Structural and biological characterisation of a novel tetra-acyl lipid A from Escherichia coli F515 lipopolysaccharide acting as endotoxin antagonist in human monocytes

We here report on the structural analysis of a novel tetra-acyl lipid A (LA(tetra)) isolated from Escherichia Loll deep rough (Re)-mutant strain F515. In addition to the biologically active hexa-acyl E, coli-type lipid A (compound 506). this incompletely acylated lipid A was found to be also present in the native LPS, Its structure was studied without further derivatisation by chemical analysis. matrix-assisted laser desorption/ionization mass spectrometry. and one- and two-dimensional H-1- acid C-13-NMR spectroscopy. It was found to be structurally distinct from the tetra-acyl lipid A biosynthetic precursor Ia (compound 406) in lacking the primary (R)-3-hydroxytetradecanoic acid 14:0(3-OH) in position 3' ester-linked to the 'non-reducing' glucosamine (GlcN II), The hydroxyl group at the (R)-3-hydroxytetradecanoic acid attached to position 2' of GlcN II was found to be substituted by dodecanoic acid (12:0), thus forming a dodecanoyloxytetradecanoyl residue 14:0[3-O(12:0)]. The acylation pattern at the 'reducing GlcN I was identical to that of compound 406 in having two primary (R)-3-hydroxy tetradecanoic acid residues [14:0(3-OH)] attached to positions 3 (ester-linked) and 2 (amide-linked), respectively, In human mononuclear cells (hMNC) the new LA(tetra) antagonized LPS-induced release of interleukine-1 (IL-1), interleukine-6 (IL-6), and tumor necrosis factor (TNF) in a dose-dependant manner with identical antagonistic potency as compared with compound 406, Also like compound 406. it was found to be an agonist in murine macrophage-like J774.1 cells.