Chemically modified "polar patch" mutants of subtilisin in peptide synthesis with remarkably broad substrate acceptance: designing combinatorial biocatalysts

A significant enhancement of the applicability of the serine protease subtilisin Bacillus lentus (SBL) in peptide synthesis was achieved by using the strategy of combined site-directed mutagenesis and chemical modification to create chemically modified mutant (CMM) enzymes. The introduction of polar and/or homochiral auxiliary substituents, such as X=oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of the primary specificity S(1) pocket created SBL CMMs S166C-S-X with strikingly broad structural substrate specificities. These CMMs are capable of catalyzing the coupling reactions of not only L-amino acid esters but also D-amino acid esters as acyl donors with glycinamide to give the corresponding dipeptides in good yields. These powerful enzymes are also applicable to the coupling of L-amino acid acyl donors with alpha-branched acyl acceptor, L-alaninamide. Typical increases in isolated yields of dipeptides of 60-80 % over SBL-WT (e.g. 0 % yield of Z-D-Glu-GlyNH(2) using SBL-WT-->74 % using S166C-S-(CH(2))(2) NMe(3) (+)) demonstrate the remarkable synthetic utility of this "polar patch" strategy. Such wide-ranging systems displaying broadened and therefore similarly high, balanced yields of products (e.g. 91 % Z-L-Ala-GlyNH(2) and 86 % yield of Z-D-Ala-GlyNH(2) using S166C-S-(3R,4S)-indenooxazolidinone) may now allow the use of biocatalysts in parallel library synthesis.     

Amperometric detection of hydrazine by cyclic voltammetry and flow injection analysis using ruthenium modified glassy carbon electrodes

Glassy carbon electrodes modified with (5-amino-1,10-phenanthroline)bis(bipyridine)ruthium(II) chloride hydrate, [(bpy)₂Ru(5-phenNH₂)]Cl₂·H₂O, are shown to oxidize hydrazine with excellent sensitivity. The presence of an amine group on the ruthenium complex facilitates electropolymerization onto the electrode surface. Using cyclic voltammetry, a large catalytic current is observed upon oxidation of hydrazine in phosphate buffer (pH 5.0), compared to the current obtained from the ruthenium-modified electrode with no hydrazine present. The sensitivity of cyclic voltammetry is sufficient for obtaining a linear calibration curve for hydrazine over the range of 10⁻⁵ to 10⁻² M. Hydrodynamic amperometry was used to determine the working potential for flow injection analysis. The limit of detection for hydrazine was determined to be 8.5 μM using FIA. The thickness of these films was shown to increase linearly with the number of electropolymerization cycles, in the range of 1000-2500 nm for 5-20 cycles, respectively, using Rutherford backscattering spectrometry (RBS). RBS analysis also suggests that the film is multilayered with the outermost layers containing a high ruthenium concentration, followed by layers where the concentration of ruthenium decreases linearly and approaches zero at the electrode surface.     

A Concise, Enantioselective Synthesis of (-)- and (+)-Hongconin

Optically pure enone 9c, available in three steps from known 6-deoxy D-galactal derivative 7b, reacts with cyanophthalide 6 to directly afford the natural product (-)-hongconin (1), a compound from traditional Chinese medicine recently shown to exhibit antianginal activity. The enantiomer of 1 and its (+)-cis-diastereomer were also synthesized in a parallel fashion from the L-sugar counterpart. The use of C-glycoside Michael acceptors, as opposed to their O-glycoside counterparts, represents a potentially useful simplification of phthalide annulation methodology in synthesizing numerous other such optically pure isochromanquinoids, since it obviates the inconvenience of additional steps late in the synthetic scheme associated with reductive manipulation of a remaining acetal moiety into the desired pyran ring substituent.     

