Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris. Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N-terminal 150 amino acids, including the zinc finger DNA-binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5'-CYCC-3' sequences, although all DNA sequences containing the 5'-CYCC-3' motif do not qualify as Mxr1p-binding sites. Key DNA-binding determinants are present outside 5'-CYCC-3' motif and Mxr1p preferably binds to DNA sequences containing 5'-CYCCNY-3' than those containing 5'-CYCCNR-3' sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p-binding sites in other methanol-inducible promoters of P. pastoris. Copyright (C) 2010 John Wiley & Sons, Ltd.

Digital simulation of galvanostatic current-potential data for gas-diffusion electrodes and estimation of electrodekinetic parameters

A computerized non-linear-least-squares regression procedure to analyse the galvanostatic current-potential data for kinetically hindered reactions on porous gas-diffusion electrodes is reported. The simulated data fit well with the corresponding measured values. The analytical estimates of electrode-kinetic parameters and uncompensated resistance are found to be in good agreement with their respective values obtained from Tafel plots and the current-interrupter method. The procedure circumvents the need to collect the data in the limiting-current region where the polarization values are usually prone to errors. The polarization data for two typical cases, namely, methanol oxidation on a carbon-supported platinum-tin electrode and oxygen reduction on a Nafion-coated platinized carbon electrode, are successfully analysed.

Dimple and nose coalescences in phase-separation processes

Coalescence processes are investigated during phase separation in a density-matched liquid mixture (partially deuterated cyclohexane and methanol) under near-critical conditions. As a result of the interplay between capillary and lubrication forces, ''nose'' coalescence appears to be always associated with the slow growth of isolated droplets (exponent almost-equal-to 1/3), whereas ''dimple'' coalescence corresponds to the fast growth of interconnected droplets (exponent almost-equal-to 1). At each stage of growth, the distribution of droplets trapped during dimple coalescence is reminiscent of all of the previous coalescence events.

Edge-sharing bioctahedral diruthenium(III) complexes containing methoxy and carboxylate bridges:x-ray structures, redox behavior, and core stability

Edge-sharing bioctahedral (ESBO) complexes [Ru-2(OMe)(O2CC6H4-p-X)3(1-MeIm)(4)](ClO4)2 (X = OMe (1a), Me (1b)) and [Ru-2(O2CC6H4-P-X)(4)(1-MeIm)(4)](ClO4)(2) (X = OMe (2a), Me (2b)) are prepared by reacting Ru2Cl(O(2)CR)(4) with 1-methylimidazole (1-MeIm) in methanol followed by treatment with NaClO4. Complex 2a and the PF6- salt (1a') of 1a have been structurally characterized. Crystal data for 1a.1.5MeCN. 0.5Et(2)O: triclinic, P (1) over bar, a = 13.125(2) Angstrom, b = 15.529(3) Angstrom, c 17.314(5) Angstrom, a; 67.03(2)degrees, beta 68.05(2)degrees, gamma = 81.38(1)degrees, V 3014(1) Angstrom(3), Z = 2. Crystal data for 2a: triclinic, P (1) over bar, a 8.950(1) Angstrom, b = 12.089(3) Angstrom, c = 13.735(3) Angstrom, alpha 81.09(2)degrees, beta = 72.27(1)degrees, gamma = 83.15(2)degrees, V = 1394(1) Angstrom(3), Z = 1. The complexes consist of a diruthenium(III) unit held by two monoatomic and two three-atom bridging ligands. The 1-MeIm ligands are at the terminal sites of the [Ru-2(mu-L)(eta(1):mu-O(2)CR)(eta(1):eta(1):mu-O(2)CR)(2)](2+) core having a Ru-Ru single bond (L = OMe or eta(1)-O(2)CR). The Ru-Ru distance and the Ru-O-Ru angle in the core of 1a' and 2a are 2.49 Angstrom and similar to 76 degrees. The complexes undergo one-electron oxidation and reduction processes in MeCN-0.1 M TBAP to form mixed-valence diruthenium species with Ru-Ru bonds of orders 1.5 and 0.5, respectively.

