High-throughput preparative process utilizing three complementary chromatographic purification technologies

A high-throughput process was developed in which wells in plates generated from parallel synthesis are automatically channeled to an appropriate purification technique using analytical data as a guide. Samples are directed to either of three fundamentally different preparative techniques: HPLC with UV-triggered fraction collection, supercritical fluid chromatography (SFC) with UV-triggered fraction collection, or HPLC with MS-triggered fraction collection. Automated analysis of the analytical data identifies the product compound mass and creates work lists based on chromatographic properties exhibited in the data so that each preparative instrument cherry picks the appropriate list of samples to purify when a preparative-scale plate is loaded.     

Protein-induced bonding perturbation of the rhodopsin chromophore detected by double-quantum solid-state NMR

We have obtained carbon-carbon bond length data for the functional retinylidene chromophore of rhodopsin, with a spatial resolution of 3 pm. The very high resolution was obtained by performing double-quantum solid-state NMR on a set of noncrystalline isotopically labelled bovine rhodopsin samples. We detected localized perturbations of the carbon-carbon bond lengths of the retinylidene chromophore. The observations are consistent with a model in which the positive charge of the protonated Schiff base penetrates into the polyene chain and partially concentrates around the C13 position. This coincides with the proximity of a water molecule located between the glutamate-181 and serine-186 residues of the second extracellular loop, which is folded back into the transmembrane region. These measurements support the hypothesis that the polar residues of the second extracellular loop and the associated water molecule assist the rapid selective photoisomerization of the retinylidene chromophore by stabilizing a partial positive charge in the center of the polyene chain.     

Contraction mappings in interval analysis

Under certain conditions, the contraction mapping fixed point theorem guarantees the convergence of the iterationx _i+1=f(x _i ) toward a fixed point of the functionf:R ⁿ →R ⁿ. When an interval extensionF off is used in a similar iteration scheme to obtain a sequence of interval vectors these conditions need not provide convergence to a degenerate interval vector representing the fixed point, even if the width of the initial interval vector is chosen arbitrarily small. We give a sufficient condition on the extensionF in order that the convergence is guaranteed. The centered form of Moore satisfies this condition.     

Kinetics and mechanism of a macrocyclic chromium(III) complex oxidation to chromium(IV) by hexacyanoferrate(III) in strongly alkaline media

Oxidation of the macrocyclic Cr(III) complex cis-[Cr(cycb)(OH)₂]⁺, where cycb=rac-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane, by an excess of the hexacyanoferrate(III) in basic solution, slowly produces Cr(V) species. These species, detected using e.p.r. spectroscopy, are stable under ambient conditions for many hours, and the hyperfine structure of the e.p.r. spectrum is consistent with the interaction of the d-electron with four equivalent nitrogen nuclei. Electro-spray ionization mass spectrometry suggests a concomitant oxidation of the macrocyclic ligand, in which double bonds and double bonded oxygen atoms have been introduced. By comparison basic chromate(III) solutions are oxidized rapidly to chromate(VI) by hexacyanoferrate(III) without any detectable generation of stable Cr(V) intermediates.Kinetics of oxidation of the macrocyclic Cr(III) complex in alkaline solution has been studied under excess of the reductant. Rate determining formation of Cr(IV) by a second order process involving the Cr(III) and the Fe(III) reactants is seen. This reaction also involves a characteristic higher order than linear dependence on the hydroxide concentration. Reaction mechanisms for the processes, including oxidation of the coordinated macrocyclic ligand – under excess of the oxidant- are proposed.     

Molecular structures of two metal tetrakis(tetrahydroborates), Zr(BH4)(4) and U(BH4)(4): equilibrium conformations and barriers to internal rotation of the triply bridging BH4 groups

