Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases

Acceptor reactivity in glycosylation reactions

The outcome of a glycosylation reaction critically depends on the reactivity of all reaction partners involved: The donor glycoside (the electrophile), the activator (that generally provides the leaving group on the activated donor species) and the glycosyl acceptor (the nucleophile). The influence of the donor on the outcome of a glycosylation reaction is well appreciated and documented. Differences in donor reactivity have led to the development of chemoselective glycosylation reactions and the reactivity of donor glycosides has been tuned to affect stereoselective glycosylation reactions. The quantification of donor reactivity has enabled the conception of streamlined one-pot glycosylation sequences. In contrast, although it has long been known that the nature and the reactivity of the nucleophile influence the outcome of a glycosylation, the knowledge of acceptor reactivity and insight into the consequences thereof are often circumstantial or anecdotal. This review documents how the reactivity impacts the glycosylation reaction outcome both in terms of chemical yield and stereoselectivity. The effect of acceptor nucleophilicity on the reaction mechanism is described and steric, conformational and electronic influences are outlined. Quantitative and computational approaches to comprehend acceptor nucleophilicity are assessed. The increasing insight into the stereoelectronic effects governing glycoside reactivity will eventually enable the conception of effective stereoselective glycosylation methodology that can be tuned to the reaction partners at hand

Dual-Participation Protecting Group Solves the Anomeric Stereocontrol Problems in Glycosylation Reactions

The 2,2-dimethyl-2-(ortho-nitrophenyl)acetyl (DMNPA) group permits, via robust neighboring group participation (NGP) or long distance participation (LDP) effects, the stereocontrolled 1,2-trans, 1,2-cis, as well as β-2,6-dideoxy glycosidic bond generation, while suppressing the undesired orthoester byproduct formation. The robust stereocontrol capability of the DMNPA is due to the dual-participation effect from both the ester functionality and the nitro group, verified by control reactions and DFT calculations and further corroborated by X-ray spectroscopy.Bio-organic Synthesi