title: Synthesis of α-Deoxymono and Difluorohexopyranosyl 1-Phosphates and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases.
abstract: 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 kcat/Kmchanges 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 pKa2values of analogues (1-3) were determined by proton decoupled31P and19F 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.
authors: College of Pharmacy, Dalhousie University , 5968 College Street, Halifax, Nova Scotia B3H 3J5, Canada.
authors: Jian-She
authors: JS
authors: Zhu
authors: College of Pharmacy, Dalhousie University , 5968 College Street, Halifax, Nova Scotia B3H 3J5, Canada.
authors: Nicole E
authors: NE
authors: McCormick
authors: Department of Chemistry, Dalhousie University , Halifax, Nova Scotia B3H 4R2, Canada.
authors: Shannon C
authors: SC
authors: Timmons
authors: College of Pharmacy, Dalhousie University , 5968 College Street, Halifax, Nova Scotia B3H 3J5, Canada.|Department of Chemistry, Dalhousie University , Halifax, Nova Scotia B3H 4R2, Canada.
authors: David L
authors: DL
authors: Jakeman
mesh_terms: D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D019791:Guanidine / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009713:Nucleotidyltransferases / Q000737:chemistry*; D010710:Phosphates / Q000138:chemical synthesis* / Q000737:chemistry; D013379:Substrate Specificity; D013936:Thymidine / Q000737:chemistry*
chemical_list: D010710:Phosphates; D009713:Nucleotidyltransferases; D019791:Guanidine; D013936:Thymidine