Synthesis and stereochemistry of some bicyclic gamma-lactones from parasitic wasps (Hymenoptera : Braconidae). Utility of hydrolytic kinetic resolution of epoxides and palladium(II)-catalyzed hydroxycyclization-carbonylation-lactonization of ene-diols

A palladium(II)-catalyzed hydroxycyclization-carbonylation-lactonization sequence with appropriate pent-4-ene-1,3-diols provides efficient access to the bicyclic gamma -lactones, 5-n-butyl- and 5-n-hexyltetrahydrofuro-[3,2-b]furan-2(3H)-ones (3) and (4), respectively, in both racemic and enantiomeric forms. Some of the substrate pent-4-ene-1,3-diols of high enantiomeric excess (ee) have been derived from racemic terminal epoxides by hydrolytic kinetic resolution (HKR) using cobalt (III)-salen complexes. (9Z,12R)-(+)-Ricinoleic acid also serves as a chiral pool source of other pent-4-ene-1,3-diols. These syntheses and enantioselective gas chromatography confirm the structures and absolute stereochemistry of the lactones in some species of parasitic wasps (Hymenoptera: Braconidae). The highly abundant 5-n-hexyltetrahydrofuro-[3,2-b]furan-2(3H)-one (4) in Diachasmimorpha kraussii and D. longicaudata is of high ee (> 99%) with (3aR,5R,6aR) stereochemistry.

An Activated O N Acyl Transfer Auxiliary: Efficient Amide-Backbone Substitution of Hindered "Difficult" Peptides

Overcoming the phenomenon known as difficult synthetic sequences has been a major goal in solid-phase peptide synthesis for over 30 years. In this work the advantages of amide backbone-substitution in the solid-phase synthesis of difficult peptides are augmented by developing an activated N-alpha-acyl transfer auxiliary. Apart from disrupting troublesome intermolecular hydrogen-bonding networks, the primary function of the activated N-alpha-auxiliary was to facilitate clean and efficient acyl capture of large or beta-branched amino acids and improve acyl transfer yields to the secondary N-alpha-amine. We found o-hydroxyl-substituted nitrobenzyl (Hnb) groups were suitable N-alpha-auxiliaries for this purpose. The relative acyl transfer efficiency of the Hnb auxiliary was superior to the 2-hydroxy-4-methoxybenzyl (Hmb) auxiliary with protected amino acids of varying size. Significantly, this difference in efficiency was more pronounced between more sterically demanding amino acids. The Hnb auxiliary is readily incorporated at the N-alpha-amine during SPPS by reductive alkylation of its corresponding benzaldehyde derivative and conveniently removed by mild photolysis at 366 nm. The usefulness of the Hnb auxiliary for the improvement of coupling efficiencies in the chain-assembly of difficult peptides was demonstrated by the efficient Hnb-assisted Fmoc solid-phase synthesis of a known hindered difficult peptide sequence, STAT-91. This work suggests the Hnb auxiliary will significantly enhance our ability to synthesize difficult polypeptides and increases the applicability of amide-backbone substitution.

Photoinduced electron transfer and electronic energy transfer in naphthyl-appended cyclams

A series of novel macrocyclic tetraaza ligands that incorporate a naphthalene moiety as a photoactive chromophore have been prepared and structurally characterized as their Cu(II) complexes. Variable-temperature photophysical studies have concluded that the luminescence quenching evident in the Cu(H) complexes is due to intramolecular electronic energy transfer (EET). In their free-base forms, these ligands undergo reductive luminescence quenching via photoinduced electron transfer (PET) reactions, with proximate amine lone pairs acting as electron donors. Consequently, the emission behavior can be modulated by variations in pH and/or the presence of other Lewis acids such as Zn(H).

Total synthesis and absolute stereochemistry of plakortone D

The first total synthesis of plakortone D is described and thereby establishes the structure and absolute stereochemistry of the most biologically active member of the marine-derived plakortone family. The sterically congested bicyclic lactone core results from a Pd(II)-induced hydroxycyclization−carbonylation−lactonization sequence on an enediol whose chirality was installed by AD-technology. Attachment of the side chain, also constructed using AD-methodology, was achieved by using a modified Julia coupling. The described approach enables acquisition of other plakortones and analogues, in the correct (natural) stereochemical series.

