Chemical Biology Tools for Modulating and Visualizing Gram-Negative Bacterial Surface Polysaccharides

Abstract

Bacterial cells present a wide diversity of saccharides that decorate the cell surface and help mediate interactions with the environment. Many Gram-negative cells express O-antigens, which are long sugar polymers that makeup the distal portion of lipopolysaccharide (LPS) that constitutes the surface of the outer membrane. This review highlights chemical biology tools that have been developed in recent years to facilitate the modulation of O-antigen synthesis and composition, as well as related bacterial polysaccharide pathways, and the detection of unique glycan sequences. Advances in the biochemistry and structural biology of O-antigen biosynthetic machinery are also described, which provide guidance for the design of novel chemical and biomolecular probes. Many of the tools noted here have not yet been utilized in biological systems and offer researchers the opportunity to investigate the complex sugar architecture of Gram-negative cells.

Synthesis and Application of Rare Deoxy Amino l-Sugar Analogues to Probe Glycans in Pathogenic Bacteria

“…Synthesis of l -FucNAz 5 starting from l -rhamnosyl 2,4-diol 9 is shown in Scheme . Diol 9 was transformed into l -fucosamine derivative 10 by C2,C4 inversion by employing a slight modification of our reported one-pot procedure. It should be noted that using a cheaper sodium nitrite (NaNO 2 ) as a nucleophile in place of originally employed and expensive tetrabutyl ammonium nitrite (TBANO 2 ) resulted in much improved yield of 10 .…”

“…To synthesize l -pneumosamine derivative 6 , we started from the known l -fucosyl 2,4-diol 13 (Scheme ). Regioselective triflation of the more reactive equatorial C2-OH group of 13 by employing controlled amount of triflic anhydride generated the corresponding C2-OTf, which upon subsequent refluxing with sodium azide at 110 °C in DMF followed by concomitant removal of benzoyl groups provided l -pneumosamine 3,4 diol derivative 14 in 71% yield over three steps.…”

“…Thus, MOE complements traditional glycan analysis tools that are stymied by rare bacterial glycan building blocks. 51,52 Although bacteria utilize over 700 monosaccharides in glycan biosynthesis, 21−23 only a small percentage of these sugars have been chemically modified into metabolic probes. 31 Thus, the knowledge learned from existing probes does not adequately capture the enormous amount of glycan epitope variability across the bacterial domain.…”

“…These studies have validated MOE, paired with intentional choice of unnatural sugars, as a powerful strategy to examine bacterial glycans even in the absence of full structural information. Thus, MOE complements traditional glycan analysis tools that are stymied by rare bacterial glycan building blocks. , …”

Expanding the Substrate Scope of a Bacterial Nucleotidyltransferase via Allosteric Mutations

“…Across bacteria, an estimated 700 distinct activated monosaccharides are used to build cell surface glycoconjugates, as well as to decorate intracellular proteins and natural products. − Bacterial cell envelopes are enriched in glycan motifs that are pivotal to survival and infection . These motifs include the cell wall layer (peptidoglycan) and surface polysaccharides that make up the exposed glycocalyx layer, such as lipopolysaccharides with attached O-antigens in the outer membrane of Gram-negative bacteria − (Figure A). Glycoconjugates are typically built by dedicated glycosyltransferases that largely use nucleoside diphosphate sugars (NDP-sugars), and to a lesser extent nucleoside monophosphate sugars, as donor substrates to react with biomolecular acceptors.…”