RESUMEN
Despite the ever-growing research interest in polyhydroxyalkanoates (PHAs) as green plastic alternatives, our understanding of the regulatory mechanisms governing PHA synthesis, storage, and degradation in the model organism Ralstonia eutropha remains limited. Given its importance for central carbon metabolism, PHA homeostasis is probably controlled by a complex network of transcriptional regulators. Understanding this fine-tuning is the key for developing improved PHA production strains thereby boosting the application of PHAs. We conducted promoter pull-down assays with crude protein extracts from R. eutropha Re2058/pCB113, followed by liquid chromatography with tandem mass spectrometry, to identify putative transcriptional regulators involved in the expression control of PHA metabolism, specifically targeting phasin phaP1 and depolymerase phaZ3 and phaZ5 genes. The impact on promoter activity was studied in vivo using ß-galactosidase assays and the most promising candidates were heterologously produced in Escherichia coli, and their interaction with the promoters investigated in vitro by electrophoretic mobility shift assays. We could show that R. eutropha DNA-binding xenobiotic response element-family-like protein H16_B1672, specifically binds the phaP1 promoter in vitro with a KD of 175 nM and represses gene expression from this promoter in vivo. Protein H16_B1672 also showed interaction with both depolymerase promoters in vivo and in vitro suggesting a broader role in the regulation of PHA metabolism. Furthermore, in vivo assays revealed that the H-NS-like DNA-binding protein H16_B0227 and the peptidyl-prolyl cis-trans isomerase PpiB, strongly repress gene expression from PphaP1 and PphaZ3, respectively. In summary, this study provides new insights into the regulation of PHA metabolism in R. eutropha, uncovering specific interactions of novel transcriptional regulators.
RESUMEN
Terminal oxygen radicals involving p- and d-block atoms are quite common, but s-block compounds with an oxygen radical character remain rare. Here, we report that alkaline-earth metal beryllium atoms react with OF2 to form the oxygen beryllium fluorides OBeF and OBeF2. These species are characterized by matrix-isolation infrared spectroscopy with isotopic substitution and quantum-chemical calculations. The linear molecule OBeF has a 2Π ground state with an oxyl radical character. The 3A2 (C2v) ground state of OBeF2 represents the unusual case of a triplet oxygen atom stabilized by a relatively weak interaction by the Lewis acidic BeF2. The interaction involves both a donor component from oxygen to empty Be orbitals and a back-bonding contribution from fluorine substituents toward oxygen.
RESUMEN
The trifluorosilylarsinidene F3SiAs in the triplet ground state has been generated through the reaction of laser-ablated silicon atoms with AsF3 in cryogenic Ne- and Ar-matrices. The reactions proceed with the initial formation of perfluorinated arsasilene FAsSiF2 in the singlet ground state by two As-F bonds insertion reaction on annealing. The trifluorosilylarsinidene F3SiAs was formed via F-migration reactions of FAsSiF2 under irradiation at UV light (λ = 275â nm). The characterization of FAsSiF2 and F3SiAs by IR matrix-isolation spectroscopy is supported by computations at CCSD(T)-F12/aug-cc-pVTZ and B3LYP/aug-cc-pVTZ levels of theory.
RESUMEN
The reaction of laser-ablated boron atoms with hexafluorobenzene (C6 F6 ) was investigated in neon and argon matrices, and the products are identified by matrix isolation infrared spectroscopy and quantum-chemical calculations. The reaction is triggered by a boron atom insertion into one C-F bond of hexafluorobenzene on annealing, forming a fluoropentafluorophenyl boryl radical (A). UV-Vis light irradiation of fluoropentafluorophenyl boryl radical causes generation of a 2-difluoroboryl-tetrafluorophenyl radical (B) via a second C-F bond activation. A perfluoroborepinyl radical (C) is also observed upon deposition and under UV-Vis light irradiation. This finding reveals the new example of a dual C-F bond activation of hexafluorobenzene mediated by a nonmetal and provides a possible route for synthesis of new perfluorinated organo-boron compounds.
