Biomimetic Formation of 2‐Tropolones by Dioxygenase‐Catalysed Ring Expansion of Substituted 2,4‐Cyclohexadienones

Substituted 2‐tropolone natural products are found in plants and fungi. Their biosynthesis is thought to occur by ring expansion from a cyclohexadienone precursor, but this reaction has not previously been demonstrated experimentally. Treatment of 6‐hydroxy‐6‐hydroxymethylcyclohexa‐2,4‐dienone with the non‐haem iron(II)‐dependent extradiol catechol dioxygenase MhpB from Escherichia coli results in the formation of the 2‐tropolone ring‐expansion product through a pinacol‐type rearrangement. Three further substituted cyclohexa‐2,4‐dienone analogues were prepared, and treatment of each analogue was found to give the substituted 2‐tropolone ring‐expansion product. This ring expansion could also be effected nonenzymatically by treatment with 1,4,7‐triazacyclononane and FeCl₂. This is a novel transformation for non‐haem iron‐dependent enzymes, and this is the first experimental demonstration of the proposed ring‐expansion reaction in tropolone biosynthesis.     

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.     

Synthesis of polymer-supported metal-ion complexes and evaluation of their catalytic activities

Polymer-supported transition-metal-ion complexes of the N,N′-bis(o-hydroxy acetophenone) propylenediamine (HPPn) Schiff base were prepared by the complexation of iron(III), cobalt(II), and nickel(II) ions on a polymer-anchored N,N′-bis(5-amino-o-hydroxy acetophenone) propylenediamine Schiff base. The complexation of iron(III), cobalt(II), and nickel(II) ions on the polymer-anchored HPPn Schiff base was 83.44, 82.92, and 89.58 wt%, respectively, whereas the unsupported HPPn Schiff base showed 82.29, 81.18, and 87.29 wt % complexation of these metal ions. The iron(III) ion complexes of the HPPn Schiff base showed octahedral geometry, whereas the cobalt(II) and nickel(II) ion complexes were square planar in shape, as suggested by spectral and magnetic measurements. The thermal stability of the HPPn Schiff base increased with the complexation of metal ions, as evidenced by thermogravimetric analysis. The HPPn Schiff base showed a weight loss of 51.0 wt % at 500°C, but its iron(III), cobalt(II), and nickel(II) ion complexes showed weight losses of 27.0, 35.0, and 44.7 wt % at the same temperature. The catalytic activity of the unsupported and supported metal-ion complexes was analyzed by the study of the oxidation of phenol and epoxidation of cyclohexene in the presence of hydrogen peroxide. The supported HPPn Schiff base complexes of iron(III) ions showed a 73.0 wt % maximum conversion of phenol and 90.6 wt % epoxidation of cyclohexene, but unsupported complexes of iron(III) ions showed 63.8 wt % conversion of phenol and 83.2 wt % epoxidation of cyclohexene. The product selectivity for catechol (CTL) and epoxy cyclohexane (ECH) was 93.1 wt % and 98.1 wt % with the supported HPPn Schiff base complexes of iron(III) ions, but it was low with the supported Schiff base complexes of cobalt(II) and nickel(II) ions. The selectivity for CTL and ECH varied with the molar ratio of the metal ions but remained unaffected by the molar ratio of hydrogen peroxide to the substrate. The energy of activation for the epoxidation of cyclohexene and oxidation of phenol with the polymer-supported Schiff base complexes of iron(III) ions was 10.0 and 12.7 kJ/mol, respectively, but it was found to be higher with the supported HPPn Schiff base complexes of cobalt(II) and nickel(II) ions and with the unsupported HPPn Schiff base complexes of iron(III), cobalt(II), and nickel(II) ions. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci 2008     

Para‐substituted 1‐Phenyl‐3‐methyl‐4‐aroyl‐5‐pyrazolones as Selective Extractants for Vanadium(V) from Acidic Chloride Solutions

