The coordination chemistry of copper(I) in liquid ammonia, trialkyl and triphenyl phosphite, and tri-n-butylphosphine solution

The coordination chemistry of the solvate complexes of the relatively soft electron-pair acceptor copper(I) has been studied in solution and solid state in seven solvents with strong electron-pair donor properties, liquid ammonia, trimethyl, triethyl, triisopropyl, tri-n-butyl and triphenyl phosphite, and tri-n-butylphosphine. The solvate complexes have been characterised by means of EXAFS and 63Cu NMR spectroscopy, and in some cases also by 65Cu NMR spectroscopy. The copper(I) ion is three-coordinated, most probably in a coplanar trigonal fashion, in liquid ammonia with a mean Cu-N bond distance of 2.00(1) Angstroms. No 63Cu NMR signal has been detected from the ammonia solvated copper(I) ion in liquid ammonia, which supports a three-coordination. The phosphite and phosphine solvated copper(I) ions are tetrahedral with Cu-P bond distances in the range 2.24-2.28 Angstrom in both solution and solid state as determined by EXAFS spectroscopy. The tetrahedral configuration of these complexes has been confirmed by 63Cu and 65Cu NMR spectroscopy through the J(63Cu-31P) and J(65Cu-31P) couplings. The fact that two of the investigated complexes, [Cu(P(OC6H5)3)4]+ and [Cu(P(C4H9)3)4]+, are 63Cu and 65Cu NMR silent is probably caused by a significantly angular distorted tetrahedral configuration.     

A general method for coulometric titration of alkylphenols with bromine

A general method for coulometric titration of alkylphenols with anodically generated bromine is described. The reaction is carried out in a water-acetic acid medium and the reactivity is governed by varying the water content and the concentration of bromide ion and by the addition of pyridine. In that way all types of alkylphenols can be titrated quantitatively. For phenols containing more than one free ortho and para position, the titration can also be carried out either to the monobromination stage or to the full bromination stage. The mean relative error is ± 1·2% for monobromination and ±1·5% for full bromination. A method for rapid determination of the number of free ortho and para positions in alkylphenols by using two coulometric titrations is also described.     

Towards molecular-imprint based SPE of local anaesthetics

Summary Solidago canadensis L., Canadian goldenrod (Asteraceae) has been used in European phytotheraphy for centuries as a component of urological and antiphlogistical remedies. High-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and online mass spectrometry (MS) has been used for the separation and quantification of phenolics (chlorogenic acid, caffeic acid, kaempferol-3-O-α-L-rutinoside (nicotiflorin), quercetin-3-O-β-D-rutinoside (rutin), quercetin-3-O-β-D-galactoside (hyperoside), quercetin-3-O-β-D-glucoside (isoquercitrin), quercetin-3-O-β-D-rhamnoside (quercitrin), kaempferol-3-O-α-L-rhamnoside (afzelin) and quercetin from Solidaginis herba. Extracts have been obtained using different technologies. Three aqueous and three alcoholic extracts were studied separately. Reversedphase high-performance liquid chromatography separation of polyphenols on octadecyl sorbent Hypersil was performed, using acetonitrile: acetic acid 2.5 v/v % as eluent in gradient elution. Our results confirm previous reports concerning the presence of several flavonoids. Quantification of the main quercetin glycosides in pharmaceuticals is also reported. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

In-capillary micro solid-phase extraction and capillary electrophoresis separation of heterocyclic aromatic amines with nanospray mass spectrometric detection

A miniaturised technique to analyse and detect heterocyclic aromatic amines (HAs) using micro solid-phase extraction (SPE) coupled on-line (in-capillary) to capillary electrophoresis (CE) separation with nanospray (nESI) mass spectrometry (MS) detection has been developed. HAs are mutagenic and carcinogenic compounds formed at low levels in protein-rich food during cooking. Due to the low concentrations of HAs and the high complexity of the matrix in which they exist, sensitive and selective analytical methods are required for quantification. SPE was performed on a packed bed of C₁₈ particles inside the CE capillary, which minimised the dead volume. The on-line coupling of SPE, CE and nESI-MS reduced the time for extraction and identification to less than half an hour, which will allow for screening of several samples per day. The new technique provides short analysis time, low sample and solvent consumption, and HAs in standard solutions were easily detected at 12–17 fmol injections, and in spiked urine samples at 750–810 fmol injections.     

Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase

Whole cells ofTrigonopsis variabilis were immobilized by entrapment in Ca²⁺-alginate and used for the production of α-keto acids from the corresponding D-amino acids. The D-amino acid oxidase within the immobilized cells has a broad substrate specificity. Hydrogen peroxide formed in the enzymatic reaction was efficiently hydrolyzed by manganese oxide co-immobilized with the cells. The amino acid oxidase activity was assayed with a new method based on reversed-phase HPLC. Oxygen requirements, bead size, concentration of cells in the beads, flow rate, and other factors were investigated in a “ trickle-bed ” reactor.     

Homogeneous sample preparation for automated high throughput analysis with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples.     

Synthesis and conformational analysis of 9,10-bis-aminomethyl-11,12-dicarboxy-dibenzobarrelene derivatives

The synthesis of bis-γ-amino acid dibenzobarrelene derivatives (9,10-bis-aminomethyl-11,12-bis-carboxy-dibenzobarrelene) is presented. Bromomethylation of anthracene followed by azide substitution gave 9,10-bis-azidomethylanthracene. Azide reduction, N-Boc protection, and Diels-Alder cycloaddition in DMAD furnished the protected 9,10-bi-aminomethyl-11,12-dicarboxy-dibenzobarrelene derivative, which was further converted into the bis-γ-amino acid methyl ester, the N-Boc-protected bis-γ-amino methyl amide, and a bis-γ-lactam. Monte Carlo simulations and X-ray analysis of the 9,10-substituted dibenzobarrelenes revealed an exposed hydrophobic surface surrounded by amino and carboxy groups.     

Unambiguous evidence for the participation of singlet oxygen ( 1 ) in photodynamic oxidation of amino acids

Abstract— A variety of experimental tests have been applied to the methylene-blue-sensitized photooxidation of amino acids to distinguish between singlet oxygen and non-singlet oxidation mechanisms. Conventional flash photolysis and laser photolysis were used to measure the rate constants for the quenching of excited triplet sensitizer and singlet oxygen by the amino acids histidine. tryptophan and methionine and the nucleotide guanosine-5′-monophosphate. In the case of histidine, the rate constants alone rule out an oxidation mechanism involving direct reaction with excited dye. With the other amino acids, and with guanosine monophosphate, the oxidation rates might be accounted for by either mechanism. The inhibition of the photo-oxidation of both tryptophan and methionine as well as histidine by the singlet-oxygen quenchers N₃^? and tetramethylethylene suggests that these reactions occur via a singlet-oxygen mechanism. A newly developed test of singlet oxygen reactions involving a comparison of photooxidation rates in normal and perdeuterated solvents has been used to establish that the photooxidation of tryptophan proceeds primarily by a singlet-oxygen mechanism. These experiments appear to constitute the first proof that singlet oxygen is involved in the photooxidation of the three amino acids tryptophan, methionine and histidine.     

Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori

Cell surface proteins of the human gastric pathogen Helicobacter pylori extracted during different in vitro growth phases were analyzed by one- and two-dimensional gelelectrophoresis (1-DE and 2-DE) and by 2-DE immunoblot. Broth-cultured H. pylori cells were stained with an acridine-orange dye to monitor the morphological status of the organism. In 2-day-cultures, 96% of the bacterial cells were spiral-shaped and four days later a morphological switch to coccoid forms occurred. In 10-day cultures spiral-shaped forms were not found. By 1-DE, proteins with the molecular masses of 87 and 120 kDa were detected in the 2-day cultures that disappeared in cells of 12-day cultures. A protein corresponding in size to the heat shock protein (GroEl homolog, Hsp60) and a 62 kDa protein, the ureaseB-subunit, were identified in extracted proteins of 2-, 8-, and 12-day cultures. 2-DE revealed an increased number of silver-stained spots of 8-day cultures (in average 250 spots) compared with protein extracted from 2-day cells (in average 160 spots). 2-DE immunoblots performed with sera containing antibodies to major H. pylori proteins such as the A- and B-subunits of urease and the Hsp60 showed similar reactivity to surface proteins extracted from 2-, 8-, and 12-day cultures, suggesting that these proteins remain immunologically intact. Pooled sera from infected patients absorbed with spiral-shaped cells showed an almost total blocking of the antibody reactivity to extracted coccoid proteins in 2-DE immunoblot. Eighteen spots were still visible, but this reactivity probably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.