Evaluation of generic chiral liquid chromatography screens for pharmaceutical analysis

Two different automated generic liquid chromatography screens for the separation of chiral compounds of pharmaceutical interest have been evaluated. The test set comprised 53 chemically diverse chiral compounds involving 55 enantiomeric pairs from the pharmaceutical industry (i.e. starting materials, synthetic intermediates and drug substances). The first screen utilised four polysaccharide-based columns with five mobile phases and showed enantioselectivity for 87% of the test compounds. The second screen employed three macrocyclic glycopeptide columns with two mobile phases and showed enantioselectivity for 65% of the test compounds. Merging of the two screening procedures resulted in an enantioselectivity for 96% of the chiral compounds. It is anticipated that the systematic use of the automated chiral HPLC screens described in this report will substantially reduce the necessary time for method development of pharmaceutically related chiral analytical methods.     

Metal complexes of macrocyclic ligands. Part XXIV. Binuclear complexes with tetraazamacrocycle-N,N′,N″,N‴-tetraacetic acids

The three ligands H₄dota, H₄teta, and H₄heta give binuclear complexes with Cu²⁺ and Ni²⁺, the spectral properties of which have been studied. The structures of Cu₂(dota)·5H₂O and Cu₂(teta)·6H₂O have been established by X-ray diffraction analysis.     

Correction of inner-filter effect in fluorescence excitation-emission matrix spectrometry using Raman scatter

Fluorescence excitation-emission matrix (EEM) spectroscopy is a useful tool for interpretation of fluorescence information from natural water samples. One of the major problems with this technique is the inner-filter effect (IFE), i.e. absorption of light at both the excitation and emission wavelengths. The common solutions are to either dilute the sample or apply some form of mathematical correction, most often based on the measured absorbance of the sample. Since dilution is not always possible, e.g. in on-line or in situ EEM recordings, and corrections based on absorbance are hampered primarily by the use of a separate absorbance instrument, neither of these solutions is optimal. In this work, we propose a mathematical correction procedure based on the intensity of Raman scatter from water. This procedure was found to reduce the error after correction by up to 50% in comparison with two absorbance correction procedures. Furthermore, it does not require the use of a separate absorbance measurement, and it is applicable to on-line and in situ EEM recordings, where the IFE would otherwise cause problems.     

What can we learn from N2O isotope data? – Analytics,processes and modelling

The isotopic composition of nitrous oxide (N₂O) provides useful information for evaluating N₂O sources and budgets. Due to the co-occurrence of multiple N₂O transformation pathways, it is, however, challenging to use isotopic information to quantify the contribution of distinct processes across variable spatiotemporal scales. Here, we present an overview of recent progress in N₂O isotopic studies and provide suggestions for future research, mainly focusing on: analytical techniques; production and consumption processes; and interpretation and modelling approaches. Comparing isotope-ratio mass spectrometry (IRMS) with laser absorption spectroscopy (LAS), we conclude that IRMS is a precise technique for laboratory analysis of N₂O isotopes, while LAS is more suitable for in situ/inline studies and offers advantages for site-specific analyses. When reviewing the link between the N₂O isotopic composition and underlying mechanisms/processes, we find that, at the molecular scale, the specific enzymes and mechanisms involved determine isotopic fractionation effects. In contrast, at plot-to-global scales, mixing of N₂O derived from different processes and their isotopic variability must be considered. We also find that dual isotope plots are effective for semi-quantitative attribution of co-occurring N₂O production and reduction processes. More recently, process-based N₂O isotopic models have been developed for natural abundance and ¹⁵N-tracing studies, and have been shown to be effective, particularly for data with adequate temporal resolution. Despite the significant progress made over the last decade, there is still great need and potential for future work, including development of analytical techniques, reference materials and inter-laboratory comparisons, further exploration of N₂O formation and destruction mechanisms, more observations across scales, and design and validation of interpretation and modelling approaches. Synthesizing all these efforts, we are confident that the N₂O isotope community will continue to advance our understanding of N₂O transformation processes in all spheres of the Earth, and in turn to gain improved constraints on regional and global budgets.     

