Furan-oxidation-triggered inducible DNA cross-linking: acyclic versus cyclic furan-containing building blocks--on the benefit of restoring the cyclic sugar backbone

Oligodeoxynucleotides incorporating a reactive functionality can cause irreversible cross-linking to the target sequence and have been widely studied for their potential in inhibition of gene expression or development of diagnostic probes for gene analysis. Reactive oligonucleotides further show potential in a supramolecular context for the construction of nanometer-sized DNA-based objects. Inspired by the cytochrome P450 catalyzed transformation of furan into a reactive enal species, we recently introduced a furan-oxidation-based methodology for cross-linking of nucleic acids. Previous experiments using a simple acyclic building block equipped with a furan moiety for incorporation into oligodeoxynucleotides have shown that cross-linking occurs in a very fast and efficient way and that substantial amounts of stable, site-selectively cross-linked species can be isolated. Given the destabilization of duplexes observed upon introduction of the initially designed furan-modified building block into DNA duplexes, we explore here the potential benefits of two new building blocks featuring an extended aromatic system and a restored cyclic backbone. Thorough experimental analysis of cross-linking reactions in a series of contexts, combined with theoretical calculations, permit structural characterization of the formed species and allow assessment of the origin of the enhanced cross-link selectivity. Our experiments clearly show that the modular nature of the furan-modified building blocks used in the current cross-linking strategy allow for fine tuning of both yield and selectivity of the interstrand cross-linking reaction.     

Characterization of fatty acid and triacylglycerol composition in animal fats using silver-ion and non-aqueous reversed-phase high-performance liquid chromatography/mass spectrometry and gas chromatography/flame ionization detection

Fatty acid (FA) and triacylglycerol (TG) composition of natural oils and fats intake in the diet has a strong influence on the human health and chronic diseases. In this work, non-aqueous reversed-phase (NARP) and silver-ion high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection and gas chromatography with flame-ionization detection (GC/FID) and mass spectrometry detection are used for the characterization of FA and TG composition in complex samples of animal fats from fallow deer, red deer, sheep, moufflon, wild boar, cock, duck and rabbit. The FA composition of samples is determined based on the GC/FID analysis of FA methyl esters. In total, 81 FAs of different acyl chain length, double bond (DB) number, branched/linear, cis-/trans- and DB positional isomers are identified. TGs in animal fats contain mainly monounsaturated and saturated FAs. High amounts of branched and trans-FAs are observed in the samples of ruminants. In NARP mode, individual TG species are separated including the separation of trans- and branched TGs. Silver-ion mode provides the separation of TG regioisomers, which enables the determination of their ratios. Great differences in the preference of unsaturated and saturated FAs in the sn-2 position on the glycerol skeleton are observed among individual animal fats. Unsaturated FAs are preferentially occupied in the sn-2 position in all animal samples except for wild boar with the strong preference of saturated FAs in the sn-2 position.     

High Performance Liquid Chromatography‐Electrochemistry‐Electrospray Ionization Mass Spectrometry (HPLC/EC/ESI‐MS) for Detection and Characterization of Roscovitine Oxidation Products

On‐line LC‐EC/ESI‐MS has been established as a fast and simple method to mimic some types of oxidation reaction of various drugs and to study the formation and structure of the resulting products. This technique has been applied to a 2,6,9‐trisubstituted purine, R‐roscovitine, which is known to be an inhibitor of some cyclin‐dependent kinases (CDKs) and a potential anticancer drug. Oxidation of R‐roscovitine in an electrochemical cell (EC), operated under various conditions, resulted in appearance of 6 major products. These were further analyzed by high‐resolution mass spectrometry, their structures were elucidated by accurate mass measurement and compared to previously identified R‐roscovitine in vitro/in vivo metabolites. Although none of the observed products was structurally identical to those identified previously in vitro/in vivo, all of them, except for the methoxylated products, resembled similarity due to appearing through the same reaction type. R‐roscovitine in the EC cell underwent N‐dealkylation of the isopropyl moiety, hydroxylation of the aromatic side‐chain, dihydroxylation, methoxylation and dimer formation. The hydroxylation product was identified as Olomoucine II, a R‐roscovitine derivative, which displays 10‐times higher CDK‐inhibiting activity than R‐roscovitine and the occurrence of which, as R‐roscovitine product, has not yet been observed in vitro/in vivo.     

Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.     

Strong Solutions to Non-stationary Channel Flows of Heat-Conducting Viscous Incompressible Fluids with Dissipative Heating

We study an initial-boundary-value problem for time-dependent flows of heat-conducting viscous incompressible fluids in channel-like domains on a time interval (0,T). For the parabolic system with strong nonlinearities and including the artificial (the so called “do nothing”) boundary conditions, we prove the local in time existence, global uniqueness and smoothness of the solution on a time interval (0,T ^∗), where 0<T ^∗≤T.     

