Recombinant cytochrome p450 immobilization for biosensor applications

As a result of the very attractive pleiotropic properties of the heme-enzymes, three P450 cytochrome isoforms (P4501A2, P4502B4, P450SCC) have been utilized to identify a general optimal procedure to biodevice assembly for sensing a wide range of organic substances. The Langmuir-Blodgett films appears to yield the best stable working conditions as shown by UV-vis spectrophotometry, nanogravimetry, circular dichroism, and electrochemical characterization, to identify the ordered nanostructures of P450 cytochromes optimal for clozapine, styrene, and cholesterol sensing. Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine sensing. By the combination of proper immobilization, transducer and nanostructured mutants of high-grade stable and selective P450-based sensors appear capable to detect the interaction with a wide range of organic substrates such as fatty acids, drugs, and toxic compounds.     

Incorporation of fullerene derivatives into smectite clays: a new family of organic-inorganic nanocomposites

Three fulleropyrrolidine derivatives, characterized by the presence of positive charges, were introduced in the interlayer space of montmorillonite. The composites were characterized by powder X-ray diffraction and differential thermal and thermogravimetric (DTA-TGA) analysis, in conjunction with FTIR, UV-Vis, Raman, and (57)Fe-M?ssbauer spectroscopies. Organophilic derivatives were intercalated into organically modified clays, while water-soluble fulleropyrrolidines were introduced into the clay galleries through ion exchange. The experiments, complemented by computer simulations, show that not all the clay-clay platelets are intercalated by the fullerene derivatives and that a sizable amount of charge transfer takes place between the host and the guests.     

Efficient and general synthesis of 5-(alkoxycarbonyl)methylene-3-oxazolines by palladium-catalyzed oxidative carbonylation of prop-2-ynylamides

A variety of prop-2-ynylamides have been carbonylated under oxidative conditions to give oxazolines, oxazolines with chelating groups, and bisoxazolines bearing an (alkoxycarbonyl)methylene chain at the 5 position in good yields. The cyclization-alkoxycarbonylation process was carried out in alcoholic media at 50-70 degrees C and under 24 bar pressure of 3:1 carbon monoxide/air in the presence of catalytic amounts of 10% Pd/C or PdI2 in conjunction with KI. Cyclization occurred by anti attack of an oxygen function on the palladium-coordinated triple bond, followed by stereospecific alkoxycarbonylation, strictly resulting in E-stereochemistry. The structures of representative oxazolines and bisoxazolines have been determined by X-ray diffraction analysis.     

Efficient synthesis of ureas by direct palladium-catalyzed oxidative carbonylation of amines

A general synthesis of symmetrically disubstituted ureas and trisubstituted ureas by direct Pd-catalyzed oxidative carbonylation of primary amines or of a mixture of a primary and a secondary amine, respectively, with unprecedented catalytic efficiencies for this kind of process, is reported. Reactions are carried out at 90-100 degrees C in DME as the solvent in the presence of PdI(2) in conjunction with an excess of KI as the catalytic system and under 20 atm of a 4:1 mixture of CO and air. In some cases, working in the presence of an excess of CO(2) (40 atm) in addition to CO and air (60 atm total) had a beneficial effect on substrate reactivity and product yield. Cyclic five-membered and six-membered ureas were easily formed from primary diamines. The methodology has been successfully applied to the synthesis of pharmacologically active ureas, such as those deriving from alpha-amino esters or urea NPY5RA-972, a potent antagonist of the neuropeptide Y5 receptor.     

The effect of mechanical interlocking on crystal packing: predictions and testing

The first statistical analyses of the X-ray crystal structures of mechanically interlocked molecular architectures, the first molecular mechanics-based solid-state calculations on such structures and atomic force microscopy (AFM) experiments are used in combination to predict and test which types of benzylic amide macrocycle-containing rotaxanes possess mobile components in the crystalline phase and thus could form the basis of solid-state devices that function through mechanical motion at the molecular level. The statistical studies and calculations show that crystals formed by rotaxanes possess similarities and unanticipated differences with respect to the crystal packing of noninterlocked molecules. Trends in the rotaxane series correlate quantities related to crystal packing, molecular size, stoichiometry, and H-bonding. In accordance with the findings of Gavezzotti et al. for conventional molecular architectures, a principal component analysis (PCA) showed that three vectors related to the size, packing parameters, and stoichiometry are sufficient to describe the crystal properties of benzylic amide macrocycle-containing rotaxanes. When hydrogen bond-related quantities are included in a second PCA, they combine with the size and the stoichiometry vectors but not with packing-related parameters, indicating that the intramolecular "saturation" of the H-bonds (between the interlocked components) takes precedence over crystal assembly (i.e., intermolecular packing) in these systems. However, cluster analyses also suggest a major role for the energy of interaction between the macrocycle and its crystal environment. The identification of such a "privileged" interaction is of fundamental importance to the development of rotaxanes with in-crystal mobility of one or more of their interlocked components, a prerequisite for the exploitation of molecular level mechanical motion in the solid state. The set of trends found, together with the calculated energies, was used to propose guidelines for which benzylic amide macrocycle-containing rotaxanes are best suited to become building blocks for systems with mobile submolecular units in the crystalline phase. An experimental test of the predictive power of such guidelines was carried out using AFM on a rotaxane and its thread, identified by the study as a promising candidate for solid-state mobility. Intuitively, the rotaxane should be less mobile in the solid state since it has multiple sets of both hydrogen bond donors and acceptors that can form strong inter- and intramolecular H-bonds. Conversely, the thread has no hydrogen bond donors and cannot form such bonds. The AFM experiments, however, confirm the statistical analysis prediction that the rotaxane is considerably more mobile in the solid than the thread.     

