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TLR4 is a key pattern recognition receptor that can sense pathogen- and danger- associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro-inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species-specific, and barely predictable structure-activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan-based lipid A-mimicking molecules that can activate human and murine TLR4-mediated signaling with picomolar affinity. Exploiting crystal structure - based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible β(1→6)-linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non-reducing βGlcN(1↔1')βGlcN scaffold. Excellent stereoselectivity in a challenging β,β-1,1' glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of β,β-1,1'-linked diglucosamine-derived glycolipids entailing multiple long-chain (R)-3- acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D-molecular shape and conformational rigidity of unnatural β,β-1,1'-linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4-mediated signaling in a species-independent manner.

Vaccines are the most effective medical intervention due to their continual success in preventing infections and improving mortality worldwide. Early vaccines were developed empirically however, rational design of vaccines can allow us to optimise their efficacy, by tailoring the immune response. Establishing the immune correlates of protection greatly informs the rational design of vaccines. This facilitates the selection of the best vaccine antigens and the most appropriate vaccine adjuvant to generate optimal memory immune T cell and B cell responses. This review outlines the range of vaccine types that are currently authorised and those under development. We outline the optimal immunological correlates of protection that can be targeted. Finally we review approaches to rational antigen selection and rational vaccine adjuvant design. Harnessing current knowledge on protective immune responses in combination with critical vaccine components is imperative to the prevention of future life-threatening diseases.

The innate immune response to lipopolysaccharide is essential for host defense against Gram-negative bacteria. In response to bacterial infection, the TLR4/MD-2 complex that is expressed on the surface of macrophages, monocytes, dendritic, and epithelial cells senses picomolar concentrations of endotoxic LPS and triggers the production of various pro-inflammatory mediators. In addition, LPS from extracellular bacteria which is either endocytosed or transfected into the cytosol of host cells or cytosolic LPS produced by intracellular bacteria is recognized by cytosolic proteases caspase-4/11 and hosts guanylate binding proteins that are involved in the assembly and activation of the NLRP3 inflammasome. All these events result in the initiation of pro-inflammatory signaling cascades directed at bacterial eradication. However, TLR4-mediated signaling and caspase-4/11-induced pyroptosis are largely involved in the pathogenesis of chronic and acute inflammation. Both extra- and intracellular LPS receptors—TLR4/MD-2 complex and caspase-4/11, respectively—are able to directly bind the lipid A motif of LPS. Whereas the structural basis of lipid A recognition by the TLR4 complex is profoundly studied and well understood, the atomic mechanism of LPS/lipid A interaction with caspase-4/11 is largely unknown. Here we describe the LPS-induced TLR4 and caspase-4/11 mediated signaling pathways and their cross-talk and scrutinize specific structural features of the lipid A motif of diverse LPS variants that have been reported to activate caspase-4/11 or to induce caspase-4/11 mediated activation of NLRP3 inflammasome (either upon transfection of LPS in vitro or upon infection of cell cultures with intracellular bacteria or by LPS as a component of the outer membrane vesicles). Generally, inflammatory caspases show rather similar structural requirements as the TLR4/MD-2 complex, so that a “basic” hexaacylated bisphosphorylated lipid A architecture is sufficient for activation. However, caspase-4/11 can sense and respond to much broader variety of lipid A variants compared to the very “narrow” specificity of TLR4/MD-2 complex as far as the number and the length of lipid chains attached at the diglucosamine backbone of lipid A is concerned. Besides, modification of the lipid A phosphate groups with positively charged appendages such as phosphoethanolamine or aminoarabinose could be essential for the interaction of lipid A/LPS with inflammatory caspases and related proteins.

