Generation of new landomycins by combinatorial biosynthetic manipulation of the LndGT4 gene of the landomycin E cluster in S. globisporus

A 3 kb DNA fragment from the Streptomyces globisporus 1912 landomycin E (LaE) biosynthetic gene cluster (lnd) was completely sequenced. Three open reading frames were identified, lndGT4, lndZ4, and lndZ5, whose probable translation products resemble a glycosyltransferase, a reductase, and a hydroxylase, respectively. Studies of generated mutants from disruption and complementation experiments involving the lndGT4 gene allowed us to determine that LndGT4 controls the terminal L-rhodinose sugar attachment during LaE biosynthesis and that LndZ4/LndZ5 are responsible for the unique C11-hydroxylation of the landomycins. Generation of the novel landomycins F, G, and H in the course of these studies provided evidence for the flexibility of lnd glycosyltransferases toward their acceptor substrates and a basis for initial structure-activity relationships within the landomycin family of antibiotics.     

Syntheses of polysubstituted pyrroles and of 2,3-disubstituted quinoxalines and substituted benzo[g]quinoxalines

Tetraacetylethylene ( 1 ), and cis-diacetylethylene ( 4 ) reacted under mild conditions with 3-amino-2-butenoic acid methyl ester ( 6 ), benzene-1,2-diamine and naphthalene-2,3-diamine to give polysubstituted pyrroles, 2,3-disubstituted quinoxalines and 2,3-disubstituted benzo[g]quinoxalines respectively. Some aspects of the reactions mechanisms are discussed.     

Composition of the black crusts from the Saint Denis Basilica, France, as revealed by gas chromatography-mass spectrometry

The organic fraction of black crusts from Saint Denis Basilica, France, is composed of a complex mixture of aliphatic and aromatic compounds. These compounds were studied by two different analytical approaches: tetramethyl ammonium hydroxide (TMAH) thermochemolysis in combination with gas chromatography-mass spectrometry (GC-MS), and solvent extraction, fractionation by silica column, and identification of the fraction components by GC-MS. The first approach, feasible at the microscale level, is able to supply fairly general information on a wide range of compounds. Using the second approach, we were able to separate the complex mixture of compounds into four fractions, enabling a better identification of the extractable compounds. These compounds belong to different classes: aliphatic hydrocarbons (nalkanes, n-alkenes), aliphatic and aromatic carboxylic acids (n-fatty acids, alpha,omega-dicarboxylic acids, and benzenecarboxylic acids), polycyclic aromatic hydrocarbons (PAH), and molecular biomarkers (isoprenoid hydrocarbons, diterpenoids, and triterpenoids). With each approach, similar classes of compounds were identified, although TMAH thermochemolysis failed to identify compounds present at low concentrations in black crusts. The two proposed methodological approaches are complementary, particularly in the study of polar fractions.     

Multimodal open-tubular capillary electrochromatographic analysis of amines and peptides

This study describes a comparison of different modes of open-tubular electrochromatography (OTCEC) in bare and etched capillaries. To carry out the investigation, the separation of impurities of two synthetic peptides and the separation of a mixture of five heterocyclic aromatic amines were studied. Three different types of stationary phase were evaluated: (i) fluorosurfactants (anionic and zwitterionic) adsorbed in the inner wall of the capillary (electrochromatography with dynamically modified stationary phases (DMS)CEC); (ii) physically adsorbed polymers (DMA-SO(3-) and DMA-N(+)(CH(3))(3)) and (iii) chemically modified capillaries (C(18), cholesteryl 10-undecanoate and diol). The results confirm that electrochromatography can be a viable alternative to capillary electrophoresis (CE) and liquid chromatography, more established separation techniques. It is possible to differentiate some minor species for the synthetic peptides that cannot be resolved by CE or high-performance liquid chromatography (HPLC). Moreover the separation of the amine mixture depends strongly on the stationary phase used.     

The first immobilization of pyridine-bis(oxazoline) chiral ligands

[formula: see text] A chiral pyridine-bis(oxazoline) ligand, functionalized with a vinyl group in the pyridine ring, can be polymerized with styrene and divinylbenzene to obtain supported chiral ligands. As proof of the usefulness of this supported ligands, the corresponding ruthenium complexes are catalysts for the cyclopropanation reaction of styrene with ethyl diazoacetate with up to 85% ee.     

