Low-temperature rapid-scan detection of reactive intermediates in epoxidation reactions catalyzed by a new enzyme mimic of cytochrome p450

The use of synthetic iron(III) porphyrins as models for heme-type catalysts in biomimetic cytochrome P450 research has provided valuable information on the nature and reactivity of intermediates produced in the "peroxide shunt" pathway. This article reports spectroscopic detection of reactive intermediates formed in the epoxidation reaction of cis-stilbene with m-chloroperoxybenzoic acid catalyzed by a new mimic of cytochrome P450 with a substituted RSO3- group (1). The application of low-temperature rapid-scan stopped-flow techniques enabled the determination of equilibrium and rate constants for the formation and decay of all intermediates in the catalytic cycle of 1, including the rate constant for the formation (1*+)FeIV=O and for oxygen transfer to the substrate. Noteworthy, the reaction of (1*+)FeIV=O with cis-stilbene leads to an almost complete re-formation (95%) of the starting complex 1. The results show that complex 1 is a valuable catalyst with promising properties for further applications in a biomimetic approach toward mimicking oxygenation reactions of cytochrome P450.     

Lipase catalyzed enantioselective desymmetrization of a prochiral pentane-1,3,5-triol derivative

The enantioselective desymmetrization of the prochiral 3-O-silyl protected pentanetriol derivative 3 was carefully investigated. At −10 °C, the bacterial lipase from Burkholderia cepacia immobilized on ceramic particles led to monoacetate (S)-4 in 52% yield and >99.9% ee. At a reaction temperature of −40 °C the yield and enantioselectivity were even higher, but the reaction time was very long. Theoretical simulations of the reaction progress indicated an enantioselectivity of 25:1 at −10 °C and 35:1 at −40 °C. (S)-4 was converted into the enantiomerically pure building block 5-azidopentane-1,3-diol (S)-7 in two steps. The absolute configuration of (S)-7 was determined by exciton-coupled circular dichroism (ECCD) of diester (S)-8.     

Asymmetric synthesis of novel spirocycles via a chiral phosphoric acid catalyzed desymmetrization

A straightforward method for the asymmetric preparation of novel lactone and lactam spirocycles is described. An initial desymmetrization via a chiral Brønsted acid yields enantioenriched lactones which readily undergo a second cyclization to give the desired spirocycle.     

Reductive desymmetrization of 2-alkyl-1,3-diketones catalyzed by optically active beta-ketoiminato cobalt complexes

[reaction: see text] The reductive desymmetrization of acyclic 1,3-diketones was achieved for the first time by catalytic borohydride reduction in the presence of optically active beta-ketoiminato cobalt(II) complex catalysts. In this reaction, various 2-substituted-1,3-diaryl-1,3-propanediones were converted into the corresponding optically active 2-substituted-1,3-diaryl-3-hydroxypropanone in good-to-high yields with excellent diastereo- and enantioselectivities and high catalytic efficiencies.     

Structural basis for a Kolbe-type decarboxylation catalyzed by a glycyl radical enzyme

4-Hydroxyphenylacetate decarboxylase is a [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in clostridia. The decarboxylation product p-cresol (4-methylphenol) is a virulence factor of the human pathogen Clostridium difficile . Here we describe the crystal structures at 1.75 and 1.81 ? resolution of substrate-free and substrate-bound 4-hydroxyphenylacetate decarboxylase from the related Clostridium scatologenes . The structures show a (βγ)(4) tetramer of heterodimers composed of a catalytic β-subunit harboring the putative glycyl/thiyl dyad and a distinct small γ-subunit with two [4Fe-4S] clusters at 40 ? distance from the active site. The γ-subunit comprises two domains displaying pseudo-2-fold symmetry that are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. The N-terminal domain coordinates one cluster with one histidine and three cysteines, and the C-terminal domain coordinates the second cluster with four cysteines. Whereas the C-terminal cluster is buried in the βγ heterodimer interface, the N-terminal cluster is not part of the interface. The previously postulated decarboxylation mechanism required the substrate's hydroxyl group in the vicinity of the active cysteine residue. In contrast to expectation, the substrate-bound state shows a direct interaction between the substrate's carboxyl group and the active site Cys503, while His536 and Glu637 at the opposite side of the active site pocket anchor the hydroxyl group. This state captures a possible catalytically competent complex and suggests a Kolbe-type decarboxylation for p-cresol formation.     

