Nucleotidylyltransferase inhibitors are designed to target enzymes responsible for one step of cell wall biosynthesis in Gram-positive, Gram-negative and mycobacteria. Glucose 1-phosphate thymidylyltransferase Cps2L/RmlA (EC 2.7.7.24) is an enzyme essential for the growth and proliferation of many bacteria, including Mycobacterium tuberculosis. Cps2L/RmlA serves to couple glucose 1-phosphate and deoxythymidine triphosphate to form deoxythymidine diphosphoglucose. dTDP-?-L-rhamnose acts as an inhibitor of RmlA. Phosphonates are synthetic analogues of natural phosphates that have shown widespread ability to probe biological systems. Several approaches were investigated toward the synthesis of dTDP-?-L-rhamnose analogues, which incorporated phosphonate functionality into their scaffolds. A series of L-rhamnose phosphonate and ketosephosphonate analogues, with varying degrees of fluorination about the 1C position, were synthesized. These compounds were evaluated as potential inhibitors of thymidylyltransferase Cps2L, the first enzyme in the L-rhamnose biosynthetic pathway, and a novel antibiotic target. Enzyme-inhibitor and enzyme-substrate binding experiments were performed using WaterLOGSY NMR spectroscopy for the phosphonate-based compounds and known enzyme sugar nucleotide substrates. IC50 values were measured and Ki values were calculated for the compounds determined to be inhibitors. New insights were gained into the binding promiscuity of various enzymes within the L-rhamnose biosynthetic pathway (Cps2L, RmlB-D) and the mechanism of inhibition for the most potent inhibitor, L-rhamnose-1C-phosphonate. Thiophosphates are analogues of natural phosphates in which the P—O bond has been replaced with a P—S bond. Methods were investigated for the preparation of O and S-glucosyl thiophosphates. A series new protected glucosyl thiophosphate compounds were synthesized and characterized as precursors to glucose-1-thiophopshate, a probable glucose 1-phosphate substrate analogue for Cps2L.