The Chemical Structure of Lipid A

Abstract

In Salmonella minnesota lipopolysaccharide the lipid A backbone, a substituted diphosphorylated p1 ,6-linked D-glucosamine disaccharide molecule, carries approximately seven residues of fatty acids : one each of dodecanoic, hexadecanoic, D-3-hydroxytetradecanoic and ~-3-O-(tetradecanoyl)-tetradecanoic acid in ester linkage and two of D-3-hydroxytetradecanoic acid in amide linkage. In the present study it is shown that treatment of the lipopolysaccharide with alkali at elevated temperature leads, through a p-elimination reaction, to the generation of amidebound A'-tetradecenoic acid. This suggested that the 3-hydroxyl group of amide-bound hydroxy fatty acids carried a substituent. To elucidate the nature of the substituent, free Salmonella lipid A was methylated with methyl iodine in the presence of silver salts followed by mild acid hydrolysis, a procedure which is known to cleave amide (and not ester) bonds selectively. In the hydrolysate, by means of combined gas-liquid chromatography/mass spectrometry the methyl esters of 3-O-(dodecanoyl)-tetradecanoic and 3-O-(hexadecanoyl)-tetradecanoic acid were identified. This shows that in lipid A amide-linked 3-hydroxytetradecanoic acid residues are 3-0-acylated by dodecanoic and hexadecanoic acid, respectively.Quantitative analyses suggest that the Salmonella lipid A backbone is substituted by four D-3-hydroxytetradecanoyl residues, two being present as esters and two as amides. The nonhydroxylated fatty acids are not bound directly to the backbone. Rather, they are attached to hydroxyl groups of 3-hydroxytet radecanoyl residues : specifically, tetradecanoic acid substitutes ester-bound and dodecanoic and hexadecanoic acid amide-bound 3-hydroxytetradecanoic acid.Lipopolysaccharides, the 0-antigens and endotoxins of gram-negative bacteria, are made up of two chemically distinct portions : a heteropolysaccharide and a covalently linked lipid, termed lipid A [l]. While the structures determining 0-antigenicity are embedded in the polysaccharide part, the endotoxic properties of lipopolysaccharides are anchored in the lipid A component [2]. Chemically, lipid A of Salmonella (and many other gram-negative bacteria) consists of a p1 ,6-linked D-glucosamine disaccharide which carries partially substituted phosphoryl ksidues in positions C-4' and C-1. The hydroxyl group at C-3' represents the attachment site of the saccharide portion in intact lipopolysaccharide. The phosphorylated glucosamine disaccharide (lipid A backbone) carries, in addition, long-chain fatty acids in both ester and amide linkage which appear to play a role in the expression of endotoxic activity. Of the acyl residues present in Salmonella lipid A approximately one each of dodecanoic, hexadecanoic, D-3-hydroxytetradecanoic and ~-3-O-(tetradecanoyI)-tetradecanoic acid (and possibly smaller amounts of ~-3 -0 -(~-2 -hydroxytetradecanoy1)-tetradecanoic acid) are involved in ester and two of D-3-hydroxytetradecanoic acid in amide linkage [3].According to our present knowledge, the Salmonella lipid A backbone pr...

Nature and location of amide‐bound (R)‐3‐acyloxyacyl groups in lipid A of lipopolysaccharides from various gram‐negative bacteria

“…E. coli) only one amide-bound 3-acyloxyacyl residue was detected. Based on chemical considerations [7] and results of soft ionization techniques [8] it appears that this residue is linked to GlcN I1 and that the hydroxyl group of the amide-linked 3-hydroxy fatty acid of GlcN I is free. In other lipid A preparations (e.g.…”

“…Methylation and N-deacylation of free lipid A preparations and the analysis of 3-acyloxyacyl residues by combined gas-liquid chromatography/mass spectrometry was performed under the conditions and with the instruments described [7, 201. The same solvents, chemicals and reference compounds were used as in these previous studies.…”

“…It has also previously been demonstrated that in these bacteria approximately 50% of the ester-linked (R)-3-hydroxy fatty acids are 3-0-acylated by straight-chain nonhydroxylated or (S)-Zhydroxy fatty acids [l, 3-61. Very recently it was shown for enterobacterial lipid A that also the amidebound (R)-3-hydroxy fatty acids (notably that at position 2') carry nonhydroxylated fatty acids at their 3-hydroxyl group in a specific distribution [7,81. From these results it appeared that (R)-3-acyloxyacyl groups are important and dominating lipophilic constituents of lipid A and it was speculated that they may be also present in lipid A of bacteria other than Enterobacteriaceae.…”

'Coxiella Burnetii' Vaccine Development: Lipopolysaccharide Structural Analysis

“…We and others (34,35) have precisely located the fatty acids and substituted fatty acids on the central core disccharide.…”

“…They can directly be analyzed by GC-MS (35,36). The positional analysis of amide-linked fatty acids in lipid-A can be carried out by means of a technique recently described (37).…”