Production of Fluorescent Oligosaccharide Substrates for the Neisseria meningitidis Serogroup W Capsule Polymerase

Abstract

Neisseria meningitidis is a Gram‐negative bacteria that causes most cases of bacterial meningitis. The six disease‐causing serogroups of N. meningitidis differ in capsular polysaccharide composition and linkage. Our long‐term goal is to develop a better understanding of the molecular mechanism of the serogroup W capsule polymerase. This enzyme synthesizes a heteropolysaccharide of galactose and sialic acid. We aim to develop a fluorescence‐based assay to aid in our studies. In this work, we describe our initial efforts to produce fluorescent substrates for the serogroup W capsule polymerase. Preliminary radioactivity assays indicate the enzyme has the ability to elongate serogroup W oligosaccharide fragments. The enzyme was more active with oligosaccharides than full length polysaccharide. These fragments, obtained by acid‐hydrolysis and heating, are the starting point for production of fluorescent substrates. Oligosaccharides were subjected to reaction with the fluorescent dye 1,2‐diamino‐4,5‐methylenedioxybenzene (DMB). Initial results indicate successful incorporation of the dye as assessed by HPLC with fluorescence detection. Alternately, copper‐catalyzed “click” chemistry is also being used to produce fluorescent oligosaccharides. Chemical reactions are being performed to add an azido group to oligosaccharides. These will be conjugated to an alkyne‐containing dye by 1,3‐cycloaddition “click” chemistry. Future work seeks to further characterize and optimize fluorescent substrates from both methods for testing with the serogroup W capsule polymerase.Funded in part by ACS Project SEED (C.W).