The molecular structures of Zr[(mu-H)(3)BH](4) and U[(mu-H)(3)BH](4) have been investigated by density functional theory (DFT) calculations and gas electron diffraction (GED). The triply bridged bonding mode of the tetrahydroborate groups in the former is confirmed, but both DFT calculations and GED structure refinements indicate that the BH(4) groups are rotated some 12 degrees away from the orientation in which the three bridging B-H bonds are staggered with respect to the opposing ZrB(3) fragment. As a result the symmetry of the equilibrium conformation is reduced from T(d) to T. Bond distances and valence angles are as follows (DFT/GED): Zr-B = 232.2/232.4(5) pm; Zr-H(b) = 214.8/214.4(6) pm; B-H(b) = 125.3/127.8(8) pm; B-H(t) = 119.4/118.8(17) pm; angle ZrBH(b) = 66.2/65.6(3) degrees; the smallest dihedral angle of type tau(BZrBH(b)) = 48/45(2) degrees. DFT calculations on Hf(BH(4))(4) indicate that the structure of this molecule is very similar to that of the Zr analogue. Matrix-isolation IR spectroscopy and DFT calculations on U(BH(4))(4) show that while the polymeric solid-state structure is characterized by terminal triply bridging and metal-metal bridging bidentate BH(4) groups, all BH(4) groups are triply bridging in the gaseous monomer. Calculations with one of the two nonbonding 5f electrons on U occupying an a(1) and the other distributed equally among the three t(2) orbitals indicate that the equilibrium conformation has T(d) symmetry, i.e. that the three B-H(b) bonds of each tetrahydroborate group are exactly staggered with respect to the opposing UB(3) fragment with tau(BUBH(b)) = 60 degrees. Calculations including spin-orbit interactions indicate that Jahn-Teller distortions from T(d) symmetry are either absent or very small. The best agreement between observed and calculated GED intensity data was obtained for a model of T(d) symmetry, but models of T symmetry with dihedral angles tau(BUBH(b)) > 42 degrees cannot be ruled out. Bond distances and valence angles are as follows (DFT/GED): U-B = 248.8/251.2(4) pm; U-H(b) = 227.7/231.5(6) pm; B-H(b) = 126.0/131.6(5) pm, B-H(t) = 119.5/117.8(11) pm; angle UBH(b) = 65.6/63.1(3) degrees. It is suggested that the different equilibrium conformations of the three molecules are determined primarily by repulsion between bridging H atoms in different tetrahydroborate groups.     

Electron-mediating Cu(A) centers in proteins: a comparative high field (1)H ENDOR study

High field (W-band, 95 GHz) pulsed electron-nuclear double resonance (ENDOR) measurements were carried out on a number of proteins that contain the mixed-valence, binuclear electron-mediating Cu(A) center. These include nitrous oxide reductase (N(2)OR), the recombinant water-soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase (COX) ba(3) (M160T9), its M160QT0 mutant, where the weak axial methionine ligand has been replaced by a glutamine, and the engineered "purple" azurin (purpAz). The three-dimensional (3-D) structures of these proteins, apart from the mutant, are known. The EPR spectra of all samples showed the presence of a mononuclear Cu(II) impurity with EPR characteristics of a type II copper. At W-band, the g( perpendicular) features of this center and of Cu(A) are well resolved, thus allowing us to obtain a clean Cu(A) ENDOR spectrum. The latter consists of two types of ENDOR signals. The first includes the signals of the four strongly coupled cysteine beta-protons, with isotropic hyperfine couplings, A(iso), in the 7-15 MHz range. The second group consists of weakly coupled protons with a primarily anisotropic character with A(zz) < 3 MHz. Orientation selective ENDOR spectra were collected for N(2)OR, M160QT0, and purpAz, and simulations of the cysteine beta-protons signals provided their isotropic and anisotropic hyperfine interactions. A linear correlation with a negative slope was found between the maximum A(iso) value of the beta-protons and the copper hyperfine interaction. Comparison of the best-fit anisotropic hyperfine parameters with those calculated from dipolar interactions extracted from the available 3-D structures sets limit to the sulfur spin densities. Similarly, the small coupling spectral region was simulated on the basis of the 3-D structures and compared with the experimental spectra. It was found that the width of the powder patterns of the weakly coupled protons recorded at g(perpendicular) is mainly determined by the histidine H(epsilon)(1) protons. Furthermore, the splitting in the outer wings of these powder patterns indicates differences in the positions of the imidazole rings relative to the Cu(2)S(2) core. Comparison of the spectral features of the weakly coupled protons of M160QT0 with those of the other investigated proteins shows that they are very similar to those of purpAz, where the Cu(A) center is the most symmetric, but the copper spin density and the H(epsilon)(1)-Cu distances are somewhat smaller. All proteins show the presence of a proton with a significantly negative A(iso) value which is assigned to an amide proton of one of the cysteines. The simulations of both strongly and weakly coupled protons, along with the known copper hyperfine couplings, were used to estimate and compare the spin density distribution in the various Cu(A) centers. The largest sulfur spin density was found in M160T9, and the lowest was found in purpAz. In addition, using the relation between the A(iso) values of the four cysteine beta-protons and the H-C-S-S dihedral angles, the relative contribution of the hyperconjugation mechanism to A(iso) was determined. The largest contribution was found for M160T9, and the lowest was found for purpAz. Possible correlations between the spin density distribution, structural features, and electron-transfer functionality are finally suggested.     

