Characterization of an O2 adduct of an active cobalt-substituted extradiol-cleaving catechol dioxygenase

The first example of an O(2) adduct of an active Co-substituted oxygenase has been observed in the extradiol ring cleavage of the electron-poor substrate 4-nitrocatechol (4NC) by Co(II)-homoprotocatechuate 2,3-dioxygenase (Co-HPCD). Upon O(2) binding to the high-spin Co(II) (S = (3)/(2)) enzyme-substrate complex, an S = (1)/(2) EPR signal exhibiting (59)Co hyperfine splitting (A = 24 G) typical of a low-spin Co(III)-superoxide complex was observed. Both the formation and decay of the new intermediate are very slow in comparison to the analogous steps for turnover of 4NC by native high-spin Fe(II)-HPCD, which is likely to remain high-spin upon O(2) binding. A similar but effectively stable S = (1)/(2) intermediate was formed by the inactive [H200N-Co-HPCD(4NC)] variant. The observations presented shed light on the key roles played by the substrate, the second-sphere His200 residue, and the spin state of the metal center in facilitating O(2) binding and activation.     

Polyfluoro-1,2-epoxy alkanes and cycloalkanes. Part IV [1]. Thermal reactions of the epoxides of the pentamer and hexamer oligomers of tetrafluoroethene

Oxirans (1) and (2), derived respectively from the pentamer and hexamer oligomers of tetrafluoroethene, were pyrolysed over pyrex glass at 300–500° alone and in the presence of cyclohexene, bromine and toluene. Thus, oxiran (1), pyrolysed alone, afforded perfluoro-2-methylbut-1-ene (3), perfluoro-2,3-dimethylpent-2-ene (4) and (E) and (Z) perfluoro-2,3-hex-3-ene (TFE tetramer) (5a, 5b). Co-pyrolysis of (1) with bromine afforded (E) and (Z) 2-bromoperfluoro-3-methylpent-2-ene (6a, 6b), whilst with toluene, (E) and (Z) 2H-perfluoro-3-methylpent-2-ene (7a, 7b) were obtained: (1) with excess cyclohexene also gave (7a, 7b). The oxiran (2), on pyrolysis alone, gave only (3). In the presence of bromine, (2) gave an equimolar mixture of 1-bromoperfluoro-3-methylpentan-2-one (8) and 3-bromoperfluoro-3-methylpentane (9). Co-pyrolysis of (2) with toluene yielded (3) and 3H-perfluoro-3-methylpentane (10). Pyrolysis of (2) with cyclohexene at 175° gave perfluoro-3-methyl-2-(1-methylpropyl)pent-2-en-1-oylfluoride (11), pentafluoroethylcyclohexane (12) and perfluoro[(1-ethyl-1-methylpropyl) (1-methylpropyl)]ketne (13).     

Reactions of tetrafluoroethylene oligomers. Part 1. Some pyrolytic reactions of the pentamer and hexaher and of the fluorine adducts of the tetramer and pentamer

Pyrolyses of these highly branched fluorocarbons over glass beads caused the preferential thermolyses of CC bonds where there is maximum carbon substitution. Fluorinations of perfluoro-3,4-dimethylhex-3-ene (tetramer) (I) and perfluoro-4-ethyl-3,4-dimethylhex- 2-ehe (pentamer) (II) over cobalt (III) fluoride at 230° and 145° respectively afforded the corresponding saturated fluorocarbons (III) and (IV), though II gave principally the saturated tetramer (III) at 250°. Pyrolysis of III alone at 500—520° gave perfluoro-2-methylbutane (V), whilst pyrolysis of III in the presence of bromine or toluene afforded 2-bromononafluorobutane (VI) and 2H-nonafluorobutane (VII) respectively. Pyrolysis of perfluoro-3-ethyl-3, 4-dimethylhexane (IV) alone gave a mixture of perfluoro-2-methylbutane (V), perfluoro-2-methylbut-1-ene (VIII), perfluoro-3-methylpentane (IX), perfluoro-3,3-dimethylpentane (X), and perfluoro-3,4- dimethylhexane (III). Pyrolysis of IV in the presence of bromine gave (VI) and 3-bromo-3-trifluoromethyl-decafluoropentane (XI): with toluene, pyrolysis gare VlI and 3H-3-trifluoromethyldecafluoropentane (XII). Pyrolysis of II at 500° over glass gave perfluoro-1,2,3-trimethylcyclobutene (XIII) and perfluoro-2,3-dimethylpenta-1,3(E)- and (Z)-diene (XIV) and (XV) respectively. The diene mixture (XIV and XV) was fluorinated with CoF₃ to give perfluoro-2,3-dimethylpentane (XVI) and was cyclised thermally to give the cyclobutene (XIII). Pyrolysis of perfluoro-2- (1′-ethyl-1′-methylpropyl)-3-methylpent-1-ene (XVII) (TFE hexamer major isomer) at 500° gave perfluoro-1-methyl-2-(1′-methylpropyl)cyclobut-1-ene (XVIII) and perfluoro-2-methyl-2-(1′-methylpropyl)buta-1,3-diene (XIX). Fluorination of XVIII over CoF₃ gave perfluoro-1-methyl-2- (1′-methylpropyl)cyclobutane (XX), which on co-pyrolysis with bromine gave VI. XIX on heating gave XVIII. Reaction of XVIII with ammonia in ether gave a mixture of E and Z 1′-trifluoromethyl-2-(1′-trifluoromethyl- pentafluoropropyliden-1′-yl)tetrafluorocyclobutylamine (XXI) which on diazotisation and hydrolysis afforded 2-(2′trifluoromethyl- tetrafluorocyclobut-1-en-1′-yl)-octafluorobutan-2-ol (XXII).     

