C-glycosidic analogues of lipid A and lipid X: synthesis and biological activities.

The synthesis of a series of novel analogues of lipid A, the lipophilic terminal of lipopolysaccharides (LPS), and lipid X, the reducing monosaccharide unit in lipid A, is reported. In these compounds, the native 1-O-phosphate group has been replaced by a "bioisosteric" CH2COOH substituent. The new N,O-acylated monosaccharide C-glycosides were obtained by Wittig reaction of suitably protected glucosamine derivatives. These lipid X analogues were recognized as substrates by the enzyme lipid A synthase and could be coupled with UDP-lipid X to afford the corresponding disaccharide analogues of the lipid A precursor on preparative scale. All compounds were characterized by NMR, MS, and elemental analysis, and were tested for their ability to enhance nonspecific resistance to infection in mice and also for endotoxicity. The results clearly show that the new compounds express biological activities similar to those of their O-phosphorylated natural counterparts. Furthermore, these compounds exhibit a better therapeutic index in mouse models than the standard LPS obtained from Salmonella abortus equi.

The production of tumor necrosis factor by mouse bone marrow-derived macrophages in response to bacterial lipopolysaccharide and a chemically synthesized monosaccharide precursor.

Lipid X, a monosaccharide biosynthetic precursor of lipid A, has been chemically synthesized and was shown to induce bone marrow-derived macrophages to release tumor necrosis factor (TNF) in vitro. However, relatively high amounts of lipid X were necessary for induction, and the levels of TNF were much less than those induced by small amounts of lipid A itself or LPS. Lipid X prepared by extraction of Escherichia coli mutants induced higher levels of TNF than the chemically synthesized material, but this is probably partially due to amounts of impurities in the extracted material. Pretreatment of macrophages with IFN-gamma resulted in the release of higher amounts of TNF on subsequent induction with either LPS or lipid X. In contrast, pretreatment of macrophages with LPS induced hyporesponsiveness for TNF production on subsequent rechallenge with LPS. Lipid X, on the other hand, was incapable of making macrophages hyporesponsive for TNF production.

SDZ MRL 953, a novel immunostimulatory monosaccharidic lipid A analog with an improved therapeutic window in experimental sepsis.

SDZ MRL 953, a new synthetic monosaccharidic lipid A, was investigated in vitro and in vivo for immunopharmacological activities. In experimental models of microbial infections, the compound was highly protective when it was administered prophylactically either once or three times to myelosuppressed or immunocompetent mice. The 50% effective doses of SDZ MRL 953 varied with the infectious agents and the route of its administration. In all cases, the 50% effective doses were about 10(3) times higher than those obtained with endotoxin from Salmonella abortus equi. SDZ MRL 953 was, however, less toxic than lipopolysaccharide by a factor of 10(4) to greater than 7 x 10(5) times in galactosamine-sensitized mice. The compound was also an effective inducer of tolerance to endotoxin. Hence, repeated dosing with the compound induced a transient resistance (greater than or equal to 1 week) to lethal challenges with endotoxin. In vitro, the compound was devoid of intrinsic antimicrobial activity, but it moderately induced the release of cytokines from monocytes and primed human neutrophils for the enhanced production of reactive oxygen metabolites in response to a soluble stimulus. The results presented here suggest that SDZ MRL 953 may be useful in a clinical setting for enhancing resistance to infections, particularly in patients undergoing myelosuppressive chemotherapy or irradiation, and for the prophylaxis of endotoxin shock.

Biosynthesis of lipid A in Escherichia coli: identification of UDP-3-O-[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine as a precursor of UDP-N2,O3-bis[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine.

The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UDP-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucosamine (UDP-2,3-diacyl-GlcN) and N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) [Ray, B. L., Painter, G., & Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859]. We have previously shown that UDP-2,3-diacyl-GlcN is generated in extracts of E. coli by fatty acylation of UDP-GlcNAc, giving UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc as the first intermediate, which is rapidly converted to UDP-2,3-diacyl-GlcN [Anderson, M. S., Bulawa, C. E., & Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536-15541; Anderson, M. S., & Raetz, C. R. H. (1987) J. Biol. Chem. 262, 5159-5169]. We now demonstrate a novel enzyme in the cytoplasmic fraction of E. coli, capable of deacetylating UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc to form UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine. The covalent structure of the previously undescribed UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine intermediate was established by 1H NMR spectroscopy and fast atom bombardment mass spectrometry. This material can be made to accumulate in E. coli extracts upon incubation of UDP-3-O-[(R)-3- hydroxymyristoyl]-GlcNAc in the absence of the fatty acyl donor [(R)-3-hydroxymyristoyl]-acyl carrier protein. However, addition of the isolated deacetylation product [UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine] back to membrane-free extracts of E. coli in the presence of [(R)-3-hydroxymyristoyl]-acyl carrier protein results in rapid conversion of this compound into the more hydrophobic products UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and O-[2-amino-2-deoxy-N2,O3- bis[(R)-3-hydroxytetradecanoyl]-beta-D-glucopyranosyl]-(1----6)-2-amino- 2-deoxy-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucopyranose 1-phosphate (tetra-acyldisaccharide-1-P), demonstrating its competency as a precursor. In vitro incubations using [acetyl-3H]UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc confirmed release of the acetyl moiety in this system as acetate, not as some other acetyl derivative. The deacetylation reaction was inhibited by 1 mM N-ethylmaleimide, while the subsequent N-acylation reaction was not. Our observations provide strong evidence that UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine is a true intermediate in the biosynthesis of UDP-2,3-diacyl-GlcN and lipid A.

