Mycobacteria possess an outer membrane rich in mycolic acids that are ester-linked to sugars such as trehalose and arabinogalactan, resulting in virulence-associated glycoconjugates such as trehalose monomycolate (TMM), trehalose dimycolate (TDM), and arabinogalactan mycolate (AGM). The mycoloyl esters in TMM and TDM can be transferred or hydrolyzed via known enzymes (e.g., Ag85 and trehalose dimycolate hydrolase) in processes associated with outer membrane biosynthesis or remodeling. While it is likely that AGM mycoloyl esters must also be broken down, the enzyme(s) that perform this task, as well as the implications for this process in mycobacterial physiology and pathogenesis, are unknown.  This study aims to develop analogues of AGM bearing fluorescence resonance energy transfer (FRET) partners on the sugar and mycoloyl group, so that upon enzymatic separation of these two moieties in cells or lysate, fluorescence signal is generated. Here, we report progress toward the synthesis of fluorescence-quenched monosaccharide and disaccharide AGM FRET probes. Upon completion of these compounds, they will be used in whole-cell assays and native gel electrophoresis assays to identify and study the activity of enzymes that are involved in AGM metabolism.<br/>

Synthesis of fluorescence resonance energy transfer (FRET)-based fluorogenic probes for the investigation of arabinogalactan mycolate metabolism in mycobacteria

255th National Meeting (2018)

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