Electric biasing of the divertor tiles in compass-D

It has been suggested that by electrically biasing the divertor tiles in a tokamak, one could create a toroidally-asymmetric potential structure in the scrape-off layer (SOL) plasma. The resultingE×B convective motion should increase the plasma transport in the SOL and thereby reduce the heat load on the divertor plates. This paper describes plans for such experiments on COMPASS-D and preliminary theoretical modelling. The COMPASS-D divertor target consists of 24 graphite tiled toroidal segments. The inner and outer strike point tiles on all these segments will be insulated from each other, and cabling will be installed to allow for biasing of individual tiles in any combination. Theoretical analysis suggests that the electrostatic potential perturbations caused by the biasing will be confined to the divertor region below the X-point, and that substantial SOL broadening can be achieved by applying modest voltages. Aside from active control of SOL transport, it is hoped that the biasing will be useful to probe other divertor and SOL physics issues. Presented at the Workshop on the Role of Electric Fields in Plasma Confinement and Exhaust, Budapest, 18–19 June, 2000. This work was jointly funded by the UK Department of Trade and Industry and Euratom, and was supported by US DoE under contract W-7405-ENG-48 at LLNL.     

Optimization of N‐methyl‐N‐[tert‐butyldimethylsilyl]trifluoroacetamide as a derivatization agent for determining isotopic enrichment of glycerol in very‐low density lipoproteins

Stable isotope kinetic studies play an important role in the study of very‐low density lipoprotein (VLDL) metabolism, including basic and clinical research. Today, [1,1,2,3,3‐²H₅]glycerol is the most cost‐effective alternative to measure glycerol and triglyceride kinetics. Recycling of glycerol from glycolysis and gluconeogenesis may lead to incompletely labelled tracer molecules. Many existing methods for the measurement of glycerol isotopic enrichment involve the production of glycerol derivatives that result in fragmentation of the glycerol molecule after ionization. It would be favourable to measure the intact tracer molecule since incompletely labelled tracer molecules may be measured as fully labelled. The number of methods available to measure the intact tracer in biological samples is limited. The aim of this project was to develop a gas chromatography/mass spectrometry (GC/MS) method for glycerol enrichment that measures the intact glycerol backbone and is suitable for electron ionization (EI), which is widely available. A previously published method for N‐methyl‐N‐[tert‐butyldimethylsilyl]trifluoroacetamide (MTBSTFA) derivatization was significantly improved; we produced a stable derivative and increased recovery 27‐fold in standards. We used the optimized MTBSTFA method in VLDL‐triglyceride and found that further modification was required to take matrix effects into account. We now have a robust method to measure glycerol isotopic enrichment by GC/EI‐MS that can be used to rule out the known problem of tracer recycling in studies of VLDL kinetics. Copyright © 2010 John Wiley & Sons, Ltd.     

Intramolecular thiomaleimide [2 + 2] photocycloadditions: stereoselective control for disulfide stapling and observation of excited state intermediates by transient absorption spectroscopy

Thiomaleimides undergo efficient intermolecular [2 + 2] photocycloaddition reactions and offer applications from photochemical peptide stapling to polymer crosslinking; however, the reactions are limited to the formation of the exo head-to-head isomers. Herein, we present an intramolecular variation which completely reverses the stereochemical outcome of this photoreaction, quantitatively generating endo adducts which minimise the structural disturbance of the disulfide staple and afford a 10-fold increase in quantum yield. We demonstrate the application of this reaction on a protein scaffold, using light to confer thiol stability to an antibody fragment conjugate. To understand more about this intriguing class of [2 + 2] photocycloadditions, we have used transient absorption spectroscopy (electronic and vibrational) to study the excited states involved. The initially formed S₂ (π₁π*) excited state is observed to decay to the S₁ (n₁π*) state before intersystem crossing to a triplet state. An accelerated intramolecular C–C bond formation provides evidence to explain the increased efficiency of the reaction, and the impact of the various excited states on the carbonyl vibrational modes is discussed.

Tethered bromomaleimides are described which undergo thiol conjugation and rapid regio- and stereoselective photocycloadditions to serve as disulfide staples; with the excited state intermediates observed by transient absorption spectroscopies.     

Early stage kinetics in a unified model of shear-induced demixing and mechanical shear banding instabilities

We present a unified model of shear-induced demixing and "mechanical" shear banding instabilities in polymeric and surfactant solutions, by combining a simple flow instability with a two-fluid approach to concentration fluctuations. Within this model, we calculate the "spinodal" limit of stability of initially homogeneous shear states to demixing/banding, and predict the selected length and time scales at which inhomogeneity first emerges after a shear start-up "quench" into the unstable region, finding qualitative agreement with experiment. Our analysis is the counterpart, for this driven phase transition, of the Cahn-Hilliard calculation for unsheared fluid-fluid demixing.