Chemoenzymatic Synthesis of C6-Modified Sugar Nucleotides To Probe the GDP-d-Mannose Dehydrogenase from Pseudomonas aeruginosa.

Sanaz Ahmadipour,Gavin J Miller,Giulia Pergolizzi,Robert A Field,Martin Rejzek

doi:10.1021/acs.orglett.9b00967

Sanaz Ahmadipour, Gavin J Miller + Show 3 more

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https://doi.org/10.1021/acs.orglett.9b00967

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Abstract

The chemoenzymatic synthesis of a series of C6-modified GDP-d-Man sugar nucleotides is described. This provides the first structure-function tools for the GDP-d-ManA producing GDP-d-mannose dehydrogenase (GMD) from Pseudomonas aeruginosa. Using a common C6 aldehyde functionalization strategy, chemical synthesis introduces deuterium enrichment, alongside one-carbon homologation at C6 for a series of mannose 1-phosphates. These materials are shown to be substrates for the GDP-mannose pyrophosphorylase from Salmonella enterica, delivering the required toolbox of modified GDP-d-Mans. C6-CH3 modified sugar-nucleotides are capable of reversibly preventing GDP-ManA production by GMD. The ketone product from oxidation of a C6-CH3 modified analogue is identified by high-resolution mass spectrometry.

Highlights

A lginate constitutes the major component of the exopolysaccharide coating within mucoidal Pseudomonas aeruginosa infections, which are deleterious for cystic fibrosis patients,[1] contributing to the biofilm environment and augmenting the antibiotic resistance profile of the bacterium
Since there is no corresponding enzyme in humans, specific inhibition of GDP-D-mannose dehydrogenase (GMD) could be envisaged as a tactic to stop alginate production and interfere with biofilm formation in chronic mucoid P. aeruginosa infections
GMD is a member of a small group of NAD+-dependent four-electron-transfer dehydrogenases, which includes UDP-glucose dehydrogenase (UGD),[6,7] and converts GDP-D-Man 1 to GDP-D-ManA 2

Summary

Organic Letters

In order to investigate this mechanism of action for GMD and to lay a foundation for the development of potential inhibitors, we report the design, synthesis, and preliminary evaluation of a series of C6-modified GDP-DMan tools (Figure 2). Using MeMgBr, we first sought to evaluate the addition of small alkyl groups to 6 (Scheme 1), to enable one-carbon homologation This reaction yielded significant amounts (up to 80% by crude 1H NMR) of a competing C4−C5 elimination product. Letter analyses (see Figures S4 and S6 in the Supporting Information), even after several small-scale repeats and a prolonged 38 h reaction time This analogue was instead synthesized chemically, using GMP-morpholidate, to ensure that sufficient material could be isolated to characterize and complete the series. Strategic chemical modifications at C6 of the pyranose ring (deuteration and one-carbon homologation) followed by enzymatic pyrophosphorylation were effected Access to these materials will enable a more-detailed mechanistic investigation of GMD and other important biochemical enzymes that utilize GDP-D-Man, including nucleotidylyltransferases, glycosyltransferases, and phosphorylases. Experimental procedures, characterization data, and 1H, 13C, and 31P NMR spectra for all new compounds, alongside enzymatic methods (PDF)

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