Abstract

Para‐substituted 4‐aroyl derivatives of 1‐phenyl‐3‐methyl‐5‐pyrazolones (HX), namely, 1‐phenyl‐3‐methyl‐4‐(4‐fluorobenzoyl)‐5‐pyrazolone (HPMFBP) and 1‐phenyl‐3‐methyl‐4‐(4‐toluoyl)‐5‐pyrazolone (HPMTP) were synthesized and examined with regard to the extraction behavior of multivalent metal ions such as magnesium(II), aluminum(III), titanium(IV), vanadium(V), chromium(III), manganese(II), iron(II), and iron(III) that are present in titania waste chloride liquors. For comparison, studies have also been carried out with 1‐phenyl‐3‐methyl‐4‐benzoyl‐5‐pyrazolone (HPMBP). The results demonstrate that vanadium(V) and iron(III) are extracted into chloroform with 4‐aroyl‐5‐pyrazolones as VO₂X · HX and FeX₃, respectively. On the other hand, magnesium(II), aluminum(III), titanium(IV), chromium(III), manganese(II), and iron(II) were not found to be extracted into the organic phase. The equilibrium constants of vanadium(V) and iron(III) with various 4‐aroyl‐5‐pyrazolones follow the order HPMFBP>HPMBP>HPMTP, which is in accordance with their pKa values. The selectivity between vanadium(V) and iron(III) increases with increasing hydrochloric acid concentration. Further, it is clear from the results that iron(III) is not getting extracted above 1.0 mol dm^?3 hydrochloric acid solution. The electronic and IR spectra of the extracted complexes of vanadium(V) and iron(III) were used to further clarify the nature of the extracted complexes. The potential of these reagents for the selective extraction and separation of vanadium(V) from titania waste chloride liquors has also been discussed.     

New precursor for the post-synthesis preparation of Fe-ZSM-5 zeolites with low iron content

A new method for preparing ZSM-5 catalysts containing highly dispersed iron species is presented. Iron(III) oxalate was used as the iron precursor in an aerobic aqueous exchange process. The presence of different iron species inside the zeolite pores was investigated by IR spectroscopy of adsorbed NO, which revealed an excellent iron dispersion. The results were compared with those obtained on a Fe-ZSM-5 catalysts prepared by iron(II) chloride sublimation and on samples prepared by controlled migration of framework iron in isomorphously substituted samples.     

人白细胞介素-18突变体的虚拟筛选及其活性

目的筛选合适的人白细胞介素-18(hIL-18)突变体,并检测其活性。方法运用分子模建工具,模建出IL-18受体及IL-18/IL-18R复合物的分子结构,分别虚拟突变IL-18的4个半胱氨酸为另外19种氨基酸,以野生型IL-18为模板进行同源模建,建立其三维结构模型,计算其IL-18/IL-18R复合物三维结构及分子间作用能变化情况,筛选几种突变方案。运用分子生物学技术构建突变体,表达纯化后进行活性检测。结果根据理论预测情况,筛选了12株突变体。突变的质粒经双酶切及测序鉴定,证明构建正确。突变体蛋白以包涵体形式表达,表达量约占菌体总蛋白的30%,纯化后纯度大于90%。纯化的C38Glu、C127Ser等几株突变体在低浓度时,活性轻微上升,与理论预测结果基本相同。结论已成功构建了多株IL-18的突变体,运用生物信息学方法进行先期筛选有助于加快IL-18的突变研究。     

Selective removal of iron from aqueous solution using ion imprinted thiocyanato-functionalized silica gel sorbents

We combined surface imprinting technique with sol-gel process for preparing a new iron(III)-imprinted thiocyanato-functionalized silica gel sorbent using 3-thiocyanatopropyltriethoxysilane as the functional monomer, and epichlorohydrin as the cross-linking agent for the selective removal of iron(III) from aqueous solution. The iron(III)-imprinted thiocyanato-functionalized silica gel was characterized by FT-IR spectra, SEM, and the static adsorptiondesorption method. Maximum binding capacity, optimum pH, and equilibrium binding time were found to be 20.31 mg·g^?1, pH 4.2, and 20min, respectively. The relative selectivity coefficients of Fe(III)-imprinted thiocyanato-functionalized silica gel sorbents for Fe(III)/Ni(II), Fe(III)/Co(II), Fe(III)/Cd(II) and Fe(III)/Pb(II) were 11.8, 10.6, 8.2 and 9.1 times greater than the non-imprinted silica gel, respectively. The iron(III)-imprinted thiocyanato-functionalized silica gel sorbents could be used repeatedly without decreasing in their adsorption capacities significantly.     

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility

Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.     

Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.     

Engineering the steroid-specificity of an anti-17beta-estradiol Fab by random mutagenesis and competitive phage panning

We have employed random mutagenesis and phage display to improve the steroid-specificity of an anti-17beta-estradiol Fab fragment. The VH domain was mutated using error-prone PCR; the mutation rate was controlled by adjusting the number of effective duplications. A phage library of 2 x 10(6) independent mutants was generated, each mutant containing on average 24 amino acid changes. We selected for decreased testosterone (TES) cross-reactivity by adding a large excess TES as a competitor to the panning reactions. After four panning rounds, the cross-reactivities of the individual mutant clones ranged from 19 to 4%, showing up to 20-fold improvement over the original value (78%). Estradiol affinities were mainly unchanged. Sequencing of the VH regions revealed two hot spots, one located around Ser32 in CDR1 and the other around Thr52A in CDR2, while no mutations were found in CDR3. Although most clones had multiple mutations, it was possible to deduce the residues relevant to the improved specificity by comparing the sequences and binding data of the mutants. We demonstrated that controlled error-prone PCR mutagenesis is a rapid method to identify such key residues, lending itself to the scanning of 'lead' positions for further mutagenesis by other methods.      

Random mutagenesis on the Pseudomonas lipase activator protein, LipB: exploring amino acid residues required for its function

LipB, lipase activator protein from Pseudomonas aeruginosa TE3285, specifically recovers the enzymatic activity of denatured inactive lipase. To find important amino acid residues of LipB in this reactivation, random mutagenesis using error-prone PCR was performed on a gene encoding the functional region of LipB. The resultant DNA library was introduced into the lipase expression system using Escherichia coli, and LipB mutants lacking lipase activity were selected by two screening procedures. First, on agar plates containing tributyrin as a substrate for lipase, single colonies lacking active lipase secretion were selected as clones missing the active LipB. Second, to exclude nonsense and frameshift mutants, the molecular size of LipB in the given clones was confirmed by Western blotting. From the selected mutants, of which multiple residues are replaced, five single- residue substituted mutants were newly prepared. Consequently, Y99C, Y99H, S102R and R115C mutants formed no detectable complex with the lipase and lost the in vitro reactivation activity. In the case of Y99C and R115C, their single cysteine residue formed the intermolecular disulfide bridge. Thus, Tyr99 and Arg115 are likely to exist on the molecular surface of LipB, and are candidates for residues that make direct interaction with the denatured lipase in the reactivation process.      

Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution

To expand the functionality of lipase B from Candida antarctica(CALB) we have used directed evolution to create CALB mutantswith improved resistance towards irreversible thermal inactivation.Two mutants, 23G5 and 195F1, were generated with over a 20-foldincrease in half-life at 70°C compared with the wild-typeCALB (WT-CALB). The increase in half-life was attributed toa lower propensity of the mutants to aggregate in the unfoldedstate and to an improved refolding. The first generation mutant,23G5, obtained by error-prone PCR, had two amino acid mutations,V210I and A281E. The second generation mutant, 195F1, derivedfrom 23G5 by error-prone PCR, had one additional mutation, V221D.Amino acid substitutions at positions 221 and 281 were determinedto be critical for lipase stability, while the residue at position210 had only a marginal effect. The catalytic efficiency ofthe mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyloctanoate was also found to be superior to that of WT-CALB. Received May 8, 2003; revised June 9, 2003; accepted June 23, 2003.     

Facile synthesis, spectroscopy and electrochemical activity of two substituted iron phthalocyanines as oxygen reduction catalysts in an acidic environment

Two substituted iron phthalocyanine macrocycles were synthesized and evaluated as oxygen reduction electrocatalysts in an acidic, or proton exchange membrane (PEM) fuel cell-like, environment. The non-noble, transition metal macrocycles iron(II) 2,9,16,23-tetra-tert-butyl-phthalocyanine (FePc(tBu)₄), and iron(II) 2,11,20,29-Tetra-tert-butyl-2,3-naphthalocyanine (FeNpPc(tBu)₄) were made via a facile synthesis. Successful synthesis was confirmed using UV-Vis spectroscopy as well as matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy. These two substituted iron phthalocyanines were characterized using cyclic voltammetry (CV) and their electrocatalytic activities towards the oxygen reduction reaction (ORR) were evaluated using rotating disk electrode (RDE) techniques. The ORR activities of these substituted iron phthalocyanines were compared to those of the unsubstituted species, iron(III) phthalocyanine (FePc). The RDE trace of FeNpPc(tBu)₄ indicates a 2 electron ORR mechanism, whereby O₂ is reduced mainly to H₂O₂. This is in contrast to FePc, FeNpPc and FePc(tBu)₄, which exhibit predominantly 4 or mixed 2 and 4, electron ORR mechanisms.     

Electrical properties of macromolecular complexes of coordinated polymers with mixed valence of Co(II), Co(III) and Co(II–III)

Electrical properties of bis‐ethylenediamine Co(II), Co(III) and Co(II–III) mixed valence complexes and polyethylenediaminecarbosilazane (PEDCSZ) [Co(II), Co(III) and Co(II–III)] macromolecules were measured at room temperature in a frequency range between 1 Hz and 10⁶ Hz. Electrical modulus, permittivity and ac conductivity were calculated using ac impedance and phase‐angle measurements. It was found that the overall conductivity of PEDCSZ macromolecules with Co is much higher than that for Co monomers. In addition, the conductivity of PEDCSZ Co(III) and Co(II–III) mixed valence compounds are higher than those containing Co(II) where the electronic conduction is dominant. The relaxation time increases from about 2.7 × 10^?6 s for Co(II) chloride to a value of 2.9 × 10^?4 s for Co(II–III) chloride monomers. Copyright © 2003 Society of Chemical Industry     

金属改性的介孔硅材料FSM-16催化苯酚与H_2O_2合成苯二酚

研究了金属改性的介孔硅材料FSM-16在该反应中的催化性能。其中Fe和Cu改性的FSM-16表现出比较好的性能。在Fe-FSM-16的作用下,苯酚的转化率达到27.8%,对苯二酚和邻苯二酚选择性达到96.4%(邻/对=1.8)。FT-IR和XRD表征证明铜离子进入了FSM-16的骨架,并形成了Si—O—Cu键。     

Catalytic destruction of malathion by metallomicelle layers

Two micellized Cu(II) and Fe(III) ion complexes were synthesized and found to possess good catalytic activity in the cleavage of the malathion/dupric (ferric) complex thionate ester. The complexes form metallomicelles, which bind the substrate by coordinating with the thionate sulfur in the malathion (which is chemically similar to the nerve agent sarin). Possible reasons for the rate acceleration include enhanced electrophilicity of the micellized metals, enhanced surface activity, and the recognized ability of cationic micelles to accelerate the cleavage of the thionate ester. The results of kinetic data (half-life times) for malathion degradation were 13.7 and 8.3 min in the presence of Cu(II) and Fe(III) metallomicelle layers, respectively.     

