Synthesis of α-Deoxymono and Difluorohexopyranosyl 1-Phosphates and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases.

Eight fluorinated isosteric α-d-glucopyranosyl 1-phosphate (Glc 1P) analogues have been synthesized. A promiscuity investigation of the thymidylyltransferase Cps2L and the guanidylyltansferase GDP-ManPP with these analogues showed that all were accepted by either enzyme, with the exception of 1,6-diphosphate 6. Kinetic parameters were determined for these analogues using a continuous coupled assay. These data demonstrated the broad substrate promiscuity of Cps2L, with k/K changes for monofluoro substitution at C-2, C-4, and C-6 and difluoro substitution at C-2 within two orders of magnitude. In contrast, the kinetic analysis of GDP-ManPP was only possible with three out of eight analogues. The pKa values of analogues (1-3) were determined by proton decoupled P and F NMR titration experiments. Counterintuitively, the axial fluoro substituent in 3 did not change chemical shift upon titration, and there was no significant increase in acidity for the difluoro analogue over the monofluoro analogues. No strong Brønsted linear free-energy correlations were observed among all five substrates (1-3, Glc 1P, and Man 1P) for either enzyme-catalyzed reactions. However, Brønsted correlations were observed among selected substrates, indicating that the acidity of the nucleophilic phosphate and the configuration of the hexose each plays a significant role in determining the substrate specificity.