Synthesis of the sialic acid (-)-KDN and certain epimers from (-)-3-dehydroshikimic acid or (-)-quinic acid

(-)-3-Dehydroshikimic acid (3-DHS, 4), a C(7)-building block now available in large quantity from corn syrup, has been converted into the sialic acid (-)-KDN (3) as well as its C-7- and C-8-epimers. (-)-Quinic acid can be used for the same purpose. [structure: see text]     

Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker’s yeast using an expanded bed adsorption system

Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers’ yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn²⁺, Ni²⁺ and Cu²⁺ and eluted using 0–50 mM EDTA gradient found that charging with Zn²⁺ gave the highest recovery and the lowest EDTA concentration required for elution. These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA. The ADH was found to elute at 5 mM EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively. Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers’ yeast diluted to 10 mg/ml total protein content with a recovery of 80–100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.     

On some hyperbolic type equations and weighted anisotropic Hardy operators

We introduce a version of weighted anisotropic Morrey spaces and anisotropic Hardy operators. We find conditions for boundedness of these operators in such spaces. We also reveal the role of these operators in solving some classes of degenerate hyperbolic partial differential equations. Copyright © 2016 John Wiley & Sons, Ltd.     

Turnover of hydrogen isotopes in lake sturgeon blood: implications for tracking movements of wild populations

Naturally occurring deuterium (²H) in biota can be used to trace movement, migration and geographic origin of a range of organisms. However, to evaluate movements of animals using δ²H measurements of tissues, it is necessary to establish the turnover time of ²H in the tissues and the extent of isotopic discrimination from different environmental ²H sources to those tissues. We investigated the turnover of ²H in lake sturgeon (Acipenser fulvescens) blood by manipulating both environmental water δ²H and diet δ²H over a four-month period. The half-life of deuterium in lake sturgeon blood was 37.9 days after an increase in the environmental water δ²H of +714?‰. However, no clear turnover in blood ²H occurred over the same period in a separate trial following a change of ?63.8?‰ or +94.2?‰ in diet. These findings suggest that environmental water ²H exchanges much faster with blood than diets and that blood δ²H values can be used to trace movements of sturgeon and other fish moving among isotopically distinct waters.     

Deriving the ultrastructure of α-crustacyanin using lower-resolution structural and biophysical methods

The low-resolution structure of α-crustacyanin has been determined to 30 ? resolution using negative-stain electron microscopy (EM) with single-particle averaging. The protein, which is an assembly of eight β-crustacyanin dimers, appears asymmetrical and rather open in layout. A model was built to the EM map using the X-ray crystallographic structure of β-crustacyanin guided by PISA interface analyses. The model has a theoretical sedimentation coefficient that matches well with the experimentally derived value from sedimentation velocity analytical ultracentrifugation. Additionally, the EM model has similarities to models calculated independently by rigid-body modelling to small-angle X-ray scattering (SAXS) data and extracted in silico from the β-crustacyanin crystal lattice. Theoretical X-ray scattering from each of these models is in reasonable agreement with the experimental SAXS data and together suggest an overall design for the α-crustacyanin assembly.     

Computer-aided Architectural Planning

This paper describes an application of on-line computing facilities to the initial stages of the planning and design of a building complex. The study was under-taken as part of a graduate thesis on the design of a university campus.     

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

Gliotoxin is produced by non-ribosomal peptide synthesis and secreted from certain fungi, including Aspergillus fumigatus. It is an epipolythiodioxopiperazine that contains an intact disulphide bridge and is the focus of intense research as a consequence of its negative immunomodulatory properties. Gliotoxin detection is generally enabled by reversed-phase–high-performance liquid chromatography (RP-HPLC), with absorbance detection (220–280 nm), or liquid chromatography-mass spectrometry, yet detection is not readily achievable by matrix-assisted laser desorption ionisation–time-of-flight mass spectrometry (MALDI-ToF MS). We have developed a single-pot derivatisation strategy which uses sodium borohydride-mediated reduction of gliotoxin followed by immediate alkylation of exposed thiols by 5′-iodoacetamidofluorescein to yield a stable product, diacetamidofluorescein-gliotoxin (GT-(AF)₂), of molecular mass 1103.931 Da ((M + H)+). This product is readily detectable by RP-HPLC and exhibits a 6.8-fold increase in molar absorptivity compared with gliotoxin, which results in a higher sensitivity of detection (40 ng; 125 pmoL). GT-(AF)₂ also fluoresces (excitation/emission, 492:518 nm). Unlike free gliotoxin, the product (>800 fmol) is detectable by MALDI-ToF MS. Sporidesmin A can also be detected by RP-HPLC and MALDI-ToF MS (>530 fmol) using this strategy. We also demonstrate that the strategy facilitates detection of gliotoxin (mean ± SD = 3.55 ± 0.07 μg 100 μL⁻¹; n = 2) produced by A. fumigatus, without the requirement for organic extraction of culture supernatants and associated solvent removal. GT-(AF)₂ is also detectable (150 ng; 460 pmol) by thin-layer chromatography.     

Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter

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Highlights? A representative small molecule library contains 12% firefly luciferase inhibitors ? Competitive, noncompetitive, uncompetitive, and MAI inhibitors were identified ? For FLuc-RGAs 65% of FLuc inhibitors with known MOIs increased the luminescence signal ? Methods to identify FLuc inhibitors that interfere with assay results are described     

Terpyridine-fused polyaromatic hydrocarbons generated via cyclodehydrogenation and used as ligands in Ru(II) complexes

A series of novel fused 4'-substituted 2,2'?:?6',2'-terpyridine ligands and their ruthenium(II) complexes were prepared. The unusual 4'-substituents comprised 2,3,4,5-pentaphenylbenzene and its tert-butyl derivative (1 and 2) and the products from oxidative cyclodehydrogenation, i.e. polyaromatic fragments consisting of ten or thirteen fused benzene rings (3 and 4). The syntheses of all the ligands are discussed in terms of the demands and limitations of the Scholl reaction. The optical properties of the ligands, along with the single-crystal X-ray structures of 1 and 2, are presented. The latter show that the pentaphenylbenzene and terpyridine appendages of 1 and 2 are perpendicular in the solid state. Despite the inclusion of the large organic chromophore the absorption and emission properties of the Ru(II) bis-terpy complexes (of ligands 1, 2 and 3) were found to be comparable to those of [Ru(terpy)(2)](2+). They are non-emissive at room temperature but emit at 77 K with excited state lifetimes of 11-12 μs.