Lipid A is a microheterogeneous Gram-negative bacterial glycolipid responsible for bacterial interaction with the innate immune system of mammalian host. Lipid A, which comprises just a small fragment (2 kDa) of a large (20 kDa) bacterial glycan lipopolysaccharide (LPS), anchors LPS in the bacterial outer membrane where it plays an important structural role. When endotoxic LPS is released into the blood circulation of the infected host, the lipid A is immediately sensed by the pattern recognition receptors of innate immune system, such as Toll-like receptor 4 and caspase-4, which instantly induce the intracellular pro-inflammatory signaling directed at bacterial clearance. Unique immunomodulating potential of LPS as well its major role in the pathogenesis of sepsis syndrome prompted intensive research in the fields of structural elucidation and synthesis of lipid A. The lipid A variants obtained by the chemical synthesis provide many advantages over the lipid A preparations obtained by isolation from bacterial cultures in terms of chemical and biological purity, defined chemical structure, and homogeneity which are extremely important for the application in the immunobiological and structure-activity relationships studies. Current review provides a survey of modern approaches toward divergent synthesis of biologically important lipid A variants. Particular emphasis is made on synthetic strategies toward classic lipid A forms corresponding to LPS from Escherichia coli, Salmonella typhimurium, Neisseria meningitidis, and Helicobacter pylori as well as lipid A variants entailing unique posttranslational modifications with unusual or branched lipid chains (Porphyromonas gingivalis and Rhizobium sin-1 lipid A) or with phosphodiester-linked amino sugars (Burkholderia and Francisella lipid A). Special attention is given to the synthesis and application of monophosphoryl lipid A and analogs as stand-alone or “inbuilt” adjuvants for fully synthetic vaccines candidates.

Lipopolysaccharides (LPS) are the main components of the external leaflet of the Gram-negative outer membrane and consist of three different moieties: lipid A, core oligosaccharide, and O-polysaccharide. The lipid A is a glucosamine disaccharide with different levels of acylation and phosphorylation, beside carrying, in certain cases, additional substituents on the sugar backbone. It is also the main immunostimulatory part of the LPS, as its recognition by the host immune system represents a fundamental event for detection of perilous microorganisms. Moreover, an uncontrolled immune response caused by a large amount of circulating LPS can lead to dramatic outcomes for human health, such as septic shock. The immunostimulant properties of an LPS incredibly vary depending on lipid A chemical structure, and for this reason, natural and synthetic variants of the lipid A are under study to develop new drugs that mimic or antagonise its natural effects. Here, we review past and recent findings on the lipid A as an antibiotic target and immune-therapeutic molecule, with a special attention on the crucial role of the chemical structure and its exploitation for conceiving novel strategies for treatment of several immune-related pathologies.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling1. Recent studies indicate that the bacterial metabolite d-glycero-β-d-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2–4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-β-d-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR–Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)–TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand–receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively. The bacterial metabolite ADP-heptose activates NF-κB in host cells via alpha-kinase 1 and the TIFA–TRAF signalling pathway.

Gram-negative bacterial lipopolysaccharide (LPS)-induced Toll-like receptor 4 (TLR4) mediated pro-inflammatory signaling plays a key role in immunoprotection against infectious challenges and boosts adaptive immunity, whereas the activation of the cytosolic LPS receptor caspase-4/11 leads to cell death by pyroptosis and is deeply implicated in the development of sepsis. Despite tremendous advances in the understanding of the LPS-TLR4 interaction, predictably regulated TLR4 activation has not yet been achieved. The structural basis for the induction of caspase-4/11 protease activity by LPS is currently unknown. The modulation of innate and adaptive immune responses through the controlled induction of TLR4 signaling without triggering caspase-4/11 activity would open novel perspectives in the development of safe vaccine adjuvants and immunotherapeutics. We report the discovery of highly potent glycan-based immunostimulants with picomolar affinity for TLR4 which interact with caspase-4/11 and promote caspase-4/11 oligomerization while abolishing caspase-11 protease activity. The rigidity and twisted molecular shape of the alpha,alpha-(1 1')-linked disaccharide core of synthetic LPS mimicking anionic glycolipids accounted for both species-independent and adjustable TLR4-mediated NF-kappa B signaling and the modulation of caspase-4/11 activation. By the use of crystal structure based design and advanced synthetic chemistry we created a set of versatile probes for studying the structural basis of caspase-4/11 activation and established a chemical strategy for controllable TLR4 mediated cytokine release which is dissociable from the induction of caspase-11 protease activity.

