Electrochemical detection of amino acids at carbon nanotube and nickel-carbon nanotube modified electrodes

The oxidation and enhanced detection of traditionally 'non-electroactive' amino acids at a single-wall carbon nanotube (SWNT) surface and at a nickel hydroxide film electrochemically deposited and generated upon the SWNT layer is reported. Different CNT are compared, with Nafion-dispersed SWNT offering the most favorable layer for constant-potential amperometric detection. Factors affecting the oxidation process, including the pH or applied potential, are assessed. The response of the SWNT-Nafion coated electrode compares favorably with that of copper and nickel disk electrodes under flow injection analysis (FIA) conditions. The electrodeposition of nickel onto the SWNT-Nafion layer (Ni-CNT) led to a dramatic enhancement of the analytical response (vs. that observed at the SWNT or nickel electrodes alone). The oxidative process at the Ni(OH)(2) layer has been studied and the increase in sensitivity rationalized. In the presence of amino acid the Ni-CNT layer undergoes an electrocatalytic process in which the amino acid reduces the newly formed NiO(OH) species. Furthermore, the attractive response of both the CNT and Ni-CNT layers has allowed these electrodes to be used for constant-potential FIA of various amino acids and indicates great promise for monitoring chromatographic effluents. Once again an improved signal was observed at the Ni-CNT electrode compared to nickel deposited upon a bare glassy carbon electrode (Ni-GC).     

Ab initio characterization of several states of nitroxylium (NO). Comparison of fragmentation energies of nitroxylium,nitroxyl (NO3), and nitrate

Ab initio calculations have been performed at the self-consistent field (HF) level, and its perturbative extensions up to fourth-order (MPn), for several electronic states of nitroxylium (NO) as well as for a large number of reference species. Geometries are optimized at the HF/DZ and HF/DZP levels (double zeta and double zeta plus polarization bases). The ground state is found to be the D_3h ¹A₁′ state, with the C_2v ¹A₁ (closed Y) state higher by 0.94 eV. The relationship between adding electrons or oxygen atoms to NO⁺ and NO is explored, especially in relation to fragmentation energies of NO (q = 0 or 1). A comparison is drawn between NO and two isoelectronic species, CO₃ and C(CH₂)₃, where no surprises are found.     

Interconversions of aryl radicals by 1,4-shifts of hydrogen atoms. A synthesis of benzo[a]corannulene

Aryl radicals generated ortho to aryl substituents by flash vacuum pyrolysis (FVP) of the corresponding aryl chlorides are shown to be capable of transferring hydrogen atoms between the ortho and ortho' positions (1,4-shifts of hydrogen atoms). In the examples described here, the rearranged aryl radicals are trapped by subsequent radical cyclization reactions. For example, FVP of 2-(o-chlorophenyl)benzo[c]phenanthrene gives 1-phenylbenzo[ghi]fluoranthene as the major product by homolysis of the C-Cl bond, 1,4-shift of a hydrogen atom out of the sterically congested cove region to the radical center, cyclization of the rearranged radical, and rearomatization of the molecule by loss of the other cove region hydrogen. In a control experiment run under the same conditions, FVP of 2-phenylbenzo[c]phenanthrene, which lacks a radical precursor, gave primarily recovered starting material. When the FVP was repeated using 2-(2,6-dichlorophenyl)benzo[c]phenanthrene as the starting material, benzo[a]corannulene was obtained as the major product, presumably by the same cascade of events to produce 1-(o-chlorophenyl)benzo[ghi]fluoranthene, which then suffers a second radical cyclization spontaneously under the high-temperature conditions to give the geodesic polyarene.     

