Concomitant polymorphic forms of 3‐cyclopropyl‐5‐(2‐hydrazinylpyridin‐3‐yl)‐1,2,4‐oxadiazole

The dipharmacophore compound 3‐cyclopropyl‐5‐(2‐hydrazinylpyridin‐3‐yl)‐1,2,4‐oxadiazole, C₁₀H₁₁N₅O, was studied on the assumption of its potential biological activity. Two concomitant polymorphs were obtained on crystallization from isopropanol solution and these were thoroughly studied. Identical conformations of the molecules are found in both structures despite the low difference in energy between the four possible conformers. The two polymorphs differ crucially with respect to their crystal structures. A centrosymmetric dimer formed due to both stacking interactions of the `head‐to‐tail' type and N—H…N(π) hydrogen bonds is the building unit in the triclinic structure. The dimeric building units form an isotropic packing. In the orthorhombic polymorphic structure, the molecules form stacking interactions of the `head‐to‐head' type, which results in their organization in a column as the primary basic structural motif. The formation of N—H…N(lone pair) hydrogen bonds between two neighbouring columns allows the formation of a double column as the main structural motif. The correct packing motifs in the two polymorphs could not be identified without calculations of the pairwise interaction energies. The triclinic structure has a higher density and a lower (by 0.60 kcal mol^?1) lattice energy according to periodic calculations compared to the orthorhombic structure. This allows us to presume that the triclinic form of 3‐cyclopropyl‐5‐(2‐hydrazinylpyridin‐3‐yl)‐1,2,4‐oxadiazole is the more stable.     

Optimization of sample preparation and ICP-MS analysis for determination of 60 elements for characterization of the plant ionome

An ICP-MS method for determination of 60 elements in plant samples is proposed based on optimization of digestion, recommending use of HF besides HNO₃ and H₂O₂ and calibration procedures, using CRMs for construction of calibration curves. Adequate choice of analytical isotopes and various measurement conditions (cold plasma for the determination of Al, Ba, Ca, Fe, K, Mg, Mn, Na, Si and Sr and DRC mode for determination of Ag, As, Ni, Pd, Pt, Se and V) as well as introduction of appropriate corrections lead to determination of as large number of elements with quadropole ICP-MS as with the more expensive SF-ICP-MS. Two measurements are performed: cold plasma and standard/DRC mode. The analytical characteristics of the method are demonstrated by analysis of five CRMs and the agreement of the experimental results with the certified/information/literature values is very good. Detection limits are low enough to permit the determination of all elements but platinum metals at background level. The applicability of the method is demonstrated by analysis of Taraxacum officinale (dandelion) samples collected from regions with different anthropogenic influence. The results indicate high degree of pollution round the Pb-Zn smelter with As, Cd, Cu, Ni, Pb and Zn and increased concentrations of B, Be, Bi, Hg, In, Mn, Sb, Se, Sn, Ti, Tl, V and Zr. The dandelion sample, collected along a highway has increased concentrations of traffic released elements: Pt, Pd, Rh, Ce, La, Pb as well as Cu, Zn, Ba and Rb.     

Controlled rate thermal analysis of hydromagnesite

The reaction of magnesium minerals such as brucite with CO₂ is important in the sequestration of CO₂. The study of the thermal stability of hydromagnesite and diagenetically related compounds is of fundamental importance to this sequestration. The understanding of the thermal stability of magnesium carbonates and the relative metastability of hydrous carbonates including hydromagnesite, artinite, nesquehonite, barringtonite and lansfordite is extremely important to the sequestration process for the removal of atmospheric CO₂. This work makes a comparison of the dynamic and controlled rate thermal analysis of hydromagnesite and nesquehonite. The dynamic thermal analysis of synthetic hydromagnesite proves that dehydration takes place in two steps at 135 and 184°C, dehydroxylation at 412°C and decarbonation at 474°C. Controlled rate thermal analysis shows the first dehydration step is isothermal and the second quasi-isothermal at 108 and 145°C, respectively. In the CRTA experiment both water and carbon dioxide are evolved in an isothermal decomposition at 376°C. CRTA technology offers better resolution and a more detailed interpretation of the decomposition processes of magnesium carbonates such as nesquehonite via approaching equilibrium conditions of decomposition through the elimination of the slow transfer of heat to the sample as a controlling parameter on the process of decomposition. Constant-rate decomposition processes of non-isothermal nature reveal partial nesquehonite structure.     

A method for rapid protease substrate evaluation and optimization

We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.     

