Iterative in situ click chemistry assembles a branched capture agent and allosteric inhibitor for Akt1

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties.     

Multivariate curve resolution modeling of liquid chromatography-mass spectrometry data in a comparative study of the different endogenous metabolites behavior in two tomato cultivars treated with carbofuran pesticide

A metabonomic study based on the application of multivariate curve resolution and alternating least squares (MCR-ALS) to three-way data sets obtained by liquid chromatography coupled to mass spectrometry detection (LC-MS) was carried out for Rambo and Raf tomato cultivars treated with carbofuran pesticide. Samples were picked up during a 21 days period after treatment and analyzed by LC-MS in scan mode, along with the corresponding blank samples. Then, MCR-ALS was applied to the three-way data sets using column wise augmented matrices, and the evolutionary profiles as a function of the time after treatment were estimated for the metabolites present in both cultivars, as well as their corresponding pure spectra estimations. A comparative study using those estimations showed that some of these metabolites followed different behavior for the different cultivars after treatment. Since all treated and untreated Rambo and Raf samples were picked up according to the same sampling protocol and in a similar state of maturation, any difference in the behavior between profiles can be interpreted as an effect due to the presence of pesticide and to the kind of cultivar. Based on this hypothesis, several PLS-DA approaches were tested to check if it would be possible to classify samples by using the metabolites MCR estimations. Results showed that PLS-DA models for classification of treated or non-treated (blank) samples were the best ones obtained (98.44% of correct classifications for the validation set), which supports the stress effects related to carbofuran treatment. In addition, excellent discrimination among the four groups could be attained (89.06% of correct classifications for the validation set).     

Borane-Tetrahydrofuran as a Useful Reagent in the N-Monoalkylation of Amines and Aminoalcohols by Carbonyl Compounds

A new procedure for the high-yield N-monoalkylation of primary aromatic and aliphatic amines is described.     

Proton NMR relaxation study of molecular dynamics of chromonic liquid crystal Edicol Sunset Yellow

Proton nuclear magnetic resonance (¹H NMR) relaxometry, over about five decades in Larmor frequency, and pulsed field gradient NMR were used to study the molecular dynamics in the chromonic nematic and isotropic phases of stacked molecules of the binary mixture composed by Edicol Sunset Yellow (ESY) and deuterated water. Our results evidence that in both phases collective motions are responsible for the spin-lattice relaxation dispersion in the Larmor frequency range below 1 MHz. In the nematic phase, the collective motion are attributed to columnar undulations within the stacked molecules, while, in the isotropic phase, the results are explained by local order fluctuations due to the formation of the stacks. The high frequency dispersion was explained by individual molecular motions like rotations around and perpendicular to the stack axis, and also self-diffusion.     

Nonequilibrium molecular dynamics of the rheological and structural properties of linear and branched molecules. Simple shear and poiseuille flows; instabilities and slip

Nonequilibrium molecular-dynamics simulations are performed for linear and branched chain molecules to study their rheological and structural properties under simple shear and Poiseuille flows. Molecules are described by a spring-monomer model with a given intermolecular potential. The equations of motion are solved for shear and Poiseuille flows with Lees and Edward's [A. W. Lees and S. F. Edwards, J. Phys. C 5, 1921 (1972)] periodic boundary conditions. A multiple time-scale algorithm extended to nonequilibrium situations is used as the integration method, and the simulations are performed at constant temperature using Nose-Hoover [S. Nose, J. Chem. Phys. 81, 511 (1984)] dynamics. In simple shear, molecules with flow-induced ellipsoidal shape, having significant segment concentrations along the gradient and neutral directions, exhibit substantial flow resistance. Linear molecules have larger zero-shear-rate viscosity than that of branched molecules, however, this behavior reverses as the shear rate is increased. The relaxation time of the molecules is associated with segment concentrations directed along the gradient and neutral directions, and hence it depends on structure and molecular weight. The results of this study are in qualitative agreement with other simulation studies and with experimental data. The pressure (Poiseuille) flow is induced by an external force F(e) simulated by confining the molecules in the region between surfaces which have attractive forces. Conditions at the boundary strongly influence the type of the slip flow predicted. A parabolic velocity profile with apparent slip on the wall is predicted under weakly attractive wall conditions, independent of molecular structure. In the case of strongly attractive walls, a layer of adhered molecules to the wall produces an abrupt distortion of the velocity profile which leads to slip between fluid layers with magnitude that depends on the molecular structure. Finally, the molecular deformation under flow depends on the attractive force of the wall, in such a way that molecules are highly deformed in the case of strong attracting walls.     