Reactions of fac-[PtMe2(OMe)(H2O)3]+ with halide ions: effect of halide trans effect on methoxide hydrolysis

A solution of fac-[PtMe2(OMe)(H2O)(3)](+) (1) in aqueous perchloric acid underwent very slow hydrolysis of the Pt-OMe bond, over many, weeks. When chloride was added to a solution of 1, two interconverting isomers of [PtMe2(OMe)Cl(H2O)(2)] (with chloride trans to methyl) were formed, and with excess chloride, [PtMe2(OMe)Cl-2(H2O)](-) (both chloride ligands trans to methyl). This solution was stable at ambient temperature, but on heating, methanol was formed and [PtMe2Cl2(H2O)(2)] (both chloride ligands cis to methyl) was produced in the solution. It is proposed that this reaction proceeds via an intermediate complex with chloride bound trans to methoxide. Concentration gave solid [{PtMe2Cl2}n], whose identity was confirmed by conversion to [PtMe(2)Cl(2)py(2)] (pyridine, py, trans to methyl). With bromide and iodide, methoxide hydrolysis occurred at ambient temperature, more slowly with bromide than with iodide, to form solid [{PtMe2X2}(n)] without significant concentrations of [PtMe2X2(H2O)(2)] formed as an intermediate. The greater tendency for Pt-OMe bond to hydrolyse trans to halide compared with 1 was ascribed to the higher trans effect of the halide ligand compared with that of water. (C) 2003 Elsevier Science B.V. All rights reserved.

Kinetic and phylogenetic characterization of an anaerobic dechlorinating microbial community

The reductive dechlorination (RD) of tetrachloroethene (PCE) to vinyl chloride (VC) and, to a lesser extent, to ethene (ETH) by an anaerobic microbial community has been investigated by studying the processes and kinetics of the main physiological components of the consortium. Molecular hydrogen, produced by methanol-utilizing acetogens, was the electron donor for the PCE RD to VC and ETH without forming any appreciable amount of other chlorinated intermediates and in the near absence of methanogenic activity. The microbial community structure of the consortium was investigated by preparing a 1 6S rDNA clone library and by fluorescence in situ hybridization (FISH). The PCR primers used in the clone library allowed the harvest of 16SrDNA from both bacterial and archaeal members in the community. A total of 616 clones were screened by RFLP analysis of the clone inserts followed by the sequencing of RFLP group representatives and phylogenetic analysis. The clone library contained sequences mostly from hitherto undescribed bacteria. No sequences similar to those of the known RD bacteria like 'Dehalococcoides ethenogenes' or Dehalobacter restrictus were found in the clone library, and none of these bacteria was present in the RD consortium according to FISH. Almost all clones fell into six previously described phyla of the bacterial domain, with the majority (56(.)6%) being deep-branching members of the Spirochaetes phylum. Other clones were in the Firmicutes phylum (18(.)5%), the Chloroflexi phylum (16(.)4%), the Bacteroidetes phylum (6(.)3%), the Synergistes genus (11(.)1%) and a lineage that could not be affiliated with existing phyla (11(.)1%). No archaeal clones were found in the clone library. Owing to the phylogenetic novelty of the microbial community with regard to previously cultured microorganisms, no specific microbial component(s) could be hypothetically affiliated with the RD phenotype. The predominance of Spirochaetes in the microbial consortium, the main group revealed by clone library analysis, was confirmed by FISH using a purposely developed probe.

Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.

A numerical approach to modeling the catalytic voltammetry of surface-confined redox enzymes

A finite difference method for simulating voltammograms of electrochemically driven enzyme catalysis is presented. The method enables any enzyme mechanism to be simulated. The finite difference equations can be represented as a matrix equation containing a nonlinear sparse matrix. This equation has been solved using the software package Mathematica. Our focus is on the use of cyclic voltammetry since this is the most commonly employed electrochemical method used to elucidate mechanisms. The use of cyclic voltammetry to obtain data from systems obeying Michaelis-Menten kinetics is discussed, and we then verify our observations on the Michaelis-Menten system using the finite difference simulation. Finally, we demonstrate how the method can be used to obtain mechanistic information on a real redox enzyme system, the complex bacterial molybdoenzyme xanthine dehydrogenase.

Synthesis in the plakortone series: Plakortone E

A Pd(II)-mediated hydroxycyclisation-carbonylation-lactonisation sequence has operated efficiently with racemic enediol (8) to furnish (four) separable diastereomers of the bicyclic lactone system assigned to the sponge-derived, bioactive plakortone E. All four are cis ring-fused, and one is identical, on the basis of H-1 and C-13 NMR spectroscopic comparisons, with plakortone E, thus confirming its constitution and relative stereochemistry about the bicyclic lactone core. This synthetic approach, when applied to stereoisomer (13), will establish the absolute stereochemistry of plakortone E, likely to be that shown for (14).