Asymmetric desymmetrization of meso-vic-diols by carbamoylation catalyzed with a chiral Cu(II) complex

Asymmetric desymmetrization of meso-vic-diols was achieved by carbamoylation in the presence of copper triflate and (S,S)-Ph-BOX as a catalyst without any use of bases. The method was successfully applied to asymmetric desymmetrization of five- to eight-membered cyclic meso-vic-diols in high enantioselectivity with up to 93% ee.     

Electrochemiluminescence for probing interactions between a mimic enzyme and biological molecules

The present work proposed a novel ECL protocol to probe the interactions between mimic enzymes and small biological molecules. Iron(II) phthalocyanine (FePc) and two imidazoles (imidazole and histidine) were chosen as model molecules of mimic enzyme and small biological molecules, respectively. The interactions between FePc and the imidazoles were probed by a sensitive luminol–O₂ ECL system. Before complexing with the imidazoles, FePc can inhibit luminol–O₂ ECL due to its electrocatalysis towards O₂, however, after complexing with the imidazoles, FePc decreases the electrocatalysis, leading to the observation of an enhanced luminol–O₂ ECL. Additionally, the proposed protocol enables detection limits of 1.0 × 10^?8 mol L^?1 and 1.0 × 10^?7 mol L^?1 to be achieved, respectively, for imidazole and histidine under the physiological pH condition (pH 7.4).     

Regulation of glutamine synthetase by enzyme catalyzed structural modification

A highly sophisticated mechanism for the regulation of glutamine metabolism in Escherichia coli involves the modification of the enzyme glutamine synthetase by the covalent attachment and release of adenylyl residues. Separate enzymes catalyze the adenylylation and deadenylylation reactions, and these enzymes are reciprocally controlled by the state of nitrogen nutrition of the cell. The resultant structural alterations in glutamine synthetase enable it to change its catalytic potential greatly in response to varying cellular demands, in order to maintain nitrogen homeostasis.     

Enantiomeric separation of a group of chiral dihydropyridines by electrokinetic chromatography

Electrokinetic chromatography (EKC) was employed to achieve the enantiomeric separation of a group of chiral 1,4-dihydropyridines (DHPs) with pharmacological activity. Micelles of bile salts alone or mixed with neutral cyclodextrins, micelles of sodium dodecyl sulfate (SDS) mixed with neutral cyclodextrins, and anionic cyclodextrin derivatives, i.e., carboxymethyl-gamma-cyclodextrin (CM-gamma-CD), carboxymethyl-beta-cyclodextrin (CM-beta-CD), and succinylated beta-cyclodextrin (Succ-beta-CD), were employed as pseudostationary phases. The enantiomeric separation ability of these chiral selectors with respect to DHPs was studied in different experimental conditions. CM-beta-CD was shown to be the best chiral selector to perform the enantiomeric separation of DHPs by EKC. Next, the influence of the CM-beta-CD concentration, the pH and nature of the buffer, the temperature, and the applied voltage on the enantiomeric resolution of DHPs was studied. The use of a 50 mM ammonium acetate buffer, pH 6.7, 25 mM in CM-beta-CD together with an applied voltage of 15 or 20 kV, and a temperature of 15 degrees C enabled the individual enantiomeric separation of twelve DHPs, each one into its two enantiomers, and their separation in multicomponent mixtures of up to six DHPs into all their enantiomers.     

Condensation of benzyl chloride catalyzed by group II metals

The polymerization of benzyl chloride in the presence of various group II metal salts has been investigated. The extent of reaction and the relative activities of various salts were determined by measurement of the evolved HCl as a function of time. Formation, in situ, of an active catalyst is proposed.     

Highly enantioselective desymmetrization of meso anhydrides by a bifunctional thiourea-based organocatalyst at low catalyst loadings and room temperature

Bifunctional (thio)urea-based cinchona alkaloid derivatives have been shown to promote highly efficient enantioselective desymmetrization reactions of meso anhydrides. The most selective of these catalysts is capable of the enantioselective methanolysis of succinic and glutaric anhydride derivatives to form hemiester products with >90% yield and enantiomeric excess at 1 mol % loading and ambient temperature.     

The reduction of ribonucleotides catalyzed by the enzyme ribonucleotide reductase

This work is devoted to the study of the radical catalytic pathway for the ribonucleotide reduction process assisted by ribonucleotide reductase. The present study is directed toward the investigation of one of the most controversial steps in the reduction pathway – the elimination of the 2′ hydroxyl group from the ribonucleotide. Several groups have made different proposals for this step, which all fit available experimental data; however, so far, it has not been possible to demonstrate clearly which is the correct pathway for the elimination. Here, we resort to high-level quantum mechanical calculations to analyze the energetics of the proposed mechanisms, as well as to propose alternative pathways, and evaluate their feasibility, according to the observed kinetics of the enzyme and other existing experimental data. Our study shows that the elimination occurs via two different simultaneous acid-/base-catalyzed pathways, depending on the protonation state of one key active site amino acid, Glu441.     

