Synthesis and Evaluation of l-Rhamnose 1C-Phosphonates as Nucleotidylyltransferase Inhibitors

Abstract

/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 ...

Kinetic evaluation of glucose 1-phosphate analogues with a thymidylyltransferase using a continuous coupled enzyme assay

“…Flash chromatography was performed with Merck silica gel 60 (230-400 mesh). 25 General methods and instrumentation: enzymatic analysis Cps2L 3 and PNP 35 were overexpressed, isolated, and quantified as previously described. NMR spectra were recorded on a Bruker 500 MHz spectrometers.…”

“…10,35 Sugar phosphates 1, 2, and 3 had K m values that were an order of magnitude larger than Glc-1P. XO was then used to catalyse the final reaction in the sequence, namely the oxidation of hypoxanthine to uric acid that produces a change in absorbance at λ 290 such that initial reaction velocities can be monitored spectrophotometrically (Scheme 5).…”

Polyphosphate-containing bisubstrate analogues as inhibitors of a bacterial cell wall thymidylyltransferase

“…29,30 The kinetic data obtained were fit to standard inhibition equations by non-linear regression, using GraFit analysis software, providing a K i value for each of the compounds (2-7) evaluated, Table 1. A Lineweaver-Burk plot was also obtained in each case in order to demonstrate the mode of inhibition best described for each compound (ESI).…”

Mechanistic Evaluation of a Nucleoside Tetraphosphate with a Thymidylyltransferase

“…HPLC instrumentation and methods were as described previously. 10 Specific activity was measured from a reaction containing p 4 dT or dTTP (1 mM), Three pathways delineating possible outcomes for the activity of p 4 dT as a Cps2L substrate are illustrated in Scheme 1. In the first proposed pathway ((ii), Scheme 1), p 4 dT is hydrolyzed, potentially enzymatically, releasing monophosphate (P i ) and dTTP, which then reacts with Glc 1-P to produce deoxythymidine 5´-diphospho glucose (dTDP-Glc) in the same manner as the physiological reaction ((i), Scheme 1).…”