly heads as an enzyme source. Most of the compounds 4 and 5 showed contact toxicity as high as the analogous methidathion insecticides, which appeared to correlate with the strong anti-AChE activity. On the other hand, all the compounds 6 showed a high activity in AChE inhibition but only a poor insecticidal activity.     

Photosynthesis,Transpiration, and Water Use Efficiency of Caragana microphylla,C. intermedia,and C. korshinskii

In the order C. microphylla — C. intermedia — C. korshinskii, compensation irradiance, saturation irradiance, and optimum temperature for photosynthesis increased, net photosynthetic rate (P _N) at low irradiance and low temperature decreased, optimum air humidity decreased, and P _N at low air humidity increased. Daily cumulative value of P _N increased while daily cumulative value of transpiration (E) decreased, and hence water use efficiency (WUE =P _N/E) increased. Diurnal course of P _N of C. microphylla was a double-peak curve, but the second peak in the curves of C. intermedia and C. korshinskii was not visible. These physiological characteristics are biological basis for the geographical distribution of these three Caragana species, and are in relation to water conditions of their habitats and distinctiveness in leaf hair of plant.     

Syntheses of daidzein-7-yl β- -glucopyranosiduronic acid and daidzein-4′,7-yl di-β- -glucopyranosiduronic acid

Syntheses of the title compounds — commonly known as ‘daidzein 7-glucuronide’ and ‘daidzein 4′,7-diglucuronide’ — are described. Selective 7-deacetylation of 4′,7-di-O-acetyldaidzein is employed.     

Physiological activities and the active constituents of potentially medicinal plants used by wild chimpanzees of the Mahale mountains,Tanzania

Potential medicinal plants for wild chimpanzees have been studied in order to discover their physiologically active compounds. Tests of the physiological activity of 3 plant species—Vernonia amygdalina, Aspilia mossambicensis, andFicus exasperata—indicate that they contain a variety of active compounds. From one species,V. amygdalina, an antitumor agent and 2 possible antitumor promoters are identified. Furthermore, steroid glucosides were isolated as the bitter substances. These structurally new compounds are expected to exhibit a number of significant physiological activities. The chemical investigation of possible medicinal plants used by chimpanzees should be helpful in recovering naturally occurring compounds of medicinal significance for human use.     

Chemolithoautotrophy in the marine,magnetotactic bacterial strains MV-1 and MV-2

Magnetite-producing magnetotactic bacteria collected from the oxic–anoxic transition zone of chemically stratified marine environments characterized by O₂/H₂S inverse double gradients, contained internal S-rich inclusions resembling elemental S globules, suggesting they oxidize reduced S compounds that could support autotrophy. Two strains of marine magnetotactic bacteria, MV-1 and MV-2, isolated from such sites grew in O₂-gradient media with H₂S or thiosulfate (S₂O₃^2–) as electron sources and O₂ as electron acceptor or anaerobically with S₂O₃^2– and N₂O as electron acceptor, with bicarbonate (HCO₃^–)/CO₂ as sole C source. Cells grown with H₂S contained S-rich inclusions. Cells oxidized S₂O₃^2– to sulfate (SO₄^2–). Both strains grew microaerobically with formate. Neither grew microaerobically with tetrathionate (S₄O₆^2–), methanol, or Fe²⁺ as FeS, or siderite (FeCO₃). Growth with S₂O₃^2– and radiolabeled ¹⁴C-HCO₃^– showed that cell C was derived from HCO₃^–/CO₂. Cell-free extracts showed ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. Southern blot analyses indicated the presence of a form II RubisCO (cbbM) but no form I (cbbL) in both strains. cbbM and cbbQ, a putative post-translational activator of RubisCO, were identified in MV-1. MV-1 and MV-2 are thus chemolithoautotrophs that use the Calvin–Benson–Bassham pathway. cbbM was also identified in Magnetospirillum magnetotacticum. Thus, magnetotactic bacteria at the oxic–anoxic transition zone of chemically stratified aquatic environments are important in C cycling and primary productivity.     

DNA sequence dependence of guanine-O alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea

After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O⁶-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules gunine-O⁶ alkylation does nor occur at random; rather, the probability of a substitution reaction at the nucleophilic O⁶ atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O⁶-2′-deoxyguanosine) monoclonal antibody, the frequency of guanine-O⁶ alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5t́ and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5t́ adenine and 5′ cytosine, provided an up to 10-fold more ‘permissive’ condition for O⁶-alkylation of the central guanine than a 5′ thymine (with a 5-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Synthesis and properties of chitosan hydrogels modified with heterocycles

Preparation and properties of chitosan modified with heterocycles in absence or presence of gluteraldehyde as a cross linker is described. New modified chitosan–heterocyclic hydrogels were prepared from chitosan and heterocyclic compounds such as N,N′-biisomaleimide, N,N′-biisophthalimide, and N,N′-phthalimidomaleimide via a crosslinking reaction. The new hydrogels chemical structure was characterized by spectral analysis (IR), X-ray diffraction, thermal gravimetric analysis (TGA), solubility, and swellability in water and different organic solvents. Evaluation of the efficiency of the new hydrogels to uptake copper and cobalt ions from aqueous systems was carried out and promising results were obtained.     