Photoinduced electron transfer in zinc porphyrin-crown ether supramolecule with Eu3+ electron acceptor in crown void

Photoinduced electron transfer (ET) reactions in the zinc porphyrin-crown ether (ZnPCE) supramolecule, in which one crown ether moiety containing Eu3+ as electron acceptor is covalently linked to zinc porphyrin (ZnP), were studied by flash photolysis. In methanol solutions, highly efficient charge separation occurs via intramolecular ET from (ZnP)-Zn-3 to Eu3+ encapsulated in the crown ether void (k(1) = (3 +/- 1) X 10(3) s(-1)) and intramolecular ET from 3ZnP to uncomplexed Eu2+ (k(2) = (2.5 +/- 0.5) X 10(3) s(-1)). Intermolecular ET from Eu2+ escaped from the crown ether void to ZnP.+ (k(tau) = (4.3 +/- 0.7) X 10(8) M(-1) s(-1)) seems to be the main pathway of charge recombination.

Constructions of 2D-Metallamacrocycles Using Half-Sandwich Ru-2(II) Precursors: Synthesis, Molecular Structures, and Self-Selection for a Single Linkage Isomer

Coordination-driven self-assembly of oxalato-bridged half-sandwich p-cymene ruthenium complex Ru-2(mu-eta(4)-C2O4)(MeOH)(2)(eta(6)-p-cymene)(2)] (O3SCF3)(2) (1a) with several ditopic donors (L-a-L-d) in methanol affords a series of bi- and tetranuclear metallamacrocycles (2a and 3-5). Similarly, the combination of 2,5-dihydroxy-1,4-benzoquinonato (dhbq)-bridged binuclear complex Ru-2(mu-eta(4)-C6H2O4)(MeOH)(2)(eta(6)-p-cymene)(2)](O3SCF3)(2) (1b) with a flexible bidentate amide linker (L-a) in 1:1 molar ratio gave the corresponding tetranuclear complex 2b. All the macrocycles were isolated as their triflate salts in high yields and were fully characterized by various spectroscopic techniques. Finally, the molecular structures of all the assemblies were determined unambiguously by single-crystal X-diffraction analysis. Interestingly, the combination of acceptor 1a or 1b with an unsymmetrical linear ditopic donor L-a results in a self-sorted linkage isomeric (head-to-tail) macrocycle (2a or 2b) despite the possibility of formation of two different isomeric macrocycles (head-to-head or head-to-tail) due to different connectivity of the donor. Molecular structures of the complexes 2a and 2b showed tetranuclear rectangular geometry with dimensions of 5.51 angstrom x 13.29 angstrom for 2a and 7.91 angstrom x 13.46 angstrom for 2b. In both cases, two binuclear Ru-2(II) building blocks are connected by a mu-N-(4-pyridyl)isonicotinamide donor in a head-to-tail fashion. Surprisingly, the macrocycle 2a loses one counteranion and cocrystallizes with monodeprotonated 1,3,5-trihydroxybenzene via strong intermolecular pi-pi stacking and hydrogen bonding. The tweezer complex 3 showed strong fluorescence in solution, and it showed fluorescence sensing toward nitroaromatic compounds. A fluorescence study demonstrated a marked quenching of the initial fluorescence intensity of the macrocycle 3 upon gradual addition of trinitrotoluene and exhibits significant fluorescence quenching response only for nitroaromatic compounds compared to various other aromatic compounds tested.

