General base catalysis for cleavage by the active-site cytosine of the hepatitis delta virus ribozyme: QM/MM calculations establish chemical feasibility

The hepatitis delta virus (HDV) ribozyme is an RNA motif embedded in human pathogenic HDV RNA. Previous experimental studies have established that the active-site nucleotide C75 is essential for self-cleavage of the ribozyme, although its exact catalytic role in the process remains debated. Structural data from X-ray crystallography generally indicate that C75 acts as the general base that initiates catalysis by deprotonating the 2'-OH nucleophile at the cleavage site, while a hydrated magnesium ion likely protonates the 5'-oxygen leaving group. In contrast, some mechanistic studies support the role of C75 acting as general acid and thus being protonated before the reaction. We report combined quantum chemical/molecular mechanical calculations for the C75 general base pathway, utilizing the available structural data for the wild type HDV genomic ribozyme as a starting point. Several starting configurations differing in magnesium ion placement were considered and both one-dimensional and two-dimensional potential energy surface scans were used to explore plausible reaction paths. Our calculations show that C75 is readily capable of acting as the general base, in concert with the hydrated magnesium ion as the general acid. We identify a most likely position for the magnesium ion, which also suggests it acts as a Lewis acid. The calculated energy barrier of the proposed mechanism, approximately 20 kcal/mol, would lower the reaction barrier by approximately 15 kcal/mol compared with the uncatalyzed reaction and is in good agreement with experimental data.     

Gas phase reaction of substituted isoquinolines to carboxylic acids in ion trap and triple quadrupole mass spectrometers after electrospray ionization and collision-induced dissociation

Within the mass spectrometric study of bisubstituted isoquinolines that possess great potential as prolylhydroxylase inhibitor drug candidates (e.g., FG-2216), unusually favored gas-phase formations of carboxylic acids after collisional activation were observed. The protonated molecule of [(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid was dissociated, yielding the 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid methyleneamide cation. Subsequent dissociation caused the nominal elimination of 11 u that resulted from the loss of HCN and concomitant addition of oxygen to the product ion, which formed the protonated 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid. The preference of this structure under mass spectrometric conditions was substantiated by tandem mass spectrometry analyses using the corresponding methyl ester (1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid methyl ester) that eliminated methylene (-14 u) upon collisional activation. Moreover, the major product ion of 1-chloro-4-hydroxy-isoquinoline-3-carboxylic acid, which resulted from the loss of water in MS3 experiments, restored the precursor ion structure by re-addition of H2O. Evidences for these phenomena were obtained by chemical synthesis of proposed gas-phase intermediates, H/D exchange experiments, high-resolution/high accuracy mass spectrometry at MSn level, and "ping-pong" analyses (MS7, in which the precursor ion was dissociated and the respective product ion isolated to regenerate the precursor ion for repeated dissociation. Based on these results, dissociation pathways for [(1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid were suggested that can be further utilized for the characterization of structurally related compounds or metabolic products in clinical, forensic, or doping control analysis.     

Thin macroporous silicon heterojunction solar cells

We demonstrate the processing of a heterojunction solar cell from a purely macroporous silicon (MacPSi) absorber that is generated and separated from a monocrystalline n‐type Cz silicon wafer by means of electrochemical etching. The etching procedure results in straight pores with a diameter of (4.7 ± 0.2) µm and a distance of 8.3 µm. An intrinsic amorphous Si (a‐Si)/p⁺‐type a‐Si/indium tin oxide (ITO) layer stack is on the front side and an intrinsic a‐Si/n⁺‐type a‐Si/ITO layer stack is on the rear side. The pores are open when depositing the layers onto the 3.92 cm²‐sized cell. The conductive layers do not cause shunting through the pores. A silicon oxide layer passivates the pore walls. The energy‐conversion efficiency of the (33 ± 2) µm thick cell is 7.2%. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Non-stationary forward flux sampling

