Identification of extracellular nanoparticle subsets by nuclear magnetic resonance

Exosomes are a subset of secreted lipid envelope-encapsulated extracellular vesicles (EVs) of 50–150 nm diameter that can transfer cargo from donor to acceptor cells. In the current purification protocols of exosomes, many smaller and larger nanoparticles such as lipoproteins, exomers and microvesicles are typically co-isolated as well. Particle size distribution is one important characteristics of EV samples, as it reflects the cellular origin of EVs and the purity of the isolation. However, most of the physicochemical analytical methods today cannot illustrate the smallest exosomes and other small particles like the exomers. Here, we demonstrate that diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) method enables the determination of a very broad distribution of extracellular nanoparticles, ranging from 1 to 500 nm. The range covers sizes of all particles included in EV samples after isolation. The method is non-invasive, as it does not require any labelling or other chemical modification. We investigated EVs secreted from milk as well as embryonic kidney and renal carcinoma cells. Western blot analysis and immuno-electron microscopy confirmed expression of exosomal markers such as ALIX, TSG101, CD81, CD9, and CD63 in the EV samples. In addition to the larger particles observed by nanoparticle tracking analysis (NTA) in the range of 70–500 nm, the DOSY distributions include a significant number of smaller particles in the range of 10–70 nm, which are visible also in transmission electron microscopy images but invisible in NTA. Furthermore, we demonstrate that hyperpolarized chemical exchange saturation transfer (Hyper-CEST) with ¹²⁹Xe NMR indicates also the existence of smaller and larger nanoparticles in the EV samples, providing also additional support for DOSY results. The method implies also that the Xe exchange is significantly faster in the EV pool than in the lipoprotein/exomer pool.

Diffusion and xenon NMR based methods to determine a very broad range of sizes and sub-sets of extracellular vesicles.     

Mass spectrometric analysis of ceramide perturbations in brain and fibroblasts of mice and human patients with peroxisomal disorders

In this study, the levels and composition of ceramides in brains of newborn mice lacking peroxisomes (Pex5-/-, Zellweger mice) were analyzed using normal-phase high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS). Total ceramide compositions were found to be comparable to that of control animals. However, a minor ceramide species, containing hexacosanoic/hexacosenoic acid as the amide fatty acid, was 9-fold increased. Also, in the sphingomyelin-derived ceramides this species was elevated. Subsequent analysis of extracts from fibroblasts of Pex5-/- mice and mice with a defective peroxisomal beta-oxidation (lacking D-specific multifunctional protein 2 (MFP2)), revealed, again, a similar rise in this particular ceramide. Further, this ceramide was elevated in human X-ALD fibroblasts as well. Whether C26:1/0-ceramide is linked to some of the pathology seen in Zellweger syndrome remains to be investigated. However, an increase in this sphingolipid can be considered as a diagnostic criterion for diseases caused by defects in peroxisome biogenesis or peroxisomal beta-oxidation.     

Breakthrough of polymers in interactive liquid chromatography

Two separate peaks are observed for narrow polymer standards in both isocratic and gradient HPLC. One peak appears around the solvent front (the "solvent-plug peak" or "breakthrough peak"), whereas the second peak is retained significantly--or even highly. Although the effect has been observed many times before, it has never been rigorously explained. In this paper we provide a detailed explanation for the breakthrough peak. The two completely separate peaks are demonstrated not to represent to different fractions of the sample (e.g., the low- and high-molecular-mass parts of the distribution). Both peaks are representative of the entire polymeric sample for narrow polymer standard. Because the amount of the polymer in the breakthrough peak may vary, the quantitative analysis of the polymers by LC is jeopardized. The effects of the sample solvent, the (initial) mobile phase composition, the injection volume, the injected sample concentration, the column temperature, and the analyte structure and molecular mass on the breakthrough peak were investigated in LC experiments involving standards of polystyrene and poly(methyl methacrylate). Three necessary and sufficient conditionsare suggested for the breakthrough phenomenon to be observed. Recommendations to avoid the breakthrough phenomenon are given, culminating in a structured method for selecting the best possible sample solvents.     

Novel analogues of 3-substituted-2,3-dihydro-1,4-dioxino[2,3-b]pyridines: modifications in the dioxane ring

The synthesis of a series of novel analogues of the 3-substituted-2,3-dihydro-1,4-dioxino[2,3-b]pyridine core, modified in the non-aromatic ring, is described. Due to the presence of a versatile hydroxymethyl group in their structure, these novel scaffolds are attractive intermediates for the preparation of potential new therapeutic agents.     