Theory of the photodissociation of ozone in the Hartley continuum; effect of vibrational excitation and O(1D) atom velocity distribution

The effect of vibrational excitation on the photodissociation cross section of ozone in the Hartley continuum is examined. The calculations make use of newly computed potential energy and transition dipole moment surfaces. The initial vibrational states of the ozone are computed using grid based techniques and the first few ab initio computed vibrational energy level spacings agree to within 10 cm(-1) with experimental values. The computed total absorption cross sections arising from different initial vibrational states of ozone are discussed in the light of the nature of the transition dipole moment surface. The computed cross section for excitation from the ground vibrational-rotational state is in good agreement with the experimentally measured cross section. Excitation of the asymmetric stretching vibration of ozone has a marked effect on both the form and magnitude of the photodissociation cross section. The velocity distributions of highly reactive O(1D) atoms arising from the photodissociation process in different wavelength ranges is also presented. The results show that the O(1D) atoms travel with a most probable translational velocity of 2.030 km s(-1) corresponding to a translational energy of 0.342 eV or 33.0 kJ mol(-1).     

The generalized Hulthén potential

A generalization of the Hulthén potential is presented on the basis of an approach that uses the factorization of a general Hamiltonian by means of a specific model of operational equations with the structure ∼β(r)∓(d/dr). To achieve this goal, the treatment of the V _Hs(r) standard Hulthén potential for bound s states is carried out by proposing a particular β_p(r) ansatz to identify V _Hs(r) by means of a particular Riccati-type relationship. Once the identification has been achieved, the generalized Hulthén potential is obtained straightforwardly with the aid of a general Riccati formula. As expected, the Hamiltonian of the generalized Hulthén potential is isospectral when compared with the corresponding standard Hamiltonian. Moreover, according to the Darboux transform there exists a modified Hulthén potential which is also isospectral. We show that the latter is just a particular case of the generalized Hulthén interaction model. Received: 14 September 1999 / Accepted: 3 February 2000 / Published online: 19 April 2000     

A new restricted access molecularly imprinted polymer capped with albumin for direct extraction of drugs from biological matrices: the case of chlorpromazine in human plasma

A new restricted access molecularly imprinted polymer coated with bovine serum albumin (RAMIP-BSA) was developed, characterized, and used for direct analysis of chlorpromazine in human plasma samples. The RAMIP-BSA was synthesized using chlorpromazine, methacrylic acid, and ethylene glycol dimethacrylate as template, functional monomer, and cross-linker, respectively. Glycerol dimethacrylate and hydroxy methyl methacrylate were used to promote a hydrophilic surface (high density of hydroxyl groups). Afterward, the polymer was coated with BSA using glutaraldehyde as cross-linker, resulting in a protein chemical shield around it. The material was able to eliminate ca. 99 % of protein when a 44-mg mL^?1 BSA aqueous solution was passed through it. The RAMIP-BSA was packed in a column and used for direct analysis of chlorpromazine in human plasma samples in an online column switching high-performance liquid chromatography system. The analytical calibration curve was prepared in a pool of human plasma samples with chlorpromazine concentrations ranging from 30 to 350 μg L^?1. The correlation coefficient obtained was 0.995 and the limit of quantification was 30 μg L^?1. Intra-day and inter-day precision and accuracy presented variation coefficients and relative errors lower than 15 % and within ?15 and 15 %, respectively. The sample throughput was 3 h^?1 (sample preparation and chromatographic analysis steps) and the same RAMIP-BSA column was efficiently used for about 90 cycles.     

Coupling Pervaporation-Gas Chromatography for Speciation of Volatile Forms of Selenium in Sediments

Abstract

A method for speciation of dimethylselenide (DMeSe), dimethyldiselenide (DMeDSe) and diethylselenide (DEtSe) in sediments based on a coupling between a pervaporation module, a preconcentration sorptive trap and a gas chromatograph-mass spectrometer is reported. The coupling is performed through a high pressure injection valve which allows two different operational modes: (a) analysis without preconcentration, in which analytes are directly driven from the pervaporation chamber to the injection port of the chromatograph, and (b) analysis with preconcentration in a trap, in which the analytes from the pervaporation chamber are first trapped on a Tenax minicolumn and then thermally desorbed and driven to the GC. This second approach improves the sensitivity compared to the direct coupling, reaching estimated absolute detection limits lower than 0.6 ng Se for each tested species. The method is applied to the determination of volatile organic selenium species in several sediments collected from different areas in the Southwest of Spain.     

Acrolein detection based on alcohol dehydrogenase inhibition

This paper presents the effect of acrolein on three dehydrogenases and proposes a fast spectrometric method for acrolein analysis. We have found that alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) are inhibited by low acrolein concentrations (0.2?mM) while inhibition of glutamate dehydrogenase (GDH) is not observed even at higher acrolein concentrations (1?mM). Acrolein is a suicide substrate for AlDH and ADH inhibition by acrolein is competitive. Cysteine (L-Cys) and glutathione (GSH) react with acrolein and thus reduce its expected inhibitory effect. ADH was chosen to develop a spectrophotometric method for acrolein analysis based on enzyme inhibition. The calibration curve is linear between 0.2 and 1.0?mM acrolein.