The bacterial metabolite and potent innate immune modulator d - glycero -β- d - manno -heptose-1,7-bisphosphate (HBP) and its α-configured counterpart d - glycero -α- d - manno -heptose-1,7-bisphosphate were synthesized via stereoselective anomeric phosphorylation of the peracetylated d , d -heptose 7-dibenzylphosphate by exploiting different nucleophilicity of equatorial and axial lactols in the d - manno -series. We also report a novel approach for anomeric phosphorylation using modified Mitsunobu reaction conditions and provide the first full structural characterization of HBP. The first chemical synthesis of HBP offers access to an anomerically pure structurally defined probe for biological studies and to a lead compound operating as a powerful stimulator of intracellular signaling for possible therapeutic immunomodulation.

International audience; During an infection, the detection of pathogens is mediated through the interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. β-Heptose 1,7-bisphosphate (βHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that βHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compare the ability of chemically synthesized βHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, βHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that βHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP-heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP-heptose sensing occurs down to 10-10 M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of βHBP in S. flexneri infection and identify ADP-heptose as a new bacterial PAMP.

During an infection, the detection of pathogens is mediated through the interactions between pathogen‐associated molecular patterns (PAMPs) and pathogen recognition receptors. β‐Heptose 1,7‐bisphosphate (βHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that βHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF‐κB activation and pro‐inflammatory gene expression. Here, we compare the ability of chemically synthesized βHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, βHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that βHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP‐heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP‐heptose sensing occurs down to 10(−10) M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of βHBP in S. flexneri infection and identify ADP‐heptose as a new bacterial PAMP.

Despite significant advances made in the last decade in the understanding of molecular mechanisms of sepsis and in the development of clinically relevant therapies, sepsis remains the leading cause of mortality in intensive care units with increasing incidence worldwide. Toll-like receptor 4 (TLR4)-a transmembrane pattern-recognition receptor responsible for propagating the immediate immune response to Gram-negative bacterial infection-plays a central role in the pathogenesis of sepsis and chronic inflammation-related disorders. TLR4 is complexed with the lipopolysaccharide (LPS)-sensing protein myeloid differentiation-2 (MD-2) which represents a preferred target for establishing new anti-inflammatory treatment strategies. Herein we report the development, facile synthesis, and biological evaluation of novel disaccharide-based TLR4-MD-2 antagonists with potent anti-endotoxic activity at micromolar concentrations. A series of synthetic anionic glycolipids entailing amide-linked beta-ketoacyl lipid residues was prepared in a straightforward manner by using a single orthogonally protected nonreducing diglucosamine scaffold. Suppression of the LPS-induced release of interleukin-6 and tumor necrosis factor was monitored and confirmed in human immune cells (MNC and THP1) and mouse macrophages. Structure-activity relationship studies and molecular dynamics simulations revealed the structural basis for the high-affinity interaction between anionic glycolipids and MD-2, and highlighted two compounds as leads for the development of potential anti-inflammatory therapeutics.

Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori and associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous hits involved in H. pylori-, but not IL-1β- and TNF-α- dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry and mutant H. pylori strains, identified the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation as well as TIFAsome formation depended on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as core of a novel innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen's T4SS.

SummaryActivation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori and associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous hits involved in H. pylori-, but not IL-1β- and TNF-α- dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry and mutant H. pylori strains, identified the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation as well as TIFAsome formation depended on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as core of a novel innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen’s T4SS.

SummaryAutotransporters deliver virulence factors to the bacterial surface by translocating an effector passenger domain through a membrane-anchored barrel structure. Although passenger domains are diverse, those found in enteric bacteria autotransporters, including AIDA-I in diffusely adhering Escherichia coli (DAEC) and TibA in enterotoxigenic E. coli, are commonly glycosylated. We show that AIDA-I is heptosylated within the bacterial cytoplasm by autotransporter adhesin heptosyltransferase (AAH) and its paralogue AAH2. AIDA-I heptosylation determines DAEC adhesion to host cells. AAH/AAH2 define a bacterial autotransporter heptosyltransferase (BAHT) family that contains ferric ion and adopts a dodecamer assembly. Structural analyses of the heptosylated TibA passenger domain reveal 35 heptose conjugates forming patterned and solenoid-like arrays on the surface of a β helix. Additionally, CARC, the AIDA-like autotransporter from Citrobacter rodentium, is essential for colonization in mice and requires heptosylation by its cognate BAHT. Our study establishes a bacterial glycosylation system that regulates virulence and is essential for pathogenesis.

