Theory of asymmetric organocatalysis of Aldol and related reactions: rationalizations and predictions

Computational studies have led to models to understand some classic and contemporary asymmetric reactions involving organocatalysts. The Hajos-Parrish-Eder-Sauer-Wiechert reaction and intermolecular aldol reactions as well as Mannich reactions and oxyaminations catalyzed by proline and other amino acids, and Diels-Alder reactions catalyzed by MacMillan's chiral amine organocatalysts have been studied with density functional theory. Quantitative predictions for several new catalysts and reactions are provided.     

Scalar transport in a milli-scale metal foam reactor

We present an investigation on species mass transfer in a milli-scale plug flow reactor using metal foams to enhance the mixing process. In the current design the reactor consists of a pipe with an inner diameter of 7 mm and metal foam inserts with different pore sizes of 20, 30 and 45 ppi. Simultaneous PIV and LIF measurements were performed in orthogonal planes normal to the radial and axial direction downstream of a foam element of 50 mm length. The investigated Reynolds numbers range from 600 to 7600 defined with the empty tube diameter and the bulk velocity. We discuss the influence of the pore sizes on the scalar mixing efficiency and compare the results to the reference empty tube case. A strongly intermittent flow caused by the metal foam was observed over the whole range of Reynolds numbers. The increased radial velocities lead to an enhanced mixing performance. Coefficients of Variation in the order of 0.1 were achieved.     

Single chain dimers of MASH-1 bind DNA with enhanced affinity

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.     

Nucleophilic Water Capture or Proton Loss: Single Amino Acid Switch Converts δ‐Cadinene Synthase into Germacradien‐4‐ol Synthase

δ‐Cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene δ‐cadinene and about 2 % germacradien‐4‐ol, which is also generated from FDP by the cyclase germacradien‐4‐ol synthase. Herein, the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule was investigated by site‐directed mutagenesis of δ‐cadinene synthase. If W279 in δ‐cadinene synthase was replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product was directly proportional to the van der Waals volume of the amino acid side chain. DCS‐W279A is a catalytically highly efficient germacradien‐4‐ol synthase (k_cat/K_M=1.4×10^?3 μm s^?1) that produces predominantly germacradien‐4‐ol in addition to 11 % δ‐cadinene. Water capture is not achieved through strategic positioning of a water molecule in the active site, but through a coordinated series of loop movements that allow bulk water access to the final carbocation in the active site prior to product release.     

Effect of placing,removing and polishing of amalgam restorations on 24-h urinary mercury concentration

Twenty-four-hour urinary mercury concentration were assessed in 43 patients before and after removing old amalgam fillings (8 pts), placing (13 pts), and polishing of amalgam (22 pts). Baseline analyses 8 days before the treatments showed on average 18.5±7.2 g mercury mass excreted per 24-h urine samples. The removal of old fillings caused a total excreted mass of 56.3±32.3 g Hg, the placing of amalgam 45.9±26.2 g Hg, and the polishing 56.25±33.77 g Hg, respectively, one day after the treatments. When compared with the baseline values, the urinary mass excreted remained significantly elevated during the 8-day follow-up. However, all Hg values measured were below the WHO recommandations for the threshold limits for urinary mercury.     

A stable miniature protein with oxaloacetate decarboxylase activity

An 18-residue miniature enzyme, Apoxaldie-1, has been designed, based on the known structure of the neurotoxic peptide apamin. Three lysine residues were introduced on the solvent-exposed face of the apamin alpha-helix to serve as an active site for decarboxylation of oxaloacetate. The oxidised form of Apoxaldie-1, in which two disulfide bonds stabilise the alpha-helix, formed spontaneously. CD spectroscopy measurements revealed that, in its oxidised form, Apoxaldie-1 adopted a stably folded structure, which was lost upon reduction of the disulfide bonds. Despite its small size and the absence of a designed binding pocket, Apoxaldie-1 displayed saturation kinetics in its oxidised form and catalysed the decarboxylation of oxaloacetate at a rate that was almost four orders of magnitude faster than that observed with n-butylamine. This rivals the performance of the best synthetic oxaloacetate decarboxylases reported to date. Unlike those, however, Apoxaldie-1 displayed significant stability. It maintained its secondary structure at temperatures in excess of 75 degrees C, in the presence of high concentrations of guanidinium chloride and at pH values as low as 2.2. Apamin-based catalysts have potential for the generation of miniature peptides that display activity under nonphysiological conditions.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases(RNase A, angiogenin and bovine seminal RNase) identifies threesurface loops that are highly variable between the three proteins.Two hypotheses were contrasted: (i) that this variation mightbe responsible for the different catalytic activities of thethree proteins; and (ii) that this variation is simply an exampleof surface loops undergoing rapid neutral divergence in sequence.Three hybrids of angiogenin and bovine pancreatic ribonuclease(RNase) A were prepared where regions in these loops taken fromangiogenin were inserted into RNase A. Two of the three hybridshad unremarkable catalytic properties. However, the RNase Amutant containing residues 63–74 of angiogenin had greatlydiminished catalytic activity against uridylyl-(3' – 5')-adenosine(UpA), and slightly increased catalytic activity as an inhibitorof translation in vitro. Both catalytic behaviors are characteristicof angiogenin. This is one of the first examples of an engineeredexternal loop in a protein. Further, these results are complementaryto those recently obtained from the complementary experiment,where residues 59–70 of RNase were inserted into angiogenin[Harper and Vallee (1989) Biochemistry, 28, 1875–1884].Thus, the external loop in residues 63–74 of RNase A appearsto behave, at least in part, as an interchangeable ‘module’that influences substrate specificity in an enzyme in a waythat is isolated from the influences of other regions in theprotein.     

Zur Auffindung von α-, β- und γ-Dehydrotocopherol in Weizenkeimöl mittels HPLC und GC/MS - ein Beitrag zur Analytik der Tocopherole

Finding of α-, β- and γ-Dehydrotocopherol in Wheat Germ Oil by HPLC and GC/MS - a Contribution to Tocopherol Analysis Fractions of β-tocotrienol/γ-tocopherol isolated from commercial wheat germ oils by preparative TLC were separated once more by HPLC. Three dehydrotocopherols (α-, β-and γ-) were found and identified by GC/MS. HPLC-retention data for plastochromanol-8 in these and corresponding fractions of Latex (Hevea brasiliensis) could be ascertained. The hypothetic 5,7-dimethyltocotrienol could not be found neither in the equivalent fractions of wheat germ oil and Latex nor in those of rude palm oil.