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To investigate the structure-activity relationships, various biological activities, including pyrogenicity, lethal toxicity, elicitation of Shwartzman reaction, mitogenicity and tumor necrosis factor (TNF)-inducing activity, were compared among natural and synthetic lipid A's differing in fatty acid composition. In all these tests, natural lipid A's from Escherichia coli and Salmonella minnesota and synthetic LA-15-PP, which carries 3-hydroxy- and 3-acyloxy-tetradecanoyl groups at the 2, 3 and 2', 3' positions, respectively, showed the strongest activities among the tested lipid A's. In contrast, LA-16-PP, in which the amide-bound 3-hydroxytetradecanoic acid at position 2 of LA-15-PP is replaced by 3-hexadecanoyloxytetradecanoic acid, exhibited lower activity than LA-15-PP and natural lipid A's. Although LA-16-PP has been assumed to have a typical Salmonella lipid A structure (and, in fact, it has a structure corresponding to one of the components of Salmonella lipid A), the activity of this synthetic compound was not comparable to that of natural Salmonella lipid A. LA-17-PP, in which tetradecanoic acid is the sole fatty acid component, exhibited relatively strong mitogenicity and TNF-inducing activity, but very low pyrogenicity. The activities of LA-18-PP, which has ester-bound tetradecanoic acid and amide-bound 3-hydroxytetradecanoic acid, were lower than those of LA-17-PP. The results indicate that the differences in fatty acid composition of lipid A's have important influences on the biological activities studied.

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Biological activities of five synthetic lipid A analogues (D-glucosamine derivatives) were examined to elucidate the structure required for expression of the biological activities of endotoxin. Proclotting enzyme of horseshoe crab activation, interferon-inducing and tumor necrosis factor-inducing activities were significantly expressed by an analogue which possesses 4-0-phosphoryl, 3-0-tetradecanoyl and N-tetradecanoyloxytetradecanoyl groups. The results obtained with different analogues show that the 4-0-phosphoryl and N-tetradecanoyloxytetradecanoyl groups are important for expression of the above activities. The effect of 6-0-acylation in preventing the expression of these biological activities is also suggested. Pyrogenic activity was not detected in any of the compounds tested.

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (p-I,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amidebound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.It is well established that lipid A component of lipopolysaccharides of gram-negative bacteria is responsible for most, if not all, various biological and endotoxic activities including lethal toxicity, pyrogenicity, immunomodulating activities, and the activities to induce various mediators [I]. The chemical structure of the lipid A backbone in many bacteria appears to be identical and consists of a (/3-1,6)-linked D-glucosamine disaccharide which carries phosphate residues in the positions of 1 and 4 as well as amide-bound and ester-bound D-3-hydroxy and/or acyloxy fatty acids [2], although a number of minor differences in structure are known in lipid A derived from various bacteria. It is also known that isolated free lipid A is a mixture of congeners.To confirm lipid A structure and to establish the structure responsible for the functions, compounds based on the proposed structure were recently chemically synthesized [3 -51 and their biological activities were investigated in many laboratories [6-121. Although some of the synthetic analogues have been reported to show significant biological activities such as interferon inducing [7] as well as mitogenic and adjuvant activities [6, 7, 1 I], the activity of the analogues, if any [6-121 were much weaker than natural lipid A.More recently, locations of ester-linked 3-hydroxytetradecanoic acid (or acyloxy fatty acid) of lipid A from Escherichia coli, Salmonella typhimurium and Proteus mirahilis were reinvestigated. The results disclosed that the locations were not at the 3,4 and 6' positions as presumed previously but at the 3 and 3' positions of diglucosamine unit 113-151. The compound with such altered structure, therefore, was hoped to enhance biological activities and represent comparable activities to natural lipid A. Based on the modified structure, analogue...