Identification of 4-amino-4-deoxychorismate synthase as the molecular target for the antimicrobial action of (6s)-6-fluoroshikimate

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.     

Automated multiresidue analysis of pesticides in olive oil by on-line reversed-phase liquid chromatography-gas chromatography using the through oven transfer adsorption-desorption interface

A multiresidue, automated and rapid method for the determination of pesticide residues in olive oil is presented. The method employs the through oven transfer adsorption-desorption interface for the on-line coupling of reversed-phase liquid chromatography and gas chromatography. In this fully automated system, olive oil is directly injected with no sample pre-treatment step other than filtration. Methanol-water is used as eluent in the liquid chromatography pre-separation step. The selected liquid chromatography fraction containing the pesticides is automatically transferred to the gas chromatography. The liquid chromatography column flow during elution is different from the flow during the transfer. Using a flame ionisation detector, pesticide detection limits varied from 0.1 to 0.3 mg/l.     

Titanocene-catalyzed cascade cyclization of epoxypolyprenes: straightforward synthesis of terpenoids by free-radical chemistry

The titanocene-catalyzed cascade cyclization of epoxypolyenes, which are easily prepared from commercially available polyprenoids, has proven to be a useful procedure for the synthesis of C(10), C(15), C(20), and C(30) terpenoids, including monocyclic, bicyclic, and tricyclic natural products. Both theoretical and experimental evidence suggests that this cyclization takes place in a nonconcerted fashion via discrete carbon-centered radicals. Nevertheless, the termination step of the process seems to be subjected to a kind of water-dependent control, which is unusual in free-radical chemistry. The catalytic cycle is based on the use of the novel combination Me(3)SiCl/2,4,6-collidine to regenerate the titanocene catalyst. In practice this procedure has several advantages: it takes place at room temperature under mild conditions compatible with different functional groups, uses inexpensive reagents, and its end step can easily be controlled to give exocyclic double bonds by simply excluding water from the medium.     

cis-Enhanced cyclopropanation catalysts: reaction chemistry of three isomers of Rh2[N(C6H5)COCH3]4

The catalytic activities of three structural isomers of Rh₂[N(C₆H₅)COCH₃]₄ in cyclopropanation reactions were surveyed. These studies showed cis cyclopropanation selectivity with bulky alkenes for 2,2-cis- and 2,2-trans-Rh₂[N(C₆H₅)COCH₃]₄.     

An evaluation of the performance of porous polymer monolith assisted electrospray

A novel electrospray interface, which has distinct advantages over conventional pulled silica emitters, has been developed. This novel interface can be easily fabricated by forming a porous polymer monolith (PPM) at the end of a fused-silica capillary that facilitates a stable electrospray over a wide range of flow rates with only a modest increase in back-pressure. A comparison was made between the PPM-assisted electrospray and a commercial nanosprayer in terms of sensitivity, stability and robustness. A PPM-filled electrospray tip produced a day-to-day signal variation of 23% relative standard deviation (RSD) over a 3-day period when spraying a 1.0 microM test peptide solution. Furthermore, three different capillaries fabricated by the same process produced a signal variation of 17% RSD, indicating that the fabrication process shows good reproducibility. The multiple flow paths of the PPM function to split the flow and reduce clogging. Even following the accumulation of debris after prolonged use, a stable spray could still be generated with the PPM-filled capillary while the commercial nanosprayer ceased to function properly. In terms of sensitivity, PPM-assisted electrospray showed an enhancement in sensitivity at infusion flow rates between 100 to 1000 nL/min while commercial nanosprayers performed slightly better at flow rates below 100 nL/min. A sample purification step can be combined with the PPM-assisted sprayer, using the PPM as a stationary phase to desalt and preconcentrate samples prior to mass spectrometric detection.     