Ethanol-dependent oxygen consumption and acetaldehyde formation during vanadyl oxidation by H2O2

Sequential addition of vanadyl sulfate to a phosphate-buffered solution of H2O2 released oxygen only after the second batch of vanadyl. Ethanol added to such reaction mixtures progressively decreased oxygen release and increased oxygen consumption during oxidation of vanadyl by H2O2. Inclusion of ethanol after any of the three batches of vanadyl resulted in varying amounts of oxygen consumption, a property also shared by other alcohols (methanol, propanol and octanol). On increasing the concentration of ethanol, vanadyl sulfate or H2O2, both oxygen consumption and acetaldehyde formation increased progressively. Formation of acetaldehyde decreased with increase in the ratio of vanadyl:H2O2 above 2:1 and was undetectable with ethanol at 0.1 mM. The reaction mixture which was acidic in the absence of phosphate buffer (pH 7.0), released oxygen immediately after the first addition of vanadyl and also in presence of ethanol soon after initial rapid consumption of oxygen, with no accompanying acetaldehyde formation. The results underscore the importance of some vanadium complexes formed during vanadyl oxidation in the accompanying oxygen-transfer reactions.

Modification of photochemical reactivity on formation of inclusion complexes: photorearrangement of benzyl phenyl ethers and methyl phenoxyacetates

The photorearrangement of benzyl phenyl ethers and methyl phenoxyacetates was investigated in methanol and in complexes with cyclodextrin in both the solid state and aqueous solutions. Irradiation in cyclodextrin media leads to a large change in product distribution with a very significant ortho selectivity different from that found in methanol where the reaction is non-selective. For meta-substituted ethers and phenoxyacetates, an impressive regioselectivity between the two ortho-rearranged isomers is observed and this is significantly enhanced by increasing the substituent chain length which acts as a spacer to induce a tight fit between the host and the guest. The observed results are rationalized on the basis of specific orientations of the unsubstituted and meta-substituted ethers and phenoxyacetates in the cyclodextrin cavity.

Helix termination and chain reversal: Crystal and molecular structure of the alpha,beta-dehydrooctapeptide Boc-Val Delta Phe-Phe-Ala-Leu-Ala-Delta Phe-Leu-OH

The crystal structure of the dehydro octapeptide Boc-Val-Delta Phe-Phe-Ala-Leu-Ala-Delta Phe-Leu-OH has been determined to atomic resolution by X-ray crystallographic methods. The crystals grown by slow evaporation of peptide solution in methanol/water are orthorhombic, space group P2(1)2(1)2(1). The unit cell parameters are a = 8.404(3), b = 25.598(2) and c = 27.946(3) Angstrom, Z = 4. The agreement factor is R = 7.58% for 3636 reflections having (\F-o\) greater than or equal to 3 sigma (\F-o\). The peptide molecule is characterised by a 3(10)-helix at the N-terminus and a pi-turn at the C-terminus. This conformation is exactly similar to the helix termination features observed in proteins. The pi-turn conformation observed in the octapeptide is in good agreement with the conformational features of pi-turns seen in some proteins. The alpha(L)-position in the pi-turn of the octapeptide is occupied by Delta Phe(7), which shows that even bulky residues can be accommodated in this position of the pi-turns. In proteins, it is generally seen that alpha(L)-position is occupied by glycine residue. No intermolecular head-to-tail hydrogen bonds are observed in solid state structure of the octapeptide. A water molecule located in the unit cell of the peptide molecule is mainly used to hold the peptide molecule together in the crystal. The conformation observed for the octapeptide might be useful to understand the helix termination and chain reversal in proteins and to construct helix terminators for denovo protein design.

Crystal and molecular structure of Boc-Phe-Val-OMe; comparison of the peptide conformation with its dehydro analogue

The crystal structure of the peptide Boc-Phe-Val-OMe determined by X-ray diffraction methods is reported in this paper. The crystals grown from aqueous methanol are orthorhombic, space group P2(1)2(1)2(1), a = 11.843(2), b = 21.493(4), c = 26.676(4)Angstrom and V = 6790 Angstrom(3). Data were collected on a CAD4 diffractometer using MoK2 radiation (lambda = 0.7107 Angstrom) up to Bragg angle theta = 26 degrees. The structure was solved by direct methods and refined by a least-squares procedure to an R value of 6.8% for 3288 observed reflections. There are three crystallographically independent peptide molecules in the asymmetric unit. All the three molecules exhibit extended conformation. The sidechain of the Val(2) residue shows two different conformations. The conformation of the peptide Boc-Phe-Val-OMe is compared with the conformation of Ac-Delta Phe-Val-OH. It is observed that while Boc-Phe-Val-OMe exhibits an extended conformation, Ac-Delta Phe-Val-OH shows a folded conformation. The results of this comparison highlight the conformation constraining property of the Delta Phe residue. Interestingly, even though Boc-Phe-Val-OMe and Ac-Delta Phe-Val-OH are conformationally different, they exhibit similar packing patterns in the solid state. (C) Munksgaard 1995.

