Low endotoxic activities of synthetic Salmonella-type lipid A with an additional acyloxyacyl group on the 2-amino group of beta (1-6) glucosamine disaccharide 1,4'-bisphosphate

Abstract

A synthetic lipid A (Salmonella type, compound 516), P(1-6)-linked D-glucosamine disaccharide 1,4'bisphosphate, with three acyloxyacyl groups and one hydroxyacyl group, i.e., (R)-3-hexadecanoyloxytetradecanoyl, (R)-3-hydroxytetradecanoyl, (R)-3-dodecanoyloxytetradecanoyl, and (R)-3-tetradecanoyloxytetradecanoyl groups at the 2-amino, 3-hydroxyl, 2'-amino, and 3'-hydroxyl groups, respectively, was less biologically active than the synthetic Escherichia coli-type lipid A (compound 506), which has only two acyloxyacyl groups at the 2' and 3' positions and is substituted with a (R)-3-hydroxytetradecanoyl group at the 2-amino group. Compound 516 exhibited considerably weaker pyrogenic and leukopenic activity than compound 506, and it scarcely prepared rabbit skin for the Shwartzman reaction and lacked lethal toxicity on chicken embryos, although its lethal toxicity in galactosamine-loaded mice was as strong as that of compound 506. Compound 516 was also less active than compound 506 or natural E. coli lipid A (from Restrain F515) in other biological test systems, such as the Limulus test, stimulation of macrophages and lymphocytes, and interferon-inducing activity but not for interleukin-1 induction or complement activation. This observation suggests that there is an optimal number of acyloxyacyl groups on the glucosamine backbone for producing the biological activities of lipid A, especially the endotoxic activities. The 4'-monophosphate analog (compound 514) of compound 516 in general had significantly weaker activity than compound 516 in the above assays, most probably because of its greater hydrophobicity and consequently lower solubility in assay systems. Bacterial R595 lipid A derived from S. minnesota Re-mutant, which is a mixture of compounds 516 and 506, their 4'-monophosphate analogs and other compounds, exerted intermediate degrees of activity between compounds 506 and 516 in the various test systems employed. We (20) previously reported the synthesis of a lipid A with biological activities -comparable to those of a natural lipid A, which is responsible for the endotoxic and most of the other biological activities of bacterial lipopolysaccharides (LPS) (7,21,38). A synthetic compound (compound 506), P(1-6)linked D-glucosamine disaccharide 1,4'-bisphosphate, having two (R)-3-hydroxytetradecanoyl groups, the (R)-3dodecanoyloxytetradecanoyl, and the (R)-3-tetradecanoyloxytetradecanoyl group at 2-amino, 3-hydroxyl, 2'-amino and 3'-hydroxyl groups, respectively, which was prepared according to the proposed structure of lipid A from an Escherichia coli Re-mutant (F515) (10, 11; Fig. 1A), exhibited full biological activities identical to or sometimes stronger than those of the reference natural products, F515 lipid A and E. coli 055:B5 LPS.

Electron Microscopic Observation of Crystals of Escherichia coli K‐12 Lipopolysaccharide

“…6). Moreover, the SDS-PAGE profiles of both materials exhibited homogeneity in accordance with the previous reports (1,8,9). As one of the endotoxic activities, the preparatory potencies of both crystal and control materials were compared in the dermal Shwartzman reaction in rabbits (Table 1).…”

“…In contrast to the heterogeneity of the lipid A components of S. minnesota LPS (1, 8 -10), E. coli LPS has been shown to consist of one main hexaacyl lipid A (1,8,9). As already stated in the previous report (7) , E. coli K-12 LPS formed hexagonal plate crystals and solid column crystals ( Figs.…”

Crystallization and Analyses of Crystals of Various Chemotypes of R‐Form Lipopolysaccharides from Salmonella Spp.

“…The other is a heptaacyl lipid A, which was originally assumed to be a typical component of Salmonella lipid A (25,26). In contrast to the hexaacyl lipid A, however, the heptaacyl lipid A exhibits only weak endotoxic activities (3,12,17). From these findings, the real endotoxic material of LPSs from Salmonella spp.…”

“…It has been shown that lipid A of Salmonella spp. exhibits heterogeneity and contains at least several components (22), among which there are two major components (3,17,20). One is a hexaacyl lipid A, which represents the main component of E. coli lipid A (4,7,9,10,18).…”

Crystallization of Synthetic Escherichia coli‐Type Lipid A

“…Therefore, it is necessary to use synthetic lipid A for determination of crystallizability of lipid A. Earlier studies using synthetic compounds have revealed that a hexaacyl lipid A (E. coli-type lipid A) exhibits a variety of endotoxic activities (3,4,15) whereas a heptaacyl lipid A, which was originally assumed to be a typical component of S. minnesota lipid A (21,22), exhibits only very weak activities (2,7,14). Therefore, the real endotoxic material of LPSs from Salmonella spp.…”