The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment.

We investigated the immunogenicity and antigenicity of synthetic lipid A and partial structures thereof. Included in the study were compounds which varied in the position of phosphate (1-mono-, 4'-mono-, and 1,4'-bisphosphates) and in the acylation (type, number, and distribution of fatty acids) and, in the case of monosaccharide compounds, the nature of the backbone sugar (D-glucosamine, D-glucose, 3-amino-3-deoxy-D-glucose, and 2,3-diamino-2,3-dideoxy-D-glucose). With the aid of the passive-hemolysis and passive-hemolysis-inhibition assays and by absorption experiments, five distinct antibody specificities were detected in polyclonal rabbit antisera raised against sheep erythrocyte-coated lipid A and lipid A incorporated into the membrane of liposomes (liposome-incorporated immunogens). Three antibody specificities reacted with disaccharide antigens specific for a 1-mono-, 4'-mono-, and 1,4'-bisphosphorylated beta-1,6-linked D-glucosamine disaccharide. Two antibodies reacted with either 1- or 4-phosphates of acylated D-gluco-configured monosaccharides and exhibited no cross-reaction with each other. However, they cross-reacted with disaccharide antigens with phosphate groups in the appropriate positions. We found that the physicochemical state and the environment of lipid A modulated its immunoreactivity. The immunogenicity was best expressed by erythrocyte-coated and liposome-incorporated immunogens. The antigenicity of lipid A was also greatly influenced by its physical surroundings. The reaction pattern of the above antibodies was highly specific in the hemolysis assay and in absorption experiments (the antibody reacted with antigen embedded in a cell membrane), whereas some cross-reactivities were observed in inhibition studies (the antibody reacts with antigen in aqueous solution). By using liposome-incorporated antigens as inhibitors, nonspecific reactions were avoided and specific ones were enhanced. Thus the antibodies described above against lipid A recognize epitopes in the hydrophilic backbone, the exposure of which depends on the intrinsic physicochemical properties of lipid A on the one hand and the physical environment on the other.

Inhibition of lipopolysaccharide activation of 7OZ/3 cells by anti-lipopolysaccharide antibodies.

We have investigated the ability of mAb against LPS to inhibit LPS-induced activation of 7OZ/3 pre-B cells. The fine specificity and relative affinity of these mAb for lipid A and LPS were also determined. We found that antibodies inhibited only the activity of glycolipids which they bound with relatively high affinity. However, two high affinity antibodies binding to non-lipid A epitopes did not block cellular activation. Some, but not all, relatively high affinity antibodies binding to the lipid A region of the LPS molecule inhibited biologic activity. The inhibitory antibodies bound to at least two distinct epitopes within the lipid A region. These data suggest that LPS interacts with 7OZ/3 cells in a highly specific fashion.

New synthetic analogs of lipid A as lipopolysaccharide agonists or antagonists of B lymphocyte activation.

We have studied the ability of synthetic analogs of lipid A to mimic lipopolysaccharide (LPS) for activation of 70Z/3 pre-B cells (expression of surface Igs) or to antagonize this effect. The results indicate that the presence of glucosamine (mono- or disaccharide) as a 'backbone' for the attachment of fatty acids is not necessary for activation of cells of the B lineage. Phosphate groups are not necessary either. Other structural features such as the configuration of particular asymmetric carbons, and the distance between an anionic group and an N-acyl chain, seem to be much more critical parameters for activation of B cells. Among the synthetic lipids which were unable to activate 70Z/3 cells, one compound, consisting of N,N-acylated and bisphosphorylated 2,3-dideoxy-2,3-diamino-D-glucose, behaved as a specific LPS antagonist and blocked also the activation triggered by the other synthetic inducers. The influence of the synthetic lipids on the entry of mature mouse B lymphocytes into the G1A phase of the cell cycle (cell enlargement) was also investigated. A high correlation was observed between the potency to activate pre-B cells and the ability to induce blast formation in mature B cells.