Improved preparative electrochromatography column design

Improved chromatography column fittings were developed for the efficient and reliable application of an electric field to a preparative chromatography column (a process termed electrochromatography). The improved fittings contained electrodes in close proximity to the column packing media and allowed uniform electric fields to be applied. Membranes in the fittings prevented mixing of the electrode and the column eluent buffers. The membranes prevented gases and electrolytic products generated in the electrode chamber from entering the column eluent buffer. An electrode buffer solution was pumped through the electrode chamber to a large external container. The circulation of buffer through the electrode chamber removed the gases and electrolytic products and ensured a uniform electric field by helping to maintain a constant buffer composition. The membranes prevented macromolecules being separated on the column from coming in contact with the electrodes.     

A new class of heterocyclic betaines: 1,1-dimethyl-3-phenylpyrazolium-5-oxide

1,1-Dimethyl-3-phenylpyrazolium-5-oxide (1) was isolated as a minor product (3%) in the reaction of ethyl benzoylacetate with 1,1-dimethylhydrazine and also was obtained as the major product (58%) from the reaction of methyl p-toluenesulfonate with ethyl benzoylacetate dimethylhydrazone. The structural assignment was made on the basis of chemical reactions and spectral data and the formula is represented by a resonance structure for which there are two principal canonical forms. Reaction of 1 with methanol at room temperature afforded methyl benzoylacetate dimethylhydrazone. Ethyl benzoylacetate dimethylhydrazone was shown to exist as a tautomeric mixture containing 65% of the imine form at room temperature.     

Potential energy surfaces for HenNe+ ions: ab initio and diatomics-in-molecule results

The potential energy surface of He2Ne+ has been reinvestigated using a combination of ab initio and diatomics-in-molecule (DIM) calculations. In contrast to the reports of two recent studies the ion is found to have an asymmetric linear He-Ne-He structure, with no barrier to formation from the separated atoms on the ground-state surface. The He-Ne+ bond lengths at the potential minimum are 1.51 and 1.81 A, and the total bonding energy is 0.717 eV. Comparing the He2Ne+ energy to that of HeNe+, the bonding energy for the second helium atom is 0.06 eV, about 10% of that of the first He atom. The saddle point between the two equivalent minima is a symmetric structure, 0.0074 eV above the potential minimum. A symmetric geometry becomes the overall potential minimum if the 2s hole on the Ne is excluded from the reference states of a multireference configuration interaction calculation. A DIM potential was created for the HenNe+ family of ions. The DIM potential is consistent with the asymmetric He2Ne+ ion serving as a core; it predicts a slightly more asymmetric geometry than the ab initio results. Additional helium atoms form five-membered rings around the bonds of the core ion to fill the first shell and then add to the ends of the cluster. The asymmetric core ion and the highly compact structure help to account for the lack of apparent shell structure in the mass spectrometry of HenNe+ clusters. Finally, we recommend that the value De=0.63+/-0.04 eV be adopted for the ground state of HeNe+.     

Porous MoS2 synthesized by ultrasonic spray pyrolysis

The hydrodesulfurization (HDS) activity of molybdenum sulfide-based catalysts is localized to the edges of this layered solid and is, therefore, highly dependent on the technique used to prepare the material. Here, ultrasonic spray pyrolysis (USP) was used to synthesize porous, nanostructured MoS2. Low surface area powders, not useful for catalysis, are generally produced by USP. This work shows that when combined with a dissolvable template, USP is capable of producing high surface area materials. An aqueous solution of ammonium tetrathiomolybdate and colloidal silica was nebulized and pyrolyzed to give a MoS2/SiO2 composite material. Leaching with HF removed the sacrificial SiO2, resulting in a highly porous MoS2 network with surface areas as high as 250 m2/g. Cobalt-promoted MoS2 networks were also synthesized. The thiophene HDS activities of these materials were substantially higher than those of unsupported MoS2 and RuS2 standards, illustrating the enhanced dispersion of the HDS active phase achieved by this synthetic technique.     