An efficient synthesis of 6-formyl-1,2-dihydro-2-oxo-3-pyridinecarboxylic acid and some carbonyl derivatives of it and its 6-acetyl homologue

Starting with 1,1-dimethoxy-2-propanone ( 1 ), 6-formyl-1,2-dihydro-2-oxo-3-pyridinecarboxylic acid ( 5a ) has been prepared in large quantities by a highly efficient, 4-step synthesis. This compound, along with its one carbon homologue, 6-acetyl-1,2-dihydro-2-oxo-3-pyridinecarboxylic acid ( 5b ) has been reacted with several carbonyl derivative forming reagents to provide a series of side chains for β-lactams. Among these carbonyl derivatives are styrylamides which were prepared from Wittig and Horner-Emmons reagents. The preparation of the phosphonium salts and phosphonate esters is also described.     

Determination of molybdenum by atomic-absorption spectrometry after separation by 5,5'-methylenedisalicylohydroxamic acid extraction and further reaction with thiocyanate and tin (II)

A method is described for the determination of molybdenum down to the microgram level, in samples of soil, steels, fertilizers and pharmaceuticals. After attack with acids, this element is separated from matrix elements by extraction of its 5,5'-methylenedisalicylohydroxamic acid (MEDSHA) complex from 4M hydrochloric acid, into methyl isobutyl ketone. Molybdenum is determined by atomic-absorption spectrometry (AAS), after conversion of the Mo-MEDSHA complex into the MoSCN(-) complex in the organic phase. The detection limit is 0.03 microg/ml, with a relative standard deviation not exceeding 1.5% at a level of 2 microg/ml. The method is highly selective and suffers only from interference by tungsten.     

Spectrophotometric determination of indium in nickel alloys and zinc ores with 1-(2-pyridylmethylideneamine)-3-(salicylideneamine)thiourea

A sensitive method for the spectrophotometric determination of indium with 1-(2-pyridylmethylideneamine)-3-(salicylideneamine)thiourea is proposed. A yellow complex is formed at pH 4.5 (succinate buffer) in a medium containing 40% dimethylformamide, and the absorbance is measured at 415 nm. The molar absorptivity is 6.2 x 10(4)l.mole(-1).cm(-1). The relative standard deviation of the procedure is 1.5%. The method has been applied to determination of indium in a nickel alloy and three zinc ores, with prior isolation of indium by co-precipitation with ammonia and extraction into n-butyl acetate from 5M hydrobromic acid.     

Selenium redox cycling in the protective effects of organoselenides against oxidant-induced DNA damage

The biological role of selenium is a subject of intense current interest, and the antioxidant activity of selenoenzymes is now known to be dependent upon redox cycling of selenium within their active sites. Exogenously supplied or metabolically generated organoselenium compounds, capable of propagating a selenium redox cycle, might therefore supplement natural cellular defenses against the oxidizing agents generated during metabolism. We now report evidence that selenium redox cycling can enhance the protective effects of organoselenium compounds against oxidant-induced DNA damage. Phenylaminoethyl selenides were found to protect plasmid DNA from peroxynitrite-mediated damage by scavenging this powerful cellular oxidant and forming phenylaminoethyl selenoxides as the sole selenium-containing products. The redox properties of these organoselenoxide compounds were investigated, and the first redox potentials of selenoxides in the literature are reported here. Rate constants were determined for the reactions of the selenoxides with cellular reductants such as glutathione (GSH). These kinetic data were then used in a MatLab simulation, which showed the feasibility of selenium redox cycling by GSH in the presence of the cellular oxidant, peroxynitrite. Experiments were then carried out in which peroxynitrite-mediated plasmid DNA nick formation in the presence or absence of organoselenium compounds and GSH was monitored. The results demonstrate that GSH-mediated redox cycling of selenium enhances the protective effects of phenylaminoethyl selenides against peroxynitrite-induced DNA damage.     

Preparative high-performance liquid chromatography of peptides on a new reversed-phase packing material, Kromasil C18

Preparative reversed-phase high-performance liquid chromatography has found wide use in the production of peptides for pharmaceutical formulations. Purity of the substance and overall economy of the chromatographic system are the most important criterias. In this sense optimized, silica particles and production process with capability to separately control parameters important to chromatography, are essential to high-performance chromatography. Kromasil C18 packing material was tested and evaluated in respect of its selectivity, flow and pressure properties, resolution, load capacity, recovery, adsorption effects, mechanical strength and chemical degradation.