Investigating the Substrate Specificity of l-Rhamnose Biosynthetic Enzymes (RmlA–D): Chemoenzymatic Synthesis of an Isosteric Nonhydrolyzable TDP-β-l-Rhamnose Phosphonate

l-Rhamnose (Rha) is widely distributed within bacteria as a component of the polysaccharides that populate bacterial cell walls. While Rha-containing polysaccharides are highly variable in both structure and function, their presence in pathogenic bacteria has provided incentive to explore inhibition of the Rha-pathway as a means toward antibiotic development. Herein, we explore the substrate scope of the Rha biosynthetic enzymes RmlB, RmlC, and RmlD, from Aneurinibacillus thermoaerophilus, with deoxyfluoro-, aminodeoxy-, and phosphonate congeners. The isosteric phosphonate was the only congener turned over by all three enzymes demonstrating conversion to 5′-deoxythymidine-C-(1-deoxy β-l-rhamnosyl bisphosphate) (TDP-1C-Rha). Scale-up of this enzymatic process enabled the isolation of TDP-1C-Rha, an isosteric nonhydrolyzable phosphonate analogue of TDP-Rha and offers an orthogonal approach to deliver phosphonate–phosphate derived enzyme probes.