An improved 15N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins

A new (15)N constant-time relaxation dispersion pulse scheme for the quantification of millisecond time-scale exchange dynamics in proteins is presented. The experiment differs from previously developed sequences in that it includes (1)H continuous-wave decoupling during the (15)N Carr-Purcell-Meiboom-Gill (CPMG) pulse train that significantly improves the relaxation properties of (15)N magnetization, leading to sensitivity gains in experiments. Moreover, it is shown that inclusion of an additional (15)N 180 degrees refocusing pulse (phase cycled +/- x) in the center of the CPMG pulse train, consisting of 1(5)N 180 degrees (y) pulses, provides compensation for pulse imperfections beyond the normal CPMG scheme. Relative to existing relaxation-compensated constant-time relaxation dispersion pulse schemes, nu(CPMG) values that are only half as large can be employed, offering increased sensitivity to slow time-scale exchange processes. The robustness of the methodology is illustrated with applications involving a pair of proteins: an SH3 domain that does not show millisecond time-scale exchange and an FF domain with significant chemical exchange contributions.     

Bioactivities and chemical constituents of a Vietnamese medicinal plant Che Vang, Jasminum subtriplinerve Blume (Oleaceae)

Five crude extracts were made from leaves and stems of Jasminum subtriplinerve Blume (Oleaceae) and investigated for antimicrobial, antioxidant and cytotoxic activities. The extractions were done with petroleum ether, ethyl acetate, ethanol, methanol or water. All extracts exhibited anti-bacterial activity except the water fraction. On the other hand, all extracts exhibit antioxidant activity except the petroleum ether fraction using the DPPH radical scavenging assay. Only the petroleum ether fraction showed a cytotoxicity activity against tested cell-lines, Hep-G2 and RD with IC(50) values of 19.2 and 20 microg mL(-1), respectively. From the petroleum ether and ethyl acetate extracts, two triterpenes namely 3beta-acetyl-oleanolic acid and lup-20-en-3beta-ol and a sterol, stigmast-5-en-3beta-ol were isolated. The structure of those compounds were elucidated by spectrometric methods IR, MS, 1D-NMR, 2D-NMR and simulated ACD/NMR spectra. The data presented here indicate that J. subtriplinerve do contain compounds with interesting biological activity.     

Rational design of iron catalysts for C–X bond activation

We have quantum chemically studied the iron-mediated C X bond activation (X = H, Cl, CH₃) by d⁸-FeL₄ complexes using relativistic density functional theory at ZORA-OPBE/TZ2P. We find that by either modulating the electronic effects of a generic iron-catalyst by a set of ligands, that is, CO, BF, PH₃, BN(CH₃)₂, or by manipulating structural effects through the introduction of bidentate ligands, that is, PH₂(CH₂)_nPH₂ with n = 6–1, one can significantly decrease the reaction barrier for the C X bond activation. The combination of both tuning handles causes a decrease of the C H activation barrier from 10.4 to 4.6 kcal mol⁻¹. Our activation strain and Kohn-Sham molecular orbital analyses reveal that the electronic tuning works via optimizing the catalyst–substrate interaction by introducing a strong second backdonation interaction (i.e., “ligand-assisted” interaction), while the mechanism for structural tuning is mainly caused by the reduction of the required activation strain because of the pre-distortion of the catalyst. In all, we present design principles for iron-based catalysts that mimic the favorable behavior of their well-known palladium analogs in the bond-activation step of cross-coupling reactions.     

Insertion of Chemical Handles into the Backbone of DNA during Solid-Phase Synthesis by Oxidative Coupling of Amines to Phosphites

Conjugation of molecules or proteins to oligonucleotides can improve their functional and therapeutic capacity. However, such modifications are often limited to the 5′ and 3′ end of oligonucleotides. Herein, we report the development of an inexpensive and simple method that allows for the insertion of chemical handles into the backbone of oligonucleotides. This method is compatible with standardized automated solid-phase oligonucleotide synthesis, and relies on formation of phosphoramidates. A unique phosphoramidite is incorporated into a growing oligonucleotide, and oxidized to the desired phosphoramidate using iodine and an amine of choice. Azides, alkynes, amines, and alkanes have been linked to oligonucleotides via internally positioned phosphoramidates with oxidative coupling yields above 80 %. We show the design of phosphoramidates from secondary amines that specifically hydrolyze to the phosphate only at decreased pH. Finally, we show the synthesis of an antibody-DNA conjugate, where the oligonucleotide can be selectively released in a pH 5.5 buffer.     

Optimization of solid phase extraction clean up and validation of quantitative determination of corticosteroids in urine by liquid chromatography-tandem mass spectrometry