Stereoselective synthesis of variably protected α- and β-l-Ara4N glycosyl H-phosphonates as key intermediates in the syntheses of β-l-Ara4N-modified LPS structures and α-l-Ara4N-containing biosynthetic precursors is reported. A facile one-pot approach toward β-l-Ara4N glycosyl H-phosphonates includes anomeric deallylation of protected 4-azido β-l-Ara4N via terminal olefin isomerization followed by ozonolysis and methanolysis of formyl groups to furnish exclusively β-configured lactols that are phosphitylated with retention of configuration. The carbohydrate epitope of β-l-Ara4N-modified Lipid A, βGlcN(1→6)αGlcN(1→P←1)β-l-Ara4N, was stereoselectively synthesized and linked to maleimide-activated bovine serum albumin.

Attachment of 4-amino-4-deoxy-L-arabinose (Ara4N) to phosphates or sugar hydroxyl groups of lipopolysaccharide (LPS) contributes to bacterial resistance against common antibiotics. For a detailed study of antigenic properties and binding interactions, Ara4N-containing inner-core ligands related to Burkholderia and Proteus LPS have been synthesized in good yields. Glycosylation at position 8 of allyl glycosides of oct-2-ulosonic acids (Ko, Kdo) has been accomplished using an N-phenyltrifluoroacetimidate 4-azido-4-deoxy-L-arabinosyl glycosyl donor followed by azide reduction and global deprotection. The β-L-Ara4N-(1 → 8)-α-Kdo disaccharide was further extended into the branched β-L-Ara4N-(1 → 8)[α-Kdo-(2 → 4)]-α-Kdo trisaccharide via a regioselective glycosylation of a protected triol intermediate. Synthesis of Ara4N-modified lipid A part structure occurring in the LPS of Burkholderia, Pseudomonas, and Klebsiellla strains was accomplished using the H-phosphonate approach. The stereocontrolled assembly of the phosphodiester linkage connecting glycosidic centers of two aminosugars was elaborated employing an anomeric H-phosphonate of cyclic silyl-ether protected 4-azido-4-deoxy-β-L-arabinose, which was coupled to the hemiacetal of the lipid A GlcN-disaccharide backbone. Conditions for global deprotection, which warrant the integrity of “double anomeric” phosphodiester linkage, were successfully developed. Introduction of thiol-terminated spacer at the synthetic ligands allows both coupling to bovine serum albumin (BSA) and immobilization on gold nanoparticles as well as generation of glycoarrays.

This review summarizes recent approaches towards the selective formation of anomeric aldose and aldulosonic acid phosphates of biological relevance, in particular as precursors for the synthesis of nucleotide-activated sugars.

Starting from l -lyxose, indium-mediated chain elongation with allyl bromide followed by acetylation and oxidative cleavage of the double bond and deprotection afforded 2-deoxy- l - galacto -heptose as a 2-deoxy analogue of the bacterial carbohydrate l - glycero - d - manno -heptose in good overall yield. For the synthesis of the ADP-activated derivative, the 2-deoxy-heptose was O-acetylated and transformed into the anomeric bromide derivative, which was then converted into the acetylated heptopyranosyl phosphate by reaction with tetrabutylammonium phosphate. Deprotection and separation of the anomeric phosphates furnished 2-deoxy-β- l - galacto -heptopyranosyl phosphate. Coupling of the acetylated heptosyl phosphate with AMP morpholidate afforded the acetylated ADP derivative in good yield. Removal of the acetyl groups gave the target compound ADP 2-deoxy- l - galacto -heptopyranose, which may serve as substrate analogue of bacterial ADP heptosyl transferases for biochemical and crystallographic studies.

