/npsi/ctrl?lang=en http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/ctrl?lang=fr Access and use of this website and the material on it are subject to the Terms and Conditions set forth at http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/jsp/nparc_cp.jsp?lang=en NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. For the publisher's version, please access the DOI link below./ Pour consulter la version de l'éditeur, utilisez le lien DOI ci-dessous.http://dx.doi.org/10.1021/jo401542sThe Journal of Organic Chemistry, 78, 19, pp. 9822-9833, 2013-09-10 ABSTRACT: We report the synthesis of a series of phosphonates and ketosephosphonates possessing an L-rhamnose scaffold with varying degrees of fluorination. These compounds were evaluated as potential inhibitors of α-D-glucose 1-phosphate thymidylyltransferase (Cps2L), the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis, and a novel antibiotic target. Enzyme−substrate and enzyme−inhibitor binding experiments were performed using water-ligand observed binding via gradient spectroscopy (WaterLOGSY) NMR for known sugar nucleotide substrates and selected phosphonate analogues. IC 50 values were measured and K i values were calculated for inhibitors. New insights were gained into the binding promiscuity of enzymes within the prokaryotic L-rhamnose biosynthetic pathway (Cps2L, RmlB−D) and into the mechanism of inhibition for the most potent inhibitor in the series, L-rhamnose 1C-phosphonate. ■ INTRODUCTIONStreptococcus pneumoniae is a prevalent, highly infectious, Gram-positive pathogen that has shown high clinical resistance to penicillin and chloramphenicol. 1 The mechanisms of resistance for S. pneumoniae and other more invasive pathogens, including Mycobacterium tuberculosis, usually entail alterations to drug target proteins involved in cell wall synthesis. 2−5 Bacterial chemo-resistance, including resistance to synergistic treatments using β-lactams and aminoglycosides, is a growing barrier to the treatment of invasive pathogens (i.e., S. pneumoniae and M. tuberculosis). 6 Cell wall biosynthesis is a commonly pursued target for a variety of bacterial enzyme inhibitors as cell wall assembly is essential for bacterial survival and virulence. L-Rhamnose (6-deoxy-L-mannose) serves as the linking unit between arabinogalactan and peptidoglycan 7,8 and is a necessary constituent of the bacterial cell wall in many bacterial species. 9 The biosynthesis of L-rhamnose begins with nucleotidylyltransferase enzymes (Cps2L/RmlA) responsible for generating activated sugars in the form of glycosylated nucleoside diphosphates (NDPs). These NDPs are generated from the enzymatic coupling of sugar 1-phosphates with nucleoside triphosphates (NTPs). Once formed, the NDPs may be further modified ...

Thymidylyltransferases (thymidine diphospho pyrophosphorylases) are nucleotidylyltransferases that play key roles in the biosynthesis of carbohydrate components within bacterial cell walls and in the biosynthesis of glycosylated natural products. They catalyze the formation of sugar nucleotides concomitant with the release of pyrophosphate. Protein engineering of thymidylyltransferases has been an approach for the production of a variety of non-physiological sugar nucleotides. In this work, we have explored chemical approaches towards modifying the activity of the thymidylyltransferase (Cps2L) cloned from S. pneumoniae, through the use of chemically synthesized 'activated' nucleoside triphosphates with enhanced leaving groups, or by switching the metal ion co-factor specificity. Within a series of phosphonate-containing nucleoside triphosphate analogues, thymidylyltransferase activity is enhanced based on the acidity of the leaving group and a Brønsted-type analysis indicated that leaving group departure is rate limiting. We have also determined IC50 values for a series of bisphosphonates as inhibitors of thymidylyltransferases. No correlation between the acidity of the inhibitors (pKa) and the magnitude of enzyme inhibition was found.

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