Position-specific isotope analysis of the methyl group carbon in methylcobalamin for the investigation of biomethylation processes

In the environment, the methylation of metal(loid)s is a widespread phenomenon, which enhances both biomobility as well as mostly the toxicity of the precursory metal(loid)s. Different reaction mechanisms have been proposed for arsenic, but not really proven yet. Here, carbon isotope analysis can foster our understanding of these processes, as the extent of the isotopic fractionation allows to differentiate between different types of reaction, such as concerted (S_N2) or stepwise nucleophilic substitution (S_N1) as well as to determine the origin of the methyl group. However, for the determination of the kinetic isotope effect the initial isotopic value of the transferred methyl group has to be determined. To that end, we used hydroiodic acid for abstraction of the methyl group from methylcobalamin (CH₃Cob) or S-adenosyl methionine (SAM) and subsequent analysis of the formed methyl iodide by gas chromatography (GC) isotope ratio mass spectrometry (IRMS). In addition, three further independent methods have been investigated to determine the position-specific δ ¹³C value of CH₃Cob involving photolytic cleavage with different additives or thermolytic cleavage of the methyl-cobalt bonding and subsequent measurement of the formed methane by GC-IRMS. The thermolytic cleavage gave comparable results as the abstraction using HI. In contrast, photolysis led to an isotopic fractionation of about 7 to 9 ‰. Furthermore, we extended a recently developed method for the determination of carbon isotope ratios of organometal(loid)s in complex matrices using hydride generation for volatilization and matrix separation before heart-cut GC and IRMS to the analysis of the low boiling partly methylated arsenicals, which are formed in the course of arsenic methylation. Finally, we demonstrated the applicability of this methodology by investigation of carbon fractionation due to the methyl transfer from CH₃Cob to arsenic induced by glutathione.

Integrated biological–chemical approach for the isolation and selection of polyaromatic mutagens in surface waters

Many environmental mutagens, including polyaromatic compounds are present in surface waters, often in complex mixtures and at low concentrations. The present study provides and applies a novel, integrated approach to isolate polyaromatic mutagens in river water using a sample from the River Elbe. The sample was taken downstream of industrial discharges using blue rayon (BR) as a passive sampler that selectively adsorbs polyaromatic compounds and was subjected to effect-directed fractionation in order to characterise the compounds causing the detected effect(s). The procedure relies on three complementary fractionation steps, the Ames fluctuation assay with strains TA98, YG1024 and YG1041 with and without S9 activation and analytical screening. Several mutagenic fractions were isolated by combining mutagenicity testing with fractionation. The enhanced mutagenicity in the nitroreductase and/or O-acetyltransferase overexpressing strains YG1024 and YG1041 strains suggested amino- and/or nitro-compounds causing mutagenicity in several fractions. Analytical screening of mutagenic fractions with LC-HRMS/MS provided a list of molecular formulas typically containing one to ten nitrogen and at least two oxygen atoms supporting the presence of amino and nitro-compounds in the mutagenic fractions.

Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Regioselective Synthesis of Trichloromethyl‐Substituted Salicylates and Cyclohexenones by One‐Pot Cyclizations of 1,3‐Bis(trimethylsilyloxy)buta‐1,3‐dienes

A variety of 6‐(trichloromethyl)salicylates (=2‐hydroxy‐6‐(trichloromethyl)benzoates) were prepared by TiCl₄‐mediated cyclization of 1,3‐bis(trimethylsilyloxy)buta‐1,3‐dienes with 1,1,1‐trichloro‐4,4‐dimethoxybut‐3‐en‐2‐one. The employment of trimethylsilyl trifluoromethanesulfonate (Me₃SiOTf) as Lewis acid resulted in the formation of trichloromethyl‐substituted cyclohexenones. The cyclizations proceeded with good‐to‐very‐good regioselectivities.     

Synthesis of benzaldehyde-functionalized LewisX trisaccharide analogs for glyco-SAM formation

LewisX (Le^x) antigen based carbohydrate–carbohydrate interactions are mediated by complexation of metal ions. Although theoretical studies about the influence of participating hydroxyl groups in the Le^x trisaccharide head group (Galβ(1-4)[Fucα(1-3)]GlcNAc) could gave same rudimental information about the basic mechanism behind this interaction, a little is known about orientation and configuration of the hydroxyl groups required for the specific interaction mediated by Ca²⁺ complexation. Therefore, there is a need of non-natural derivatives to provide detailed information about the requirements for hydroxyl group arrangement in Le^x head group surface plasmon resonance and gold nanoparticle techniques have shown to be powerful tools to investigate carbohydrate–carbohydrate interactions. Benzaldehyde-functionalized glycans can be used for attachment to both gold nanoparticles and surface plasmon resonance sensor surfaces. Therefore, seven benzaldehyde equipped Le^x analogs including the natural trisaccharide were synthesized utilizing convergent approach. The derivatives were applied in ongoing carbohydrate–carbohydrate interaction studies by surface plasmon resonance experiments to prove theoretical postulate about the structural requirements of hydroxyl group arrangements in Le^x trisaccharides.     

An efficient and convenient synthesis of unsymmetrical disulfides from thioacetates

We have developed convenient methods for the synthesis of functionalized unsymmetrical dialkyl disulfides under mild conditions in very good yields. The designed method is based on the reaction of (5,5-dimethyl-2-thioxo-1,3,2-dioxaphosphorinan-2-yl)-disulfanyl derivatives 1 with functionalized alkyl thiolate anions, generated in situ from thioacetates 2 and sodium methoxide or butylamine. The developed method allows the preparation of unsymmetrical disulfides bearing additional hydroxy, carboxy, amino, azido, biotin, or maleimide functionalities.