C-Glycosides of l - glycero - d - manno - and d - glycero - d - manno -heptose containing either ( S )- or ( R )-2-hydroxypropyl aglycons are easily accessible compounds via condensation of reducing heptoses with pentane-2,4-dione. 2′,3′-Di- O -acetyl adenosine was transformed into the corresponding 5′- O -cyanoethyl N , N -diisopropylaminophosphoramidite derivative, which was coupled in fair yields to the O-acetylated diastereoisomeric C-glycosidic alcohols. Oxidation of the phosphite triesters followed by deprotection furnished four ADP-heptose analogues, wherein the heptosyl phosphate moiety had been replaced by a three carbon-skeleton. The compounds serving as substrate analogues will be used for co-crystallization experiments with ADP heptosyl transferases.

Immune stimulatory pathogen associated molecular patterns (PAMPs) are major drivers of infection pathology. Infections with Gram-negative bacteria or negatively polar and single stranded RNA influenza virus are prominent causes of morbidity and mortality. Toll-like receptor (TLR) 4 is a major host sensor for both of the two infections. In order to inhibit TLR4 driven immune activation we recently developed synthetic tetra-acylated lipid A mimetics based on a conformationally restricted βGlcN(1↔1)αGlcN disaccharide scaffold (DA-compounds) that antagonized ectopically overexpressed human and murine TLR4/MD-2 complexes. Here we comparatively analyzed human peripheral blood mononuclear cell (hPBMC) and murine bone marrow derived macrophage (mBM) activation upon 30 min of preincubation in vitro with six variably acylated DA-compounds. 16 h subsequent to consequent LPS challenge, we sampled culture supernatants for cytokine and NO concentration analysis. Four compounds significantly inhibited release of both TNF and IL-6 by hPBMCs upon LPS challenge. In contrast, three compounds effectively inhibited mBM production of MIP-2 and KC, and even five of them inhibited IL-6 and NO production. LPS driven like other TLR ligand driven mBM TNF release was largely unimpaired. The inhibitory effect was specific in that Clo75 driven cytokine release by both hPBMCs and mBMs was unimpaired by the compounds analyzed. Our results indicate biological species specificity of LPS antagonism by variably tetraacylated lipid A mimetics and validate three out of six DA-antagonists as promising candidates for development of therapeutically applicable anti-inflammatory compounds.

Reducing heptopyranoses of the d -glycero- d -manno- and l -glycero- d -manno-configuration were converted in good overall yields into β-anomeric C-glycosides via condensation with pentane-2,4-dione, followed by sodium methoxide-induced enrichment of the β-anomeric products and subsequent O-acetylation. The resulting 2-oxo-propyl glycosides were further transformed into separable ∼1:1 diastereomeric mixtures of (S)-and (R)-2-hydroxy derivatives. The configuration of the additional stereogenic centre was assigned on the basis of NMR data obtained from the corresponding Dale–Mosher esters. Finally, phosphorylation of the d -glycero- d -manno-heptose analogues using the phosphoramidite procedure and deprotection furnished the (S)- and (R)-2-phosphate derivatives in good yields. The compounds serve as substrate analogues for enzymes involved in bacterial ADP heptose biosynthesis and glycosyl transfer reactions.

Based on structural data of lipid A from Chlamydia trachomatis strains, chemically pure tetra- and pentaacyl 1,4′-bisphosphoryl as well as the related 4′-monophosphoryl derivatives of lipid A were synthesized. (R)-3-Hydroxyicosanoic acid as a chiral constituent was prepared via Noyori-reduction of methyl-3-oxoicosanoic acid. Synthetic intermediates were O-acylated with myristoic acid residues at positions 3 and 3′ and N-acylated with (R)-3-hydroxyicosanoic acid at both glucosamine units. Efficient purification methods for highly hydrophobic long-chain tri-, tetra- and pentaacyl progenitors of lipid A have been developed. Purity and homogeneity of the synthetic target compounds were confirmed by NMR and MS-data as well as a sensitive immunostaining approach. The tetra- and pentaacyl species serve as biomedical probes to investigate the endotoxic potential of chlamydial lipid A and to clarify its role in Chlamydia associated infections.

