Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595. Purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination

Abstract

The monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of Salmonella minnesota R595 was fractionated on a silicic acid column to yield the heptaacyl, hexaacyl, and pentaacyl MLA. Each of these MLAs was methylated with diazomethane to yield the dimethyl derivative and purified to homogeneity by reverse-phase high performance liquid chromatography. The molecular ions obtained by positive ion fast atom bombardment mass spectrometry of purified dimethyl heptaacyl MLA allowed us to establish the molecular formula and Mr of C112H211N2O23P and 1983.3, respectively. Cleavage at the glycosidic linkage yielded an oxonium ion of mass 1115, which showed that the distal sugar unit contained one phosphate (dimethyl), two hydroxymyristates, one laurate, and one myristate, while the reducing sugar unit contained two hydroxymyristates and one palmitate. By utilizing two-dimensional NMR spectroscopy, we were able to assign all of the protons of dimethyl heptaacyl MLA. This assignment included the beta protons of the three acyloxyacyl groups. A substantial downfield shift of the protons at the 3- and 3' -carbons was observed, which indicated that these two positions are occupied by ester groups. Fast atom bombardment mass spectral analysis of the hexaacyl and pentaacyl MLAs showed that these structures were identical to the previously designated TLC-3 and TLC-5 fractions, respectively, from Salmonella typhimurium. From this study, the complete structures of the MLA series found in the LPS of S. minnesota can now be described.