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.

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.

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 103 times higher than those obtained with endotoxin from Salmonella abortus equi. SDZ MRL 953 was, however, less toxic than lipopolysaccharide by a factor of 104 to >7 x 105 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 (.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.Lipopolysaccharides (LPS) are common constituents of cell walls of gram-negative bacteria. They can cause a whole array of pathophysiological effects and are also the most powerful immunostimulants known. It is generally accepted that the lipid A moiety, the terminal acylated P(1-6)glucosamine disaccharide-1,4'-diphosphates of endotoxin, is responsible for immunopharmacological activity and induction of endotoxicity, such as changes in leukocyte count, disseminated intravascular coagulation, and multiorgan failure leading to irreversible shock and death (5,21,25).Extensive studies have unsuccessfully addressed the possibility of harnessing the immunopharmacological activities of endotoxins by using various detoxifying approaches (17,20). The elucidation of the correct structure of lipid A (8, 26) and the subsequent success in the total synthesis of biologically active lipid A and analogs (9-12, 27) have rekindled interest in the possibility of separating the immunostimulatory and toxic moieties of endotoxin. Efforts to identify beneficial immunostimulatory lipid A derivatives have concentrated on synthetic analogs representing both the nonreducing (10-12) and reducing sugar moieties, such as lipids X and Y (15,27). Synthetic lipid A subunits of the nonreducing sugar moiety such as GLA-27 and GLA-60 were reported to activate B cells and macrophages and to induce release of mediators including gamma interferon and tumor necrosis factor (TNF) at nontoxic doses (for a review, see reference 5). The analogs were also found to be active in enhancing host resistance to microbial and viral infections in normal and myelosuppressed mice (5-7). Synth...

, and 4,6-0-benzylidene-~-~-galactose (see 42) were prepared by ozonolysis of the corresponding nitrones which were obtained from the acid-catalyzed reaction of p-nitrobenzaldehyde with the hydroxylamine 4, the unprotected oximes 3 and 5-9 and the 4,643 -benzylidenc oximes 3537, respectively (Schemes 1-3). Thegluco-and manno-nitrones 10 and 12 were isolated, and their ring size and their anomeric and ( E / Z ) configurations were determined by NMR spectroscopy and by their transformation into their corresponding nitro derivatives. The structure of the deoxynitroaldoses were determined by NMR spectroscopy, polarimetry, and, in the case of 14, 16, and 17, by formation of the 4,6-O-benzylidene (14+40) or 4,6-0-isopropylidene (16+43, 17-23) derivatives (Scheme 3). Acetylation of the nitroglucopyranose 14, the 2-acetamido-nitroglucopyranose 17, and the nitrogalactofuranose 19 gave the crystalline peracetylated nitroaldoses 22, 24, and 45, respectively (Scheme 4 , Figs. I and 3); acetylation of the nitromannopyranose 16 gave the nitro-arabino-glycal44 (Scheme 4 ) . The structure of the peracetylated nitroglucopyranose 22, the nitroglucosamine 25, the nitrogalactofuranose 45, and the nitroribofuranose 20 were confirmed by X-ray analysis (Figs. 1 4 ) . In all cases, including the b-u-glucopyranose derivative 22, considerably shortening of the (endocyclic) C( 1)-0 bond was observed. Basecatalyzed anomerization of the 8-D-configurated nitroglucopyranose 14, the nitromannopyranose 16, the benzylidene acetal 40 of nitroglucose, and the 2,3,4,6-tetraacetylated glucosamine derivative 24 gave the corresponding nitro-cc-u-aldoses 15, 26,47, and 25, respectively (Scheme 4).

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