Monooxygenase stereoselectivity in the biosynthesis of stereoisomeric spiroacetals in the cucumber fly, Bactrocera cucumis

The stereoselectivity of hydroxylation of alkyltetrahydropyran-2-ols (or their biological equivalents) in the formation of stereoisomers of 2,8-dimethyl-1,7-dioxaspiro[5.5]undecanes in male Bactrocera cucumis has been investigated. Racemic, (6R)-, and (6S)-6-methyl-2-[5-H-2(1)]-n-pentyltetrahydropyran-2-ol was administered under an [O-18(2)]-enriched atmosphere. The stereochemistry and isotopic composition of generated spiroacetals were monitored by combined enantioselective GC-MS. The monooxygenase(s) strongly prefers the (6S)-substrate and furnishes predominantly the (S)-alcohol and then the (2S,6R,8S)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. The (2S,6S,8R) and (2R,6S,8S) (E,Z)-isomers appear to be derived in vivo predominantly from (R)-hydroxylation of the (6S)-tetrahydropyranol.

Linear ketenimines. Variable structures of C,C-dicyanoketenimines and C,C-bis-sulfonylketenimines

C,C-Dicyanoketenimines 10a-c were generated by flash vacuum thermolysis of ketene NS-acetals 9a-c or by thermal or photochemical decomposition of alpha-azido-,beta-cyanocinnamonitrile 11. In the latter reaction, 3,3-dicyano-2-phenyl-1-azirine 12 is also formed. IR spectroscopy of the keteniminines isolated in Ar matrixes or as neat films, NMR spectroscopy of 10c, and theoretical calculations (B3LYP/6-31G*) demonstrate that these ketenimines have variable geometry, being essentially linear along the CCN-R framework in polar media (neat films and solution), but in the gas phase or Ar matrix they are bent, as is usual for ketenimines. Experiments and calculations agree that a single CN substituent as in 13 is not enough to enforce linearity, and sulfonyl groups are less effective that cyano groups in causing linearity. C,C-Bis(methylsulfonyl)ketenimines 4-5 and a C-cyano-C-(methylsulfonyl)ketenimine 15 are not linear. The compound p-O2NC6H4N=C= C(COOMe)2 previously reported in the literature is probably somewhat linearized along the CCNR moiety. A computational survey (B3LYP/6-31G*) of the inversion barrier at nitrogen indicates that electronegative C-substituents dramatically lower the barrier; this is also true of N-acyl substituents. Increasing polarity causes lower barriers. Although N-alkylbis(methylsulfonyl)ketenimines are not calculated to be linear, the barriers are so low that crystal lattice forces can induce planarity in N-methylbis(methylsulfonyl)ketenimine 3.

Calorimetric demonstration of the potential of molecular crowding to emulate the effect of an allosteric activator on pyruvate kinase kinetics

A method based on isothermal calorimetry is described for the direct kinetic assay of pyruvate kinase. In agreement with earlier findings based on the standard coupled assay system for this enzyme in the presence of a fixed ADP concentration, the essentially rectangular hyperbolic dependence of initial velocity upon phosphoenolpyruvate concentration is rendered sigmoidal by the allosteric inhibitor phenylalanine. This effect of phenylalanine can be countered by including a high concentration of a space- filling osmolyte such as proline in the reaction mixtures. This investigation thus affords a dramatic example that illustrates the need to consider potential consequences of thermodynamic nonideality on the kinetics of enzyme reactions in crowded molecular environments such as the cell cytoplasm.

Lanczos subspace time-independent wave packet calculations of S(D-1)+H-2 reactive scattering

In this paper. we present the results of quantum dynamical simulations of the S (D-1) + H-2 insertion reaction on a newly developed potential energy surface (J. Chem. Phys. 2001, 114, 320). State-to-state reaction probabilities. product state distributions, and initial-state resolved cumulative reaction probabilities from a given incoming reactant channel are obtained from a time-independent wave packet analysis, performed within a single Lanczos subspace. Integral reaction cross sections are then estimated by J-shifting method and compared with the results from molecular beam experiment and QCT calculations.

Calculation of resonances and product state distributions for the unimolecular dissociation of H2S

Resonance phenomena associated with the unimolecular dissociation of H2S --> SH + H have been investigated quantum mechanically by the Lanczos homogeneous filter diagonalization method using a newly developed potential energy surface (J. Chem. Phys. 2001, 114, 320). Resonance energies, widths (rates), and product state distributions have been obtained. Both dissociation rates and product state distributions of SH show, strong fluctuations, indicating that the dissociation of H2S is essentially irregular. Statistical analysis of neighboring level spacing and width distributions also confirms this behavior. The dissociation rates and product state distributions are compared to the predictions of quantum phase space theory.

