Investigation of the quantitative properties of the quadrupole orthogonal acceleration time-of-flight mass spectrometer with electrospray ionisation using 3,4-methylenedioxymethamphetamine

This paper describes the investigation of the potential of a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF) equipped with an atmospheric pressure ionisation interface for quantitative measurements of small molecules separated by reversed phase liquid chromatography. To this end, the detection limits and linear dynamic range in particular were studied in an LC/MS/MS experiment using 3,4-methylenedioxymethamphetamine standards and 3,4-methylenedioxyethylamphetamine for internal standardisation. In a second phase, the experiment was repeated with real biological extracts (whole blood, serum, and vitreous humour). A calibration for 3,4-methylenedioxymethamphetamine and its metabolite 3,4-methylenedioxyamphetamine was prepared in each of these matrices again using 3,4-methylenedioxyethylamphetamine as internal standard. The resulting quantitative data were compared with those obtained by liquid chromatography with fluorescence detection for the same extracts. The Q-TOF results revealed excellent sensitivity and a linear dynamic range of nearly four decades (2-10 000 pg on-column, r(2) = 0.9998, 1/x weighting). Furthermore, all the calibration curves prepared in biological material were superimposable, LC/MS/MS and LC-fluorescence, and the quantitative results for actual samples compared very favourably. It was concluded that the Q-TOF achieves a linear dynamic range for quantitative LC/MS/MS work exceeding that of fluorescence detection and at much better absolute sensitivity. Copyright 1999 John Wiley & Sons, Ltd.     

Target Directed Drug Synthesis: The Aminoacyl-tRNA Synthetases as Possible Targets

The “tailor-made” pharmaceutical has been an old dream ever since Paracelsus' days. His saying “Dosis sola facit venenum” is still valid. The ideal pharmacon should inhibit the pathological process or the parasitical organism to a maximum while causing as little harm as possible to the human organism. In order to achieve this goal one must try to make use of metabolic differences between the metabolism of the pathological organism and normal human metabolism in a rational way. With today's improved knowledge of enzymatic processes this seems to be a possible and highly promising approach. The pharmaceutical should act upon a process of central importance, such as the process of protein biosynthesis, where the required highly accurate construction of the macromolecules is achieved by a “proofreading” process. It is shown that this “proofreading” mechanism exhibits specific difference in different species.     

Studies Related to Norway Spruce Galactoglucomannans: Chemical Synthesis,Conformation Analysis,NMR Spectroscopic Characterization,and Molecular Recognition of Model Compounds

Galactoglucomannan (GGM) is a polysaccharide mainly consisting of mannose, glucose, and galactose. GGM is the most abundant hemicellulose in the Norway spruce (Picea abies), but is also found in the cell wall of flax seeds, tobacco plants, and kiwifruit. Although several applications for GGM polysaccharides have been developed in pulp and paper manufacturing and the food and medical industries, attempts to synthesize and study distinct fragments of this polysaccharide have not been reported previously. Herein, the synthesis of one of the core trisaccharide units of GGM together with a less‐abundant tetrasaccharide fragment is described. In addition, detailed NMR spectroscopic characterization of the model compounds, comparison of the spectral data with natural GGM, investigation of the acetyl‐group migration phenomena that takes place in the polysaccharide by using small model compounds, and a binding study between the tetrasaccharide model fragment and a galactose‐binding protein (the toxin viscumin) are reported.     

Toward bioinspired galectin mimetics: identification of ligand-contacting peptides by proteolytic-excision mass spectrometry

Clinically relevant bioactivities of human galectins (adhesion/growth-regulatory galactoside-specific lectins) inspired the design of peptides as new tools to elicit favorable effects (e.g., in growth control) or block harmful binding (e.g., in tissue invasion). To obtain the bioinspired lead compounds, we combined a proteolytic fragmentation approach without/with ligand contact (excision) with mass spectrometric identification of affinity-bound protein fragments, using galectin-1 and -3 as models. Two peptides from the carbohydrate recognition domains were obtained in each case in experimental series rigorously controlled for specificity, and the [157-162] peptide of galectin-3 proved to be active in blocking lectin binding to a neoglycoprotein and to tumor cell surfaces. This approach affords peptide sequences for structural optimization and intrafamily/phylogenetic galectin comparison at the binding-site level with a minimal requirement of protein quantity, and it is even amenable to mixtures.     

Synthesis and Conformational Analysis of (α‐D‐Galactosyl)phenylmethane and α‐,β‐Difluoromethane Analogues: Interactions with the Plant Lectin Viscumin

To bend about : The conformations of three phenyl‐C‐galactosides in solution were evaluated by using theoretical calculations and NMR spectroscopic studies. The α‐CF₂ derivative (see scheme) showed significant flexibility of the pyranose ring and around the pseudoanomeric center, whereas the other two analogues more closely resemble the natural galactosides. Regardless, all three compounds bind to a plant lectin.

     

Symmetric dithiodigalactoside: strategic combination of binding studies and detection of selectivity between a plant toxin and human lectins

Thioglycosides offer the advantage over O-glycosides to be resistant to hydrolysis. Based on initial evidence of this recognition ability for glycosyldisulfides by screening dynamic combinatorial libraries, we have now systematically studied dithiodigalactoside on a plant toxin (Viscum album agglutinin) and five human lectins (adhesion/growth-regulatory galectins with medical relevance e.g. in tumor progression and spread). Inhibition assays with surface-presented neoglycoprotein and in solution monitored by saturation transfer difference NMR spectroscopy, flanked by epitope mapping, as well as isothermal titration calorimetry revealed binding properties to VAA (K(a): 1560 ± 20 M(-1)). They were reflected by the structural model and the affinity on the level of toxin-exposed cells. In comparison, galectins were considerably less reactive, with intrafamily grading down to very minor reactivity for tandem-repeat-type galectins, as quantitated by radioassays for both domains of galectin-4. Model building indicated contact formation to be restricted to only one galactose moiety, in contrast to thiodigalactoside. The tested glycosyldisulfide exhibits selectivity between the plant toxin and the tested human lectins, and also between these proteins. Therefore, glycosyldisulfides have potential as chemical platform for inhibitor design.