Agonistic and antagonistic properties of a Rhizobium sin-1 lipid A modified by an ether-linked lipid

LPS from Rhizobium sin-1 (R. sin-1) can antagonize the production of tumor necrosis factor alpha (TNF-alpha) by E. coli LPS in human monocytic cells. Therefore these compounds provide interesting leads for the development of therapeutics for the prevention or treatment of septic shock. Detailed structure activity relationship studies have, however, been hampered by the propensity of these compounds to undergo beta-elimination to give biological inactive enone derivatives. To address this problem, we have chemically synthesized in a convergent manner a R. sin-1 lipid A derivative in which the beta-hydroxy ester at C-3 of the proximal sugar unit has been replaced by an ether linked moiety. As expected, this derivative exhibited a much-improved chemical stability. Furthermore, its ability to antagonize TNF-alpha production induced by enteric LPS was only slightly smaller than that of the parent ester modified derivative demonstrating that the ether-linked lipids affect biological activities only marginally. Furthermore, it has been shown for the first time that R. sin-1 LPS and the ether modified lipid A are also able to antagonize the production of the cytokine interferon-inducible protein 10, which arises from the TRIF-dependent pathway. The latter pathway was somewhat more potently inhibited than the MyD88-dependent pathway. Furthermore, it was observed that the natural LPS possesses much greater activity than the synthetic and isolated lipid As, which indicates that di-KDO moiety is important for optimal biological activity. It has also been found that isolated R. sin-1 LPS and lipid A agonize a mouse macrophage cell line to induce the production of TNF-alpha and interferon beta in a Toll-like receptor 4-dependent manner demonstrating species specific properties.     

Chemical synthesis of bacterial lipoteichoic acids: an insight on its biological significance

During infections caused by Gram-negative bacteria, lipopolysaccharide (LPS, endotoxin) has a dominant role leading to fulminant pro-inflammatory reactions in the host. As there is no LPS in Gram-positive bacteria, other microbial cell wall components have been identified to be the causative agent for the pro-inflammatory activity since also Gram-positive bacterial infections lead to comparable clinical symptoms and reactions. On search for the "Gram-positive endotoxin" a widely accepted hypothesis has been raised in that the lipoteichoic acids (LTAs) serve as pathogen-associated molecular patterns (PAMPs) during Gram-positive sepsis, although the amount necessary for a pro-inflammatory in vitro response is several orders of magnitude higher than that for LPS. Therefore, LTA cannot be considered to be "the (endo)toxin of Gram-positive bacteria". Although LPS and LTA show structural relatedness (amphiphilic, negatively charged glycophospholipids), they are structurally quite different from each other and one might expect that they are also recognized by different receptors of the innate immune system, the so called toll-like receptors 4 and 2 (TLR4 and TLR2), respectively. Based on their chemical structure, the LTAs were classified into four types (type I-IV) of which we have carefully investigated the LTA of Staphylococcus aureus (type I), Lactococcus garvieae (type II) and Streptococcus pneumoniae (type IV). Hence, these LTAs have been synthesized in our group and biologically evaluated with respect to their potency to activate cytokines in transiently TLR2/CD14-transfected human endothelial kidney cells (HEK 293) or human macrophages and whole blood cells. Although LTA of type I and IV are structurally quite different, especially in their hydrophilic moiety, they originally were believed to interact with the same receptor (TLR2). Hence, the chemical syntheses leading to structurally defined, non-contaminated stimuli have a major impact on the outcome and interpretation of these biological studies of the innate immune system. With this material, it became evident that synthetic LTA from S. aureus and S. pneumoniae are not recognized by TLR2. Instead, another receptor of the innate immune system, the lectin pathway of the complement, known since many years to interact with LTA in quite a specific way, has gained increasing attractivity. With the help of synthetic LTA we obtained first evidences that this receptor is indeed the pathogen recognition receptor (PRR) for LTA.     