A nearly idealized 6 '-O-methylated t-carrageenan from the Australian red alga Claviclonium ovatum (Acrotylaceae, Gigartinales)

The polysaccharides extracted from Claviclonium ovatum were studied by a combination of compositional assays, reductive partial hydrolysis, linkage analysis, Fourier Transform infrared (FTIR) spectroscopy, and C-13, H-1, and C-13/H-1 heteronuclear multiple quantum correlation (HMQC) two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The chemical and spectroscopic data showed that the alkali-modified C. ovatum polysaccharides are composed of a nearly idealized repeating unit of 6'-O-methylcarrabiose 2,4'-disulfate (the repeating unit of 6-O-methylated iota-earrageenan), although some minor components were also present. The C. ovatum galactans are the most highly methylated carrageenans reported. (C) 2004 Elsevier Ltd. All rights reserved.

Diversity in the disulfide folding pathways of cystine knot peptides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides; poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence in directing the folding pathway.

Tetra- and tribromopbenoxyanisoles in Marine Samples from Oceania

Some methoxylated polybrominated diphenyl ethers (MeO-BDEs) are known halogenated natural products (HNPs) and are frequently detected in higher organisms of the marine environment. In this study we demonstrate that a prominent MeO-BDE, previously detected in marine mammals from Australia, is identical to 3,5-dibromo-2-(2',4'-dibromo)phenoxyanisole(BC-3,6-MeO-BDE47). Up to 1.9mg/ kg of 6-MeO-BDE 47 was present in cetaceans from Australia, 0.2-0.3 mg/kg in two crocodile eggs from Australia, but concentrations of 1 or 2 orders of magnitude lower were found in shark liver oil from New Zealand and in marine mammals from Africa and the Antarctic. Concentrations of 6-MeO-BDE47 in samples from Australia were in the same range as anthropogenic pollutants such as PCB 153 and p,p'-DDE. Along with 6-MeO-BDE 47 and the known HNP 4,6-dibromo-2-(2',4'-dibromo)phenoxyanisole (BC-2,2'-MeO-BDE 68), several tribromophenoxyanisoles (MeO-triBDE) were present in tissue of Australian cetaceans. To determine their structure, abiotic debromination experiments were performed using 6-MeO-BDE 47 and 2'-MeO-BDE 68 and superreduced di cyanocobalamine. These experiments resulted in formation of eight MeO-triBDEs, all of which were detected in the cetacean samples. Five of these eight MeO-triBDEs could be identified based on two standard compounds as well as gas chromatographic and mass spectrometric features. It was also shown that the first eluting isomer (compound 1), 6-MeO-BDE 17 (compound 2), and 2-MeO-BDE 39 (compound 5) were the most prominent MeO-triBDEs in the Australian cetacean samples. The concentrations of the MeO-triBDEs in two cetacean samples were 0.20 and 0.36 mg/kg, respectively. Although the reductive debromination with dicyanocobalamine resulted in a different congener pattern than was found in the marine mammals, it could not be excluded that the tribromo congeners of 6-MeO-BDE 47 and 2'-MeO-BDE 68 in the samples were metabolites of the latter.

Effects of acetate and propionate on the performance of a photosynthetic biofilm reactor for sulfide removal

The effects of acetate and propionate on the performance of a recently proposed and characterized photosynthetic biological sulfide removal system have been investigated with a view to predicting this concept's suitability for removing sulfide from wastewater undergoing or having undergone anaerobic treatment. The concept relies on substratum-irradiated biofilms dominated by green sulfur bacteria (GSB), which are supplied with radiant energy in the band 720 - 780 nm. A model reactor was fed for 7 months with a synthetic wastewater free of volatile fatty acids (VFAs), after which time intermittent dosing of the wastewater with acetate or propionate was begun. Such dosing suppressed the areal net sulfide removal rate by similar to50%, and caused the principal net product of sulfide removal to switch from sulfate to elemental-S. Similarly suppressed values of this rate were observed when the wastewater was dosed continuously with acetate, and this rate was not significantly affected by changes in the concentration of ammonia-N in the feed. The main net product of sulfide removal was again elemental-S, which was scarcely released into the liquid, however. Sulfate reduction and sulfur reduction were observed when the light supply was interrupted and were inferred to be occurring within the irradiated biofilm. A preexisting conceptual model of the biofilm was augmented with both of these reductive processes, and this augmented model was shown to account for most of the observed effects of VFA dosing. The implications of these findings for the practicality of the technology are considered. (C) 2004 Wiley Periodicals, Inc.

Knots in rings - The circular knotted protein momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys(1)-Cys(18) disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.