A polymorphic pocket at the P10 position contributes to peptide binding specificity in class II MHC proteins

Peptides bind to class II major histocompatibility complex (MHC) proteins in an extended conformation. Pockets in the peptide binding site spaced to accommodate peptide side chains at the P1, P4, P6, and P9 positions have been previously characterized and help to explain the obtained peptide binding specificity. However, two peptides differing only at P10 have significantly different binding affinities for HLA-DR1. The structure of HLA-DR1 in complex with the tighter binding peptide shows that the peptide binds in the usual polyproline type II conformation, but with the P10 residue accommodated in a shallow pocket at the end of the binding groove. HLA-DR1 variants with polymorphic residues at these positions were produced and found to exhibit different side chain specificity at the P10 position. These results define a new specificity position in HLA-DR proteins.     

Citrate selectivity of poly(neutral red) electropolymerized films

The synthesis and the spectroscopic and potentiometric characterization of electropolymerized films obtained from neutral red (NR) on Pt surface are described. FT Raman and NMR spectroscopy were used for evaluation of the resulting poly(NR) layers. Then potentiometry was applied to study the characteristics of the complexation of carboxylates with the poly(NR) films. A potentiometric poly(NR) sensor showing high selectivity for citrate anion was employed for its determination in soft drinks.     

Epitaxial electrodeposition of Prussian blue thin films on single-crystal Au(110)

Epitaxial Prussian blue (PB) films are deposited electrochemically onto a Au(110) substrate. High-resolution X-ray diffraction shows that the PB films have a [111] out-of-plane orientation. The very large lattice mismatch of 148% is reduced to about 1% by the formation of (1 x 2)PB(111)[onemacr;10]//(6 x 5)Au(110)[onemacr;10] and (1 x 2)PB(111)[01onemacr;]//(6 x 5)Au(110)[onemacr;10] epitaxial relationships. Peaks in the cyclic voltammogram of PB on Au(110) are sharper than those on polycrystalline Au, consistent with higher structural order and a single out-of-plane orientation. The development of epitaxial films of PB and PB analogues will allow the measurement of the orientation-dependent properties of these molecular magnets. It will also open the door to the development of novel molecular spintronic devices, such as those which exhibit spin-dependent electron transfer.     

Gene expression analysis for high throughput screening applications

To meet growing needs for high throughput gene expression profiling, we established a new automated high throughput TaqMan RT-PCR method for quantitative mRNA expression analysis. In this method, the Allegro( trade mark ) (Zymark) system conducts all sample tracking and liquid handling steps, and ABI PRISM 7900 HT (Applied Biosystems) is used to conduct real-time determination of the C(t) value when amplification of PCR products is first detected and accumulation of inhibitory PCR products is unlikely to occur. The ABI PRISM 7900 HT Sequence Detection System features a real-time PCR instrument with 384-well-plate compatibility and robotic loading, and continuous wavelength detection, which enables the use of multiple fluorophores in a single reaction. The Allegro System offers an assembly line approach with a modular design that allows reconfiguration of the components to accommodate variations in the assay flow. In the present study, we have established and validated a new automated High Throughput (HT) TaqMan RT-PCR- based method for quantitative mRNA expression analysis. The data demonstrate that HT-Taqman PCR is a powerful tool that can be used for measuring low concentrations of mRNA, and is highly accurate, reproducible, and amenable to high throughput analysis. Results suggest that HT-TaqMan is a reliable method for the quantification of low-expression genes and a powerful tool with HT capability for target identification/validation, structure-activity relationship (SAR) study, compound selection for efficacy studies, and biomarker identification in drug discovery and development.     

Theoretical study of the spin trapping of hydroxyl radical by cyclic nitrones: a density functional theory approach

