Synthesis and fluorescent labelling of model mycolic acids

Abstract

Mycolic Acids (MAs) are long chain α-alkyl-β-hydroxy fatty acids that form part of the cell wall of Mycobacterium species and a few other genera. They play an important role in steering the host-pathogen relationship to establish active TB disease. These compounds are recognized by antibodies and therefore show potential for use in new diagnostic techniques such as biosensor assays. Previous studies have shown that MA-methyl esters were not antigenic, while fluorescein labelled-MA maintained antigenicity. It was proposed that this was due to the presence of the carboxylic acid group on the fluorescein label that substituted for the one on MA that became esterified during conjugation. However the existence of the free carboxylic acid on fluorescein after labelling of MA was uncertain as fluorescein labelling of palmitic acid apparently produced two structural forms, the free acid and the lactone, as a mixture of tautomers. Furthermore the original biological studies were not done on characterized material and activity may have been due to the presence of some unreacted MA. Here, the synthesis of a corynomycolic acid homologue is reported via two routes: (a) Claisen condensation followed by reduction or (b) aldol condensation. Due to the cost and poor quality of commercial “5-BromoMethylFluorescein”, this reagent had to be synthesised in the laboratory. The synthetic Corynomycolic acid homologue made in this study and a mixture of naturally occurring bovine mycolic acids obtained from Sigma-Aldrich were labelled with freshly prepared 4(5)-BMF. NMR characterization of the fluorescein labelled-MA showed the presence of a carboxylic acid group on the fluoresceinsuggesting that it is likely to maintain biological activity.