The study of many of glycosyltransferases is limited due to an inadequate access to sugar nucleotides. Preparation of sugar nucleotides through the use of nucleotidylyltransferases with broad substrate specificities is gaining significant interest and offers high yields and stereospecificity. Physiologically, the glucose 1-phosphate thymidylyltransferase catalyzes the condensation of ?-D-glucose 1-phosphate and deoxythymidine triphosphate to yield deoxythymidine diphospho glucose. Exploiting and targeting these enzymes also has the potential of yielding new therapeutics. Cps2L is a thymidylyltransferase isolated from Streptococcus pneumoniae, with broad substrate flexibility. The substrate specificity of Cps2L was evaluated with new sugar 1-phosphate analogues to gain further insight into substrate and inhibitor requirements. Several sugar 1-phosphate analogues including sugar 1C-phosphonates (and analogues thereof), 2-deoxy-2-fluorosugar 1-phosphates, and glucopyranose 1- boranophosphates have been used to probe the sugar 1-phosphate modification tolerance of Cps2L. In addition, NMR spectroscopy was used to determine the anomeric stereochemistry of 2-deoxy-2-fluorosugars nucleotide products. For those substrates that were accepted by Cps2L, steady-state kinetic parameters were determined. The enzyme is able to almost equally form Michaelis complexes with different sugar substrates, whereas the turnover values for obtaining the corresponding sugar nucleotide were different. The evaluation of the substrate tolerance of Cps2L, as well as the synthesis of ?-D-glucose-1C-thiophosphonate, a difluoro and a bisphosphono analogue of ?-D-glucose 1C-phosphonate will be described.

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