Organometallic chemistry of diphosphazanes .13. Palladium complexes of unsymmetrical diphosphazanes Ph(2)PN(Pr-i)PYY'

Unsymmetrical diphosphazanes Ph(2)PN(Pr-i)PYY' [YY' = O2C12H8 (L(1)), O2C20H12 (L(2)); Y = Ph and Y' = OC6H4Br-4 (L(3)), OC(6)H(4)Me-4 (L(4)), OC(6)H(3)Me(2)-3,5 (L(5)), N(2)C(3)HMe(2)-3,5 (L(6))] react with cis-[PdCl2(COD)] (COD = cycloocta-1,5-diene) giving the chelate complexes of the type cis-[PdCl2{eta(2)-Ph(2)PN(Pr-i)PYY'}] [YY' = O2C12H8 (1), O2C20H12 (2), Y = Ph and Y' = OC6H4Br-4 (3), OC(6)H(4)Me-4 (4), OC(6)H(3)Me(2)-3,5 (5), N(2)C(3)HMe(2)-3,5 (6)]. The P-N bond in 3 and 5 undergoes a facile cleavage in methanol solution to give cis-[PdCl2{eta(1)Ph(2)P(OMe)}{eta(1)-PhP(NHPri)(Y')}] [Y' = OC6H4Br-4 (7), OC(6)H(3)Me(2)-3,5 (8)]. Reactions of Pd-2(dba)(3) . CHCl3 (dba = dibenzylideneacetone) with the diphosphazanes Ph(2)PN(Pr-i)PPhY' [Y' = OC(6)H(4)Me-4 (L(4)), N(2)C(3)HMe(2)-3,5 (L(6)), N2C3H3 (L(7))] in the presence of MeI yields cis-[PdI2{eta(2)-Ph(2)PN(Pr-i)PPhMe}] (9); the P-O or P-N(pyrazolyl) bond of the starting ligands is cleaved and a p-C(Me) bond is formed. An analogous oxidative addition reaction in the presence of Ph(2)PN(Pr-i)PPh(2) (L(8)) yields cis-[PdI(Me)(eta(2)-L(8))] (10) and cis-[PdI2(eta 2-L(8))] (11). The structures of 8 and 9 have been determined by X-ray diffraction. Copyright (C) 1996 Elsevier Science Ltd

Reduction of quinolines to 1,2,3,4-tetrahydro derivatives employing a combination of NaCNBH3 and BF3.OEt(2)

A regiospecific reduction of quinolines (and 1,10-phenanthroline) into the corresponding 1,2,3,4-tetrahydro derivatives using a combination of sodium cyanoborohydride and boron trifluoride etherate in refluxing methanol is described. Under the same conditions indole and acridine reduced to the corresponding dihydroderivatives, whereas acyl group transfer from oxygen to nitrogen atom is also noticed in the case of 8-acyloxyquinolines.

