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

Eight fluorinated isosteric α-d-glucopyranosyl 1-phosphate (<b>Glc 1P</b>) 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 <b>6</b>. Kinetic parameters were determined for these analogues using a continuous coupled assay. These data demonstrated the broad substrate promiscuity of Cps2L, with <i>k</i>_cat/<i>K</i>_m 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 p<i>K</i>a₂ values of analogues (<b>1</b>–<b>3</b>) were determined by proton decoupled ³¹P and ¹⁹F NMR titration experiments. Counterintuitively, the axial fluoro substituent in <b>3</b> 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 (<b>1</b>–<b>3</b>, <b>Glc 1P</b>, and <b>Man 1P</b>) 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

Legislative History: An Act to Amend the Charter of the Winterport Water District (HP1719)(LD 2207)

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