Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase.

The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The Km values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for kcat were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed.