Implications of active site constraints on varied DNA polymerase selectivity

DNA polymerase selectivity often varies significantly depending on the DNA polymerase. The origin of this varying error propensity is elusive. It is assumed that DNA polymerases form nucleotide binding pockets that differ in properties such as shape and tightness. We tested this prediction and studied HIV-1 RT by employment of size-augmented nucleotides and site-directed mutagenesis of the enzyme. New valuable insights into the mechanism of DNA polymerase fidelity were obtained. The presented study provides experimental evidence that variations of steric constraints within the nucleotide binding pocket of at least two DNA polymerases cause variations in nucleotide incorporation selectivity. Thus, our results support the concept of active site tightness as a causative in differential fidelity among DNA polymerases.     

Analysis of ecstasy with a monolithic reversed-phase column

Summary A new, rapid, and precise liquid chromatographic method has been developed for simultaneous identification and quantification of amphetamine,N-methyl-3,4-methylenedioxymetamphetamine,N-ethyl-3,4-methylenedioxymetamphetamine, andN-methyl-1-(3,4-methyl-enedioxyphenyl)-2-butanamine in the presence of other constituents. The compounds were separated on a monolithic column with a gradient prepared from acetonitrile and 20mm monobasic potassium buffer at a flow rate of 1.5 mL min⁻¹. Quantitation was performed with metoclopramide as internal standard. Use of different flow rates was investigated and enabled reduction of the separation time from 11 to 3.5 min for seven substances. The method was then applied to ten seized tablets to identify and quantify their active ingredients.     

Multiple sprayer system for high-throughput electrospray ionization mass spectrometry

A multiple sprayer electrospray ion source for high-throughput analysis is described. The ion source is comprised of multiple electrospray capillaries, each with an ion lens located near the tip. The electric potentials applied to the ion lenses are used to control the sprayers. The use of ion lenses eliminates the need for mechanical blocking devices to selectively enable or disable the sprayers, and results in a less expensive and more reliable set-up. Sprayers can be enabled or disabled within approximately 50-250 ms when the lens potentials are controlled manually. For simultaneous operation of multiple electrospray capillaries, it is advantageous to orient the capillaries so that the spray from each passes directly in front of the entrance aperture of the mass spectrometer.     

Pyrolysis-gas chromatography of polymers obtained by Friedel-Crafts reactions of poly(vinyl chloride) with aromatic compounds

Pyrolysis, in combination with gas-chromatography technique, was used in the determination of the structure and study of the thermal degradation mechanism of the condensation polymers obtained by Friedel-Crafts reactions of poly(vinyl chloride) with benzene, toluene, and naphthalene. The separation of the pyrolysis products was made using a column packed with chromosorb W (80–100 mesh) coated with 15% silicone SE-52. The identification of the pyrolysis products resulted as a consequence of the thermal decomposition of condensation polymers and their semiquantitative estimation led to the final conclusion that the initial normal chlorine substitution in the macromolecular chain of poly(vinyl chloride) is followed by an important intramolecular cyclization reaction yielding 1,3-methyleneindan units.     

Electrospray ionization mass spectrometric studies of nucleophilic carbenes derived from pyrazolium and indazolium carboxylates

Pyrazolium-3-carboxylate and indazolium-3-carboxylate, which belong to the class of pseudo-cross-conjugated mesomeric betaines and which represent the electronically relevant partial structures of the betaine alkaloid Nigellicin, were examined by electrospray ionization mass spectrometry. These compounds decarboxylate to pyrazol-3-ylidene and indazol-3-ylidene. The formation of adducts of these new nucleophilic carbenes under the measurement conditions was examined.     

Conformational structure of N-methylpropionamide and N-methylisobutyroamede. NMR shift reagent and IR study

The structures of the stable conformers of N-methyjpropionamide and N-methyliso-butyroamide in CCl₄, solution were determined by a combination of IR spectroscopy and NMR spectroscopy with lanthanide shift reagents. N-methyl-propionamide was found to exist in the form of two rotational isomers, 1 and 2, with the ethyl group twisted out of the plane of the amide bond by the angles Ψ = 140 and 20°, respectively. For these two conformers, the enthalpy difference is ΔH = 2.13 ± 0.08 kcal mole^?1 and the entropy difference ΔS = 7.81 ± 0.55 cal mole^?1 grad^?1. N-methylisobutyroamide exists in a single form, with the two C-methyl group positions very close to those found in the two isomers of N-methylpropionamide.     