Genetic linkage relationships between two enzyme loci,Est-2 and acph,in the mosquito Aedes (Finlaya) togoi

An esterase locus (Est-2), coding for carboxylesterase, and an acid phosphatase locus (Acph) were genetically studied by agar gel electrophoresis in the mosquito Aedes (Finlaya) togoi. The Est-2 and Acph variants occur as a monomer and a dimer, respectively. Both enzyme loci are linked to the sex locus (M) and s (straw-colored larva); the gene arrangement and recombination distances were Est-2—12.6%—s—31.7%—M—2.9%—Acph—3.2%—Est-3. The Est-3 locus was previously shown to code for carboxylesterase.This work was supported by Grant AI 16983-02 from the National Institutes of Health, Bethesda, Md.     

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis

The last few steps in the biosynthesis of the anticancer drug Taxol in yew (Taxus) species are thought to involve the attachment of β-phenylalanine to the C13-O-position of the advanced taxane diterpenoid intermediate baccatin III to yield N-debenzoyl-2′-deoxytaxol, followed by hydroxylation on the side chain at the C2′-position to afford N-debenzoyltaxol, and finally N-benzoylation to complete the pathway. A cDNA encoding the N-benzoyl transferase that catalyzes the terminal step of the reaction sequence was previously isolated from a family of transferase clones (derived from an induced Taxus cell cDNA library) by functional characterization of the corresponding recombinant enzyme using the available surrogate substrate N-debenzoyl-2′-deoxytaxol [K. Walker, R. Long, R. Croteau, Proc. Nat. Acad. Sci. USA 99 (2002) 9166–9171]. Semi-synthetic N-debenzoyltaxol was prepared by coupling of 7-triethylsilybaccatin III and (2R,3S)-β-phenylisoserine protected as the N-Boc N,O-isopropylidene derivative by means of carbodiimide activation and formic acid deprotections. The selectivity of the recombinant N-transferase for N-debenzoyltaxol was evaluated, and the enzyme was shown to prefer, by a catalytic efficiency factor of two, N-debenzoyltaxol over N-debenzoyl-2′-deoxytaxol as the taxoid co-substrate in the benzoyl transfer reaction, consistent with the assembly sequence involving 2′-hydroxylation prior to N-benzoylation. Selectivity for the acyl/aroyl-CoA co-substrate was also examined, and the enzyme was shown to prefer benzoyl-CoA. Transfer from tigloyl-CoA to N-debenzoyltaxol to afford cephalomannine (Taxol B) was not observed, nor was transfer observed from hexanoyl-CoA or butanoyl-CoA to yield Taxol C or Taxol D, respectively. These results support the proposed sequence of reactions for C13-O-side chain assembly in Taxol biosynthesis, and suggest that other N-transferases are responsible for the formation of related, late pathway, N-acylated taxoids.     

d-Amino acid production by E. coli co-expressed three genes encoding hydantoin racemase, d-hydantoinase and N-carbamoyl-d-amino acid amidohydrolase

Three genes respectively encoding d-specific hydantoinase (DHHase), N-carbamoyl-d-amino acid amidohydrolase (DCHase) and hydantoin racemase (HRase) were co-expressed in E. coli in a system designed for the efficient enzymatic production of d-amino acids via a combination of hydantoin hydrolysis and hydantoin racemization. With the use of whole cells, the d-forms of eight amino acids – d-phenylalanine, d-tyrosine, d-tryptophan, O-benzyl-d-serine, d-valine, d-norvaline, d-leucine and d-norleucine – were efficiently converted from the corresponding dl-5-monosubtituted hydantoin compounds.     

Synthesis and glycogen phosphorylase inhibitory activity of N-(β-d-glucopyranosyl)amides possessing 1,4-benzodioxane moiety

A series of N-(β-d-glucopyranosyl)amides 5d–i were synthesized by PMe₃ mediated Staudinger reaction of O-peracetylated β-d-glucopyranosyl azide (1) followed by acylation with carboxylic acids 3d–i and subsequent Zemplén deacetylation. The new compounds were tested for their inhibitory activity against rabbit muscle glycogen phosphorylase and the structure–activity relationships of these compounds are also discussed in this paper.