Enhancing the Peroxygenase Activity of a Cofactor-Independent Peroxyzyme by Directed Evolution Enabling Gram-Scale Epoxide Synthesis

Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H₂O₂ with water being the sole by-product. These biocatalysts can provide ‘green’ routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4-oxalocrotonate tautomerase (4-OT), catalysing the epoxidation of a variety of α,β-unsaturated aldehydes with H₂O₂, is outstanding because of its independence from any cost-intensive cofactor. However, its low-level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,β-epoxy-aldehydes under preparative-scale conditions is limiting the potential of 4-OT. Herein we report the directed evolution of a tandem-fused 4-OT variant, which showed an ∼150-fold enhanced peroxygenase activity compared to 4-OT wild type, enabling the synthesis of α,β-epoxy-aldehydes in milligram- and gram-scale with high enantiopurity (up to 98 % ee) and excellent conversions. This engineered cofactor-independent peroxyzyme can provide new opportunities for the eco-friendly and practical synthesis of enantioenriched epoxides at large scale.     

Thermal behaviour of erythromycin-active substance and tablets

The application of thermal methods is of great importance in the solution of pharmaceutical problems, such as the control of raw materials, the determination of purity, the qualitative and quantitative analysis of drug formulation, tests of thermal stability and compatibility, the determination of kinetic parameters etc. The evaluation of thermal stability in the solid state is mostly made by analyzing their decomposition under isothermal and non-isothermal conditions. This study reports the study on the thermal behaviour of erythromycin-active substance and tablets, respectively, the determination of the kinetic parameters for the decomposition process under non-isothermal conditions. For the determination of kinetic parameters from the TG/DTG curves, were utilized the following methods: Friedman isoconversional, Flynn–Wall–Ozawa, Kissinger–Akahira–Sunose, Li–Tang, and Kissinger, respectively, a dynamic nitrogen atmosphere and different heating rates: 2.5, 5, 7.5, 10, and 15 °C min^?1. Thermoanalytical curves showed that the active substance is thermally more stable than the tablets and the values of activation energy indicate a considerable thermal stability of active substance. The decrease in stability was attributed to the presence of excipients.     

Kinetics and mechanism of the acid-catalysed hydrolysis of the carbonatotetraimidazolecobalt(III) ion

Summary The kinetics of the acid-catalysed hydrolysis of the [(imidazole)₄Co(CO₃)]⁺ ion was found to follow the rate law -dln[complex]/dt = k ₁ K[H⁺](1 + K[H ⁺]) in the 25–45 °C range, [H⁺] 0.05–1.0 m range and I = 1.0m. The reaction sequence consists of a rapid protonation equilibrium followed by the one-end dissociation of the coordinated carbonato ligand (rate-determining step) and subsequent fast release of the monodentate carbonato ligand. The rate parameter values, k ₁ and ITK, at 25 °C are 6.48 × 10⁻³s⁻¹ and 0.31m ⁻¹, respectively, and activation parameters for k ₁ are ΔH ₁ ^≠ = 86.1 ± 1.2kJ mol⁻¹ and ΔS ₁ ^≠ = 2.1 ± 6.3 J mol⁻¹K⁻¹. The hydrolysis rate increases with increase in ionic strength. The different ways of dealing with the data fit are presented and discussed. The kinetic results are compared with those for the similar cobalt(III) complexes.     

Determination of Cd,Pb, and Cu in the Atmospheric Aerosol of Central East Antarctica at Dome C (Concordia Station)

Trace heavy metals Cd, Pb, and Cu were determined (by square wave anodic stripping voltammetry) in aerosol samples collected at Dome C (the Italo-French Station Concordia), a remote site of the Central East Antarctic plateau, for which no data are available until now. During the Austral Summer 2005–2006, three PM10 high-volume impactors were installed in two locations nearby of Concordia station: the first one very close and downwind of the station (about 50 m north), the other two (very close to each other) in a ‘distant’ site, upwind of the station and close to the astrophysics tent (not used in that expedition) at ~800 m south of Station Concordia. For each sample, the availability of the mass of the aerosol collected (obtained by differential weighing carried out on site), in addition to the volume of the filtered air, allowed us to express results both in terms of metal mass fractions in the aerosol and in the usual way of metal atmospheric concentrations. Metal contents increased in the order Cd < Pb < Cu with the following ranges of values: Cd 1.0–8.4 µg g⁻¹ (0.09–3.1 pg m⁻³), Pb 96–470 µg g⁻¹ (12–62 pg m⁻³), and Cu 0.17–20 mg g⁻¹ (0.027–2.4 ng m⁻³). From the metal temporal profiles obtained we estimated the following background values for the area of Dome C, expressed both in mass fractions and in atmospheric concentrations: Cd 1.2 ± 0.2 µg g⁻¹ (0.24 ± 0.13 pg m⁻³), Pb (here fixed as upper limit) 113 ± 13 µg g⁻¹ (21 ± 8 pg m⁻³), and Cu 0.91 ± 0.48 mg g⁻¹ (0.12 ± 0.07 ng m⁻³). The highest values were observed in the first part of the season, and particularly for the site close to the station, possibly related to sample contamination linked to intense activity at the Concordia station connected with the beginning of the expedition, including aircraft arrivals/departures. Increments of up to 10 times (and even 20 times for Cu) were recorded with respect to the background values. The metal excesses of the contaminated over background samples were found approximately, except for Cu, in the same proportion of the metal contents of the special Antarctic blend (SAB) diesel fuel, which is used almost exclusively at Concordia Station. The effect of the wind direction was also observed. Thus in the intermediate period of the campaign, when the wind direction reversed for several days with respect to the prevailing one, Cd and Pb metal contents decreased at the sampling point installed close to the station, now upwind of Concordia station, and increased at the ‘clean’ site astrophysics tent, turned downwind at the main station. No simple and easily interpretable effect of the wind direction was observed for Cu, which suggests that some other extemporaneous and not clearly identified factor may have intervened in this case. These results suggest that the human impact at Dome C influences mainly the zone very close to the station, but also the area in the neighborhood, including the supposed clean site of the astrophysics tent (about 800 m far from the station), when the wind direction reverses with respect to the prevailing one, leaving the site downwind of the station Concordia. Since no other data are reported for the Dome C area, our results are compared with literature data referred to the South Pole Station (the only other plateau site for which data are available) and several other coastal Antarctic sites, observing that our results (excluding Cu) are the lowest ever observed for Antarctic aerosol.     

Photoredox‐Catalyzed Reductive Coupling of Aldehydes,Ketones, and Imines with Visible Light

Ketyl radical and amino radical anions, valuable reactive intermediates for C? C bond‐forming reactions, are accessible through a C?O/C?NR umpolung. However, their utilization in catalysis remains largely underdeveloped owing to the high reduction potential of carbonyl compounds and imines. In the context of photoredox catalysis, tertiary amines are commonly employed as sacrificial co‐reducing agents. Herein, an additional role of the amine is proposed, in which it is essential for the organocatalytic substrate activation. The combination of photoredox catalysis and carbonyl/imine activation enables the reductive coupling of aldehydes, ketones, and imines under mild reaction conditions.     

Synthesis, characterization, cytotoxic activity and crystal structures of tri- and di-organotin(IV) complexes constructed from the β-{[(E)-1-(2-hydroxyaryl)alkylidene]amino}propionate and β-{[(2Z)-(3-hydroxy-1-methyl-2-butenylidene)]amino}propionate skeletons

