Quantification of AICAR-ribotide concentrations in red blood cells by means of LC-MS/MS

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) arguably provides performance-enhancing properties even in the absence of physical exercise and, therefore, the substance is banned in elite sports since 2009. Due to the natural presence of AICAR in human blood and urine, uncovering the misuse by direct qualitative analysis is not possible. Entering the circulation, the riboside is immediately incorporated into red blood cells (RBCs) and transformed into the corresponding ribotide (5′-monophosphate) form. Within the present study, an analytical method was developed to determine AICAR-ribotide concentrations in RBC concentrates by means of liquid chromatography-tandem mass spectrometry. The method was validated enabling quantitative result interpretation considering the parameters specificity, precision (intra- and interday), linearity, recovery, accuracy (LOD/LOQ), stability and ion suppression. By analysing 99 RBC samples of young athletes, normal physiological levels of AICAR-ribotide were determined (10–500 ng/mL), and individual levels were found to be stable for several days. Employing in vitro incubation experiments with AICAR riboside in fresh whole blood samples, the ribotide concentrations were observed to increase significantly within 30 min from baseline to 1–10 μg/mL. These levels are considered conserved for the lifetime of the erythrocyte and, thus, the results of the in vitro model strongly support the hypothesis that measuring abnormally high AICAR-ribotide concentrations in RBC of elite athletes has the potential to uncover the misuse of this substance for a long period of time.     

Solid phase microextraction fills the gap in tissue sampling protocols

Metabolomics and biomarkers discovery are an integral part of bioanalysis. However, untargeted tissue analysis remains as the bottleneck of such studies due to the invasiveness of sample collection, as well as the laborious and time-consuming sample preparation protocols. In the current study, technology integrating in vivo sampling, sample preparation and global extraction of metabolites – solid phase microextraction was presented and evaluated during liver and lung transplantation in pig model. Sampling approaches, including selection of the probe, transportation, storage conditions and analyte coverage were discussed. The applicability of the method for metabolomics studies was demonstrated during lung transplantation experiments.     

Small changes—Huge influences: NMR chemical shifts of Ni(II) complexes with polar substrates

Neutral Ni(II) complexes have been shown to be highly valuable as robust and versatile catalysts in olefin polymerization. But they show reduced reactivity when the polar monomers methyl acrylate and vinyl acetate are incorporated. To get further insight into this behavior, NMR chemical shift calculations were performed on the system [(N,O) Ni (H) (PMe3)] 1 (N,O = ‐N,O‐{2,6‐(3,5‐(F3C)2C6H3)2C6H3) NC(H)‐3,5‐I2‐2‐O‐C6H2}). The chemical shifts show reasonable agreement with experiment but are also extremely influenced by geometrical features of the complex as well as the inserted substrate. The first prominent feature, the low‐field shift of the C_carbonyl in the incorporated monomer, can only be reproduced when it is in close proximity to the Ni and in this way hinders the attack of a new monomer. Second, the almost 100 ppm difference in the chemical shift of the carbon of the two substrates directly bound to Ni can be reasoned by the different directionality of polarization as disclosed by natural bond orbital (NBO) analysis. © 2013 Wiley Periodicals, Inc.     

Synthesis and characterization of novel carbene complexes of phosphorus(V) fluorides with potential liquid-crystalline properties

A series of novel push–pushI and push–pullII carbene-stabilized complexes of phosphorus(V) fluorides bearing substituents with liquid-crystalline properties were synthesized by the oxidative addition of difluoroamines to phosphorus(III) halides. These octahedral complexes were characterized by NMR spectroscopy and X-ray analysis.     

On the interaction of two different types of ligands binding to the same molecule part I: basics and the transfer of the decoupled sites representation to systems with n and one binding sites

The decoupled sites representation (DSR) for one type of ligand allows to regard complex overall titration curves as sum of classical Henderson-Hasselbalch (HH) titration curves. In this work we transfer this theoretical approach to molecules with different types of interacting ligands (e.g. protons and electrons), prove the existence of decoupled systems for n ₁ and one binding sites for two different ligands, and point out some difficulties and limits of this transfer. A major difference to the DSR for one type of ligand is the loss of uniqueness of the decoupled system. However, all decoupled systems share a unique set of microstate probabilities and each decoupled system corresponds to a certain permutation of these microstate probabilities. Moreover, we show that the titration curve of a certain binding site in the original system can be regarded as linear combination of the titration curves of the individual sites of the decoupled system if the weights of the linear combination are substituted by functions in the activity of the second ligand. In the underlying model with only pairwise interaction, an important observation of our theoretical investigation is the following: Even though the binding sites of ligand L ₁ may not interact directly, they can show secondary interaction due to the interaction with the second type of ligand. This means, if the activity of the second ligand is fixed and we regard the 1-dimensional titration curve of an individual binding site for ligand L ₁ depending on its activity, we may observe a strong deviation from the classical HH shape in spite of non-interacting sites for ligand L ₁.     

