Stabilisation of the transition state of phosphodiester bond cleavage within linear single-stranded oligoribonucleotides

The effect of base sequence on the stability of the transition state (TS) of phosphodiester bond cleavage within linear single-stranded oligoribonucleotides has been studied in order to better understand why the reactivity of some phosphodiester bonds is enhanced compared to an unconstrained linkage. Molecular dynamics simulations of 3.0 ns were carried out for 14 oligonucleotides that contain in the place of the scissile phosphodiester bond a phosphorane structure mimicking the TS of the bond cleavage. The hydrolytic stability of the same oligonucleotides had previously been reported. Both the non-bridging oxyanions and the leaving 5[prime or minute]-oxygen of the pentacoordinated phosphorane moiety were observed to form hydrogen bonds with solvent water molecules in a similar way with all the compounds studied. In addition, water mediated hydrogen bonds between the phosphorane non-bridging oxyanions and the bases of the 3[prime or minute]-flanking sequence were detected with some of the compounds, but not with the most labile ones. Hence, it seems that the enhanced cleavage of some internucleosidic linkages does not result from the TS stabilisation by hydrogen bonding. With heterooligomers, the stacking of bases next to the cleavage site was observed to be enhanced on going from the initial state to the TS, whereas within uracil homooligomer, having initially negligible stacking, no change in the magnitude of stacking was seen. Accordingly, while strong stacking in the initial state is known to retard the phosphodiester bond cleavage, it may in the TS accelerate the reaction. Therefore, enhanced stacking on going from the initial state to transition state appears to be a factor that markedly contributes to the hydrolytic stability of phosphodiester bonds within oligonucleotides and may, at least partly, explain accelerated cleavage compared to fully unconstrained bonds, such as those in polyuridylic acid.     

Influence of Chelating Groups on the Luminescence Properties of Europium(III) and Terbium(III) chelates in the 2,2′-bipyridine series

Eight different 2,2′-bipyridine derivatives, i.e. 2, 5, 8, 10, 12, 13, 15 , and 19 (Schemes 1 and 2), were prepared to study the influence of the chelating groups on the luminescence properties of their Eu^III and Tb^III chelates. According to our luminescence results, 2,2′-(methylenenitrilo)bis(acetic acid) as well as (methylenenitrilo)bis-(methylphosphonic acid) in 6- and 6′-position of 2,2′-bipyridine is a suitable group when developing luminescent markers for bioaffinity assays based on time-resolved luminescence measurement.     

A gate to organokrypton chemistry: HKrCCH

An organic molecule containing krypton, HKrCCH, is reported. The preparation of HKrCCH includes 193-nm photolysis of H2C2/Kr solid mixtures at 8 K and subsequent thermal mobilization of hydrogen atoms at >/=30 K. The identification is based on infrared absorption spectroscopy and supported by ab initio calculations which show ionic and covalent contributions to the bonding. We believe that a series of similar organokrypton molecules can be prepared as computationally demonstrated for HKrC4H and HKrC3H3. These results feature a generally novel way for activating chemically the H-CC- group, which can find practical applications of the krypton catalysis.     

Sensitive miniature single-particle immunoassay of prostate-specific antigen using time-resolved fluorescence

The present study describes the development of a quantitative miniaturized single microparticle immunoassay. The main objective of the study was to evaluate the performance of a miniature heterogeneous immunoassay on a single microparticle in respect to assay kinetics, volume, and sensitivity, binding capacity of microparticles and sensitivity using europium(III) nanoparticle labels. The performance of the single microparticle assay of prostate-specific antigen (PSA) was investigated using different-sized microparticles (60-920 μm in diameter) and microtiter well as a solid-phase. Equilibration time of the assay was shown to be dependent in a linear manner on surface-to-volume ratio, i.e. larger surface-to-volume translated to a faster reaction. However, no correlation between PSA binding capacity and equilibration time was observed in these kinetic studies. Only moderate improvement in assay kinetics was found when PSA binding capacity was increased on a microparticle. Using europium(III) nanoparticle labels, 107 nm in diameter, coated with streptavidin a detection sensitivity of 30 ng l⁻¹ (0.1 amol) was achieved in 1 μl total assay volume per microparticle. This was 50-fold higher compared to the same assay performed with intrinsically fluorescent europium(III) labels.     

Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout

Direct measurement of time-resolved fluorescence from a washed surface of an immunoassay well constitutes an advantage compared with label development options involving signal generation in solution. Epi-fluorometric detection collects the signal from only a small part of the microtiter well’s bottom surface and it is inadequate for the optimal assay sensitivity when using binding surfaces introduced by large coating volume. This study reports on the use of streptavidin-coated spots intended to condense the binding of the labeled antibodies to coincide with the excitation beam. The spots were generated in special microtiter wells containing 2.5-mm, 3.5-mm, and 4.5-mm diameter indentations by adsorption from liquid droplets containing either native (SAv) or modified high-capacity (GA-SAv) streptavidin. The SAv-coated and GA-SAv-coated spots exhibited maximum Eu–biotin binding densities of 0.080 and 0.47 pmol/mm², respectively. A sandwich-type immunoassay of thyroid-stimulating hormone (TSH) provided a fivefold to sixfold increase in the signal-to-background ratios of the spot assay and an equivalent improvement in the detection limit (DL < 0.01 mU/L) compared with a reference assay. Figure The condensation of the binding area into a spot (right) results in a denser collection of the labeled antibodies and more favorable signal-to-background ratios compared with a regular approach using a large binding area (left)     

Neighboring group participation in the additions of iodonium and bromonium ions to N-alkoxycarbonyl-2-azabicyclo[2.2.n]alk-5-enes (n = 1,2)

Additions of iodonium-X reagents to N-alkoxycarbonyl-2-azabicyclo[2.2.1]hept-5-enes and the homologous 2-azabicyclo[2.2.2]oct-5-enes have been found to mirror the outcomes of additions of bromonium-X reagents. Only rearranged products were observed for reactions of either of these halonium ion reagents with the azabicylo[2.2.1]hept-5-enes. For the azabicyclo[2.2.2]oct-5-enes, nitrogen participation in addition of IOH or BrOH was dependent on the N-alkoxycarbonyl group. With larger N-Boc, N-Cbz, or N-Troc protecting groups, unrearranged 5-anti-hydroxy-6-syn-I(or Br)-2-azabicyclo[2.2.2]octanes were formed by nucleophilic attack at C(5) on syn-halonium ions. The structure of N-methyl-8-anti-bromo-4-anti-hydroxy-2-azabicyclo[3.2.1]octane has been reassigned by X-ray analysis.     

Exposure to increased ambient ultraviolet B radiation has negative effects on growth, condition and immune function of juvenile Atlantic salmon (Salmo salar)

Atlantic salmon (Salmo salar) parr were exposed in two outdoor experiments, ranging in duration from 52 to 137 days, to spectral treatments: (1) natural sunlight (=present ambient UVB level), (2) solar radiation supplemented with enhanced UVB radiation from lamps simulating 20% or 8% stratospheric ozone loss or (3) UVB-depleted sunlight achieved by screening with Mylar-D film. The growth, condition and immune function of the salmon were quantified after treatments. Exposure to enhanced UVB radiation retarded growth, and decreased hematocrit value and plasma protein concentration. Further, enhanced UVB radiation affected plasma immunoglobulin concentration. The results demonstrate that juvenile Atlantic salmon are not able to fully adapt to increased ambient UVB levels in long-term exposures, and the interference with immune system function suggests a negative effect of UVB on disease resistance in Atlantic salmon.     

Some aspects of quantitative 2D NMR

We have studied the application of 2D HSQC for quantitative measurements and propose some improvements to the previously published Q-HSQC method. Application of CPMG-INEPT for polarization transfer period suppresses the evolution of J(HH), and thus corrects the shape of the cross-peaks. The better cross-peak shape makes phase correction and integration of the cross-peaks easier. Further, the (13)C resonance offset dependency can have a significant influence to the results. The offset effects can be compensated either by correcting results with a proper coefficient, or using 90 degrees composite (13)C pulses in the pulse sequence. The results show that these modifications improve the applicability of 2D HSQC for quantitative analysis when studying molecules possessing large J(HH) couplings and wide (13)C chemical shift range.     

Interpretation of Lagrange multipliers in nonlinear pricing problem

We present well-known interpretations of Lagrange multipliers in physical and economic applications, and introduce a new interpretation in nonlinear pricing problem. The multipliers can be interpreted as a network of directed flows between the buyer types. The structure of the digraph and the fact that the multipliers usually have distinctive values can be used in solving the optimization problem more efficiently. We also find that the multipliers satisfy a conservation law for each node in the digraph, and the non-uniqueness of the multipliers are connected to the stability of the solution structure.     

Synthesis of 3′,5′‐Cyclic Phosphate and Thiophosphate Esters of 2′‐C‐Methyl Ribonucleosides

2′‐C‐Methylnucleosides are known to exhibit antiviral activity against Hepatitis C virus. Since the inhibitory activity depends on their intracellular conversion to 5′‐triphosphates, dosing as appropriately protected 5′‐phosphates or 5′‐phosphorothioates appears attractive. For this purpose, four potential pro‐drugs of 2′‐C‐methylguanosine, i.e., 3′,5′‐cyclic phosphorothioate of 2′‐C‐methylguanosine and 2′‐C,O⁶‐dimethylguanosine, 1 and 2 , respectively, the S‐[(pivaloyloxy)methyl] ester of 2′‐C,O⁶‐dimethylguanosine 3′,5′‐cyclic phosphorothioate and the O‐methyl ester of 2′‐C,O⁶‐dimethylguanosine 3′,5′‐cyclic phosphate, 3 and 4 , respectively, have been prepared.