A solid phase extraction (SPE) method for extraction and clean up of 9 synthetic corticosteroids was optimized for quantification by reversed-phase high-performance liquid chromatography/negative electrospray ionisation mass spectrometry. Clean up was accomplished using a mixed mode polymeric strong anion exchange SPE column. The final method was validated according to EU regulations for determination of residues of veterinarian drugs in products of animal origin. Initial results showed a large difference in ion suppression between samples of porcine and bovine urine. The aim of optimisation was to design a procedure that minimised this difference while using a single SPE procedure and a fast HPLC method that enabled sufficient separation of the epimers beta- and dexamethasone. To include conjugated corticosteroids in the analysis, the sample was hydrolysed with Helix Pomatia beta-glucuronidase/aryl sulfatase. For the final method, which included fluocinolone acetonid, triamcinolone acetonid, beclomethasone, flumethasone, dexamethasone, betamethasone, 6alpha-methylprednisolone, prednisone and prednisolone, a quantification based on spiked samples carried through the entire analytical procedure was used. For quantification of triamcinolone acetonid an internal standard (triamcinolone acetonid-D6) was used. Relative average recoveries from 96 to 103% were found, except for beclomethasone (113%). Absolute average recoveries were 81-99%. Quantification limits (decision limits, CCalpha) were demonstrated to be not higher than 1 microg L(-1) (3 microg L(-1) for prednisone and prednisolone). The internal reproducibility, determined by triplicates from spiking at three different levels in six analytical series was 7-19% (at 2-4 microg L(-1)) except for prednisone and prednisolone (26-27% at 3-6 microg L(-1)).     

Comparison of algorithms for the calculation of molecular vibrational level densities

Level densities of vibrational degrees of freedom are calculated numerically with formulas based on the inversion of the canonical vibrational partition function. The calculated level densities are compared with other approximate equations from literature and with the exact Beyer-Swinehart values, for which a simplified but equivalent version is given. All approximate equations agree at high excitation energies, but our results are vastly superior at low energies for large molecules. The results presented here are therefore of particular relevance for thermal processes of very large molecules, e.g., of biological nature, for which the exact state counting can be prohibitively slow. Furthermore, it is valid for situations where anharmonic motion significantly influences the thermal properties.     

Large exposure estimation through automatic business group identification

Large exposure rules are considered critical for financial institutions, as they directly restrict the lending activity of banks to clients. However, empirical evidence suggests that those rules are difficult both for regulators to enforce and for financial institutions to implement. We present a data-driven analytical model that automatically and algorithmically creates groups of related parties based on ownership information, financial dependencies, business associations, and family ties. We develop a methodology based on linear algebra and networks to group clients, highlight missing critical information, and identify unreported business partners. The approach can be used both prospectively by banking institutions analyzing credit risk and by regulators. We include a case study, applying the methodology retrospectively to highlight large exposure violations and systemic risk leading up to the 2008 banking crises in Iceland.     

Liouville Property,Wiener’s Test and Unavoidable Sets for Hunt Processes

Let \((X,\mathcal {W})\) be a balayage space, \(1\in \mathcal {W}\), or – equivalently – let \(\mathcal W\) be the set of excessive functions of a Hunt process on a locally compact space X with countable base such that \(\mathcal {W}\) separates points, every function in \(\mathcal {W}\) is the supremum of its continuous minorants and there exist strictly positive continuous \(u,v\in \mathcal {W}\) such that u/v → 0 at infinity. We suppose that there is a Green function G > 0 for X, a metric ρ for X and a decreasing function \(g\colon [0,\infty )\to (0,\infty ]\) having the doubling property such that G ≈ g ° ρ. Assuming that the constant function 1 is harmonic and balls of (X, ρ) are relatively compact, it is shown that every positive harmonic function on X is constant (Liouville property) and that Wiener’s test at infinity shows, if a given set A in X is unavoidable, that is, if the process hits A with probability one, wherever it starts. An application yields that locally finite unions of pairwise disjoint balls B(z, r_z), z ∈ Z, which have a certain separation property with respect to a suitable measure λ on X are unavoidable if and only if, for some/any point x₀ ∈ X, the series \({\sum }_{z\in Z} g(\rho (x_{0},z))/g(r_{z}) \) diverges. The results generalize and, exploiting a zero-one law for hitting probabilities, simplify recent work by S. Gardiner and M. Ghergu, A. Mimica and Z. Vondra?ek, and the author.     

High-order splitting schemes for semilinear evolution equations

We first derive necessary and sufficient stiff order conditions, up to order four, for exponential splitting schemes applied to semilinear evolution equations. The main idea is to identify the local splitting error as a sum of quadrature errors. The order conditions of the quadrature rules then yield the stiff order conditions in an explicit fashion, similarly to that of Runge–Kutta schemes. Furthermore, the derived stiff conditions coincide with the classical non-stiff conditions. Secondly, we propose an abstract convergence analysis, where the linear part of the vector field is assumed to generate a group or a semigroup and the nonlinear part is assumed to be smooth and to satisfy a set of compatibility requirements. Concrete applications include nonlinear wave equations and diffusion-reaction processes. The convergence analysis also extends to the case where the nonlinear flows in the exponential splitting scheme are approximated by a sufficiently accurate one-step method.     

CE frontal analysis based on simultaneous UV and contactless conductivity detection: a general setup for studying noncovalent interactions

CE frontal analysis (CE-FA) has been established as a powerful tool to study noncovalent interactions between macromolecules and small molecules such as drug substances or pharmaceutical excipients. However, when using traditional commercial CE instrumentation, a serious drawback is related to the fact that only UV-active compounds can be studied. In recent years, contactless conductivity detection has become an attractive alternative to UV detection in CE due to its high versatility. In this study, we combine contactless conductivity detection and UV detection in a highly versatile setup for profiling noncovalent interactions between low-molecular-weight molecules and macromolecules. In the case of molecules having a chromophore the setup allows determination of binding constants using two independent detectors. The new contactless conductivity detection cell is compatible with commercial CE instrumentation and is therefore easily implemented in any analysis laboratory with CE expertise.