The chemoenzymatic synthesis of the rare bacterial sugar pseudaminic acid, and its utilisation in the study of a potential pseudaminidase

Pseudaminic acid is a non-mammalian nonulosonic acid and a component in a number of bacterial surface structures, including Pseudomonas aeruginosa lipopolysaccharide and pili. It has been shown to play a role in virulence in pathogens such as influencing motility in Campylobacter jejuni whose flagellin is glycosylated with pseudaminic acid structures. Therefore pseudaminic acid processing enzymes have been identified as putative drug targets but are yet to be fully characterised. Although generally harmless to healthy individuals, Pseudomonas aeruginosa is the most prevalent lung disease in sufferers of cystic fibrosis and is implicated in the majority of cystic fibrosis deaths. Chronic infections are associated with progression into a mucoid phenotype whereby eradication of the pathogen is almost impossible. An enzyme associated with the mucoid phenotype (PA2794) has been putatively assigned as a pseudaminidase and this project aimed to unequivocally assign this enzyme using pseudaminic acid analogues. However strategies to synthesise pseudaminic acid are currently unsuitable for large scale (< 50 mg) production and hence chemical probes for the characterisation of pseudaminic acid processing enzymes are not currently available. It was attempted to attain a PA2794 crystal structure with pseudaminic acid in complex. However this was inconclusive in elucidating the PA2794 natural substrate and further investigations prevented due to the lack of availability of the putative ligand. Therefore efforts were turned towards the design of a strategy for the synthesis of pseudaminic acid on a large scale, to allow for the pseudaminic acid chemical probes to be developed. The proposed synthesis utilised the Campylobacter jejuni pseudaminic acid biosynthetic enzymes to convert UDP-GlcNAc into pseudaminic acid in one-pot. Expression was optimised for each enzyme and modified to ensure enzyme solubility. A coupled PseB, PseC activity assay was implemented to follow conversion to the unwanted PseB by-product, and conditions adjusted to perturb this reaction. Additionally co-factor substitutes were explored in order to make large scale synthesis economically viable. A route for the large scale enzymatic production of pseudaminic acid was optimised, allowing for an economically viable, efficient and high yielding synthesis

Mechanistic and structural studies into the biosynthesis of the bacterial sugar pseudaminic acid (Pse5Ac7Ac)

The non-mammalian nonulosonic acid sugar pseudaminic acid (Pse) is present on the surface of a number of human pathogens including Campylobacter jejuni and Helicobacter pylori and other bacteria such as multidrug resistant Acinetobacter baumannii. It is likely important for evasion of the host immune sysyem, and also plays a role in bacterial motility through flagellin glycosylation. Herein we review the mechanistic and structural characterisation of the enzymes responsible for the biosynthesis of the Pse parent structure, Pse5Ac7Ac in bacteria

Synthetic Approaches for Accessing Pseudaminic Acid (Pse) Bacterial Glycans

Sugars to order: A summary of work in the field of pseudaminic acid (Pse) synthesis is provided. This non‐mammalian sugar is of increasing biological importance as an essential component in cell‐surface glycoconjugates of a number of pathogenic bacteria. Pioneering studies into biosynthesis of Pse5Ac7Ac have provided inspiration to carbohydrate chemists

Biocatalytic Transfer of Pseudaminic Acid (Pse5Ac7Ac) Using Promiscuous Sialyltransferases in a Chemoenzymatic Approach to Pse5Ac7Ac-Containing Glycosides

Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host–pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein, we describe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors, enabling the enzymatic synthesis of Pse5Ac7Ac-containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late-stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot two-enzyme process using biosynthetic enzymes. Finally, screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac-terminated glycosides.</p

Chemoenzymatic synthesis of 3-deoxy-3-fluoro-L-fucose and its enzymatic incorporation into glycoconjugates

The first synthesis of 3-deoxy-3-fluoro-L-fucose is presented, which employs a D- to L-sugar translation strategy, and involves an enzymatic oxidation of 3-deoxy-3-fluoro-L-fucitol. Enzymatic activation (FKP) and glycosylation using an a-1,2 and an a-1,3 fucosyltransferase to obtain two fluorinated trisaccharides demonstrates its potential as a novel versatile chemical probe in glycobiology

A ‘glyco-fluorine’ code revealing differential recognition by glycan binding partners

Biosensing or diagnostics using glycan sequences as targets is limited by glycan cross-reactivities. As binding sites of different proteins that all recognise a given glycan will not be identical, we introduce application of a library of synthetic analogues of a single glycan ligand as a powerful approach to obtain fingerprint binding profiles. We report the enzymatic synthesis of a 150-member library of fluorinated Lewisx analogues (‘glycofluoroforms’) using naturally occurring enzymes and fluorinated monosaccharide building blocks, and the incorporation of a subset into lipid-linked glycan probes or into glyconanoparticles for probing protein binding both in solid-phase high-throughput glycan microarray screening analyses and in solution-phase nanoparticle-based interaction studies. These fluorinated Lewisx analogues, which NMR studies showed to have very similar 3D structures compared to the nonfluorinated Lewisx, gave variously increased or decreased binding with a set of proteins, the different proteins having different preferences and tolerances for binding