Synthesis,characterization, and ecotoxicity of CeO<Subscript>2</Subscript> nanoparticles with differing properties

CeO₂ nanoparticles with various characteristics find an increasing number of applications in the electronic, medical, and other industries and are therefore likely released in the environment. This calls for investigations linking the physicochemical properties of these particles with their potential environmental impacts. In this study, CeO₂ nanoparticle powders were prepared using three different precursors [Ce(NO₃)₃, CeCl₃, and Ce(CH₃COO)₃] and annealing temperatures (300, 500, and 700 °C). This procedure resulted in nine different types of nanoparticles with differing size (5–90 nm), morphology, surface Ce³⁺/Ce⁴⁺ ratio, and slightly different crystal structures as characterized using transmission electron microscopy, dynamic light scattering, X-ray photoelectron spectroscopy, and X-ray diffraction measurements with Rietveld refinement. These CeO₂ nanoparticles underwent toxicity testing at concentrations up to 64 mg L^?1 using Daphnia magna. Toxic effects were observed for three particle types with EC50 values between 5 and 64 mg L^?1. No clear correlation was observed between the physicochemical properties (size, shape, oxygen occupancy, Ce³⁺/Ce⁴⁺ ratio) of the nanoparticles and their toxicity. However, toxicity was correlated with the amount of Ce remaining suspended in the test medium after 24 h. This indicated that toxic effects may depend on the colloidal stability of CeO₂ nanoparticles during the first day of exposure. Therefore, being readily suspended and remaining stable for several days in the aquatic media increases the likelihood that CeO₂ nanoparticles will cause unwanted adverse effects.     

Simulating the proton transfer in gramicidin A by a sequential dynamical Monte Carlo method

The large interest in long-range proton transfer in biomolecules is triggered by its importance for many biochemical processes such as biological energy transduction and drug detoxification. Since long-range proton transfer occurs on a microsecond time scale, simulating this process on a molecular level is still a challenging task and not possible with standard simulation methods. In general, the dynamics of a reactive system can be described by a master equation. A natural way to describe long-range charge transfer in biomolecules is to decompose the process into elementary steps which are transitions between microstates. Each microstate has a defined protonation pattern. Although such a master equation can in principle be solved analytically, it is often too demanding to solve this equation because of the large number of microstates. In this paper, we describe a new method which solves the master equation by a sequential dynamical Monte Carlo algorithm. Starting from one microstate, the evolution of the system is simulated as a stochastic process. The energetic parameters required for these simulations are determined by continuum electrostatic calculations. We apply this method to simulate the proton transfer through gramicidin A, a transmembrane proton channel, in dependence on the applied membrane potential and the pH value of the solution. As elementary steps in our reaction, we consider proton uptake and release, proton transfer along a hydrogen bond, and rotations of water molecules that constitute a proton wire through the channel. A simulation of 8 mus length took about 5 min on an Intel Pentium 4 CPU with 3.2 GHz. We obtained good agreement with experimental data for the proton flux through gramicidin A over a wide range of pH values and membrane potentials. We find that proton desolvation as well as water rotations are equally important for the proton transfer through gramicidin A at physiological membrane potentials. Our method allows to simulate long-range charge transfer in biological systems at time scales, which are not accessible by other methods.     

Favorable entropy of aromatic clusters in thermophilic proteins

Aromatic clusters, found with increased frequency in a set of thermophilic proteins, confer an entropic advantage over mesophilic analogues through introduction of large-amplitude oscillations; this is a source of improved free energy at high temperatures.     

New NH2H4 far-infrared laser lines and their frequencies

We report 25 new far-infrared laser lines and 26 heterodyne frequency measurements in hydrazine. The frequencies range from 1.0 to 5.5 THz with most of the frequencies between 2.5 and 4.0 THz. The lines were generated in a high frequency, far-infrared Fabry-Perot laser cavity pumped by a CO₂ laser. The cavity has a high Q for wavelengths below 150 µm and uses variable coupling to optimize the power for each line.This work has been financed by the Brazilian Government - Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and by the United States Government - NASA grant W-18, 623.Contribution of NIST, not subject to U.S. copyright.