RESUMEN
The molecular IrF5 -, IrF6 - anions and M[IrF6] (M=Na, K, Rb, Cs) ion pairs were prepared by co-deposition of laser-ablated alkali metal fluorides MF with IrF6 and isolated in solid neon or argon matrices under cryogenic conditions. The free anions were obtained as well by co-deposition of IrF6 with laser-ablated metals (Ir or Pt) as electron sources. The products were characterized in a combined analysis of matrix IR spectroscopy and electronic structure calculations using two-component quasi-relativistic DFT methods accounting for spin-orbit coupling (SOC) effects as well as multi-reference configuration-interaction (MRCI) approaches with SOC. Inclusion of SOC is crucial in the prediction of spectra and properties of IrF6 - and its alkali-metal ion pairs. The observed IR bands and the computations show that the IrF6 - anion adopts an Oh structure in a nondegenerate ground state stabilized by SOC effects, and not a distorted D4h structure in a triplet ground state as suggested by scalar-relativistic calculations. The corresponding "closed-shell" M[IrF6] ion pairs with C3v symmetry are stabilized by coordination of an alkali metal ion to three F atoms, and their structural change in the series from M=Na to Cs was proven spectroscopically. There is no evidence for the formation of IrF7, IrF7 - or M[IrF7] (M=Na, K, Rb, Cs) ion pairs in our experiments.
RESUMEN
Lewis acidic boron compounds are ubiquitous in chemistry due to their numerous applications, yet tuning and optimizing their properties towards different purposes is still a challenging field of research. In this work, the boron-based Lewis acid B[OTeF3(C6F5)2]3 was synthesized by reaction of the teflate derivative HOTeF3(C6F5)2 with BCl3 or BCl3 â SMe2. This new compound presents a remarkably high thermal stability up to 300 °C, as well as one of the most sterically encumbered boron centres known in the literature. Theoretical and experimental methods revealed that B[OTeF3(C6F5)2]3 exhibits a comparable Lewis acidity to that of the well-known B(C6F5)3. The affinity of B[OTeF3(C6F5)2]3 towards pyridine was accessed by Isothermal Titration Calorimetry (ITC) and compared to that of B(OTeF5)3 and B(C6F5)3. The ligand-transfer reactivity of this new boron compound towards different fluorides was demonstrated by the formation of an anionic Au(III) complex and a hypervalent iodine(III) species.
RESUMEN
We report on an unexpected reaction between ammonia and potassium ozonide dissolved in liq. NH3 resulting in the formation of peroxynitrite, [ONOO]-, which exclusively happens in the presence of a specific partially fluorinated aniline-based ammonium cation. High-resolution structural data of the peroxynitrite anion in cis-conformation have been obtained. We further studied this molecule anion by single crystal Raman spectroscopy. The cis and trans isomers of peroxynitrite were analysed computationally with respect to their relative energies, the cis-trans transition barrier and their decomposition pathways to the nitrate anion. By experimentally examining cations decorated with fluorine ligands to different degrees, we demonstrate that fluorine specific interactions play a crucial role in the unexpected formation of peroxynitrite and as a conspicuously structure directing factor for the underlying crystalline solid phases, exhibiting distinct micro-separations of fluorine and hydrogen enriched regions.
RESUMEN
In this work, we analyzed trifluoromethyl fluorosulfonate (CF3OSO2F) and trifluoromethoxy sulfur pentafluoride (CF3OSF5) regarding their potential use as dielectrics by investigating some of their intrinsic and extrinsic properties. Both compounds show a higher breakdown voltage than SF6 with averaged relative breakdown voltages of 1.3±0.2 for CF3OSO2F and 1.4±0.2 for CF3OSF5 compared to SF6 with 1.0. Like the dielectric (CF3)2CFCN, both compounds decompose during the breakdown process. The decomposition products were analyzed by IR spectroscopy and GCIR methods. Furthermore, the molecular structures of both gaseous compounds CF3OSO2F and CF3OSF5 have been determined by inâ situ crystallization, and their physical properties were determined as well.
RESUMEN
Herein we report a general access to silver(i) perfluoroalcoholates, their structure in the solid state and in solution, and their use as transfer reagents. The silver(i) perfluoroalcoholates are prepared by the reaction of AgF with the corresponding perfluorinated carbonyl compounds in acetonitrile and are stable for a prolonged time at -18 °C. X-ray analysis of single crystals of perfluoroalcoholate species showed that two Ag(i) centers are bridged by the alcoholate ligands. In acetonitrile solution, Ag[OCF3] forms different structures as indicated by IR spectroscopy. Furthermore, the silver(i) perfluoroalcoholates can be used as easy-to-handle transfer reagents for the synthesis of Cu[OCF3], Cu[OC2F5], [PPh4][Au(CF3)3(OCF3)], and fluorinated alkyl ethers.