Preparation and characterization of a composite of silver iodide and synthetic zeolite ZSM5

A composite material made up of AgI and the potassium form of the synthetic zeolite ZSM5 has been prepared by treating the silver form of ZSM5 with potassium iodide. The composite has been characterised by X-ray powder diffractometry, IR and X-ray photoelectron spectroscopy, electron microsonde analyses and ac conductivity measurements. The Agl content in the composite material is 11.5%. On the basis of the conductivity data obtained for the composite material as well as for K-ZSM5, Ag-ZSM5, microcrystalline AgI and a physical mixture of AgI and K-ZSM5, it has been inferred that, in the composite material, AgI forms a thin conductive shell on the surface of the K-ZSM5 particles.     

Versatile Functionalization of Carbohydrate Hydroxyl Groups through Their O-Cyanomethyl Ethers

O-Cyanomethyl ethers of carbohydrates are shown to be versatile intermediates for the preparation of sugar amines, carboxylic acids, amides, and amidine salts. This methodology for the functionalization of carbohydrates can thus provide a new array of analogs for the study of carbohydrate binding proteins. In addition, the resulting O-aminoethyl and O-carboxymethyl carbohydrates can be coupled to amino acids under standard conditions used in solid-phase peptide synthesis, providing a method for the construction of glycopeptides in which the carbohydrate moiety can be linked through any of its hydroxyl groups to the C- or the N-terminus of a given peptide.     

ON THE DNA BENDING BY PSORALEN INTERSTRAND CROSSLINKING. A GEL ELECTROPHORETIC STUDY

Abstract— A synthetic, partially double stranded decadeoxyribonucleotide with cohesive ends, containing one potential psoralen photo-crosslinking site centrally positioned (5'-d(CGGGCTACCC) + 3'-d(CCGATGGGGC)), has been ligated to double stranded DNA oligomers, which were subsequently photoreacted with 4,5',8-trimethylpsoralen. It was found that psoralen DNA interstrand crosslinking does not significantly alter the electrophoretic mobility of these DNA molecules in polyacrylamide gels. Based on this, we conclude that any bends in the DNA helix that may be induced by psoralen DNA interstrand crosslinking must be significantly less than the 45^° proposed by Tomic et at. (1987) (Science, 238, 1722) and/or of a different nature than the DNA sequence dependent bends due to d(A)_n tracts.     

UV absorption spectra and kinetics of the self reaction of CFCl2CH2O2 and CF2ClCH2O2 radicals in the gas phase at 298 K

The ultraviolet absorption spectra of the peroxy radicals derived from hydrochlorofluorocarbons 141b and 142b, (CFCl₂CH₂O₂ and CF₂ClCH₂O₂, respectively), and the kinetics of their self reactions have been studied in the gas phase at 298 K using a pulse radiolysis technique. Absorption cross sections were quantified over the wavelength range 220–300 nm. Measured absorption cross sections at 250 nm were indistinguishable within the experimental uncertainties (≈10%) and yield; Errors represent the sum of statistical uncertainty and our estimate of potential systematic errors. Our absorption cross section data were then used to derive the observed self reaction rate constants for reactions (1) and (2), defined as ?d[RO₂]/dt = 2k[RO₂]² (R = CFCl₂CH₂ or CF₂ClCH₂), of k_1obs = (4.36 ± 0.64) × 10^?12 and k_2obs = (4.13 ± 0.58) × 10^?12 cm³ molecule^?1 s^?1, quoted errors represent 2σ. These results are discussed with respect to previous studies of the absorption spectra and kinetics of peroxy radicals.