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

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-CH 3 modified sugar-nucleotides are capable of reversibly preventing GDP-ManA production by GMD. The ketone product from oxidation of a C6-CH 3 modified analogue is identified by high-resolution mass spectrometry.

Inhibition of the GDP-d-Mannose Dehydrogenase from Pseudomonas aeruginosa Using Targeted Sugar Nucleotide Probes

“…Taken together, these findings present an important development in structure-activity information for this pyrophosphorylase in accessing non-native sugar nucleotides (Figure 2), building on previous reports. 11 Replacement of mannose C4-OH with fluorine is well tolerated, similarly at C6-OH for fluorine and azide, but not for gem difluorination. A C5 modification with axial Me is also possible, but turnover is sluggish and inefficient.…”

Chemical and enzymatic synthesis of the alginate sugar nucleotide building block: GDP-d-mannuronic acid

“…3 Following this, an intricate, multi-enzyme mediated polymerisation process assembles the β-D-mannuronate polymer, which is then further modified by epimerisation, acetylation and truncation before export. As part of a program to investigate the enzymes involved in the biosynthesis of alginate, 4 were interested to chemically synthesise 1 and deliver an enabling sugar nucleotide tool to support elucidation of the alginate polymerisation process. A chemical synthesis of 1 was recently completed by Codée et al 5 using P III -amidite-P V chemistry to accomplish the key pyrophosporylation step in forming 1.…”

6R/S-deutero-α-d-mannopyranoside 1-phosphate

“…Our synthetic route began from d-mannose which was appropriately transformed into C6-aldehyde thioglycoside (1) using established procedures [19]. We next completed a reduction of (1) with NaBD 4 to deliver (2) in 65% yield (Scheme 1).…”

“…To a stirred solution of (1) [19] (0.75 g, 1.38 mmol, 1.0 equiv.) in dry MeOH (6.0 mL) at −15 • C was added a solution of NaBD 4 (60 mg, 1.44 mmol, 2.0 equiv.)…”

“…Carbohydrates diastereoselectively deuterated at C6 have provided important chemical tools [17], illustrated for mannose by a study reported by Tanner concerning the synthesis of ADP-[6 -2 H]-D,D-Hep for elucidating the mechanism of ADP-l-glycero-d-manno-heptose 6-epimerase [18]. As part of wider project concerning the chemical synthesis of modified mannose 1-phosphates and derived sugar-nucleotides [19], we required access to the title compound to establish a proof of concept methodology for incorporating C6 deuterium. Herein we provide our record of its synthesis and full characterisation from S-phenyl thioglycoside C6 aldehyde (1).…”