Modulation of innate immune responses with synthetic lipid A derivatives

The lipid A moiety of lipopolysaccharides (LPS) initiates innate immune responses by interacting with Toll-like receptor 4 (TLR4), which results in the production of a wide range of cytokines. Derivatives of lipid A show potential for use as immuno-modulators for the treatment of a wide range of diseases and as adjuvants for vaccinations. Development to these ends requires a detailed knowledge of patterns of cytokines induced by a wide range of derivatives. This information is difficult to obtain by using isolated compounds due to structural heterogeneity and possible contaminations with other inflammatory components. To address this problem, we have developed a synthetic approach that provides easy access to a wide range of lipid A's by employing a common disaccharide building block functionalized with a versatile set of protecting groups. The strategy was employed for the preparation of lipid A's derived from E. coli and S. typhimurium. Mouse macrophages were exposed to the synthetic compounds and E. coli 055:B5 LPS, and the resulting supernatants were examined for tumor necrosis factor alpha (TNF-alpha), interferon beta (IFN-beta), interleukin 6 (IL-6), interferon-inducible protein 10 (IP-10), RANTES, and IL-1beta. It was found that for each compound, the potencies (EC50 values) for the various cytokines differed by as much as 100-fold. These differences did not follow a bias toward a MyD88- or TRIF-dependent response. Instead, it was established that the observed differences in potencies of secreted TNF-alpha and IL-1beta were due to differences in the processing of respective pro-proteins. Examination of the efficacies (maximum responses) of the various cytokines showed that each synthetic compound and E. coli 055:B5 LPS induced similar efficacies for the production of IFN-beta and IP-10. However, lipid A's 1-4 gave lower efficacies for the production of RANTES and IL-6 as compared to LPS. Collectively, the presented results demonstrate that cytokine secretion induced by LPS and lipid A is complex, which can be exploited for the development of immuno-modulating therapies.     

Lipid A-Mediated Bacterial–Host Chemical Ecology: Synthetic Research of Bacterial Lipid As and Their Development as Adjuvants

Gram-negative bacterial cell surface component lipopolysaccharide (LPS) and its active principle, lipid A, exhibit immunostimulatory effects and have the potential to act as adjuvants. However, canonical LPS acts as an endotoxin by hyperstimulating the immune response. Therefore, LPS and lipid A must be structurally modified to minimize their toxic effects while maintaining their adjuvant effect for application as vaccine adjuvants. In the field of chemical ecology research, various biological phenomena occurring among organisms are considered molecular interactions. Recently, the hypothesis has been proposed that LPS and lipid A mediate bacterial–host chemical ecology to regulate various host biological phenomena, mainly immunity. Parasitic and symbiotic bacteria inhabiting the host are predicted to possess low-toxicity immunomodulators due to the chemical structural changes of their LPS caused by co-evolution with the host. Studies on the chemical synthesis and functional evaluation of their lipid As have been developed to test this hypothesis and to apply them to low-toxicity and safe adjuvants.     

Synthesis of serine-based glycolipids as potential TLR4 activators

A new series of monosaccharide-based glycolipids devoid of phosphate groups and with two lipid chains were rationally designed by varying the lipid chain lengths and saccharide structure of a α-GalCer-derived lead compound (CCL-34) that is a potent TLR4 agonist. The NF-κB activity of a 60-membered galactosyl serine-based synthetic library containing compounds with various lipid chain lengths was measured in a HEK293 cell line that stably expressed human TLR4, MD2, and CD14 (293-hTLR4/MD2-CD14). The results showed that the optimal carbon chain lengths for the lipid amine and fatty acid to activate TLR4 were 10-11 and 12, respectively. Evaluation of a 20-membered synthetic glycosyl serine-based lipid library containing compounds with various saccharide moieties and fixed lipid chain lengths revealed that the galactose moiety in CCL-34 could be replaced by glucose without loss of activity (CCL-34-S3 and CCL-34-S16). Changing the orientation of the anomeric glycosidic bond of CCL-34 resulted in a complete loss of activity (β-CCL34). Surprisingly, a change in configuration of the anomeric glycosidic bond in a glucosyl glycolipid is tolerable (CCL-34-S14). Another noteworthy observation is that the activity of a l-fucosyl derived glycolipid (CCL-34-S13) was comparable to that of CCL-34. In sum, this study determines the structural features that are crucial for an optimal TLR4-stimulating activity. It also provides several molecules with immunostimulating potential.     

