Synthesis and screening of alkyne-tagged ribitol-5-phosphate as metabolic labelling tools to study α-dystroglycan

α-Dystroglycan (α-DG) is a cell surface protein found on the membranes of peripheral nerve, skeletal muscle, and brain tissues. Heavily glycosylated, one of its glycans, known as core M3, is essential for mediating interactions between the cell and laminins in the extracellular matrix. Disruptions in the normal biosynthesis of this glycan lead to a range of congenital muscular dystrophies, categorised as α-dystroglycanopathies. In 2016, two research groups, independently elucidated the structure of a linker consisting of a tandem ribitol-5-phosphate (Rbo5P) unit, crucial for maintaining the extracellular interactions. This discovery marked the first and, to date, only identification of Rbo5P in mammalian tissue, making it a unique and valuable target for labelling α-DG. This work details the challenging synthesis of multiple alkyne-tagged ribitol and Rbo5P metabolic labelling tools. These tools were successfully synthesised using a novel methodology for the installation of cell-labile protected phosphates, avoiding the need for hazardous phosphoramidite intermediates. The scope of this reaction was also explored and applied to a range of carbohydrate and amino acid derivatives. These innovative metabolic labelling tools were employed in mammalian cells, and their biological activity was assessed through bioorthogonal reactions with azide-containing reporter groups and monitored with in-gel fluorescence and mass spectroscopy. This represents the first chemical tool developed to fluorescently label overexpressed α-DG. Additionally, this work demonstrates the first probing of mammalian cells for the presence of unidentified glycoproteins modified with ribitol or Rbo5P.