Enzyme-catalyzed synthesis of isosteric phosphono-analogues of sugar nucleotides

Beaton, Stephen A.; Huestis, Malcolm P.; Sadeghi-Khomami, Ali; Thomas, Neil R.; Jakeman, David L.

doi:10.1039/b808078j

Cited by 29 publications

(38 citation statements)

References 31 publications

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“…The K m value for 1 matched closely with previously reported studies using HPLC based assays. 37 The K m value for 2 with Cps2L has not been reported previously. Inhibitor 11 was found to be competitive with respect to 1 (K i = 536 μM) and uncompetitive with respect to 2 (K i = 497 μM) using the coupled assay method.…”

Section: ■ Introductionmentioning

confidence: 92%

“…Enzyme Inhibition Studies. Compounds 9 and 11 were first evaluated as Glc-1-P (1) analogue substrates for Cps2L given that the enzyme had previously demonstrated a broad substrate specificity, 34,37,38 including poor conversion (3−10%) observed for β-L-fucosyl phosphate. 34 Negligible turnover (<1%) to sugar nucleotide products were observed by HPLC over 24 h using 2−10 EU of Cps2L with a 8:1 ratio of 9 or 11 relative to dTTP substrate (2) (Supporting Information).…”

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2013

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/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 ...

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Section: ■ Introductionmentioning

confidence: 92%

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2013

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“…27 Substrate specificity studies were conducted with AtUSP to confirm activities with the phosphono analog of α-D-galactopyranosyl 1-phosphate and UTP (Supplementary Data, Table S1), using an identical methodology to that we have reported previously. 28,30,43 The coupling of the phosphono analog of α-D-galactopyranosyl 1-phosphate and UTP was catalyzed by AtUSP.…”

Section: Enzymatic Preparation Of the Phosphono Analog Of Udp-ch 2 -Galpmentioning

confidence: 99%

“…27,28 We next explored the substrate specificity of a recombinant Arabidopsis thaliana UDP-sugar pyrophosphorylase (AtUSP) with an apparently broad substrate specificity toward monosaccharide 1-phosphates 29 to determine whether it would accept UTP and the phosphonate analog of galactose 1-phosphate. Approximately 7 mg of recombinant protein was obtained from a 1-L culture.…”

Section: -Galpmentioning

confidence: 99%

Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

Partha

Sadeghi-Khomami

Slowski

et al. 2010

Journal of Molecular Biology

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“…A prevalent, pathogenic organism, S. pneumoniae has shown widespread clinical resistance to penicillin and chloramphenicol, as well as to synergistic treatments involving β-lactams and aminoglycosides. 5,6 Furthermore, they have been used to prepare sugar nucleotide analogues for enzymatic glycodiversification studies, [7][8][9] and used to prepare phosphonate 10 and carbacyclic 11 sugar nucleotide analogues that have been put forward as putative glycosyltransferase inhibitors. Cps2L (EC 2.7.7.24) is a bacterial thymidylyltransferase (nucleotidylyltransferase) cloned from S. pneumoniae that catalyses the first step in the biosynthesis of L-rhamnose (Scheme 1), 3 an essential constituent of the cell wall in many bacterial species.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.

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Enzyme-catalyzed synthesis of isosteric phosphono-analogues of sugar nucleotides

Abstract: Efficient enzymatic syntheses of isosteric phosphono analogues of sugar nucleotides have been accomplished using a thymidylyltransferase.

Cited by 29 publications

References 31 publications

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

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

Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

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

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