Characterization of redox centers in dimethyl sulfide dehydrogenase from Rhodovulum sulfidophilum

Dimethyl sulfide dehydrogenase from the purple phototrophic bacterium Rhodovulum sulfidophilum catalyzes the oxidation of dimethyl sulfide to dimethyl sulfoxide. Recent DNA sequence analysis of the ddh operon, encoding dimethyl sulfide dehydrogenase (ddhABC), and biochemical analysis (1) have revealed that it is a member of the DMSO reductase family of molybdenum enzymes and is closely related to respiratory nitrate reductase (NarGHI). Variable temperature X-band EPR spectra (120122 K) of purified heterotrimeric dimethyl sulfide dehydrogenase showed resonances arising from multiple redox centers, Mo(V), [3Fe-4S](+), [4Fe-4S](+), and a b-type heme. A pH-dependent EPR study of the Mo(V) center in (H2O)-H-1 and (H2O)-H-2 revealed the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(v)-anion, and Mo(V)-OH. Above pH 8.2 the dominant species was Mo(V)-OH. The maximum specific activity occurred at pH 9.27. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other molybdenum enzymes of the DMSO reductase family showed that it was most similar to the low-pH nitrite spectrum of Escherichia coli nitrate reductase (NarGHI), consistent with previous sequence analysis of DdhA and NarG. A sequence comparison of DdhB and NarH has predicted the presence of four [Fe-S] clusters in DdhB. A [3Fe-4S](+) cluster was identified in dimethyl sulfide dehydrogenase whose properties resembled those of center 2 of NarH. A [4Fe-4S](+) cluster was also identified with unusual spin Hamiltonian parameters, suggesting that one of the iron atoms may have a fifth non-sulfur ligand. The g matrix for this cluster is very similar to that found for the minor conformation of center 1 in NarH [Guigliarelli, B., Asso, M., More, C., Augher, V., Blasco, F., Pommier, J., Giodano, G., and Bertrand, P. (1992) Eur. J. Biochem. 307,63-68]. Analysis of a ddhC mutant showed that this gene encodes the b-type cytochrome in dimethyl sulfide dehydrogenase. Magnetic circular dichroism studies revealed that the axial ligands to the iron in this cytochrome are a histidine and methionine, consistent with predictions from protein sequence analysis. Redox potentiometry showed that the b-type cytochrome has a high midpoint redox potential (E-o = +315 mV, pH 8).

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: Metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(l) per CopZ and two copper(l) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(1)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(1)(2)CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(l)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper. from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(l) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange: a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.

Cloud-point extraction and preconcentration of cyanobacterial toxins (microcystins) from natural waters using a cationic surfactant

A new cloud-point extraction and preconcentration method, using a cationic, surfactant, Aliquat-336 (tricaprylyl-methy;ammonium chloride), his-been developed for the determination of cyanobacterial toxins, microcystins, in natural waters. Sodium sulfate was used to induce phase separation at 25 degreesC. The phase behavior of Aliquat-336 with respect to concentration of Na2SO4 was studied. The cloud-point system revealed a very high phase volume ratio compared to other established systems of nonionic, anionic, and cationic surfactants: At pH 6-7, it showed an outstanding selectivity in ahalyte extraction for anionic species. Only MC-LR and MC-YR, which are known to be predominantly anionic, were extracted (with averaged recoveries of 113.9 +/- 9% and 87.1 +/- 7%, respectively). MC-RR, which is likely to be amphoteric at the above pH range, was. not cle tectable in.the extract. Coupled to HPLC/UV separation and detection, the cloud-point extraction method (with 2.5 mM Aliquat-336 and 75 mM Na2SO4 at 25 degreesC) offered detection limits of 150 +/- 7 and 470 +/- 72 pg/mL for MC-LR and MC-YR, respectively, in 25 mL of deionized water. Repeatability of the method was 7.6% for MC-LR and 7.3% for MC-YR: The cloud-point extraction process can be. completed within 10-15 min with no cleanup steps required. Applicability of the new method to the determination of microcystins in real samples was demonstrated using natural surface waters, collected from a local river and a local duck pond spiked with realistic. concentrations of microcystins. Effects of salinity and organic matter (TOC) content in the water sample on the extraction efficiency were also studied.

