Measurement uncertainty and the use of reference materials

The use of (certified) reference materials and quality control materials can form a suitable basis for evaluating measurement uncertainty of routine measurements. In particular when these materials are used for quality control purposes, it is not always evident how the quality control data can be used in the uncertainty budget of a routine measurement. Current guidance documents on the evaluation of measurement uncertainty and the use of reference materials treat this subject only in part, or in very generic terms. ISO/REMCO has established a new working group that will provide practical guidance and examples on how to use quality control data in the evaluation of measurement uncertainty. A short introduction to the subject is given.

Dynamic imaging of single DNA-protein interactions using atomic force microscopy

Atomic force microscopy (AFM) imaging of static DNA-protein complexes, in air and in liquid, can be used to directly obtain quantitative and qualitative information on the structure of different complexes. For example, DNA length, the location of preferential binding sites for proteins and bending of DNA as a result of the complexation can all be measured. Recording consecutive AFM images of DNA and protein molecules under conditions that they are still able to move and interact, or dynamic AFM imaging, however, can reveal information on the dynamic aspects of the interactions between these molecules. Here, an overview is given of the technical challenges that need to be considered for successful dynamic AFM imaging studies of individual DNA-protein interactions. Necessary technical improvements to the AFM set-up and the development of new sample preparation methods are described in this paper.     

Natural deep eutectic solvents as new potential media for green technology

Developing new green solvents is one of the key subjects in Green Chemistry. Ionic liquids (ILs) and deep eutectic solvents, thus, have been paid great attention to replace current harsh organic solvents and have been applied to many chemical processing such as extraction and synthesis. However, current ionic liquids and deep eutectic solvents have still limitations to be applied to a real chemical industry due to toxicity against human and environment and high cost of ILs and solid state of most deep eutectic solvents at room temperature. Recently we discovered that many plant abundant primary metabolites changed their state from solid to liquid when they were mixed in proper ratio. This finding made us hypothesize that natural deep eutectic solvents (NADES) play a role as alternative media to water in living organisms and tested a wide range of natural products, which resulted in discovery of over 100 NADES from nature. In order to prove deep eutectic feature the interaction between the molecules was investigated by nuclear magnetic resonance spectroscopy. All the tested NADES show clear hydrogen bonding between components. As next step physical properties of NADES such as water activity, density, viscosity, polarity and thermal properties were measured as well as the effect of water on the physical properties. In the last stage the novel NADES were applied to the solubilization of wide range of biomolecules such as non-water soluble bioactive natural products, gluten, starch, and DNA. In most cases the solubility of the biomolecules evaluated in this study was greatly higher than water. Based on the results the novel NADES may be expected as potential green solvents at room temperature in diverse fields of chemistry.     

Hyphenation of optimized microfluidic sample preparation with nano liquid chromatography for faster and greener alkaloid analysis

A glass liquid–liquid extraction (LLE) microchip with three parallel 3.5 cm long and 100 μm wide interconnecting channels was optimized in terms of more environmentally friendly (greener) solvents and extraction efficiency. In addition, the optimized chip was successfully hyphenated with nano-liquid chromatography with ultraviolet and mass spectrometric detection (nanoLC–UV–MS) for on-line analysis. In this system, sample pretreatment, separation and detection are integrated, which significantly shortens the analysis time, saves labor and drastically reduces solvent consumption. Strychnine was used as model analyte to determine the extraction efficiency of the optimized 3-phase chip. Influence of organic solvent, pH of feed phase, type of alkaloid, and flow rates were investigated. The results demonstrated that the 3-phase chip nanoLC–UV/MS hyphenation combines rapid (∼25 s) and efficient (extraction efficiency >90%) sample prep, with automated alkaloid analyses. The method was applied to real samples including Strychnos nux-vomica seeds, Cephaelis ipecacuanha roots, Atropa belladonna leaves, and Vinca minor leaves.     

A new approach to untargeted integration of high resolution liquid chromatography–mass spectrometry data

Because of its high sensitivity and specificity, hyphenated mass spectrometry has become the predominant method to detect and quantify metabolites present in bio-samples relevant for all sorts of life science studies being executed. In contrast to targeted methods that are dedicated to specific features, global profiling acquisition methods allow new unspecific metabolites to be analyzed. The challenge with these so-called untargeted methods is the proper and automated extraction and integration of features that could be of relevance. We propose a new algorithm that enables untargeted integration of samples that are measured with high resolution liquid chromatography–mass spectrometry (LC–MS). In contrast to other approaches limited user interaction is needed allowing also less experienced users to integrate their data. The large amount of single features that are found within a sample is combined to a smaller list of, compound-related, grouped feature-sets representative for that sample. These feature-sets allow for easier interpretation and identification and as important, easier matching over samples. We show that the automatic obtained integration results for a set of known target metabolites match those generated with vendor software but that at least 10 times more feature-sets are extracted as well. We demonstrate our approach using high resolution LC–MS data acquired for 128 samples on a lipidomics platform. The data was also processed in a targeted manner (with a combination of automatic and manual integration) using vendor software for a set of 174 targets. As our untargeted extraction procedure is run per sample and per mass trace the implementation of it is scalable. Because of the generic approach, we envision that this data extraction lipids method will be used in a targeted as well as untargeted analysis of many different kinds of TOF-MS data, even CE- and GC–MS data or MRM. The Matlab package is available for download on request and efforts are directed toward a user-friendly Windows executable.     