Lipopolysaccharides fromMastigocladus laminosus

A lipopolysaccharide (LPS) has been isolated by phenol-water extraction from the cells of the blue-green algaMastigocladus laminosus. It has been shown that the LPS contains polysaccharide and lipid components. The polysaccharide component includes a rhamnan fragment constructed of β-1,3- and, possibly, -1,2-bound L-rhamnose residues. The lipid component is constructed of glucosamine, glucose, and fatty acid residues, among which palmitic acid predominates.     

Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire

The gingipains are cell surface Arg- and Lys-specific proteinases of the bacterium Porphyromons gingivalis, which has been associated with periodontitis, a disease that results in the destruction of the teeth-s supporting tissues. The proteinases are encoded by three genes designated rgpA, rgpB and kgp. Arg-specific proteolytic activity is encoded by rgpA/B and the Lys-specific activity by kgp. RgpA and Kgp are polyproteins comprising proteinases with C-terminal adhesin domains that are proteolytically processed. After processing, the domains remain non-covalently associated as complexes on the cell surface. RgpB is also a cell surface proteinase but does not associate with adhesin domains. Using gene knockout P. gingivalis mutants, the proteolytic processing of the gingipain domains has been shown to involve the gingipains themselves as well as C-terminal processing by a carboxypeptidase. A motif in the C-terminal domain of each protein/polyprotein has been identified that is suggested to be involved in attachment to LPS on the cell surface. RgpB lacks a C-terminal adhesin binding motif found in the catalytic domains of RgpA and Kgp. This adhesin binding motif is proposed to be responsible for the non-covalent association of the RgpA and Kgp catalytic domains into the cell surface complexes with the processed adhesin domains. The RgpA-Kgp proteinase-adhesin complexes, through the adhesin domains A1 and A3, have been implicated in colonization of P. gingivalis by binding to other bacteria in subgingival plaque and also binding to crevicular epithelial cells. The RgpA-Kgp complexes also bind to fibrinogen, laminin, collagen type V, fibronectin and hemoglobin. Amino acid sequences likely to be involved in binding to these host proteins have been identified in adhesin domains A1 and A3. It is proposed that these adhesins target the proteolytic activity to host cell surface matrix proteins and receptors. The continual cycle of binding and degradation of the surface proteins/receptors on epithelial, fibroblast and endothelial cells by the RgpA-Kgp complexes in the gingival tissue leading to cell death would contribute to inflammation, tissue destruction and vascular disruption (bleeding). P. gingivalis has an obligate growth requirement for iron and protoporphyrin IX, which it preferentially utilizes in the form of hemoglobin. Kgp proteolytic activity is essential for rapid hydrolysis of hemoglobin and it is suggested therefore that a major role of the RgpA-Kgp complexes is in vascular disruption and the binding and rapid degradation of hemoglobin for heme assimilation by P. gingivalis. The RgpA-Kgp complexes also have a major role in the evasion and dysregulation of the host-s immune response. It is proposed that host pro-inflammatory cytokines and cellular receptors close to the infection site may be rapidly and efficiently degraded by the gingipains while the proteinases at lower concentrations distally could result in the promotion of an inflammatory response through activation of proteinase-activated receptors and cytokine release. The culmination of this dysregulation would be tissue destruction and bone resorption. In animal models of disease the RgpA-Kgp complex when used as a vaccine to produce a high titre antibody response protects against challenge with P. gingivalis. Using recombinant domains of RgpA and Kgp as vaccines, it has been demonstrated that the A1 and A3 domains confer protection.     

