Synthesis, Structural Elucidation, And Biochemical Analysis of Immunoactive Glucuronosyl Diacylglycerides of Mycobacteria and Corynebacteria

Abstract

Glucuronosyl diacylglycerides (GlcAGroAc₂) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc₂ bearing both acylation patterns of (<i>R</i>)-tuberculostearic acid (C_19:0) and palmitic acid (C_16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop “rules” allowing the assignment of the acylation pattern as C_19:0 (<i>sn</i>-1), C_16:0 (<i>sn</i>-2) in the natural product from <i>Mycobacterium smegmatis</i>, and the structural assignment of related C_18:1 (<i>sn</i>-1), C_16:0 (<i>sn</i>-2) GlcAGroAc₂ glycolipids from <i>M. smegmatis</i> and <i>Corynebacterium glutamicum</i>. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from <i>C. glutamicum</i> that extends GlcAGroAc₂. This enzyme is an Mg²⁺/Mn²⁺-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol