Improving the Thermostability of a Methyl Parathion Hydrolase by Adding the Ionic Bond on Protein Surface

The thermostability of the methyl parathion hydrolase (MPH_OCH) from Ochrobactrum sp. M231 was improved using site-directed mutagenesis. Two prolines (Pro76 and Pro78) located on the protein surface were selected for mutations after inspection of the sequence alignment of MPH_OCH and OPHC2, a thermostable organophosphorus hydrolase from Pseudomonas pseudoalcaligenes C2-1. The temperature of the double-point mutant (P76D/P78K) at which the mutant lost 50% of its activity (T50) was approximately 68 °C, which is higher than that of WT enzyme (64 °C), P76D (67 °C), and P78K (59 °C). Structural analysis of P76D/P78K indicated that the substituted residues (Asp76 and Lys78) could generate an ionic bond and increase the structural electrostatic energy, which could then increase the stability of the protein. These results also suggest that the thermal stability of proteins could be improved by adding the ionic bond on protein surface.     

Synthesis and X-ray or NMR/DFT structure elucidation of twenty-one new trifluoromethyl derivatives of soluble cage isomers of C76, C78, C84, and C90

Adding 1% of the metallic elements cerium, lanthanum, and yttrium to graphite rod electrodes resulted in different amounts of the hollow higher fullerenes (HHFs) C76-D2(1), C78-C2v(2), and C78-C2v(3) in carbon-arc fullerene-containing soots. The reaction of trifluoroiodomethane with these and other soluble HHFs at 520-550 degrees C produced 21 new C76,78,84,90(CF3)n derivatives (n = 6, 8, 10, 12, 14). The reaction with C76-D2(1) produced an abundant isomer of C2-(C76-D2(1))(CF3)10 plus smaller amounts of an isomer of C1-(C76-D2(1))(CF3)6, two isomers of C1-(C76-D2(1))(CF3)8, four isomers of C1-(C76-D2(1))(CF3)10, and one isomer of C2-(C76-D2(1))(CF3)12. The reaction with a mixture of C78-D3(1), C78-C2v(2), and C78-C2v(3) produced the previously reported isomer C1-(C78-C2v(3))(CF3)12 (characterized by X-ray crystallography in this work) and the following new compounds: C2-(C78-C2v(3))(CF3)8; C2-(C78-D3(1))(CF3)10 and C(s)-(C78-C2v(2))(CF3)10 (both characterized by X-ray crystallography in this work); C2-(C78-C2v(2))(CF3)10; and C1-C78(CF3)14 (cage isomer unknown). The reaction of a mixture of soluble higher fullerenes including C84 and C90 produced the new compounds C1-C84(CF3)10 (cage isomer unknown), C1-(C84-C2(11))(CF3)12 (X-ray structure reported recently), D2-(C84-D2(22))(CF3)12, C2-(C84-D2(22))(CF3)12, C1-C84(CF3)14 (cage isomer unknown), C1-(C90-C1(32))(CF3)12, and another isomer of C1-C90(CF3)12 (cage isomer unknown). All compounds were studied by mass spectrometry, (19)F NMR spectroscopy, and DFT calculations. An analysis of the addition patterns of these compounds and three other HHF(X) n compounds with bulky X groups has led to the discovery of the following addition-pattern principle for HHFs: In general, the most pyramidal cage C(sp(2)) atoms in the parent HHF, which form the most electron-rich and therefore the most reactive cage C-C bonds as far as 1,2-additions are concerned, are not the cage C atoms to which bulky substituents are added. Instead, ribbons of edge-sharing p-C6(X)2 hexagons, with X groups on less pyramidal cage C atoms, are formed, and the otherwise "most reactive" fullerene double bonds remain intact.     

Expanding the world of endohedral fullerenes--the Tm3N@C2n (39< or =n< or =43) clusterfullerene family

A new family of endohedral fullerenes, based on an encaged trithulium nitride (Tm(3)N) cluster, was synthesised, isolated and characterised by HPLC, mass spectrometry, and visible-NIR and FTIR spectroscopy. Tm(3)N clusterfullerenes with cages as small as C(76) and as large as C(88) were prepared and six of them were isolated. Tm(3)N@C(78) is a small clusterfullerene. The two isomers of Tm(3)N@C(80) (I and II) were the most abundant structures in the fullerene soot. Tm(3)N@C(82), Tm(3)N@C(84), and Tm(3)N@C(86) represent a new series of higher clusterfullerenes. All six isolated Tm(3)N clusterfullerenes were classified as large energy-gap structures with optical energy gaps between approximately 1.2 and approximately 1.75 eV. Tm(3)N@C(80) (I) and Tm(3)N@C(80) (II) were assigned to the C(80) cages C(80):7 (I(h)) and C(80):6 (D(5h)). For Tm(3)N@C(78), the analysis pointed to an elliptical carbon cage with C(78):1 (D(3)) or C(78):4 (D(3h)) being the probable structures.     

Trifluoromethyl derivatives of insoluble small-HOMO-LUMO-gap hollow higher fullerenes. NMR and DFT structure elucidation of C2-(C74-D3h)(CF3)12, Cs-(C76-Td(2))(CF3)12, C2-(C78-D3h(5))(CF3)12, Cs-(C80-C2v(5))(CF3)12, and C2-(C82-C2(5))(CF3)12

Reaction of a mixture of insoluble higher fullerenes with CF3I at 500 degrees C produced a single abundant isomer of C74(CF3)12, C76(CF3)12, and C80(CF3)12, two abundant isomers of C78(CF3)12 and C82(CF3)12, and an indeterminant number of isomers of C84(CF3)12. Using a combination of 19F NMR spectroscopy, DFT calculations, and the structures and spectra of previously reported fullerene(CF3)n compounds, the most-probable structures of six of the seven isolated compounds were determined to be specific isomers of C2-(C74-D3h)(CF3)12, Cs-(C76-Td(2))(CF3)12), C2-(C78-D3h(5))(CF3)12), Cs-(C80-C2v(5))(CF3)12), C2-(C82-C2(5))(CF3)12), and C2-(C82-C2(3))(CF3)12) containing ribbons and/or loops of edge-sharing para-C6(CF3)2 hexagons. The seventh isolated compound is a C1 isomer of C78(CF3)12 containing two such ribbons. This set of compounds represents only the second reported isolable compound with the hollow C74-D3h cage and the first experimental evidence for the existence of the hollow fullerenes C76-Td(2), C78-D3h(5), C80-C2v(5), and C82-C2(5) in arc-discharge soots.     

Size-selective formation of C78 fullerene from a three-dimensional polyyne precursor

Multicyclic cagelike cyclophanes 2 a and 2 b containing cyclobutene rings have been prepared as precursors of three-dimensional polyynes C78H18 (1a) and C78H12Cl6 (1b), respectively. Laser irradiation of 2a and 2b induced expulsion of the aromatic fragment, indane, to give the three-dimensional polyyne anions C78H18- and C78H12Cl6-, respectively. Whereas the former anion lost only four hydrogen atoms to form C78H14-, complete loss of all hydrogen and chlorine atoms was observed from the latter anion, to yield a C78- ion that has a fullerene structure which was proven by its characteristic fragmentation pattern.     

Fullerene anions of different sizes and shapes: a 13C NMR and density-functional study

A combined experimental and theoretical study was conducted on numerous higher fullerene anions with different sizes and shapes, C76-D2, C78-C2v, C78-D3, C84-D2, and C84-D2d. The corresponding fullerenes were reduced by lithium metal to diamagnetic multiply charged anions. The centers of gravity of the 13C NMR spectra of all the multiply charged anions were deshielded, relative to those of the neutral fullerenes. The results of density functional (DFT) computations of the 13C NMR spectra and the molecular orbitals (MOs) of possible polyanion reduction products suggest that hexaanions were the species formed.     

Increasing stability of the fullerenes with the number of carbon atoms: the experimental evidence

The values of the molar standard enthalpies of formation, Delta(f)H(o)(m)(C(76), cr) = (2705.6 +/- 37.7) kJ x mol(-1), Delta(f)H(o)(m)(C(78), cr) = (2766.5 +/- 36.7) kJ x mol(-1), and Delta(f)H(o)(m)(C(84), cr) = (2826.6 +/- 42.6) kJ x mol(-1), were determined from the energies of combustion, measured by microcombustion calorimetry on a high-purity sample of the D(2) isomer of fullerene C(76), as well as on a mixture of the two most abundant constitutional isomers of C(78) (C(2nu)-C(78) and D(3)-C(78)) and C(84) (D(2)-C(84), and D(2d)-C(84). These values, combined with the published data on the enthalpies of sublimation of each cluster, lead to the gas-phase enthalpies of formation, Delta(f)H(o)(m)(C(76), g) = (2911.6 +/- 37.9) kJ x mol(-1); Delta(f)H(o)(m)(C(78), g) = (2979.3 +/- 37.2) kJ x mol(-1), and Delta(f)H(o)(m)(C(84), (g)) = (3051.6 +/- 43.0) kJ x mol(-1), results that were found to compare well with those reported from density functional theory calculations. Values of enthalpies of atomization, strain energies, and the average C-C bond energy were also derived for each fullerene. A decreasing trend in the gas-phase enthalpy of formation and strain energy per carbon atom as the size of the cluster increases is found. This is the first experimental evidence that these fullerenes become more stable as they become larger. The derived experimental average C-C bond energy E(C-C) = 461.04 kJ x mol(-1) for fullerenes is close to the average bond energy E(C-C) = 462.8 kJ x mol(-1) for polycyclic aromatic hydrocarbons (PAHs).     

Discrimination against 13C during degradation of simple and complex substrates by two white rot fungi

Changes in isotopic 13C signatures of CO2-C evolved during decomposition of a sugar (glucose), a fatty acid (palmitic acid), a protein (albumin), a structural biopolymer (lignin) and bulk plant tissue (aerial shoots from Lolium perenne) were monitored over a period of 76 days. All materials were sterilized and inoculated with either of two different species of white rot fungi, Phanerochaete chrysosporium or Coriolus versicolor, and incubated in sealed bottles at 28 degrees C. The CO2 concentration in the jars was periodically determined using an infrared gas analyzer and its isotopic (13C) signature was assessed using a trace gas (ANCA TGII) module coupled to an isotope ratio mass spectrometer (IRMS, Europa 20-20). L. perenne material inoculated with C. versicolor showed the highest C mineralization activity with approximately 70% of total C evolved as CO2 after 76 days of incubation, followed by glucose. Substrates inoculated with C. versicolor generally decomposed faster than when degraded by P. chrysosporium, except for lignin, where no significant differences between the two fungi types were found and CO2-C released was less than 2% of the initial C. Considerable 13C isotopic fractionation during the degradation of plant tissue and of pure biochemical compounds was revealed as well as progressive shifts in cumulative CO2-13C isotopic signatures over time. During the first stages of decomposition, the CO2-C released was usually depleted in 13C as compared with the initial solid substrate, but with ongoing decomposition the CO2-C evolved became progressively more enriched in 13C. P. chrysosporium usually showed a slightly higher 13C fractionation than C. versicolor during the first decomposition phase. At posterior decomposition stages isotopic discrimination was often stronger by C. versicolor. These findings on isotopic 13C discrimination during microbial degradation both of simple biochemical compounds and of complex vegetal tissue confirmed not only the existence of significant 13C isotopic fractionation during plant residue decomposition, but also the existence of non-random isotopic distribution within substrates. They also demonstrated the ability of microorganisms to selectively discriminate against 13C even when degrading an isolated simple substrate.     

A gate mechanism indicated in the selectivity filter of the potassium channel KscA

Classical molecular dynamics (MD) and non-equilibrium steered molecular dynamics (SMD) simulations were performed on the molecular structure of the potassium channel KcsA using the GROMOS 87 force fields. Our simulations focused on mechanistic and dynamic properties of the permeation of potassium ions through the selectivity filter of the channel. According to the SMD simulations a concerted movement of ions inside the selectivity filter from the cavity to extracellular side depends on the conformation of the peptide linkage between Val76 and Gly77 residues in one subunit of the channel. In SMD simulations, if the carbonyl oxygen of Val76 is positioned toward the ion bound at the S3 site (gate-opened conformation) the net flux of ions through the filter is observed. When the carbonyl oxygen leaped out from the filter (gate-closed conformation), ions were blocked at the S3 site and no flux occurred. A reorientation of the Thr75-Val76 linkage indicated by the CHARMM-based MD simulations performed Berneche and Roux [(2005) Structure 13:591–600; (2000) Biophys J 78:2900–2917] as a concomitant process of the Val76-Gly77 conformational interconversion was not observed in our GROMOS-based MD simulations.     

A microwave and quantum chemical study of cyclopropaneselenol

The microwave spectrum of cyclopropaneselenol, C 3H 5SeH, has been investigated in the 21.9-80 GHz frequency range. The microwave spectra of the ground vibrational state of five isotopologues of cyclopropaneselenol (C 3H 5 (82)SeH, C 3H 5 (80)SeH, C 3H 5 (78)SeH, C 3H 5 (77)SeH, and C 3H 5 (76)SeH) of one conformer, as well as the spectra of two vibrationally excited states of each of the C 3H 5 (80)SeH and C 3H 5 (78)SeH isotopologues of this rotamer, have been assigned. The H-C-Se-H chain of atoms is synclinal in this conformer, and there is no indication of further rotameric forms in the microwave spectrum. The b-type transitions of the ground vibrational state of the more abundant species C 3H 5 (80)SeH and C 3H 5 (78)SeH were split into two components, which is assumed to arise from tunneling of the proton of the selenol group between two equivalent synclinal potential wells. The tunneling frequencies were 0.693(55) MHz for C 3H 5 (80)SeH and 0.608(71) MHz for C 3H 5 (78)SeH. The microwave study has been augmented by high-level density functional and ab initio quantum chemical calculations, which indicate that the H-C-Se-H dihedral angle is approximately 75 degrees from synperiplanar (0 degrees).     

C76Si2异构体的分子设计和光谱研究

用INDO系列方法对C76Si2的17种可能异构体进行系统理论研究, 表明最稳定异构体是由C78(C2V)沿X方向椭球长轴所穿过的六元环上的2个C原子(29, 30)被Si取代所形成, 异构体稳定性随2个Si原子沿Z方向距离增加而降低, 且取代场所附近易成为进一步反应中心; C76Si2(29, 30)电子光谱吸收峰与C78相比发生红移, 主要是由于其对称性降低和LUMO- HOMO能隙变小。     

The thermal decomposition of hydronium jarosite and ammoniojarosite

The thermal decomposition of hydronium jarosite and ammoniojarosite was studied using thermogravimetric analysis and mass spectrometry, in situ synchrotron X-ray diffraction and infrared emission spectroscopy. There was no evidence for the simultaneous loss of water and sulfur dioxide during the desulfonation stage as has previously been reported for hydronium jarosite. Conversely, all hydrogen atoms are lost during the dehydration and dehydroxylation stage from 270 to 400 °C and no water, hydroxyl groups or hydronium ions persist after 400 °C. The same can be said for ammoniojarosite. The first mass loss step during the decomposition of hydronium jarosite has been assigned to the loss of the hydronium ion via protonation of the surrounding hydroxyl groups to evolve two water molecules. For ammoniojarosite, this step corresponds to the protonation of a hydroxyl group by ammonium, so that ammonia and water are liberated simultaneously. Iron(II) sulfate was identified as a possible intermediate during the decomposition of ammoniojarosite (421–521 °C) due to a redox reaction between iron(III) and the liberated ammonia during decomposition. Iron(II) ions were also confirmed with the 1,10-phenanthroline test. Iron(III) sulfate and other commonly suggested intermediates for hydronium and ammoniojarosite decomposition are not major crystalline phases; if they are formed, then they most likely exist as an amorphous phase or a different low temperature phases than usual.     