RESUMEN
The perfluorinated silylphosphinidene, F3SiP, in the triplet ground state is generated by the reaction of laser-ablated silicon atoms with PF3 in solid neon and argon matrices. The reactions proceed with the initial formation of a silicon trifluorophosphine complex, F3PSi, in the triplet ground state, and a more stable inserted phosphasilene, FPSiF2, in the singlet ground state upon deposition. The trifluorosilylphosphinidene was formed through F-migration reactions of FPSiF2 and F3PSi following a two-state mechanism under irradiation with visible light (λ = 470 nm) and full arc light (λ > 220 nm), respectively. High-level quantum-chemical methods support the identification of F3PSi, FPSiF2, and F3SiP by matrix-isolation IR spectroscopy.
RESUMEN
Radical trifluoromethoxylation is an attractive approach to prepare compounds featuring the important OCF3 group, however most existing methods have focused on aromatic substrates. Here, we report novel methodologies with alkenyl substrates employing bis(trifluoromethyl)peroxide (BTMP) as a practical and comparatively atom economical trifluoromethoxylating reagent. With silyl enol ether substrates, switching reaction solvent allows for the synthesis of either α-(trifluoromethoxy)ketone products or unprecedented alkenyl-OCF3 species. Furthermore, allyl silanes have been employed as substrates for the first time, affording allyl(trifluoromethyl)ether products in good yields. In each case, the methods operate at room temperature without large excesses of the alkene substrate while, in contrast to previous radical trifluoromethoxylation reactions, no catalyst, light or other activators are required.
RESUMEN
A new concept for the synthesis of dialkyl chloronium cations [RâClâR]+ is described (R = CH3, CH2CF3), that allows the formation of fluorinated derivatives. By utilizing the xenonium salt [XeOTeF5][M(OTeF5)n] (M = Sb, n = 6; M = Al, n = 4) chlorine atoms of chloroalkanes or the deactivated chlorofluoroalkane CH2ClCF3 are oxidized and removed as ClOTeF5 leading to the isolation of the corresponding chloronium salt. Since the resulting highly electrophilic cation [Cl(CH2CF3)2]+ is able to alkylate weak nucleophiles, this compound can be utilized for the introduction of a fluorinated alkyl group to those. In addition, the fluorinated alkyl chloronium cation displays a high hydride ion affinity, enabling the activation of linear hydrocarbons by hydride abstraction even at low temperatures ultimately leading to the formation of branched carbocations.
RESUMEN
Mercuric oxide is a well-known and stable solid, but the diatomic molecule Hg-O is very fragile and does not survive detection in the gas phase. However, laser ablation of Hg atoms from a dental amalgam alloy target into argon or neon containing about 0.3 % of 16 O2 or of 18 O2 during their condensation into a cryogenic matrix at 4â K allows the formation of O atoms which react on annealing to make ozone and new IR absorptions in solid argon at 521.2â cm-1 for Hg-16 O or at 496.4â cm-1 for Hg-18 O with the oxygen isotopic frequency ratio 521.2/496.4=1.0499. Solid neon gives a 529.0â cm-1 absorption with a small 7.8â cm-1 blue shift. CCSD(T) calculations found 594â cm-1 for Hg16 O and 562â cm-1 for Hg18 O (frequency ratio=1.0569). Such calculations usually produce harmonic frequencies that are slightly higher than the anharmonic (observed) values, which supports their relationship. These observed frequencies have the isotopic shift predicted for Hg-O and are within the range of recent high-level frequency calculations for the Hg-O molecule. Spectra for the related mercury superoxide and ozonide species are also considered for the first time.
RESUMEN
Group 13 oxyfluorides OMF2 were produced by the reactions of laser-ablated group 13 atoms M (M=B, Al, Ga and In) with OF2 and isolated in excess neon or argon matrices at 5â K. These molecules were characterized by matrix-isolation infrared spectroscopy and isotopic substitution experiments in conjunction with quantum-chemical calculations. The calculations indicate that the OMF2 molecules have a 2 B2 ground state with C2v symmetry. The computed molecular orbitals and spin densities show that the unpaired electron is mainly located at the terminal oxygen atom. Oxo monofluorides OMF were only observed in solid argon matrices and exhibit a linear structure in the singlet ground state. The M-O bonding in the OMF molecules can be rationalized as highly polar multiple bonds based on the calculated bond lengths and natural resonance theory (NRT) analyses. In particular, the molecular orbitals of OBF exhibit the character of a triple bond B-O resulting from two degenerate electron-sharing π bonds and an OâB dative σ bond formed by the oxygen 2p lone pair which donates electron density to the boron empty 2p orbital.