We present a method, Non-Stationary Forward Flux Sampling, that allows efficient simulation of rare events in both stationary and non-stationary stochastic systems. The method uses stochastic branching and pruning to achieve uniform sampling of trajectories in phase space and time, leading to accurate estimates for time-dependent switching propensities and time-dependent phase space probability densities. It is suitable for equilibrium or non-equilibrium systems, in or out of stationary state, including non-Markovian or externally driven systems. We demonstrate the validity of the technique by applying it to a one-dimensional barrier crossing problem that can be solved exactly, and show its usefulness by applying it to the time-dependent switching of a genetic toggle switch.     

Weak Arene Stabilization of Bulky Amido-Germanium(II) and Tin(II) Monocations

Guilty as charged: Germanium(II) and tin(II) monocations which are stabilized by an extremely bulky amido ligand and a very weakly coordinating anion are reported (see picture; E=Ge, Sn; PF=[Al{OC(CF(3) )(3) }(4) ](-) ). The metal centers exhibit weak intramolecular η(2) -arene interactions, and preliminary reactivity studies highlight the electrophilicity of the cations.     

Continuous Synthesis of Peralkylated Imidazoles and their Transformation into Ionic Liquids with Improved (Electro)Chemical Stabilities

A versatile and efficient method to synthesize tetrasubstituted imidazoles via a one‐pot modified Debus–Radziszewski reaction and their subsequent transformation into the corresponding imidazolium ionic liquids is reported. The tetrasubstituted imidazoles were also synthesized by means of a continuous flow process. This straightforward synthetic procedure allows for a fast and selective synthesis of tetrasubstituted imidazoles on a large scale. The completely substituted imidazolium dicyanamide and bis(trifluoromethylsulfonyl)imide salts were obtained via a metathesis reaction of the imidazolium iodide salts. The melting points and viscosities are of the same order of magnitude as for their non‐substituted analogues. In addition to the superior chemical stability of these novel ionic liquids, which allows them to be applied in strong alkaline media, the improved thermal and electrochemical stabilities of these compounds compared with conventional imidazolium ionic liquids is also demonstrated by thermogravimetrical analysis (TGA) and cyclic voltammetry (CV). Although increased substitution of the ionic liquids does not further increase thermal stability, a definite increase in cathodic stability is observable.     

Solid-phase synthesis of oxaliplatin-TAT peptide bioconjugates

Platinum-based drugs play a crucial role in the fight against cancer. Oxaliplatin, which is used in the treatment of colorectal carcinoma, was the last platinum-based agent to be approved worldwide. However, the efficiency of the therapy is limited for example by a low accumulation of the drug in cancer cells. Cell-penetrating peptides (CPPs) are known to ease the cellular membrane transport and are used as vectors for low-molecular-weight drugs and drug carriers; of them, TAT peptides are the best-studied group. In this work, a TAT-peptide fragment (YGRKKRRQRRR) was for the first time conjugated to a platinum(IV) analog of oxaliplatin as a vehicle for membrane penetration. Solid-phase peptide synthesis and subsequent coupling with the platinum complex afforded mono- and difunctionalized conjugates, which were separated by preparative HPLC and characterized by analytical HPLC, ESI-MS, and (1)H NMR spectroscopy. Both conjugates are active in the low micromolar range in CH1 and SW480 human cancer cells, requiring much lower concentrations than the untargeted analogs for equal effects.     

