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

Structural and functional insight into human O-GlcNAcase

O-GlcNAc hydrolase (OGA) removes O-linked N-acetylglucosamine (O-GlcNAc) from a myriad of nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments, we determined the three-dimensional structure of human OGA, revealing an unusual helix-exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors that have clinical value

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

Phosphates are regularly incorporated into bioactive small molecules, for example in sugar-1-phosphate derivatives that are used for metabolic oligosaccharide engineering. To enable efficient cellular uptake, the phosphate groups are commonly masked with biolabile S-acyl-2-thioethyl (SATE) protecting groups that are removed once the molecule is inside the cell. Typically, SATE-protected monophosphates are synthesised through phosphoramidite chemistry, which suffers from issues with hazardous and unstable reagents and can give unreliable yields. Here, we report the development of an alternative approach that makes use of an easy to synthesise tri(2-bromoethyl)phosphotriester precursor, providing access to bis-SATE-protected mixed phosphotriesters in two steps. We demonstrate the viability of our strategy on tetrabenzylated glucose as a model monosaccharide, onto which a bis-SATE-protected phosphate is introduced at either the anomeric position or at C6. We also show compability with various protecting groups and further explore the scope and limitations of the approach on different substrates, including N-acetylhexosamines and amino acid derivatives

Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates

The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells has proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here we report on the creation of fluorescent uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time and dose dependent manner. Since no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular phys-iology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-colour pulse chase ex-periments and monitoring OGT activity on specific protein substrate in live cells