Chemoenzymatic synthesis of sugar-nucleotide analogues: tools to probe a strategic dehydrogenase from Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterium, responsible for causing life threatening infections amongst fibrosis (CF) patients. Mucoid phenotypes of P. aeruginosa are characterised by overproduction of alginate, an extra-cellular polysaccharide (EPS) composed of ß-D-mannuronic acid and a-L-guluronic acid, which is involved in bacterial biofilm formation and results in a multi-drug antibiotic resistance profile.
Guanosine diphosphate-mannose dehydrogenase (GMD), a NAD+-dependent dehydrogenase responsible for production of the feedstock sugar-nucleotide building block guanosine diphosphate-mannuronic acid (GDP-ManA), has been identified as a strategic biosynthetic enzyme to investigate potential inhibition of alginate production. This oxidation is mediated by a catalytic Cys268 residue, providing a template for rational structure-based design of sugar-nucleotide tools.
This thesis describes the synthesis of GDP-ManA, the native tool for studies into alginate biosynthesis. A chemical strategy employing a P(V)-P(V) pyrophosphorylation approach towards the target was complemented by an enzymatic synthesis using the native enzyme, GMD.
To permit further mechanistic study of GMD, the synthesis of a series of structurally defined GDP-Man analogues was presented via a chemoenzymatic approach, to investigate substrate specificity and identify functionalities capable of interacting with Cys268. Modified GDP-Man analogues were accessed chemically from differentially protected C6-OH and C4-OH mannose building blocks. Following a series of functional group interconversions, transformation to the respective sugarnucleotides was evaluated using guanosine diphosphate-mannose pyrophosphorylase (GDP-ManPP). The synthesised GDP-Man analogues were then evaluated as substrates of GMD, providing a foundation for the future design and synthesis of probes to target and inhibit GMD.