Transparent inorganic/organic copolymers by the sol-gel process: Thermal behavior of copolymers of tetraethyl orthosilicate (TEOS), vinyl triethoxysilane (VTES) and (meth)acrylate monomers

Three types of inorganic/organic copolymers have been prepared in a one-step sol-gel process, and their thermal stabilities have been studied. The one-step sol gel process was carried out in mixtures of three monomeric components: HEMA (hydroxyethyl methacrylate)-VTES (vinyl triethoxysilane)-TEOS (tetraethyl orthsilicate), HDDA (hexanediol diacrylate)-VTES-TEOS and GPTA (glycerol propoxy triacrylate)-VTES-TEOS. Copolymers with HEMA, which is able to form a linear organic polymer, were the least thermally stable materials. They lost the highest proportion of weight during heat treatment to 600°C and exhibited the lowest decomposition temperatures. Copolymers with HDDA and GPTA, which are able to form crosslinked organic polymers, had higher decomposition temperatures, and their weight loss during heat treatment to 600°C was small. The skeletal densities of all copolymers increased slightly during heat treatment.     

异质富勒烯C76BN的UV和IR光谱研究

Twenty-tow possible isomers for C76BN were studied by INDO methods. The two most stable geometries are 52,53-C76BN and 29,28-C76BN, in which boron and nitrogen atoms are connected with each other and located at the 6/6 bond near the longest axis of C78(C2v). Electronic spectra of C76BN were investigated with INDO/SCI method. UV absorptions of C76BN are red-shifted compared with those of C78(C2v). The structures and IR spectra for the four stable isomers of C76BN were calculated by AM1 method. It was indicated that the substitution of the BN unit weakens the conjugation of carbon atoms, leading to the decrease of IR frequencies.     

Determination of oxytetracycline in salmon by liquid chromatography with metal-chelate fluorescence detection

A liquid chromatography (LC) method is described for the determination of oxytetracycline (OTC) in farmed Atlantic salmon muscle tissue. The method involves homogenization of salmon tissue, extraction of OTC into Mcllvaine-EDTA buffer, acid precipitation of proteins, cleanup through tandem solid-phase extraction cartridges (Strata-X and aminopropyl), elution with mobile phase containing slightly alkaline buffer and Mg2+, and LC separation with metal-chelate induced fluorescence detection. Salmon tissue was fortified with 0.10, 0.25, 0.50, 0.75, and 1.0 microg/g (ppm) oxytetracycline. Average absolute recoveries were 84, 76, 70, 76, and 85%, respectively, with relative standard deviation (RSD) values all less than 9%. The interassay average recovery was 78%, with a 4.2% RSD. Determination was based on a standard graph using peak areas with standard solutions equivalent to 0.0625, 0.125, 0.25, 0.50, and 1.0 ppm in tissue. A set of 5 matrix controls (unfortified salmon tissue) were also analyzed, in which no OTC was detected. The lowest standard was used as the limit of quantitation.     

Electron transfer collisions between isolated fullerene dianions and SF6

Electron transfer collisions of trapped doubly charged fullerene anions C76(2-), C(78)2-, and C84(2-) with SF6 are studied in a Fourier transform ion cyclotron resonance mass spectrometer at center-of-mass collisional energies ranging from thermal energy to 77 eV. Collision energy dependencies manifest threshold energies for (nominally exoergic) single electron transfer onto SF6 of 1.46+/-0.3 eV, 1.56+/-0.3 eV, and 1.63+/-0.3 eV for C(76)2-, C78(2-), and C(84)2-, respectively. Kinetics studies reveal charge-transfer cross sections of up to 430+/-200 A2 for C84(2-) at a collision energy of 77 eV. The mechanism and the energetics are discussed in terms of classical electrostatic model calculations. Additionally, we rationalize the collision energy dependencies of the charge-transfer cross sections using the two-state Landau-Zener formalism to describe the associated resonant electron tunneling probability.     

Mass-spectrometric monitoring of the thermally induced decomposition of trimethylgallium,tris(<Emphasis Type="Italic">tert</Emphasis>-butyl)gallium,and triethylantimony at low pressure conditions

The thermal decomposition of trimethylgallium (GaMe(3)), tris(tert-butyl)gallium (Ga(t)Bu(3)) and triethylantimony (SbEt(3)) was investigated in a tubular hot-wall reactor coupled with a molecular-beam sampling mass spectrometer, and decomposition mechanisms were proposed. The obtained results confirm the predominance of the surface reactions and reveal that the radical decomposition path of Ga(t)Bu(3) and SbEt(3), responsible for the formation of butane and ethane respectively, is restricted to a narrow temperature range in contrast to the molecular route that is responsible for the formation of the corresponding alkenes. GaMe(3) decomposes above 480 degrees C, forming essentially methane and also ethane to a lesser extent, whereas Ga(t)Bu(3) decomposes starting 260 degrees C to form predominantly i-butane and i-butene as major species. The decomposition of SbEt(3) starts at 400 degrees C and forms n-butane, ethane, and ethene. The selectivity to n-butane increases with the thermolysis temperature. The resulting activation energies of the relevant decomposition paths show good agreement with those among them that have been measured before by temperature-programmed desorption techniques.     

Quinolactacins revisited: from lactams to imide and beyond

Chemical analysis of a solid phase fermentation of an Australian Penicillium citrinum strain has returned all known examples of a rare class of N-methyl quinolone lactams, quinolactacins A2 (1), B2 (2), C2 (3) and A1 (4), together with the new quinolactacins B1 (5), C1 (6), D1 (7) and D2 (8), and the novel derivatives quinolonimide (9) and quinolonic acid (10). Complete stereostructures were assigned to all these compounds by detailed spectroscopic analysis and chemical interconversion. Carefully controlled and monitored decomposition studies have confirmed that quinolactacins readily undergo C-3 epimerization and oxidation, and under appropriate conditions convert to quinolonimide and quinolonic acid. Mechanisms for key transformations are proposed. The decomposition studies suggested that only quinolactacins A2 (1) and B2 (2) are genuine natural products, with all other isolated compounds being decomposition artefacts. Quinolactacins C1 (6), C2 (3), and the racemic mixture of quinolactacins D1/D2 (8/7) all displayed notable cytotoxic activity.     

Low temperature rehydration of thermally dehydroxylated Bayer–gibbsite,evolution and transformation of phases

The hexahydrate of europium nitrate hexahydrate Eu(NO₃)₃·6H₂O shows no phase transitions in the range of ?40 to 76 °C when it melts in its own water of crystallization. It was shown that the thermal decomposition is a complex step-wise process, which starts with the simultaneous condensation of 6 mol of the initial monomer Eu(NO₃)₃·6H₂O into a cyclic cluster 6[Eu(NO₃)₃·6H₂O]. This hexamer gradually loses water and nitric acid, and a series of intermediate amorphous oxynitrates is formed. The removal of HNO₃ azeotrope is essentially a continuous process occurring in the liquid phase. At higher temperatures, oxynitrates undergo further degradation, lose water, nitrogen dioxide, and oxygen, and finally, after having lost lattice water, are transformed into europium oxide. All mass losses are satisfactorily accounted for under the proposed scheme of thermal decomposition.     

Thermal decomposition studies of the polyhedral oligomeric silsesquioxane, POSSh, and when it is impregnated with the metallocene bis(eta5-cyclopentadienyl)zirconium (IV) dichloride or immobilized on silica

Thermal decomposition studies of the free polyhedral oligomeric silsesquioxane, POSS(h), and when this compound has been impregnated with Cp(2)ZrCl(2) (Cp=eta(5)-C(5)H(5)) or immobilized on SiO(2) were conducted using infrared emission spectroscopy (IES) over a 100-1000 degrees C temperature range and by thermogravimetric analysis (TGA). The organic groups in POSS(h) apparently decompose thermally into Si-CH(3), Si-H and other fragments. Upon impregnation with Cp(2)ZrCl(2), however, a different thermal decomposition pathway was followed and new infrared emission bands appeared in the 1000-900cm(-1) region suggesting the formation of Si-O-Zr moieties. When immobilized on SiO(2) and subjected to thermal decomposition, the POSS(h) compound lost its organic groups and the inorganic structure remaining was incorporated into the SiO(2) framework.