Significant kinetic solvent isotope effects in folding of the catalytic RNA from the hepatitis delta virus

The exchange of deuterium for hydrogen in water often produces solvent kinetic isotope effects (KSIEs) on the rate constants associated with enzyme reactions, including those catalyzed by RNA. Recently, KSIEs have been used to show that proton transfer occurs in the rate-limiting step of cleavage by the hepatitis delta virus (HDV) ribozyme and other catalytic RNAs. To test the underlying assumption that KSIEs are related to the chemistry step of ribozyme-mediated cleavage reactions, we developed fluorescence resonance energy transfer assays to measure KSIEs on the rate constants of conformational changes associated with substrate binding and dissociation by a trans-acting HDV ribozyme. We observe comparable KSIEs ( approximately 2-2.5-fold) of rate constants of conformational change and cleavage, while proton inventory experiments are consistent with a shift in the ensemble of transition states upon increase of D2O in the solvent. Taken together, these results challenge the common assumption that pL profiles of RNA-catalyzed reactions yielding a pKa and KSIE necessarily provide evidence for an ionization (chemistry) step to be rate-limiting. They also suggest that an unusual proton inventory may provide a signature for a conformational change contributing to the rate-limiting step.     

The effect of hydration/dehydration on the AC electrical conductivity of H4PVMo11O40 and some of its cesium salts used as catalysts

Changes in AC electrical conductance G of vanadium substituted 12-molybdophosphoric acid and of its (mono)-acid and neutral cesium salts were studied. Depending on the environment, the electrical conductivity is a combination of protonic and electronic conduction, the protonic one dominating at low temperature.     

Dilated Weyl theory applied to a set of N-coupled singular second-order differential equations

Weyl's theory for a set of N-coupled singular second-order differential equations is analyzed in relation to S-matrix theory and a dilated version is presented. Applications of this theory to two single channel scattering model problems and a two-channel model problem are given. Some implications of the present theory are discussed.     

Synthesis of various 5-substituted 2′-deoxy-3′-5′-di-O-acetyluridines

The Pd(0)-catalyzed coupling reaction of β-5-iodo-2′-deoxy-3′,5′-di-O-acetyluridine with various heteroaryltrimethylstannyl compounds gave the corresponding β-5-heteroaryl-2′-deoxy-3′,5′-di-O-acetyluridines in moderate yields. This direct coupling approach for nucleosides represented an interesting alternative to the 5-heteroaryl functionalization of pyrimidines followed by the Hilbert-Johnson glycosylation reaction which often yields mixtures of the α and β anomers.     

Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions

Several prototypes of multi-modal ligands suitable for the capture of negatively charged proteins from high conductivity (28 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal anion-exchangers were found by screening a diverse library of multi-modal ligands and selecting anion-exchangers resulting in elution of test proteins at high ionic strength. Candidates were then tested with respect to breakthrough capacity of BSA in a buffer adjusted to a high conductivity (20 mM Piperazine and 0.25 M NaCl, pH 6.0). The recovery of BSA was also tested with a salt step (from 0.25 to 2.0 M NaCl using 20 mM Piperazine as buffer, pH 6.0) or with a pH-step to pH 4.0. We have found that non-aromatic multi-modal anion-exchange ligands based on primary or secondary amines (or both) are optimal for the capture of proteins at high salt conditions. Furthermore, these new multi-modal anion-exchange ligands have been designed to take advantage not only of electrostatic but also hydrogen bond interactions. This has been accomplished through modification of the ligands by the introduction of hydroxyl groups in the proximity of the ionic group. Experimental evidence on the importance of the relative position of the hydroxyl groups on the ligand in order to improve the breakthrough capacity of BSA has been found. Compared to strong anion-exchangers such as Q Sepharose Fast Flow the new multi-modal weak anion-exchangers have breakthrough capacities of BSA at mobile phases of 28 mS/cm and pH 6.0 that are 20-30 times higher. The new multi-modal anion-exchangers can also be used at normal anion-exchange conditions and with either a salt step or a pH-step to acidic pH can accomplish the elution of proteins. In addition, the functional performance of the new anion-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 1 week. A number of multi-modal anion-exchange ligands based on aromatic amines exhibiting high breakthrough capacity of BSA have been found. With these ligands recovery was often found to be low due to strong non-electrostatic interactions. However, for phenol derived anion-exchange media the recovery can be improved by desorption at high pH.     

Evaporation driven pumping for chromatography application

A continuous transport process for liquids in micro-channels is reported. Flow was generated by evaporation at the channel end plus capillary forces. The micro-channels integrated into a two-glass-layer device were 110 microm wide, 28 microm deep and 4 or 10 cm long. A continuous liquid transport velocity of up to 2.25 mm s(-1) was observed for aqueous solutions. The flow velocity is shown to increase when an air stream is guided over the evaporation zone.     

Über einige nebenprodukte der thermolyse von bis(trimethylsilyl)-diimin

In the course of decomposition of bis(trimethylsilyl)diimine (BSD), which leads mainly to five products, the tris(trimethylsilyl)hydrazyl radical is formed among other intermediates. This radical reacts with hydrogen donors HR (e.g. HR = solvent) to tris(trimethylsilyl)hydrazine and radicals ·R, which on the other hand react further with BSD to by-products of BSD thermolysis. The types of these by-products and mechanisms of their formation are discussed. The thermolysis of BSD in toluene, for example, produces tris- and bis(trimethylsilyl)benzylhydrazine and bis(trimethylsilyl)benzalhydrazone.