The separation of the enantiomers of diquats by capillary electrophoresis using randomly sulfated cyclodextrins as chiral selectors

Diquats, derivatives of the widely used herbicide diquat, represent a new class of functional organic molecules. A combination of their special electrochemical properties and axial chirality could potentially result in their important applications in supramolecular chemistry, chiral catalysis, and chiral analysis. However, prior to their practical applications, the diquats have to be prepared in enantiomerically pure forms and the enantiomeric purity of their P- and M-isomers has to be checked. Hence, a chiral capillary electrophoresis (CE) method has been developed and applied for separation of P- and M-enantiomers of 11 new diquats. Fast and better than baseline CE separations of enantiomers of all 11 diquats within a short time 5–7 min were achieved using acidic buffer, 22 mM NaOH, 35 mM H₃PO₄, pH 2.5, as a background electrolyte, and 6 mM randomly sulfated α-, β-, and γ-cyclodextrins as chiral selectors. The most successful selector was sulfated γ-cyclodextrin, which baseline separated the enantiomers of all 11 diquats, followed by sulfated β-cyclodextrin and sulfated α-cyclodextrin, which baseline separated enantiomers of 10 and nine diquats, respectively. Using this method, a high enantiopurity degree of the isolated P- and M-enantiomers of three diquats with a defined absolute configuration was confirmed and their migration order was identified.     

Electrocatalytic monitoring of metal binding and mutation-induced conformational changes in p53 at picomole level

We developed an innovative electrochemical method for monitoring conformational transitions in proteins using constant current chronopotentiometric stripping (CPS) with dithiothreitol-modified mercury electrodes. The method was applied to study the effect of oncogenic mutations on the DNA-binding domain of the tumor suppressor p53. The CPS responses of wild-type and mutant p53 showed excellent correlation with structural and stability data and provided additional insights into the differential dynamic behavior of the proteins. Further, we were able to monitor the loss of an essential zinc ion resulting from mutation (R175H) or metal chelation. We envisage that our CPS method can be applied to the analysis of virtually any protein as a sensor for conformational transitions or ligand binding to complement conventional techniques, but with the added benefit that only relatively small amounts of protein are needed and instant results are obtained. This work may lay the foundation for the wide application of electrochemistry in protein science, including proteomics and biomedicine.     

In‐situ enrichment of phosphopeptides on MALDI plates modified by ambient ion landing

We report substantial in‐situ enrichment of phosphopeptides in peptide mixtures using titanium and zirconium dioxide‐coated matrix assisted laser desorption‐ionization (MALDI) plates prepared by recently reported ambient ion landing deposition technique. The technique was able to modify four common materials currently used for MALDI targets (stainless steel, aluminum, indium‐tin oxide glass and polymeric anchor chip). The structure of the deposited dioxide was investigated by electron microscopy, and different surfaces were compared and discussed in this study. Two standard proteins were used to test the enrichment capabilities of modified MALDI plates: casein and in‐vitro phosphorylated trehalase. The enrichment of casein tryptic digest resulted in identification of 20 phosphopeptides (including miscleavages). Trehalase was used as a suitable model of larger protein that provided more complex peptide mixture after the trypsin digestion. All four possible phosphorylation sites in trehalase were identified and up to seven phosphopetides were found (including methionine oxidations and miscleavages). Two different mass spectrometers, MALDI‐Fourier transform ion cyclotron resonance (FTICR) and MALDI‐time of flight, were used to detect the phosphopeptides from modified MALDI plates after the enrichment procedure. It was observed that the desorption‐ionization phenomena on the modified surfaces are not critically influenced by the parameters of the different MALDI ion sources (e.g. different pressure, different extraction voltages), and thus the presence of dioxide layer on the standard MALDI plate does not significantly interfere with the main MALDI processes. The detection of phosphopeptides after the enrichment could be done by both instruments. Desorption electrospray ionization coupled to the FTICR was also tested, but, unlike MALDI, it did not provide satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.     

Lagrangian analysis of consecutive images: Quantification of mixing processes in drops moving in a microchannel

A tracking method and statistical analysis is introduced to quantify the mixing of moving droplets in the Lagrangian reference frame. Aqueous microrheology samples are produced as droplets in immiscible oil using a microfluidic T-junction. Samples from initially unmixed streams of the same viscosity-fluids (water/water) or different viscosity-fluids (water/glycerin solution) are dyed with different colors to visualize their internal motions and to quantify the extent of their mixing as a function of the age in the channel. The homogeneity of the material distribution in the drop is quantified by computing skewness of pixel intensity profiles or Shannon entropy index. Such analysis is important to ensure that samples are uniformly mixed for high-throughput rheological measurements using microrheology. Samples with a high viscosity ratio mix more rapidly than those with the same viscosities and the mixing length in traversing drops in the microchannel decays exponentially with traveling displacement until the drop reaches a diffusion limit.