Sensitive redox speciation of neptunium by CE–ICP–MS

Capillary electrophoresis (CE) was used to separate the neptunium oxidation states Np(IV) and Np(V), which are the only oxidation states of Np that are stable under environmental conditions. The CE setup was coupled to an inductively coupled plasma mass spectrometer (Agilent 7500ce) using a Mira Mist CE nebulizer and a Scott-type spray chamber. The combination of the separation capacity of CE with the detection sensitivity of inductively coupled plasma mass spectrometry (ICP-MS) allows identification and quantification of Np(IV) and Np(V) at the trace levels expected in the far field of a nuclear waste repository. Limits of detection of 1?×?10^-9 and 5?×?10^-10 mol L^-1 for Np(IV) and Np(V), respectively, were achieved, with a linear range from 10^-9 to 10^-6 mol L^-1. The method was applied to study the redox speciation of the Np remaining in solution after interaction of 5?×?10^-7 mol L^-1 Np(V) with Opalinus Clay. Under mildly oxidizing conditions, a Np sorption of 31% was found, with all the Np remaining in solution being Np(V). A second sorption experiment performed in the presence of Fe²⁺ led to complete sorption of the Np onto the clay. After desorption with HClO₄, a mixture of Np(IV) and Np(V) was found in solution by CE–ICP–MS, indicating that some of the sorbed Np had been reduced to Np(IV) by Fe²⁺.     

Nonaqueous capillary electrophoresis-mass spectrometry: a versatile, straightforward tool for the analysis of alkaloids from psychoactive plant extracts

In this study we show that a nonaqueous capillary electrophoresis mass spectrometry (NACE-MS) method carefully optimized by a design of experiment can be applied to a very large number of alkaloids in different plant extracts. It is possible to characterize the pattern of the psychoactive alkaloids in several plant samples and preparations thereof, each presenting different challenges in their analysis. The method is shown to be able to separate structurally closely related substances, diastereomers and further isobaric compounds, to separate members of different alkaloid classes within one run and to tolerate significant matrix load. A comparison with methods presented in the literature reveals that a near-generic NACE-MS method for the fast profiling of alkaloids in forensically relevant plant samples has been developed.     

Site-selective X-ray spectroscopy on an asymmetric model complex of the [FeFe] hydrogenase active site

The active site for hydrogen production in [FeFe] hydrogenase comprises a diiron unit. Bioinorganic chemistry has modeled important features of this center, aiming at mechanistic understanding and the development of novel catalysts. However, new assays are required for analyzing the effects of ligand variations at the metal ions. By high-resolution X-ray absorption spectroscopy with narrow-band X-ray emission detection (XAS/XES = XAES) and density functional theory (DFT), we studied an asymmetrically coordinated [FeFe] model complex, [(CO)(3)Fe(I)1-(bdtCl(2))-Fe(I)2(CO)(Ph(2)P-CH(2)-NCH(3)-CH(2)-PPh(2))] (1, bdt = benzene-1,2-dithiolate), in comparison to iron-carbonyl references. Kβ emission spectra (Kβ(1,3), Kβ') revealed the absence of unpaired spins and the low-spin character for both Fe ions in 1. In a series of low-spin iron compounds, the Kβ(1,3) energy did not reflect the formal iron oxidation state, but it decreases with increasing ligand field strength due to shorter iron-ligand bonds, following the spectrochemical series. The intensity of the valence-to-core transitions (Kβ(2,5)) decreases for increasing Fe-ligand bond length, certain emission peaks allow counting of Fe-CO bonds, and even molecular orbitals (MOs) located on the metal-bridging bdt group of 1 contribute to the spectra. As deduced from 3d → 1s emission and 1s → 3d absorption spectra and supported by DFT, the HOMO-LUMO gap of 1 is about 2.8 eV. Kβ-detected XANES spectra in agreement with DFT revealed considerable electronic asymmetry in 1; the energies and occupancies of Fe-d dominated MOs resemble a square-pyramidal Fe(0) for Fe1 and an octahedral Fe(II) for Fe2. EXAFS spectra for various Kβ emission energies showed considerable site-selectivity; approximate structural parameters similar to the crystal structure could be determined for the two individual iron atoms of 1 in powder samples. These results suggest that metal site- and spin-selective XAES on [FeFe] hydrogenase protein and active site models may provide a powerful tool to study intermediates under reaction conditions.