Synthesis of heparin saccharides III. Synthesis of derivatives of D-glucosamine as starting materials for disaccharides.

Derivatives of benzyl 2-[1-(benzyloxy)formamido]-2-deoxy-α-D-glucopyranoside with various protecting groups at C(3) (benzoyl, benzyl and N-phenylcarbamoyl) and C(6) (benzoyl, benzylsulfonyl, N-phenylcarbamoyl and tosyl) have been synthesized as starting materials for disaccharides. The C(4) and C(6) hydroxyl groups of the amino sugar were initially blocked by an acetal group. After introduction of the protecting group at C(3), the acetal group was removed by acid hydrolysis, and the C(6) hydroxyl group was selectively acylated or sulfonylated. The 3,6-di-O-benzoate has also been prepared by dimolar benzoylation of the amino sugar, whereby the 4,6-isomer was obtained as a by-product.     

Synthesis and biological evaluation of a lipid A derivative that contains an aminogluconate moiety

A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed. The synthetic derivatives are 2-aminogluconate 3 and 2-aminogluconolactone 4, both of which lack C-3 acylation. These derivatives were obtained by the preparation of disaccharides in which the two amino groups and the C-3' hydroxy group could be modified individually with acyl or beta-hydroxy fatty acyl groups. Detailed NMR spectroscopy and MS analysis of 3 and 4 revealed that, even under neutral conditions, the two compounds equilibrate. The synthetic compounds lack the proinflammatory effects of Escherichia coli lipopolysaccharide (LPS), as indicated by an absence of tumor necrosis factor production. Although 3 and 4 were able to antagonize E. coli LPS, they were significantly less potent than the synthetic compound 2, which is acylated at C-3, and R. sin-1 LPS; these results indicate that the beta-hydroxy fatty acyl group at C-3 contributes to the antagonistic properties of R. sin-1 LPS. Based on a comparison of the biological responses of the synthetic lipid A derivatives with those of the R. sin-1 LPS and lipid A, the 3-deoxy-D-manno-octulosonic moieties appear to be important for the optimal antagonization of enteric LPS-induced cytokine production.     

Mechanism regulating cell surface expression and activation of Toll-like receptor 4

The Toll family of receptors senses microbial invasion and activates defense responses. Toll-like receptor 4 (TLR4) is a member of the Toll family that senses lipopolysaccharide (LPS), a principal membrane component from Gram-negative bacteria. LPS is known as an endotoxin that strongly activates immune cells such as macrophages and dendritic cells. LPS recognition by TLR4 requires an additional accessory molecule, MD-2. MD-2 is associated with the extracellular portion of TLR4, directly binds to LPS, and regulates subsequent LPS-induced TLR4 clustering. LPS recognition occurs on the cell surface. The subcellular distribution of TLR was shown to influence TLR responses. An endoplasmic reticulum (ER) chaperone, glycoprotein 96, is required for the stability of TLR4 and the formation of a TLR4/MD-2 complex in ER. MD-2 facilitates TLR4 glycosylation and its trafficking to the cell surface. Recently, another molecule, a protein associated with Toll-like receptor 4 (PRAT4A), was shown to play a critical role in cell surface expression of TLR4. These molecules control LPS responsiveness by regulating the subcellular distribution of TLR4.     

Impaired responses of leukemic dendritic cells derived from a human myeloid cell line to LPS stimulation