Synthesis of macrocyclic peptide nucleic acid derivatives via intramolecular chemical ligation

Macrocyclic peptide nucleic acids can be obtained by direct or indirect intramolecular cyclisation of N-terminal cysteine functionalised PNA-thioesters     

Stereochemistry of intramolecular Diels-Alder furan (IMDAF) reactions of furyl-substituted chiral ethanolamides

This paper describes the stereoselective outcome of the intramolecular Diels-Alder furan (IMDAF) reaction of substituted (2S,3S)-ethanolamides 9-13, which were synthesised from a furyl substituted cyanohydrin. The latter was obtained from 2-furaldehyde with high enantioselectivity by an oxynitrilase catalysed addition of hydrogen cyanide. The stereochemistry of the IMDAF products was shown to be dependent on the size of the ethanolamide substituents R. Small substituents (H, Me, CN) gave exclusively exo-cycloaddition, whereas more bulky ones (Ph, Et) gave both evo- and endo-addition, the larger phenyl substituent giving a high endo-exo-ratio.     

Biosynthesis of anthecotuloide,an irregular sesquiterpene lactone from Anthemis cotula L. (Asteraceae) via a non-farnesyl diphosphate route

Retrobiosynthetic analysis of the allergenic sesquiterpene lactone, anthecotuloide, suggested that this natural product could be formed either by head to head condensation of geranyl diphosphate with dimethylallyl diphosphate, or from farnesyl diphosphate (FPP), the accepted regular sesquiterpene precursor via the rearrangement of a germacranolide precursor. Isotopic labelling of anthecotuloide has now been achieved by feeding [1-(13)C]-glucose, [U-13C6]-glucose and [6,6-(2)H2]-glucose to aseptically grown plantlets of Anthemis cotula(family Asteraceae). Analysis of labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that the isoprene building blocks of this sesquiterpene are formed exclusively via the MEP terpene biosynthetic pathway. This was supported by results from an experiment using [U-13C6]-glucose. A deuterium labelling experiment using [6,6-(2)H2]-glucose supported the original proposal and showed that anthecotuloide is formed from a non FPP precursor. Isotope ratio mass spectrometry suggested that there were two pathways for sesquiterpene biosynthesis in A. cotula.     

Alcoholysis of acylpalladium(II) complexes relevant to the alternating copolymerization of ethene and carbon monoxide and the alkoxycarbonylation of alkenes: the importance of Cis-coordinating phosphines

The mechanism and kinetics of the solvolysis of complexes of the type [(L-L)Pd(C(O)CH(3))(S)](+)[CF(3)SO(3)](-) (L-L = diphosphine ligand, S = solvent, CO, or donor atom in the ligand backbone) was studied by NMR and UV-vis spectroscopy with the use of the ligands a-j: SPANphos (a), dtbpf (b), Xantphos (c), dippf (d), DPEphos (e), dtbpx (f), dppf (g), dppp (h), calix-6-diphosphite (j). Acetyl palladium complexes containing trans-coordinating ligands that resist cis coordination (SPANphos, dtbpf) showed no methanolysis. Trans complexes that can undergo isomerization to the cis analogue (Xantphos, dippf, DPEphos) showed methanolyis of the acyl group at a moderate rate. The reaction of [trans-(DPEphos)Pd(C(O)CH(3))](+)[CF(3)SO(3)](-) (2e) with methanol shows a large negative entropy of activation. Cis complexes underwent competing decarbonylation and methanolysis with the exception of 2j, [cis-(calix-diphosphite)Pd(C(O)CH(3))(CD(3)OD)](+)[CF(3)SO(3)](-). The calix-6-diphosphite complex showed a large positive entropy of activation. It is concluded that ester elimination from acylpalladium complexes with alcohols requires cis geometry of the acyl group and coordinating alcohol. The reductive elimination of methyl acetate is described as a migratory elimination or a 1,2-shift of the alkoxy group from palladium to the acyl carbon atom. Cis complexes with bulky ligands such as dtbpx undergo an extremely fast methanolysis. An increasing steric bulk of the ligand favors the formation of methyl propanoate relative to the insertion of ethene leading to formation of oligomers or polymers in the catalytic reaction of ethene, carbon monoxide, and methanol.     

1H detection in MAS solid-state NMR spectroscopy of biomacromolecules employing pulsed field gradients for residual solvent suppression

In this communication, we demonstrate the feasibility of 1H detection in MAS solid-state NMR for a microcrystalline, uniformly 2H,15N-labeled sample of a SH3 domain of chicken alpha-spectrin, using pulsed field gradients for suppression of water magnetization. Today, B0 gradients are employed routinely in solution-state NMR for coherence order selection and solvent suppression. We suggest to use gradients to purge water magnetization which cannot be suppressed using conventional water suppression schemes. The achievable gain in sensitivity for 1H detection is in the order of 5 compared to the 15N detected version of the experiment (at a MAS rotation frequency of 13.5 kHz). We expect that this labeling concept which achieves high sensitivity due to 1H detection, in combination with the possibility to measure long range 1H-1H distances as we have shown previously, to be a useful tool for the determination of protein structures in the solid state.