TLR4 is a key pattern recognition receptor that can sense pathogen‐ and danger‐ associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro‐inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species‐specific, and barely predictable structure‐activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan‐based lipid A‐mimicking molecules that can activate human and murine TLR4‐mediated signaling with picomolar affinity. Exploiting crystal structure ‐ based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible β(1→6)‐linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non‐reducing βGlcN(1↔1′)βGlcN scaffold. Excellent stereoselectivity in a challenging β,β‐1,1′ glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of β,β‐1,1′‐linked diglucosamine‐derived glycolipids entailing multiple long‐chain (R)‐3‐ acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D‐molecular shape and conformational rigidity of unnatural β,β‐1,1′‐linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4‐mediated signaling in a species‐independent manner.
The detection of metabolites of the anti-estrogenic substance cyclofenil, listed on the World Anti-Doping Agency (WADA) Prohibited List since 2004 is described. Target substances are hydroxylated metabolites, bearing an aliphatic hydroxyl group either in the 2-, 3-or 4-position of the aliphatic ring, in addition to the phenolic functions on the aromatic rings. Structural identification used NMR as well as high-resolution mass spectrometry after nano-electrospray ionisation (ESI). Unambiguous detection of all three synthesised cyclofenil metabolites M1-M3 was done using gas chromatography for separation and electron ionisation mass spectrometry for detection of the per-silylated compounds in comparison with a reference urine deriving from an excretion study within the WADA 2007 Educational Programme.
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