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 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 andF 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.

Stereoselective Synthesis of Fluorinated Galactopyranosides as Potential Molecular Probes for Galactophilic Proteins: Assessment of Monofluorogalactoside–LecA Interactions

“…Compound 24 was isolated together with an unidentifiable impurity, and was directly subjected to acetolysis under acidic conditions. The concomitant removal of the MOM protecting group proceeded as expected, generating intermediate 25 in 52 % yield over two steps (α/β=3:1) . Finally, the anomeric groups were installed as described above, allowing the isolation of products 27 and 28 via bromide intermediate 26 .…”

Recent advances in the enzymatic synthesis of sugar-nucleotides using nucleotidylyltransferases and glycosyltransferases

“…Most recently the group have extended their investigation of Cps2L promiscuity (and a GDP-ManPP) against a panel of eight fluorinated isosteric Glc 1-phosphates, broadly reaffirming a defined utility for Cps2L in the advancement of enzymatic synthesis of mimetic sugar nucleotides and indicating that the acidity of the phosphate nucleophile and the hexose configuration each contribute to substrate specificity. 29 The Lowary group also used Cps2L for an enzymatic synthesis of dTDP-Galf, obtaining the sugar-nucleotide in 50% yield from Galf 1-phosphate using Cps2L immobilised on a Ni-NTA agarose resin. 12a=12a In 2012, Linhardt et al reported a chemical synthesis of eight different GlcNAc 1phosphate and GalNAc 1-phosphate analogues and tested their capability with GlmU (Scheme 11).…”

A Chiron approach towards the stereoselective synthesis of polyfluorinated carbohydrates

“… 27 Fluorinated carbohydrates also play interesting roles in biological systems as mechanistic probes or to modulate lectin−carbohydrate interactions. 28 – 30 Almost two decades ago, the group of DiMagno synthesized the hexafluorinated analog 3 31 (Fig. 1a ) and this compound crosses red blood cell membrane at a tenfold higher rate than glucose.…”

“…Consequently, systematic biological investigations of heavily fluorinated carbohydrates have often been impeded by the weak efficiency of the multistep sequences used in de novo approaches. 28 − 30 , 33 , 34 , 38 , 39 In addition, these long synthetic sequences generally give rise to enantiomeric or diastereomeric mixtures of products, leading to difficult silica gel chromatographies for isolation of pure products. Herein, we proposed a Chiron approach for the preparation of heavily fluorinated sugars shown in Fig.…”