RESUMEN
A new synthetic access to the Lewis acid [Au(OTeF5 )3 ] and the preparation of the related, unprecedented anion [Au(OTeF5 )4 ]- with inorganic or organic cations starting from commercially available and easy-to-handle gold chlorides are presented. In this first extensive study of the Lewis acidity of a transition-metal teflate complex by using different experimental and quantum chemical methods, [Au(OTeF5 )3 ] was classified as a Lewis superacid. The solid-state structure of the triphenylphosphine oxide adduct [Au(OPPh3 )(OTeF5 )3 ] was determined, representing the first structural characterization of an adduct of this highly reactive [Au(OTeF5 )3 ]. Therein, the coordination environment around the gold center slightly deviates from the typical square planar geometry. The [Au(OTeF5 )4 ]- anion shows a similar coordination motif.
RESUMEN
We report on a comprehensive reactivity study starting from [AuF3 (SIMes)] to synthesize different motifs of monomeric gold(III) fluorides. A plethora of different ligands has been introduced in a mono-substitution yielding trans-[AuF2 X(SIMes)] including alkynido, cyanido, azido, and a set of perfluoroalkoxido complexes. The latter were better accomplished via use of perfluorinated carbonyl-bearing molecules, which is unprecedented in gold chemistry. In case of the cyanide and azide, triple substitution gave rise to the corresponding [AuX3 (SIMes)] complexes. Comparison of the chemical shift of the carbene carbon atom in the 13 C{1 H}â NMR spectrum, the calculated SIMes affinity and the Au-C bond length in the solid state with related literature-known complexes yields a classification of trans-influences for a variety of ligands attached to the gold center. Therein, the mixed fluorido perfluoroalkoxido complexes have a similar SIMes affinity to AuF3 with a very low Gibbs energy of formation when using the perfluoro carbonyl route.
RESUMEN
The tetraoxido ruthenium(VIII) radical cation, [RuO4 ]+ , should be a strong oxidizing agent, but has been difficult to produce and investigate so far. In our X-ray absorption spectroscopy study, in combination with quantum-chemical calculations, we show that [RuO4 ]+ , produced via oxidation of ruthenium cations by ozone in the gas phase, forms the oxygen-centered radical ground state. The oxygen-centered radical character of [RuO4 ]+ is identified by the chemical shift at the ruthenium M3 edge, indicative of ruthenium(VIII), and by the presence of a characteristic low-energy transition at the oxygen K edge, involving an oxygen-centered singly-occupied molecular orbital, which is suppressed when the oxygen-centered radical is quenched by hydrogenation of [RuO4 ]+ to the closed-shell [RuO4 H]+ ion. Hydrogen-atom abstraction from methane is calculated to be only slightly less exothermic for [RuO4 ]+ than for [OsO4 ]+ .
RESUMEN
Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5'-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1-90.1 g L-1 were observed together with an overproduction of ApMTAP in a 1.9%-3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg-1 (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.
RESUMEN
An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µs ' with CDW was observed in the range of 5-69 g L-1 (R2 = 0.98). The growth rates calculated based on µs ' were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µs ' as a function of the absorption coefficient µa allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.
Asunto(s)
Reactores Biológicos , Escherichia coli , Biomasa , Análisis Espectral , Fenómenos QuímicosRESUMEN
The pentafluoroorthotellurate group (teflate, OTeF5) is considered as a bulky analogue of fluoride, yet its coordination behavior in transition metal complexes is not fully understood. By reaction of [CoCl4]2- and neat ClOTeF5, we synthesized the first cobalt teflate complex, [Co(OTeF5)4]2-, which exhibits moisture-resistant Co-OTeF5 bonds. Through a combined experimental and theoretical (DFT and NEVPT2) study, the properties and electronic structure of this species have been investigated. It exhibits a distorted tetrahedral structure around the cobalt center and can be described as a d7 system with a quartet (S = 3/2) ground state. A comparative bonding analysis of the (pseudo)tetrahedral [CoX4]2- anions (X = OTeF5, F, Cl) revealed that the strength of the Co-X interaction is similar in the three cases, being the strongest in [Co(OTeF5)4]2-. In addition, an analysis of the charge of the Co center reinforced the similar electron-withdrawing properties of the teflate and fluoride ligands. Therefore, the [Co(OTeF5)4]2- anion constitutes an analogue of the polymeric [CoF4]2- in terms of electronic properties, but with a monomeric structure.