Synthesis of biolabile thioalkyl-protected phosphates from an easily accessible phosphotriester precursor.

Robust methods for the synthesis of mixed phosphotriesters are essential to accelerate the development of novel phosphate-containing bioactive molecules. To enable efficient cellular uptake, phosphate groups are commonly masked with biolabile protecting groups, such as S-acyl-2-thioethyl (SATE) esters, that are removed once the molecule is inside the cell. Typically, bis-SATE-protected phosphates are synthesised through phosphoramidite chemistry. This approach, however, suffers from issues with hazardous reagents and can give unreliable yields, especially when applied to the synthesis of sugar-1-phosphate derivatives as tools for metabolic oligosaccharide engineering. Here, we report the development of an alternative approach that gives access to bis-SATE phosphotriesters in two steps from an easy to synthesise tri(2-bromoethyl)phosphotriester precursor. We demonstrate the viability of this strategy using glucose as a model substrate, onto which a bis-SATE-protected phosphate is introduced either at the anomeric position or at C6. We show compability with various protecting groups and further explore the scope and limitations of the methodology on different substrates, including N-acetylhexosamine and amino acid derivatives. The new approach facilitates the synthesis of bis-SATE-protected phosphoprobes and prodrugs and provides a platform that can boost further studies aimed at exploring the unique potential of sugar phosphates as research tools.

A Multiplexing Activity-Based Protein-Profiling Platform for Dissection of a Native Bacterial Xyloglucan-Degrading System.

Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a "low-tech", easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.