On the apparent molar volumes of nonelectrolytes in water

Apparent molar volumes of aqueous solutions of argon and xenon have been calculated using a previously developed comprehensive equation of state for nonelectrolyte systems. The equation consists of a virial expansion truncated after the fourth virial coefficient and a closed-form term approximating higher coefficients. Mixing rules are based on the composition dependence of virial coefficients, which is known from statistical mechanics. The equation accurately represents vapor-liquid and gas-gas equilibria for the Ar+H₂O and Xe+H₂O systems over wide ranges of pressure and temperature using two binary parameters. With the binary parameters determined from phase equilibrium data, the equation accurately predicts apparent molar volumes V in the near-critical and far-from-critical regions. Apart from reproducing experimental V data, the equation reveals remarkable maxima of V as a function of pressure and temperature in the near-critical region. The implications of this equation with respect to the Ar–H₂O potential are discussed via the second virial coefficient.     

Aggregation patterns in alpha,alpha'-stabilized carbanions: assembly of a sodium cage polymer by slip-stacking of dimers

The alpha,alpha'-stabilized carbanion complexes [PhSO(2)CHCNNa.THF], 3, [t-BuSO(2)CHCNNa], 4, [PhSO(2)CHCNK], 5, [t-BuSO(2)CHCNK], 6, and [MeSO(2)CHCNLi.TMEDA], 7, have been synthesized via the metalation of the parent (organo)sulfonylacetonitriles by BuLi, BuNa, or BnK in THF solution (or THF/TMEDA in the case of 7). In addition, complexes 3 and 7 have been characterized by single-crystal X-ray analyses and have been found to adopt related structures in the solid state. Complex 7 is a molecular dimer containing a central 12-membered (OSCCNLi)(2) ring core, with each metal rendered tetracoordinate by binding to a chelating TMEDA molecule. As found in related complexes, no direct carbanion to lithium contacts are present in the structure of 7. Complex 3 forms a polymeric cage structure composed of associated "dimeric" (OSCCNNa)(2) rings, similar to those found in 7. The larger sodium cations, and the presence of only one THF molecule/metal, allow additional contacts with the anions, leading to hexacoordination at the metal centers. These contacts include long-range transannular Na-N interactions (2.8042(14) A) across the central dimeric ring and "interdimer" Na-C connections (2.8718(15) A). Dissolution of complexes 3-6 and their lithiated derivatives [PhSO(2)CHCNLi.TMEDA], 1, and [t-BuSO(2)CHCNLi.THF], 2, in DMSO-d(6) results in almost identical chemical shifts for each type of ligand. This suggests that charge-separated complexes of the form [RSO(2)CHCN](-)[M(DMSO-d(6))(n)()](+) are formed in highly polar solution.     

Unusual ligand coordination for cesium

When complexed by tetrabenzo-24-crown-8, the cesium ion can accommodate unprecedented ligation. The structures of the following complexes are presented. [Cs(tetrabenzo-24-crown-8)(eta 1-NCMe)2(eta 2-NCMe)][NO3] (1): triclinic P1, a = 12.0119(14) A, b = 13.3680(15) A, c = 13.7859(12) A, alpha = 89.124(8) degrees, beta = 66.928(9) degrees, gamma = 71.536(10) degrees, V = 1916.7(4) A3, Z = 2. [Cs(tetrabenzo-24-crown-8)(eta 1-NCMe)2(eta 2-CH2Cl2)][NO3] (2): triclinic, P1, a = 12.295(3) A, b = 13.295(3) A, c = 13.782(3) A, alpha = 89.105(17) degrees, beta = 66.096(18) degrees, gamma = 71.980(19) degrees, V = 1929.5(8) A3, Z = 2. These structures are the first reported examples of linear eta 2-acetonitrile coordination to any metal ion and the first structures illustrating eta 2-acetonitrile and dichloromethane ligation to an alkali metal ion. Possible steric and electronic origins of these unusual metal-ligand interactions are discussed.