Synthesis and adhesive property study of polyoxetanes grafted with catechols via Cu(I)-catalyzed click chemistry

Mussel-inspired catechol-containing polymers have drawn great attention due to their outstanding adhesive properties. Catechol-containing polyethylene glycol (cPEG) is a well-studied catechol-containing polymer used for tissue repair. Nevertheless, catechols can only be attached to the chain ends of polyethylene glycols thus the bonding strength of the resulting polymers is limited. Aiming at solving the problem, a series of clickable polyoxetane copolymers with grafted catechol moieties were synthesized in an efficient manner. Upon addition of FeCl₃ as the cross-linker, strong bonding strength of the adhesive was achieved. Polymer containing 15.5 molar percent of catechol showed the strongest bonding strength up to 5.59 MPa on sanded stainless steel. It was found that the triazole groups also contributed to the overall adhesive performance. This polyoxetane-based adhesive also displayed strong bonding ability to a variety of other substrates including porcine skin.     

The interaction of iron species with pyrite surfaces

The development of iron oxide films on pyrite has been investigated using linear potential sweep voltammetry. Voltammograms for a fresh pyrite surface indicated the presence of an iron(II) hydroxyoxide. After brief exposure to air, the surface layer oxidized to iron(III) hydroxy-oxide. Immersion in iron(II) solutions containing small amounts of iron(III) resulted in an increase in the thickness of the oxide layer to a limiting value; the oxide was present mainly in the iron(III) state. An iron hydroxy-oxide also developed on gold during immersion in iron(II) solution. A pyrite surface abraded on iron powder in deoxygenated water also became coated with iron hydroxy-oxide. Immersion of pyrite in cyanide solution resulted in a decrease in the quantity of oxide on the surface.     

A semi‐aza‐crown‐based chemosensor for colorimetric detection of iron(III) and iron(II) in aqueous media with large bathochromic shift

A colorimetric chemosensor, EDDAN (1,1′‐((1E,11E)‐5,8‐dioxa‐2,11‐diazadodeca‐1,11‐diene‐1,12‐diyl)bis(naphthalen‐2‐ol)), based on a semi‐aza‐crown structure, was designed and synthesised to sense iron(III) and iron(II). EDDAN showed a unique colour change in the presence of either iron(III) or iron(II), turning from pale yellow to deep purple with large bathochromic shifts. The detection limits of iron(III) and iron(II) were 2.70 and 3.43 μm , respectively. EDDAN displayed a high selectivity for iron ions in the presence of other metal cations except copper(II). In addition, EDDAN showed excellent reversibility by using ethylenediaminetetraacetic acid. Moreover, EDDAN was applied to test strips as well. Based on ultraviolet‐visible titrations, Job plots, electron paramagnetic resonance study and electrospray ionisation mass spectrometry analyses, a mechanism for the sensing of iron(III) and iron(II) was also proposed.     

Benzyl and Methyl Fatty Hydroxamic Acids Based on Palm Kernel Oil as Chelating Agent for Liquid-Liquid Iron(III) Extraction

Liquid-liquid iron(III) extraction was investigated using benzyl fatty hydroxamic acids (BFHAs) and methyl fatty hydroxamic acids (MFHAs) as chelating agents through the formation of iron(III) methyl fatty hydroxamate (Fe-MFHs) or iron(III) benzyl fatty hydroxamate (Fe-BFHs) in the organic phase. The results obtained under optimized conditions, showed that the chelating agents in hexane extract iron(III) at pH 1.9 were realized effectively with a high percentage of extraction (97.2% and 98.1% for MFHAs and BFHAs, respectively). The presence of a large amount of Mg(II), Ni(II), Al(III), Mn(II) and Co(II) ions did affect the iron(III) extraction. Finally stripping studies for recovering iron(III) from organic phase (Fe-MFHs or Fe-BFHs dissolved in hexane) were carried out at various concentrations of HCl, HNO(3) and H(2)SO(4). The results showed that the desired acid for recovery of iron(III) was 5 M HCl and quantitative recovery of iron(III) was achieved from Fe(III)-MFHs and Fe(III)-BFHs solutions in hexane containing 5 mg/L of Fe(III).