Alginate, an anionic polysaccharide composed of acetylated, β-1,4 linked Dmannuronic acid and α-1,4 linked L-guluronic acid residues, constitutes the major exopolysaccharide component of mucoid Pseudomonas aeruginosa infections. Such infections are particularly deleterious for cystic fibrosis patients, 1 contributing to the establishment of a biofilm environment and augmenting the antibiotic resistance profile of the bacterium. The molecular genetics and regulation of alginate biosynthesis in P. aeruginosa have been wellcharacterised. 2,3 However, our understanding of the chemical biology underpinning polysaccharide production and modification is still incomplete. This represents a significant opportunity to comprehend and exploit such pathways, especially within the global obligation to develop new anti-bacterial strategies; epitomised by recent work developing small molecule inhibitors for c-di-GMP binding to the receptor protein Alg44, which activates alginate production in P. aeruginosa. 4 Pivotal to the alginate biosynthetic pathway is GDP-D-mannose dehydrogenase (GMD), which is required to form the alginate sugar nucleotide feedstock, guanosine diphosphate mannuronic acid (GDP-D-ManA). 5,6 Since there is no corresponding enzyme in humans, specific inhibition of GMD could be envisaged as a tactic to stop alginate production in chronic, mucoid P. aeruginosa infections. 7 GMD belongs to a small group of NAD + -dependent fourelectron-transfer dehydrogenases, 8,9 and converts GDP-D-Man 1 to GDP-D-ManA 2 (Figure 1a); the NAD + -mediated oxidation is proposed to have four discrete steps, facilitated by an active site Cys 268 residue. 10 We recently reported the first series of C6-modified sugar nucleotides to investigate this oxidative mechanism, establishing evidence of a C6-ketone intermediate from oxidation of a C6-CH3 modified analogue. 11 Pursuant of a substrate-based inhibitor of GMD and to further expand structure-activity relationship knowledge (to underpin future small molecule inhibition strategies), we report herein the design, synthesis and evaluation of a matrix of C4-, C5-and C6-modified GDP-D-Man analogues (Figure 1b). Our strategy focused on analogues of the pyranose component within the sugar nucleotide, specifically: i) C6-OH replacements, removing capability for oxidation, but retaining the potential to bind GMD ii) C6 modifications to mimic the product carboxylic acid in 2 and iii) C4 and C5 functional group changes to probe steric and electronic effects involved in substrate binding.