Reactions of potassium β-{[(E)-1-(2-hydroxyaryl)alkylidene]amino}propionates (L¹HK-L³HK) and potassium β-{[(2Z)-(3-hydroxy-1-methyl-2- butenylidene)]amino}propionate (L⁴HK) with R₃SnCl (R = Ph and ⁿBu) and ⁿBu₂SnCl₂ yielded complexes of composition Ph₃SnL¹H (1), Ph₃SnL²H (2), Ph₃SnL⁴H (3), ⁿBu₃SnL¹H (4), and {[ⁿBu₂Sn(L²H)]₂O}₂ (5) and {[ⁿBu₂Sn(L³H)]₂O}₂ (6), respectively. These complexes have been characterized by ¹H, ¹³C, ¹¹⁹Sn NMR, ESI-MS, IR and ^119mSn Mössbauer spectroscopic techniques in combination with elemental analyses. The crystal structures of 1, 4, 5 and 6 were determined. In the solid state, compound 1 is a one-dimensional polymer built from SnPh₃ moieties bridged by single carboxylate ligands, but two alternating modes of bridging are present along the polymeric chain. Compound 4 is also a one-dimensional polymer built from SnBu₃ moieties bridged by the two carboxylate O-atoms of a single ligand, but only one mode of bridging is present. Di-n-butyltin compounds 5 and 6 are centrosymmetric tetranuclear bis(dicarboxylatotetrabutyldistannoxane) complexes containing a planar Sn₄O₂ core in which two μ₃-oxo O-atoms connect an Sn₂O₂ ring to two exocyclic Sn-atoms. The four carboxylate ligands display two different modes of coordination where both modes involve bridging of two Sn-atoms. The solution structures were predicted by ¹¹⁹Sn NMR spectroscopy. The in vitro cytotoxic activity of compound 5 against WIDR, M19 MEL, A498, IGROV, H226, MCF7 and EVSA-T human tumor cell lines is reported.     

Optimising open porous foam for acoustical and vibrational performance

A computational method for designing optimal arrangements of multilayer noise and vibration treatments in general and porous open cell foam in particular is discussed. The method uses finite element solutions to Biot's equations for poroelastic materials and provides data to evaluate cost functions and gradients. The porous material is parameterised using scaling laws linking the microscopic properties to the classical parameters, i.e. averaged elasticity, flow resistivity and characteristic viscous and thermal lengths. The cost function is either in terms of weight or in terms of the pressure response in a finite cavity, complemented with constraints on the other. However, care must be taken when choosing the cost function, as this will greatly affect the outcome of the optimisation. Observations made during the optimisation process indicate a limited number of minima within the parameter range of interest as well as beneficial continuity around these minima, thus enabling a meaningful optimisation. The results suggest that if alterations of the microscopic properties of the foam are made, the foam may be adapted to specific environmental conditions and thereby achieve improved acoustic behaviour as well as reduced weight.     

Ergodic properties of infinite-dimensional stochastic systems

The ergodic properties of two stochastic models _I and _II are investigated. Each model is described by a fieldx(t),t > 0, on the lattice =Z ^d,d < . For _I,x(t) evolves according to the equations wherex _s (t) R for eachs eF. Here the {w_s(t): s } are independent, one-dimensional Wiener processes, ₂ is a bounded interaction between adjacent lattice sites, and the potentials ₁ and ₂ satisfy appropriate regularity conditions. It is shown that for each model,x(t) is a Markov process on an infinite-dimensional phase spaceX. The probability measures onX that satisfy the Dobrushin-Lanford-Ruelle (DLR) conditions are stationary for this process and have a mixing property. Moreover, for _I any stationary, time-reversal-invariant probability measure that has certain regularity properties must satisfy the DLR conditions.This paper is based on a portion of the author's Ph.D. thesis.⁽²⁾     

In-source fragmentation and analysis of polysaccharides by capillary electrophoresis/mass spectrometry

In order to develop a robust and easy-to-use technique for characterization of bacterial polysaccharides, a pseudo-hydrolysis strategy was investigated. Based on in-source collision-induced dissociation, polysaccharide molecular ions were fragmented within the orifice-skimmer region of an electrospray ionization (ESI) mass spectrometer. The fragment ions thus generated were then analyzed similarly to the conventional ESI mass spectrometry approach. MS/MS scanning was applied to obtain product-ion spectra of the primary fragments for sequencing. To further improve the sensitivity and separation of polysaccharides from other components in the samples, a pressure-assisted capillary electrophoresis/mass spectrometry (CE/MS) system was employed. Using bacterial polysaccharides as model compounds, the mass spectra obtained for polysaccharide repeating units generated through chemical hydrolysis and in-source fragmentation were directly compared, both in positive and negative ion modes. With the additional separation of impurities provided by CE, the success of this technique has been demonstrated for structural analysis of O-chain polysaccharides (O-PS) and capsular polysaccharides (CPS). In-source fragmentation was applied to promote the formation of structurally relevant repeating units of heterogeneous CPS that would remain undetected using conventional ESI conditions. This approach was proven to be particularly useful for probing the subtle structural differences in monosaccharide composition and functionalities arising across bacterial serotypes.