The hydroxyl radical (*OH) is an important mediator of biological oxidative stress, and this has stimulated interest in its detection. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) and its alkoxycarbonyl and alkoxyphosphoryl analogues have been employed as spin traps for electron paramagnetic resonance (EPR) spectroscopic radical detection. Energies of optimized geometries of nitrones and their corresponding *OH adducts were calculated using density functional theory (DFT) at the B3LYP/6-31+G//B3LYP/6-31G level. Calculations predict that the trans adduct formation is favored in alkoxycarbonyl nitrones, while cis adducts with intramolecular H-bonding is favored for alkoxyphosphoryl nitrones. Addition of *OH to a phosphoryl-substituted nitrone is more exoergic than the carbonylated nitrones. Charge and spin densities on the nitrone spin traps were correlated with their rates of addition with *OH, and results show that the charge density on the nitronyl C, the site of *OH addition, is more positive in phosphorylated nitrones compared to DMPO and the alkoxycarbonyl nitrones. The dihedral angle between the beta-H and nitroxyl O bonds is smaller in phosphorylated nitrones, and that aspect appears to account for the longer half-lives of the spin adducts compared to those in DMPO and alkoxycarbonyl nitrones. Structures of nitrones with trifluoromethyl-, trifluoromethylcarbonyl-, methylsulfonyl-, trifluoromethylsulfonyl-, amido-, spiropentyl-, and spiroester substituents were optimized and their energies compared. Amido and spiroester nitrones were predicted to be the most suitable nitrones for spin trapping of *OH due to the similarity of their thermodynamic and electronic properties to those of alkoxyphosphoryl nitrones. Moreover, dimethoxyphosphoryl substitution at C-5 was found to be the most efficient substitution site for spin trapping of *OH, and their spin adducts are predicted to be the most stable of all of the isomeric forms.     

Spectroscopy and reactivity of a photogenerated tryptophan radical in a structurally defined protein environment

Near-UV irradiation of structurally characterized [Re(I)(CO)3(1,10-phenanthroline)(Q107H)](W48F/Y72F/H83Q/Y108W)AzM(II) [Az = Pseudomonas aeruginosa azurin, M = Cu, Zn]/[Co(NH3)5Cl]Cl2 produces a tryptophan radical (W108*) with unprecedented kinetic stability. After rapid formation (k = 2.8 x 106 s-1), the radical persists for more than 5 h at room temperature in the folded ReAzM(II) structure. The absorption spectrum of ReAz(W108*)M(II) exhibits maxima at 512 and 536 nm. Oxidation of K4[Mo(CN)8] by ReAz(W108*)Zn(II) places the W108*/W108 reduction potential in the protein above 0.8 V vs NHE.     

Mechanistic studies on the formation and reactivity of dioxygen adducts of diiron complexes supported by sterically hindered carboxylates

Dioxygen activation by enzymes such as methane monooxygenase, ribonucleotide reductase, and fatty acid desaturases occurs at a nonheme diiron active site supported by two histidines and four carboxylates, typically involving a (peroxo)diiron(III,III) intermediate in an early step of the catalytic cycle. Biomimetic tetracarboxylatodiiron(II,II) complexes with the familiar "paddlewheel" topology comprising sterically bulky o-dixylylbenzoate ligands with pyridine, 1-methylimidazole, or THF at apical sites readily react with O(2) to afford thermally labile peroxo intermediates that can be trapped and characterized spectroscopically at low temperatures (193 K). Cryogenic stopped-flow kinetic analysis of O(2) adduct formation carried out for the three complexes reveals that dioxygen binds to the diiron(II,II) center with concentration dependences and activation parameters indicative of a direct associative pathway. The pyridine and 1-methylimidazole intermediates decay by self-decomposition. However, the THF intermediate decays much faster by oxygen transfer to added PPh(3), the kinetics of which has been studied with double mixing experiments in a cryogenic stopped-flow apparatus. The results show that the decay of the THF intermediate is kinetically controlled by the dissociation of a THF ligand, a conclusion supported by the observation of saturation kinetic behavior with respect to PPh(3), inhibition by added THF, and invariant saturation rate constants for the oxidation of various phosphines. It is proposed that the proximity of the reducing substrate to the peroxide ligand on the diiron coordination sphere facilitates the oxygen-atom transfer. This unique investigation of the reaction of an O(2) adduct of a biomimetic tetracarboxylatodiiron(II,II) complex provides a synthetic precedent for understanding the electrophilic reactivity of like adducts in the active sties of nonheme diiron enzymes.