We here report on the structural analysis of a novel tetra-acyl lipid A (LA (tetra)) isolated from Escherichia coli 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 (1)H- and (13)C-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 interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in a dose-dependent 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.

Lipopolysaccharide, Escherichia coli, lipid A, mass spectrometry, spectroscopy

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12	C13	C14
0,2	lR3HOMyr	171.66	44.13	67.10	?	?	?	?	?	?	?	?	?	?	?
0,3	lR3HOMyr	171.33	42.90	67.24	?	?	?	?	?	?	?	?	?	?	?
0,6,2,3	lXLau	172.3	?	?	?	?	?	?	?	?	?	?	?
0,6,2	lR3HOMyr	169.13	40.75	71.11	33.41	?	?	?	?	?	?	?	?	?	?
0,6,4	P
0,6	bDGlcpN	100.73	55.81	73.72	73.45	75.93	61.30
0	aDGlcpN	92.88	52.08	73.72	68.24	73.45	67.52
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9	H10	H11	H12	H13	H14
0,2	lR3HOMyr	-	2.10-?	3.71	?	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	0.86
0,3	lR3HOMyr	-	2.31-2.36	3.81	?	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	0.86
0,6,2,3	lXLau	-	2.24	1.55	1.65	1.25	1.25	1.25	1.25	1.25	1.25	1.25	0.86
0,6,2	lR3HOMyr	-	2.44-?	5.12	1.55	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	0.86
0,6,4	P
0,6	bDGlcpN	4.66	3.47	3.56	3.71	3.21	3.58
0	aDGlcpN	5.30	3.88	4.99	3.36	3.87	3.82
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9	C10/H10	C11/H11	C12/H12	C13/H13	C14/H14
0,2	lR3HOMyr		44.13/2.10-?	67.10/3.71	?/?	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/0.86
0,3	lR3HOMyr		42.90/2.31-2.36	67.24/3.81	?/?	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/0.86
0,6,2,3	lXLau		?/2.24	?/1.55	?/1.65	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/0.86
0,6,2	lR3HOMyr		40.75/2.44-?	71.11/5.12	33.41/1.55	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/1.25	?/0.86
0,6,4	P
0,6	bDGlcpN	100.73/4.66	55.81/3.47	73.72/3.56	73.45/3.71	75.93/3.21	61.30/3.58
0	aDGlcpN	92.88/5.30	52.08/3.88	73.72/4.99	68.24/3.36	73.45/3.87	67.52/3.82
	P

The spectrum also has 43 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CCCCCCCCCCCC(=O)O[C@H](CCCCCCCCCCC)CC(=O)N[C@H]1[C@H](OC[C@H]2O[C@H](OP(=O)(O)O)[C@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H]2O)O[C@H](CO)[C@@H](OP(=O)(O)O)[C@@H]1O

1361.674 g/mol (C₆₆H₁₂₆N₂O₂₂P₂)