Development of a colourimetric assay for glycosynthases

The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration. In the present paper, we describe a new version of a glycosynthase activity assay using a silyl ether of p-nitrophenol, allowing fast reliable detection of fluoride even at concentrations of 4 mM and higher. This improvement of detection allows not only screening and identification but also kinetic characterisation of glycosynthases and synthetic reactions in a fast microtiter plate format. The applicability of the assay was successfully demonstrated by the biochemical characterisation of the mesophilic β-glucosynthase of Abg-E358S (Rhizobium radiobacter) and psychrotolerant β-glucosynthase BglU-E377A (Micrococcus antarcticus). The limitation of hyperthermophilic glycosidases as potential glycosynthases, when using glycosyl fluoride donors, was also illustrated by the example of the putative β-galactosidase GalPf from Pyrococcus furiosus

Enantioselective total synthesis of altersolanol A and N

The development of the first enantioselective total synthesis of altersolanol N is reported. The decisive step of the synthesis is the enantioselective formation of the tetrahydroanthraquinone nucleus by a [4 + 2]-cycloaddition in high yield and with excellent diastereo- and enantioselectivity (>95:5 dr and 95:5 er). In addition, a demanding selective monoacetylation of the OH group at the C-2 position was achieved: an epoxide ring opening with the participation of a neighbouring acetyl group could be established. The route proved to be an efficient alternative to also access enantiomerically pure altersolanol A

Metabolic resistance of the D-peptide RD2 developed for direct elimination of amyloid-β oligomers

Abstract Alzheimer’s disease (AD) is a neurodegenerative disorder leading to dementia. Aggregation of the amyloid-β peptide (Aβ) plays an important role in the disease, with Aβ oligomers representing the most toxic species. Previously, we have developed the Aβ oligomer eliminating therapeutic compound RD2 consisting solely of D-enantiomeric amino acid residues. RD2 has been described to have an oral bioavailability of more than 75% and to improve cognition in transgenic Alzheimer’s disease mouse models after oral administration. In the present study, we further examined the stability of RD2 in simulated gastrointestinal fluids, blood plasma and liver microsomes. In addition, we have examined whether RD2 is a substrate for the human D-amino acid oxidase (hDAAO). Furthermore, metabolite profiles of RD2 incubated in human, rodent and non-rodent liver microsomes were compared across species to search for human-specific metabolites that might possibly constitute a threat when applying the compound in humans. RD2 was remarkably resistant against metabolization in all investigated media and not converted by hDAAO. Moreover, RD2 did not influence the activity of any of the tested enzymes. In conclusion, the high stability and the absence of relevant human-specific metabolites support RD2 to be safe for oral administration in humans