Several myeloid leukemia-derived cells have been reported to possess the ability to differentiate into dendritic cells (DC). MUTZ-3, a myeloid leukemia cell line, responds to GM-CSF, IL-4 and TNF-alpha, and acquires a phenotype similar to immature monocyte-derived DC (MoDC). In the present study, MUTZ-3-derived DC (MuDC) showed high level expression of HLA class II molecules, CD80 and CD86, and were able to function as potent antigen presenting cells as previously reported. Interestingly, MuDC maturation was induced by CD40- mediated stimulation, but not by LPS stimulation. We analyzed CCR1, CCR7 and Toll-like receptor (TLR) expressions in MuDC, and measured IL-10 and IL-12 production after maturation stimuli. Although MuDC expressed the mRNA for TLR4, a major component of the LPS receptor system, they did not show an enhanced level of CCR7 or cytokine production after LPS stimulation. In contrast, they responded to CD40 stimulation, which resulted in increased levels of CD83, CD86 and CCR7. Moreover, while LPS- stimulated MoDC could potently stimulate NK cells in a DC-NK cell co-culture, LPS-stimulated MuDC failed to stimulate primary NK cells. Taken together, our findings suggest that, although MuDC express TLR4, unlike TNF-alpha and IL-1beta, LPS does not stimulate MuDC to acquire mature phenotypes, and they may have impaired activity to initiate innate immune response.     

Chemistry of Lipid A: At the Heart of Innate Immunity

In many Gram‐negative bacteria, lipopolysaccharide (LPS) and its lipid A moiety are pivotal for bacterial survival. Depending on its structure, lipid A carries the toxic properties of the LPS and acts as a potent elicitor of the host innate immune system via the Toll‐like receptor 4/myeloid differentiation factor 2 (TLR4/MD‐2) receptor complex. It often causes a wide variety of biological effects ranging from a remarkable enhancement of the resistance to the infection to an uncontrolled and massive immune response resulting in sepsis and septic shock. Since the bioactivity of lipid A is strongly influenced by its primary structure, a broad range of chemical syntheses of lipid A derivatives have made an enormous contribution to the characterization of lipid A bioactivity, providing novel pharmacological targets for the development of new biomedical therapies. Here, we describe and discuss the chemical aspects regarding lipid A and its role in innate immunity, from the (bio)synthesis, isolation and characterization to the molecular recognition at the atomic level.     

Myeloid differentiation primary response protein 88 blockade upregulates indoleamine 2,3-dioxygenase expression in rheumatoid synovial fibroblasts

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNAmediated knockdown of MyD88.     

Chemical Synthesis and Immunological Evaluation of the Inner Core Oligosaccharide of Francisella tularensis

Francisella tularensis , which is a Gram negative bacterium that causes tularemia, has been classified by the Center for Disease Control and Prevention (CDC) as a category A bioweapon. The development of vaccines, immunotherapeutics, and diagnostics for F. tularensis requires a detailed knowledge of the saccharide structures that can be recognized by protective antibodies. We have synthesized the inner core region of the lipopolysaccharide (LPS) of F. tularensis to probe antigenic responses elicited by a live and subunit vaccine. The successful preparation of the target compound relied on the use of a disaccharide which was modified by the orthogonal protecting groups diethylisopropylsilyl (DEIPS), 2-naphthylmethyl (Nap), allyl ether (All), and levulinoyl (Lev) ester. The ability to remove the protecting groups in different orders made it possible to establish the optimal glycosylations sequence to prepare a highly crowded 1,2,3-cis configured branching point. A variety of different methods were exploited to control anomeric selectivities of the glycosylations. A comparison of the (1)H NMR spectra of isolated material and the synthetic derivative confirmed the reported structural assignment of the inner core oligosaccharide of F. tularensis . The observation that immunizations with LPS lead to antibody responses to the inner core saccharides provides an impetus to further explore this compound as a vaccine candidate.     

Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry

We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LPS, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.     

Selective cleavage of peptides in trifluoroethanol with acid-labile amino-protecting groups]

In building up large polypeptides, it has become the established practice to use acide-labile protecting groups of the t-butyl type. Up to now, only one step of selectivity under acidic conditions has been used, consisting in the cleavage of Trt, Bpoc or Ppoc from N(α) without attacking the t-butyl protecting groups. We have found that the use of 90% trifluoroethanol as solvent permits the selective cleavage of Trt in the presence of Bpoc or Ppoc under controlled acidolytic conditions. This additional selectivity-step may be utilized when two-chained polypeptides are to be constructed. The procedure consists in acidolytic cleavage with hydrochloric acid at a constant potential as measured by the glass electrode (pH-stat). The automatic protonation of the freshly deprotected amino groups permits the evaluation of kinetic and quantitative data of the cleavage reaction. The selectivity ratios and cleavage conditions of Trt, Bpoc and Ppoc in N(α) are demonstrated here by reference to a series of model dipeptides. The successful application of this technique in the total synthesis of human insulin, a polypeptide with two peptida chains, has recently been described [8].     