Direct determination of selenium in urine samples by electrothermal atomic absorption spectrometry using a Zr plus Rh-treated graphite tube and co-injection of Rh as chemical modifier

Different chemical modifiers for use with electrothermal atomic absorption spectrometry (ET AAS) were investigated in relation to determining the selenium in human urine samples. The samples were diluted in a solution containing 1% v/v HNO₃ and 0.02% m/v cetyltrimethylammonium chloride (CTAC). Studying the modifiers showed that the use of either Ru or Ir as the permanent modifier gave low sensitivity to Se and the peak shape was very noisy, while Zr or Rh gave no peak at all. The same occurred when Zr was used in solution. For mixtures of permanent modifiers, Ir plus Rh or Zr plus Rh gave very low sensitivity, Zr plus Rh with co-injection of Ir in solution was also not efficient, Zr plus Rh in solution gave good sensitivity, but the best results were obtained with a mixture of Zr and Rh as the permanent modifier and co-injection of Rh in solution. Using this last modifier, the following dilutions with the HNO₃ and CTAC were studied: 1:1, 1:2, 1:3 and 1:4. The best dilution was 1:1, which promoted good sensitivity and a more defined peak shape and made it possible to correct for the background using a deuterium arc lamp. Under these conditions, a characteristic mass of 26±0.2 pg was obtained for Se in aqueous solution. Six certified urine samples were analyzed using matrix matching calibration and the measured concentrations were in agreement with the certified values, according to a t-test at the 95% confidence level. Recovery tests were carried out and the recoveries were in the range 100–103%, with relative standard deviation better than 9%. The limit of detection (LOD, 3 sd, n=10) was 3.0 μg L⁻¹ in the sample. The treated graphite tube could be used for at least 600 atomization cycles without significant alteration of the analytical signal.     

A versatile new strategy for the synthesis of tropolones

Swern-type oxidation of various 7-halogenobicyclo[4.1.0]heptane-2,3- or -3,4-diols affords the corresponding bicyclic diketones which undergo ^{in situ} ring expansion and loss of hydrogen halide to give α-tropolones in high yield. The quantitative conversion of the isolable 1,4-diketone 26 into the γ-tropolone acetate 27 has been achieved.     

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.     

Influence of reduction conditions on H2 adsorption in high-surface Rh/CeO2 catalysts as deduced by volumetry, calorimetry, and 1H NMR techniques

1H NMR spectra corresponding to H2 adsorption on high-surface Rh/CeO2 catalysts (S(BET) approximately 55 m2/g) are formed by two lines, attributed to hydrogen adsorbed on ceria (resonance line A) and rhodium-metal particles (upfield-shifted line B). The evolution of 1H NMR spectra as a function of temperature, time, and type of reduction (static or dynamic) allows the study of the progressive establishment of the strong metal-support interaction (SMSI) in Rh/CeO2 catalysts. As the reduction progresses, the mean adsorption heat and the amount of hydrogen adsorbed on the metal, deduced from volumetry, NMR, and calorimetry techniques, decrease considerably. As a consequence of the decrease in metal activity, the amount of hydrogen transferred to the support CeO2 is also reduced (spill-over processes). Outgassing of samples at 773 K eliminates hydrogen species retained at the metal-support surface, and oxidation treatments at 473 and 673 K eliminate the electronic effect and physical blocking of metal particles. The oxidation at 673 K recuperates the total adsorption capacity of metal particles. On the basis of these treatments, the contribution of different processes to the SMSI effect is analyzed. Electronic perturbation of rhodium particles is higher when reductions are performed in dynamic conditions; however, the importance of physical blocking of metal particles increases in static reductions. High reducibility of ceria strengthens electronic effects in Rh/CeO2 compared to those observed in Rh/TiO2 catalysts.     

Experimental and computational investigation of the energetics of the three isomers of monochloroaniline

The standard (p degrees = 0.1 MPa) molar enthalpies of formation of 2-, 3-, and 4-chloroaniline were derived from the standard molar energies of combustion, in oxygen, at T = 298.15 K, measured by rotating bomb combustion calorimetry. The Calvet high-temperature vacuum sublimation technique was used to measure the enthalpies of vaporization or sublimation of the three isomers. These two thermodynamic parameters yielded the standard molar enthalpies of formation of the three isomers of chloroaniline, in the gaseous phase, at T = 298.15 K, as 53.4 +/- 3.1 kJ.mol(-1) for 2-chloroaniline, 53.0 +/- 2.8 kJ.mol(-1) for 3-chloroaniline, and 59.7 +/- 2.3 kJ.mol(-1) for 4-chloroaniline. These values, which correct previously published data, were used to test the computational methodologies used. Therewith, gas-phase acidities, proton affinities, electron donor capacities, and N-H bond dissociation enthalpies were calculated and found to compare well with available experimental data for these parameters.