Transformation processes, pathways, and possible sources of distinctive polychlorinated dibenzo-p-dioxin signatures in sink environments

In recent years, studies on environmental samples with unusual dibenzo-p-dioxin (PCDD) congener profiles were reported from a range of countries. These profiles, characterized by a dominance of octachlorinated dibenzodioxin (OCDD) and relatively low in dibenzofuran (PCDF) concentrations, could not be attributed to known sources or formation processes. In the present study, the processes that result in these unusual profiles were assessed using the concentrations and isomer signatures of PCDDs from dated estuarine sediment cores in Queensland, Australia. Increases in relative concentrations of lower chlorinated PODS and a relative decrease of OCDD were correlated with time of sediment deposition. Preferred lateral, anaerobic dechlorination of OCDD represents a likely pathway for these changes. In Queensland sediments, these transformations result in a distinct dominance of isomers fully chlorinated in the 1,4,6,9-positions (1,4-patterns), and similar 1,4-patterns were observed in sediments from elsewhere. Consequently, these environmental samples may not reflect the signatures of the original source, and a reevaluation of source inputs was undertaken. Natural formation of PCDDs, which has previously been suggested, is discussed; however, based on the present results and literature comparisons, we propose an alternative scenario. This scenario hypothesizes that an anthropogenic PCDD precursor input (e.g. pentachlorophenol) results in the contamination. These results and hypothesis imply further investigations are warrented into possible anthropogenic sources in areas where natural PCDD formation has been suggested.

Structural changes in mixed Langmuir-Blodgett films upon nanoparticle formation

The formation of CdS nanoparticles by reacting mixed Langmuir-Blodgett films of arachidic acid and either octadecylamine or dimethyldioctadecylammonium nitrate on a cadmium-containing subphase with hydrogen sulfide gas has resulted in the identification of a number of structural changes, observed using grazing incidence X-ray diffraction. In the case of octadecylamine, the structure after reaction is a hexagonal close-packed array of surfactant-stabilized nanoclusters, with a lattice constant of a = 17.65 Angstrom. In both octadecylamine and dimethyldioctadecylammonium nitrate films, the presence of a unit cell tilted at 38degrees to the plane of the substrate was found. Despite these changes, the average nanoparticle size was unaffected by the addition of either second component to the film.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

Molecular orbital theory calculations of the H2O-carbon reaction

Carbon gasification with steam to produce H-2 and CO is an important reaction widely used in industry for hydrogen generation. Although the literature is vast, the. mechanism for the formation of H-2 is still unclear. In particular, little has, been done to investigate the potential of molecular orbital theory to distinguish different mechanism possibilities. In this work, we used molecular orbital theory to demonstrate a favorable energetic pathway where H2O is first physically adsorbed on the virgin graphite surface with negligible change in molecular structure. Chemisorption occurs via O approaching the carbon edge site with one H atom stretching away from the O in the transition state. This is followed by a local minimum. state in which the stretching H is further disconnected from the O atoms and the remaining OH group is still on the carbon edge site. The disconnected H then pivot around the OH group to bond with the H of the OH group and forms H-2. The O atom remaining on the carbon edge site is subsequently desorbed as CO. The reverse occurs when H-2 reacts with the surface oxygen to produce H2O.

Adsorption of aromatic compounds onto activated carbons: Effects of the orientation of the adsorbates

Adsorption of model aromatic compounds onto two untreated activated carbons with similar physical and chemical properties is investigated. The solution pH of all experiments was lowered so that all solutes were in their molecular forms. It is shown that the difference in the maximum adsorption capacities of the solutes was mainly attributed to the difference in the sizes of the molecules. This new experimental finding is significant to gaining insight into the orientation of the adsorbed phase and hence the adsorption mechanism of aromatic compounds in aqueous solutions. It is shown that the adsorption of aromatic compounds in a stacked motif for pi-pi interactions is unlikely, and in the absence of physical restrictions such as pore width, a T-shaped motif is the preferred orientation.

Effects of the solute ionization on the adsorption of aromatic compounds from dilute aqueous solutions by activated carbon

Adsorption of four dissociating aromatic compounds and one nondissociating compound on a commercial activated carbon is investigated systematically. All adsorption experiments were carried out in pH-controlled aqueous solutions. The adsorption isotherms are fitted to the binary homogeneous Langmuir model, where the concentrations of the molecular and the ionic species in the liquid phase are expressed in terms of the sum of the two and the degree of solute ionization. Examination of the relationships between the solution pH, the degree of ionization of the solutes, and the model parameters is found to give new insights into the adsorption process. Furthermore, this is used to correlate the variation of the monolayer capacity with the solution pH.

Role of oxygen in the nitrous oxide/carbon reaction

An investigation of the role of oxygen in the nitrous oxide/carbon reaction was carried out on various carbon samples (both graphitic and nongraphitic) over a range of temperatures and partial pressures. Previous work reported that oxygen strongly inhibited the nitrous oxide/carbon reaction. Large ratios of O-2/N2O were used in all previous work. In this work, the O-2/N2O ratio was kept below 1, and we found that oxygen did not inhibit the rate of the C + N2O reaction. Instead, the rate of the reaction in the presence of oxygen was essentially that predicted by the two independent reactions, nitrous oxide/carbon and oxygen/carbon, occurring simultaneously. A simple theoretical explanation is given for the observations, both past and present, on the basis of competitive chemisorption of nitrous oxide and oxygen on active sites.