The Mitsunobu Inversion Reaction of Sterically Hindered 17-Hydroxy Steroids

The Mitsunobu inversion reaction of 3-methoxyestra-1,3,5(10)-trien-17-ol is dramatically influenced by the acidic component. There appears to be a relationship between the dissociation constant of the electron-withdrawing substituent on the aryl acid and the overall effectiveness of the reaction, with more acidic species generally providing a higher yield of inverted product.     

Chemical reaction studies in CH4/Ar and CH4/N2 gas mixtures of a dielectric barrier discharge

Chemical reactions in a dielectric barrier discharge at medium pressure of 250-300 mbar have been studied in CH(4)/Ar and CH(4)/N(2) gas mixtures by means of mass spectrometry. The main reaction scheme is production of H(2) by fragmentation of CH(4), but also production of higher order hydrocarbon molecules such as C(n)H(m) with n up to 9 including formation of different functional CN groups is observed. Formation of C(2)H(2), C(2)H(4), and C(2)H(6) molecules has been investigated in some detail. Significant differences are noted in comparison to a theoretical estimate.     

Force field and 13C-NMR investigations of substituted cyclopentanes. A concept for the adaption of 13C NMR shifts to varying torsional arrangements in flexible conformers

MM2 Exploration of the conformational space for methylcyclopentane, in contrast to cyclopentanone yields more and flatter minima than known previously. Calculations of cyclopentanes with substituents X = F, Cl, CHMe₂, and CMe₃ with two stable conformations indicate <2° torsional angle changes with the different substituents. Cyclopentanes bearing not more than 2 substituents can accommodate all groups in pseudoequatorial positions without changing the basic envelope and twist chair geometries significantly. A model for ¹³C-shift calculation is proposed in which shift increments for the different torsional arrangements are obtained by linear interpolation between corresponding cyclohexane values. After correction for the nonequivalent carbon shifts in the hydrocarbon itself, again using the linear interpolation, a significant improvement of the shift correlations is observed. For disubstituted cyclopentanes these predict the shifts within ± 1.7 ppm with Me, CHMe₂, CMe₃, Cl, Br and OH as substituents. Configurational assignments are difficult with 1,3-di-substituted cyclopentanes, but straightforward with 1,2-di- and trisubstituted compounds. Thus, due to the presence of smaller torsional angles between, e.g. diequatorial vicinal substituents in the 1,2- cis series as compared to the trans compounds, the latter show deshielding, particularly at C2, by 1–4 ppm. Several epimers are stereo-selectively prepared by suitable ketone reduction and displacement methods.     

Metal complexes with macrocyclic ligands. Part XXII. Synthesis two of bis-tetraaza-macrocycles and study of the structures,electrochemistry, VIS and EPR spectra of their binuclear Cu2+ and Ni2+ complexes

The two bis-macrocycles 4 and 5 in which the tetraaza units are separated by a chain of different length, have been synthesized using 1,4,7-tritosyl-1,4,7,11-tetraazacyclotetradecane as starting compound and bifunctional alkylating agents. The bis-macrocycles give binuclear complexes with Ni²⁺ and Cu²⁺, the properties of which have been studied to obtain information about the interaction of the two subunits as a function of the distance. The VIS spectra of the Ni²⁺ and Cu²⁺ complexes indicate that both metal ions are in a square-planar geometry as expected from the results of the analogous complexes with 1,4,7,11-tetraazacyclotetradecane 7 . Cyclic voltammetry and differential pulse polarography of the binuclear Ni²⁺complexes in CH₃CN show a single two-electron step for ligand 5 , whereas two distinct one-electron redox processes can be observed for ligand 4 , indicating that the two metal ions interact with each other when the chain length is shorter. Similarly, the EPR studies of frozen solutions of the binuclear Cu²⁺ complexes clearly show that a magnetic dipolar interaction between the two paramagnetic centers exists, and that the strength of it depends upon the length of the bridge. Finally, from the X-ray structures of the binuclear Ni²⁺ complexes with 4 and 5 , it is seen that the two rings are kept apart as far as possible, the distances between the two metal ions determined in the solid correlate well with the observations in solution.