Theoretical insights into heme-catalyzed oxidation of cyclohexane to adipic acid

Adipic acid is a key compound in the chemical industry, where it is mainly used in the production of polymers. The conventional process of its generation requires vast amounts of energy and, moreover, produces environmentally deleterious substances. Thus, there is interest in alternative ways to gain adequate amounts of adipic acid. Experimental reports on a one-pot iron-catalyzed conversion of cyclohexane to adipic acid motivated a theoretical investigation based on density functional theory calculations. The process investigated is interesting because it requires less energy than contemporary methods and does not produce environmentally harmful side products. The aim of the present contribution is to gain insight into the mechanism of the iron-catalyzed cyclohexane conversion to provide a basis for the further development of this process. The rate-limiting step of the process is discussed, but considering the accuracy of the calculations, it is difficult to ensure whether the rate-limiting step is in the substrate oxidation or in the generation of the catalytically active species. It is shown that the slowest step in the substrate oxidation is the conversion of cyclohexanol to cyclohexane-1,2-diol. Hydrogen-atom transfer from one of the OH groups of cyclohexane-1,2-diol makes the intradiol cleavage occur spontaneously.     

How is methane formed and oxidized reversibly when catalyzed by Ni-containing methyl-coenzyme M reductase?

Ni-containing methyl-coenzyme M reductase (MCR) is capable of catalyzing methane formation and has recently been observed to also be able to catalyze the reverse reaction, the anaerobic oxidation of methane. The forward reaction has been extensively studied theoretically before and was found to consist of two steps. The first step is rate-limiting and the second step was therefore treated at a lower level. For an accurate treatment of the reverse reaction, both steps have to be studied at the same level. In the present paper, the mechanisms for the reversible formation and oxidation of methane catalyzed by MCR have been investigated using hybrid density functional theory with recent developments, in particular including dispersion effects. An active-site model was constructed based on the X-ray crystal structure. The calculations indicate that the MCR reaction is indeed reversible and proceeds via a methyl radical and a Ni-S(CoM) intermediate with reasonable reaction barriers in both directions. In a competing mechanism, the formation of the crucial Ni-methyl intermediate, was found to be strongly endergonic by over 20?kcal mol(-1) (including a barrier) with dispersion and entropy effects considered, and thus would not be reachable in a reasonable time under natural conditions.     

O-O bond cleavage in dinuclear peroxo complexes of iron porphyrins: a quantum chemical study

To gain insight into the mechanisms of O2 activation and cleavage in metalloenzymes, biomimetic metal complexes have been constructed and experimentally characterized. One such model complex is the dinuclear peroxo complex of iron porphyrins observed at low temperature in a non-coordinating solvent. The present theoretical study examines the O-O bond cleavage in these complexes, experimentally observed to occur either at increased temperature or when a strongly coordinating base is added. Using hybrid density functional theory, it is shown that the O-O bond cleavage always occurs in a state where two low-spin irons (S = +/-1/2) are antiferromagnetically coupled to a diamagnetic state. This state is the ground state when the strong base is present and forms an axial ligand to the free iron positions. In contrast, without the axial ligands, the ground state of the dinuclear peroxo complex has two high-spin irons (S = +/-5/2) coupled antiferromagnetically. Thus, the activation barrier for O-O bond cleavage is higher without the base because it includes also the promotion energy from the ground state to the reacting state. It is further found that this excitation energy, going from 10 unpaired electrons in the high-spin case to 2 in the low-spin case, is unusually difficult to determine accurately from density functional theory because it is extremely sensitive to the amount of exact exchange included in the functional.     

Automated high content screening for phosphoinositide 3 kinase inhibition using an AKT 1 redistribution assay

High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we established a medium to high throughput HCS assay in the 384-well format to measure cellular type I phosphoinositide 3 kinase (PI3K) activity. Type I PI3K is involved in several intracellular pathways such as cell survival, growth and differentiation as well as immunological responses. As a cellular model system we used Chinese Hamster Ovary (CHO) cells that had been stably transfected with human insulin receptor (hIR) and an AKT1-enhanced green fluorescent protein (EGFP) fusion construct. Upon stimulation of the hIR with insulin-like growth factor-1 (IGF-1), PI3K was activated to phosphorylate phosphatidylinositol (PtdIns)-4,5-bisphosphate at the 3-position, resulting in the recruitment of AKT1-EGFP to the plasma membrane. The AKT1-EGFP redistribution assay was robust and displayed little day-to-day variability, the quantification of the fluorescence intensity associated with plasma membrane spots delivered good Z' statistics. A novel format of compound dose-response testing was employed using serial dilutions of test compounds across consecutive microtiter plates (MTPs). The dose response testing of a PI3K inhibitor series provided reproducible IC50 values. The profiling of the redistribution assay with isoform-selective inhibitors indicates that PI3Kalpha is the main isoform activated in the CHO host cells after IGF-1 stimulation. Toxic compound side effects could be determined using automated image analysis. We conclude that the AKT1-EGFP redistribution assay represents a solid medium/high throughput screening (MTS/HTS) format to determine the cellular activity of PI3K inhibitors under conditions of growth factor stimulation.