G-quadruplex based impedimetric 2-hydroxyfluorene biosensor using hemin as a peroxidase enzyme mimic

We describe a sensitive and selective biosensor for the environmental metabolite 2-hydroxyfluorene (2-HOFlu). It is based on electrochemical impedance spectroscopy and was obtained by assembling a thiolated single-stranded DNA on a gold electrode via S-Au covalent bonding. It is then transformed to a K⁺-stabilized G-quadruplex-hemin complex which exhibits peroxidase-like activity to catalyze the oxidation of 2-HOFlu by H₂O₂. This results in the formation of insoluble products that are precipitated on the gold electrode. As a result, the charge transfer resistance (R _CT) between the solution and the electrode surface is strongly increased within 10 min as demonstrated by using the ferro/ferricyanide system as a redox probe. The difference in the charge transfer resistances (ΔR _CT) before and after incubation of the DNA film with 2-HOFlu and H₂O₂ serves as the signal for the quantitation of 2-HOFlu with a 1.2. nM detection limit in water of pH 7.4. The assay is highly selective over other selected fluorene derivatives. It was exploited to determine 2-HOFlu in spiked lake water samples where it displayed a detection limit of 3.6 nM. Conceivably, this method has a wide scope in that it may be applied to other analytes for which respective G-quadruplexes are available.

A G-quadruplex DNAzyme based impedimetric biosensor for sensitive detection of 2-hydroxyfluorene using hemin as a peroxidase enzyme mimic was constructed with a detection limit of 1.2 nM in water and 3.6 nM in spiked lake water samples.

     

Determination of yttrium in rocks by neutron activation and a simple group separation

A neutron-activation method is proposed for the determination of yttrium in rocks by separation of the rare-earth group and beta-counting. Interferences from rare-earth nuclides and the necessary corrections are discussed, and results for some standard rocks are presented. The method is suitable for combination with rare-earth determinations by group separation and Ge(Li) gamma-spectrometry.     

Optimization of the separation of a group of antifungals by capillary zone electrophoresis

Two simple, rapid, and efficient methods for the analysis of seven antifungal compounds have been developed by capillary zone electrophoresis. Resolutions higher than 1.5 were obtained using 0.025 M phosphate buffer (pH 2.30) (analysis time close to 9 min) or 0.2 M formic acid (pH 2.15) (analysis time close to 6 min), with an applied voltage of 20 kV and a temperature of 30 degrees C. The highest sensitivity and selectivity can be obtained using phosphate buffer but the shortest analysis times are achieved in the formic system. The analytical characteristics of the optimized methods were investigated. The reproducibility obtained for migration times (RSD(n = 10) < or = 1.0%) and peak areas (RSD(n = 10) < or = 4.3%) was acceptable, but better reproducibilities were obtained when verapamil was used as internal standard (RSD(n = 10) < 0.4% for relative migration times and RSD(n = 10) < or = 2.2% for peak area ratios). The lowest limit of detection was obtained for clotrimazole (0.12 microg/ml) and the highest for fluconazole and voriconazole (0.90 microg/ml). The lowest and the highest limits of quantitation were, respectively, 0.40 microg/ml for clotrimazole and 3.00 microg/ml for fluconazole and voriconazole.     

Capillary electrophoresis as a nanoreactor of separation capability: on-capillary reaction catalyzed by transition metal ions

The catalytic oxidation of 1,2-dihydroxy-benzene-3,6-disulfonate (tiron) by metal ions has been studied for detection of the metal ions in capillary electrophoresis (CE). Although Co(2+) shows the strongest catalytic capability, some other metal ions also catalyze this reaction. If metal ions encounter a H(2)O(2 )zone after electrophoretic separation in the running buffer containing tiron, tiron is catalytically oxidized while the metal ion passes through the H(2)O(2) zone. Anionic tiron radicals produced by the reaction are finally measured by the detector; in this scheme, the capillary acts as a nano- or microreactor as well as a microseparator. The effective capillary length can be controlled by changing the interval between metal ion and H(2)O(2) injections. This scheme has been successfully applied to the detection of Co(2+), Cu(2+), Mn(2+), and VO(2+). The detectability is discussed from several viewpoints, such as the intrinsic catalyst ability of metal ions, the kinetics of the catalytic reaction, and reaction times determined by the mobility of the zone of the metal ion. Some strange behaviors, which cannot be predicted by batch experiments, are also reported.