Synthesis and properties of allenic natural products and pharmaceuticals

Nowadays, about 150 natural products comprising an allenic or cumulenic structure are known. The chemistry of these compounds has turned out to be a very attractive and prolific area of interest: advances in the isolation and characterization of new allenic natural products have led to the establishment of efficient synthetic procedures which in many cases also open up an access to enantiomerically pure target molecules. Inspired by the intriguing biological activities of many allenic natural products, allene moieties are now systematically introduced in pharmacologically active classes of compounds (steroids, prostaglandins, amino acids, nucleosides). The functionalized allenes thus obtained often exhibit impressive activities as mechanism-based enzyme inhibitors, cytotoxic, or antiviral agents. A prerequisite for further developments in this field is the efficient stereoselective synthesis of allene derivatives.     

Electron microscopic phase analysis of BN-thin films

A series of BN films was deposited by means of r.f. magnetron sputtering of a h-BN target onto Si(1OO) surfaces. Hereby, the substrate bias voltage was varied. Special interest is focussed to the influence of the deposition parameters on the orientation of the growing hexagonal BN film with respect to the substrate. For structural investigation, cross section samples were prepared. In addition to HRTEM and diffraction investigations, especially electron energy loss spectroscopy (EELS) was applied successfully for phase identification. For negative bias voltages of U _B =–300 V and U _B =–350V, we found a phase system consisting of a first-grown 25 nm thick layer of hexagonal structure with the c axis parallel to the substrate surface followed by the cubic phase.Dedicated to Professor Dr. rer. nat. Dr. h.c. Hubertus Nickel on the occasion of his 65th birthday     

Chiral bidentate aminophosphine ligands: synthesis, coordination chemistry and asymmetric catalysis

Chiral aminophosphines Ph2PN(R)(CH2)nN(R)PPh2 1-4 [n= 2, R = CH(CH3)(Ph) 1; n= 3, R = CH(CH2CH3)(Ph) 2, n= 2, R = CH(CH3)(1-naphthyl) 3; n= 2, R = CH(CH3)(C6H11) 4] were synthesized by the reaction of ClPPh2 with the appropriate easily accessible enantiopure amine building blocks. For compounds 1 and 2, the corresponding selenides 5 and 6 were prepared to determine the electronic character of the phosphine moieties. By reaction of 1 with either PdCl2(cod) or PdCl(CH3)(cod) the cis-complexes 7 and 8 were obtained. The molecular structure for complex 7, cis-[PdCl2(1)], was determined by X-ray crystallography. Reaction of PtCl2(cod) with 1 or 2 yielded the corresponding monomeric cis-isomers 9 and 10. The rhodium derivative [RhCl(CO)(1)] (11) was obtained as a mixture of cis and trans-isomers. Preliminary results in the rhodium catalyzed hydroformylation of styrene and vinyl acetate, with ee's up to 51% and high regioselectivities, showed the potential of these chiral aminophosphines for homogeneous catalysis.     

A new approach for fluorine determination by solid sampling graphite furnace molecular absorption spectrometry

This work deals with the determination of fluorine by solid sampling graphite furnace molecular absorption spectrometry. The molecular absorbance of aluminum monofluoride (AlF), which is produced in the vapor phase in the presence of Al³⁺, is measured at 227.5 nm, a non-resonant platinum line. A conventional graphite furnace program has been used with pyrolysis and vaporization temperatures of 800 and 2300 °C, respectively. Solutions of Ba²⁺ and Al³⁺ have been used to avoid fluorine losses during the pyrolysis stage and to produce AlF in the vaporization stage, respectively. Certified coal and alumina samples were analyzed using aqueous standards for calibration. The agreement between the found concentration and the certified value, or the value obtained by another method ranged from 92 to 105%, with a relative standard deviation less than 8.5%. The limit of detection and the characteristic mass was 0.17 μg g^− 1 and 205 pg, respectively.     

Perfluormethyl-Element-Liganden. XXIX. Darstellung und spektroskopische Untersuchungen von M(CO)4L2-Komplexen (M = Cr,Mo, W; L = Me2PSMe,Me2PSeMe, (CF3)2PSMe, (CF3)2PSeMe)

Perfluoromethyl Element Ligands. XXIX. Preparation and Spectroscopic Investigation of M(CO)₄L₂ Complexes (M ? Cr, Mo, W; L ? Me₂PSMe, Me₂PSeMe, (CF₃)₂PSMe, (CF₃)₂PSMe) The complexes M(CO)₄L₂ (see Inhaltsübersicht) have been prepared by the reaction of tetracarbonyl norbornadiene metal compounds M(CO)₄NBD with L at room temperature or 35°C, respectively. The cis-complexes formed in the first step undergo rearrangement to trans-isomers at higher temperatures. New compounds have been characterized by analytical and spectroscopic (IR, NMR, MS) methods.