Mechanistic studies of ethylene hydrophenylation catalyzed by bipyridyl Pt(II) complexes

Cationic platinum(II) complexes [((t)bpy)Pt(Ph)(L)](+) [(t)bpy =4,4'-di-tert-butyl-2,2'-bipyridyl; L = THF, NC(5)F(5), or NCMe] catalyze the hydrophenylation of ethylene to generate ethylbenzene and isomers of diethylbenzene. Using ethylene as the limiting reagent, an 89% yield of alkyl arene products is achieved after 4 h at 120 °C. Catalyst efficiency for ethylene hydrophenylation is diminished only slightly under aerobic conditions. Mechanistic studies support a reaction pathway that involves ethylene coordination to Pt(II), insertion of ethylene into the Pt-phenyl bond, and subsequent metal-mediated benzene C-H activation. Studies of stoichiometric benzene (C(6)H(6) or C(6)D(6)) C-H/C-D activation by [((t)bpy)Pt(Ph-d(n))(THF)](+) (n = 0 or 5) indicate a k(H)/k(D) = 1.4(1), while comparative rates of ethylene hydrophenylation using C(6)H(6) and C(6)D(6) reveal k(H)/k(D) = 1.8(4) for the overall catalytic reaction. DFT calculations suggest that the transition state for benzene C-H activation is the highest energy species along the catalytic cycle. In CD(2)Cl(2), [((t)bpy)Pt(Ph)(THF)][BAr'(4)] [Ar' = 3,5-bis(trifluoromethyl)phenyl] reacts with ethylene to generate [((t)bpy)Pt(CH(2)CH(2)Ph)(η(2)-C(2)H(4))][BAr'(4)] with k(obs) = 1.05(4) × 10(-3) s(-1) (23 °C, [C(2)H(4)] = 0.10(1) M). In the catalytic hydrophenylation of ethylene, substantial amounts of diethylbenzenes are produced, and experimental studies suggest that the selectivity for the monoalkylated arene is diminished due to a second aromatic C-H activation competing with ethylbenzene dissociation.     

Enhanced comprehensive two-dimensional gas chromatographic resolution of polychlorinated biphenyls on a non-polar polysiloxane and an ionic liquid column series

A total of 196 out of 209 polychlorobiphenyl (PCB) congeners were resolved using GC×GC-TOFMS with a non-polar/ionic liquid column series consisting of poly(50%-n-octyl-50%-methyl)siloxane and (1,12-di(tripropylphosphonium)dodecane bis(trifluoromethansulfonyl)amide) in the first and second dimension, respectively. It has been found that 13 PCB congeners overlap in five doublets (CB12+CB13, CB62+CB75, CB70+CB76, CB97+CB125 and CB153+CB168) and one triplet (CB90+CB101+CB113). All toxic, "dioxin like" congeners were separated with no interferences from any PCB congener. The 109 PCBs present in Aroclor 1242 and the 82 PCBs present in Aroclor 1260 were resolved GC×GC-TOFMS analysis on this column set.     

Effects of finite size nuclei in relativistic four-component calculations of hyperfine structure

The effect of a finite size model for both the nuclear charge and magnetic moment distributions on calculated EPR hyperfine structure have been studied using a relativistic four-component method based on density functional theory. This approach employs a restricted kinetically balanced basis (mDKS-RKB) and includes spin-polarization using noncollinear spin-density exchange-correlation functionals in the unrestricted fashion. Benchmark calculations have been carried out for a number of small molecules containing Zn, Cd, Ag, and Hg. The present results are compared with those obtained at the Douglas-Kroll-Hess second order (DKH-2) method. The dependence of the results on the quality of the orbital and auxiliary basis sets has been studied. It was found that some basis sets contain irregularities that deteriorate the results. Especial care has to be taken also on the construction of the auxiliary basis for fitting the total electron and spin-densities.     

Femtosecond dynamics of the ring closing process of diarylethene: a case study of electrocyclic reactions in photochromic single crystals

The cyclization reaction of the photochromic diarylethene derivative 1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)perfluorocyclopentene was studied in its single crystal phase with femtosecond transient absorption spectroscopy. The transient absorption measurements were performed with a robust acquisition scheme that explicitly exploits the photoreversibility of the molecular system and monitors the reversibility conditions. The crystalline system demonstrated 3 × 10(4) repeatable cycles before significant degradation was observed. Immediately following photoexcitation, the excited state absorption associated with the open-ring conformation undergoes a large spectral shift with a time constant of approximately 200 fs. Following this evolution on the excited state potential energy surface, the ring closure occurs with a time constant of 5.3 ps, which is significantly slower than previously reported measurements for similar derivatives in the solution phase. Time resolved electron diffraction studies were used to further demonstrate the assignment of the transient absorption dynamics to the ring closing reaction. The mechanistic details of the ring closing are discussed in the context of prior computational work along with a vibrational mode analysis using density functional theory to give some insight into the primary motions involved in the ring closing reaction.