Structure-activity relationships in hydroxy-2,3-diarylxanthone antioxidants. Fast kinetics spectroscopy as a tool to evaluate the potential for antioxidant activity in biological systems

A structure-activity relationship has been established for eight hydroxy-2,3-diarylxanthones (XH) bearing hydroxy groups on the two aryl rings. One-electron oxidation by superoxide radical-anions (˙O(2)(-)) and ˙Trp radicals as well as reaction with ˙CCl(3)O(2) and ˙CHCl(2)O(2) radicals demonstrates that two OH groups are required for efficient antioxidant reactivity in cetyltrimethylammonium bromide micelles. Hydroxy groups at the meta and para positions on either of the two phenyl rings confer enhanced reactivity, but XH bearing an OH at the para position of either phenyl ring is unreactive. While oxidation is favoured by OH in both meta and para positions of 2-aryl xanthone substituents, addition of a third and/or fourth OH enhances electron-donating capacity. In Cu(2+)-induced lipid peroxidation of human LDL, the lag period preceding the commencement of lipid peroxidation in the presence of XH bearing OH at meta and para positions on the 3-phenyl ring is extended to twice that observed with a comparable concentration of quercetin, a reference antioxidant. These antioxidants are also superior to quercetin in protecting human skin keratinocytes against tert-butylhydroperoxide-induced oxidative stress. While XH antioxidant activity in model biological systems is consistent with the structure-activity relationship, their response is also modulated by the localization of XH and by structural factors.     

Synthesis and biological evaluation of Rhizobium sin-1 lipid A derivatives

A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed. The approach employed the advanced intermediate 3-O-acetyl-6-O-(3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-beta-d-glucopyrano-syl)-2-azido-4-O-benzyl-2-deoxy-1-thio-alpha-d-glucopyranoside (5), which is protected in such a way that the anomeric center, the C-2 and C-2' amino groups, and the C-3 and C-3' hydroxyls can be selectively functionalized. The synthetic strategy was used for the preparation of 2-deoxy-6-O-[2-deoxy-3-O-[(R)-3-hydroxy-hexadecanoyl]-2-[(R)-3-octacosanoyloxy-hexadecan]amido-beta-d-glucopyranosyl]-2-[(R)-3-hydroxy-hexadecan]amido-3-O-[(R)-3-hydroxy-hexadecanoyl]-alpha-d-glucopyranose (11) and 2-deoxy-6-O-[2-deoxy-3-O-[(R)-3-hydroxy-hexadecanoyl]-2-[(R)-3-octacosanoyloxy-hexadecan]amido-beta-d-glucopyranosyl]-2-[(R)-3-hydroxy-hexadecan]amido-3-O-[(R)-3-hydroxy-hexadecanoyl]-d-glucono-1,5-lactone (13), which contain an unusual octacosanoic acid moiety and differ in the oxidation state of the anomeric center. The results of biological studies indicate that 11 and 13 lack the proinflammatory effects of Escherichia coli lipopolysaccharides (LPS). Furthermore, 13 emulated the ability of heterogeneous R. sin-1 LPS to antagonize enteric LPS, providing evidence for the critical role of the gluconolactone moiety of R. sin-1 LPS in mediating this antagonistic effect. Compound 13 is the first example of a lipid A derivative that is devoid of phosphate but possesses antagonistic properties, making it an attractive lead compound for development of a drug to use in the treatment of Gram-negative septicemia.