Coexpression of CPR from Various Origins Enhances Biotransformation Activity of Human CYPs in S. pombe

Cytochrome P450 enzymes (CYPs or P450s) are the most important enzymes involved in the phase I metabolism of drugs (and other xenobiotics) in humans, and the corresponding drug metabolites are needed as reference substances for their structural confirmation and for pharmacological or toxicological characterization. We have previously shown that biotechnological synthesis of such metabolites is feasible by whole-cell biotransformation with human CYPs recombinantly expressed in the fission yeast Schizosaccharomyces pombe. It was the aim of this study to compare the activity of seven human microsomal CYPs (CYP2C9, CYP2D6, CYP3A4, CYP3A5, CYP3A7, CYP17, and CYP21) upon coexpression with NADPH-cytochrome P450 oxidoreductases (CPRs) from various origins, namely, human CPR (hCPR) and its homologues from fission yeast (ccr1) and the bishop’s weed Ammi majus (AmCPR), respectively. For this purpose, 28 recombinant strains were needed, with five of them having been constructed previously and 23 strains being newly constructed. Bioconversion experiments showed that coexpression of a CPR does not only influence the reaction rate but, in some cases, also exerts an influence on the metabolite pattern. For CYP3A enzymes, coexpression of hCPR yielded the best results, while for another two, hCPR was equally helpful as ccr1 (both CYP17 and CYP21) or AmCPR (CYP17 only), respectively. Interestingly, CYP2D6 displayed its highest activity when coexpressed with ccr1 and CYP2C9 with AmCPR. These results corroborate the view of CPR as a well-suited bio-brick in synthetic biology for the construction of artificial enzyme complexes.     

Valence‐to‐Core X‐Ray Emission Spectroscopy of Iron–Carbonyl Complexes: Implications for the Examination of Catalytic Intermediates

Valence‐to‐core X‐ray emission spectroscopy (V2C XES) has been applied to a series of compounds relevant to both homogeneous catalysts and intermediates in heterogeneous reactions, namely [Fe(CO)₅], [Fe₂(CO)₉], [Fe₃(CO)₁₂], [Fe(CO)₃(cod)] (cod=cyclo‐octadienyl), [Fe₂Cp₂(CO)₄] (Cp=cyclo‐pentadienyl), [Fe₂Cp*₂(CO)₄] (Cp*=tetramethylcyclopentadienyl), and [FeCp(CO)₂(thf)][B(ArF)₄] (ArF=pentafluorophenyl). DFT calculations of the V2C XES spectra show very good agreement with experiment, which allows for an in depth analysis of the origins of the observed spectral signatures. It is demonstrated that the observed spectral features can be broken down into specific ligand and metal fragment contributions. The relative intensities of the observed features are further explained through a quantitative investigation of the metal 3p and 4p contributions to the spectra. The ability to use V2C XES to separate carbonyl, hydrocarbon, and solvent contributions is highlighted.     

Chemoselectivity Control: Gold(I)‐Catalyzed Synthesis of 6,7‐Dihydrobenzofuran‐4(5H)‐ones and Benzofurans from 1‐(Alkynyl)‐7‐oxabicyclo[4.1.0]heptan‐2‐ones

New and chemoselective gold(I)‐catalyzed transformations of 1‐(arylethynyl)‐7‐oxabicyclo[4.1.0]‐ heptan‐2‐ones were developed. Two completely different products—6,7‐dihydrobenzofuran‐4(5H)‐ones and benzofurans—could be obtained from the same starting material. The selectivity is determined by the ligand of the gold catalyst: triphenylphosphine delivers 6,7‐dihydrobenzofuran‐4(5H)‐ones, and 1,3‐bis(diisopropylphenyl)imidazol‐2‐ylidene leads to benzofurans. Eleven examples of each case are provided. The mechanistic suggestions for the pathways to both product types are supported by isotope labeling experiments.