Structure, Spectra and Redox Behaviour of Copper(11) Complexes of Bis( benzimidazoly1)diamine Ligands

The linear quadridentate ligand N,N'-bis(benzimidazoI-2-ylethyl)ethane-l,2-diamine (L') and its 1 - methylbenzimidazole analogue (L2) and homologues form 1 : 1 complexes with Cu(CIO,),; L' also forms complexes of the types CuL'X, where X = NO,, PF,, Br or CI and CuL'(X)Y where X = CI or Br and Y = CIO, or Br. Deep blue CuL1Br,*2H20 crystallizes in the monoclinic space group C2/c with Z = 4, a = 9.91 9(2), b = 16.626(3), c = 14.1 02(3) le\ and p = 94.39(2)". The structure was solved by Patterson and Fourier difference methods and refined by the least-squares technique to R = 0.064 for 2195 independent reflections with / > 1.50(/). The molecule lies on a two-fold axis symmetrically around Cu". The co-ordination around Cu" is found to be square planar with two amino nitrogens and two benzimidazole nitrogens forming the equatorial plane [CU-N 1.983(3) and 2.037(4) A]. The bromides are at longer distances [3.349(1) A] in axial sites. Ligand field and EPR spectra indicate that one bromide or chloride ion is axially co-ordinated to Cu" in [CuL1l2+. This ion exhibits quasi-reversible redox behaviour. Electrochemical studies of the dihalides in methanol have established the presence of [CuL'X,], [CuL'(X)]+ and [CuL'I2+ in equilibrium. In complexes with 565 [CuL4I2+ [L4 = N,Nbis( benzimidazol-2-ylmethyl)ethane-l,2-diamine] and 555 [CuL3] [L3 = N,N'-bis(1 -methylbenzimidazol- 2-ylmethyl)propane-l,3-diamine] chelate rings, Cull does not seem to lie in the N, square plane, as revealed by their low A values and irreversible electrochemical behaviour. The Cu"-Cu' redox potentials in methanol are in the order [CuL1I2+ < [CuL3I2+ < [CuL4I2+; this illustrates that sixmembered chelate rings are suitable to stabilize Cu", when CU-N 0 interactions are favourable.

Crystal structure of Boc-LAla-Delta Phe-Delta Phe-Delta Phe-Delta Phe-NHMe: a left-handed helical peptide

alpha,beta-Dehydrophenylalanine residues constrain the peptide backbone to beta-bend conformation. A pentapeptide containing four consecutive (Delta Phe) residues has been synthesised and crystallised. The peptide Boc-LAla-Delta Phe-Delta Phe-Delta Phe-Delta Phe-NHMe (C45H46N6O7, MW = 782.86) was crystallised from an acetonitrile/methanol mixture. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) With a = 19.455(6), b = 20.912(9), c = 11.455(4) Angstrom and Z = 4. The X-ray (MoKalpha, lambda = 0.7107 Angstrom) intensity data were collected using the Rigaku-AFC7 diffractrometer. The crystal structure was determined by direct methods and refined using the least-squares technique, R = 8.41% for 1827 reflections with \F-o\ > 4 sigma\F-o\. The molecule contains the largest stretch of consecutive dehydrophenylalanine residues whose crystal structure has been determined so far. The peptide adopts left-handed 3(10)-helical conformation despite the presence of LAla at the N-terminus. The mean phi, psi values, averaged across the last four residues are 56.8 degrees and 17.5 degrees, respectively. There are four 4-->1 intramolecular hydrogen bonds, characteristic of the 3(10)-helix. In the crystal each molecule interacts with four crystallographically symmetric molecules with one hydrogen bond each.

Enhancing the mesoporosity of pillared clay catalyst - a study of pillar aggregates and deactivation in alkylation reaction

A sample of montmorillonite was pillared with aluminium polyoxycations in presence of different amounts of tween-80, a nonionic surfactant, ranging from 0.01 to 0.20 mmol/meq of clay. The amount of aluminium sorbed was found to vary with the amount of surfactant added during pillaring. Vapour phase catalytic activity of the samples for alkylation of toluene with methanol in a fixed bed down flow reactor showed that the rate of deactivation, in general, increased with decrease in the pillar density. The samples treated with 0.06 to 0.08 mmol/meq of surfactant showed the lowest deactivation and also an enhancement in the mesopores which did not change on calcining to 540°C. Suppression of deactivation is attributed to the distribution of pillars by the surfactant in such a way as to decrease the coke formation.