GDP d -glycero-α- d -manno-Heptopyranose has been prepared in good overall yield from 2,3,4,6,7-penta-O-acetyl- d -glycero- d -manno-heptopyranose by a short-step synthesis. Phosphitylation using the phosphoramidite procedure afforded the α-anomer in high selectivity. Subsequent oxidation and partial deprotection gave the acetylated phosphate derivative, which was subjected to the coupling reaction with GMP-morpholidate to furnish the acetylated heptose nucleoside diphosphate in good yield. De-O-acetylation and final purification afforded the target GDP d -glycero-α- d -manno-heptopyranose, which serves as the substrate of the heptosyl transferase in Aneurinibacillus thermoaerophilus DSM 10155 and occurs as an intermediate in the biosynthesis of GDP 6-deoxy-heptose in Yersinia pseudotuberculosis.

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram-negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4-amino-4-deoxy-β-l-arabinose (β-l-Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro-inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by β-l-Ara4N at the anomeric phosphate and its Ara4N-free counterpart. The double glycosyl phosphodiester was assembled by triazolyl-tris-(pyrrolidinyl)phosphonium-assisted coupling of the β-l-Ara4N H-phosphonate to α-lactol of β(1→6) diglucosamine, pentaacylated with (R)-(3)-acyloxyacyl- and Alloc-protected (R)-(3)-hydroxyacyl residues. The intermediate 1,1′-glycosyl-H-phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, β-l-Ara4N-substituted Burkholderia Lipid A. The β-l-Ara4N modification significantly enhanced the pro-inflammatory innate immune signaling of otherwise non-endotoxic Burkholderia Lipid A.

The endotoxic portion of lipopolysaccharide (LPS), a glycophospholipid Lipid A, initiates the activation of the Toll-like Receptor 4 (TLR4)–myeloid differentiation factor 2 (MD-2) complex, which results in pro-inflammatory immune signaling. To unveil the structural requirements for TLR4·MD-2-specific ligands, we have developed conformationally restricted Lipid A mimetics wherein the flexible βGlcN(1→6)GlcN backbone of Lipid A is exchanged for a rigid trehalose-like αGlcN(1↔1)αMan scaffold resembling the molecular shape of TLR4·MD-2-bound E. coli Lipid A disclosed in the X-ray structure. A convergent synthetic route toward orthogonally protected αGlcN(1↔1)αMan disaccharide has been elaborated. The α,α-(1↔1) linkage was attained by the glycosylation of 2-N-carbamate-protected α-GlcN-lactol with N-phenyl-trifluoroacetimidate of 2-O-methylated mannose. Regioselective acylation with (R)-3-acyloxyacyl fatty acids and successive phosphorylation followed by global deprotection afforded bis- and monophosphorylated hexaacylated Lipid A mimetics. αGlcN(1↔1)αMan-based Lipid A mimetics (α,α-GM-LAM) induced potent activation of NF-κB signaling in hTLR4/hMD-2/CD14-transfected HEK293 cells and robust LPS-like cytokines expression in macrophages and dendritic cells. Thus, restricting the conformational flexibility of Lipid A by fixing the molecular shape of its carbohydrate backbone in the “agonistic” conformation attained by a rigid αGlcN(1↔1)αMan scaffold represents an efficient approach toward powerful and adjustable TLR4 activation.

L- Glycero-D- manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L- glycero-D- manno-heptopyranose to Re-LPS and Rd 2-LPS, respectively. It had been proposed that both reactions involve ADPL- glycero-D- manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL- glycero-D- manno-heptose was isolated from a heptosyltransferase-deficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL- glycero-β-D- manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro .

Chemical syntheses of inner core determinants have been performed to provide defined artificial antigens (BSA-glycoconjugates) for characterization of monoclonal antibodies directed against important epitopes residing in the inner core of bacterial lipopolysaccharides. Efficient block synthesis of Kdo oligosaccharides has been employed to prepare the allyl glycoside [5] corresponding to the Chlamydia-specific Kdo trisaccharide epitope, to be used in crystallization and NMR (transfer NOe) experiments. Human pathogenic strains of Pseudomonas aeruginosa of RNA group I contain a highly phosphorylated heptose region with a 7- O-carbamoyl L- glycero-D- mannoheptose moiety which may be exploited as immunochemical marker for pathogenic Pseudomonas species. The 7- O-carbamoyl-substituted heptoside [12] as well as the disaccharides 7- O-carbamoyl-L- gro-α-D- manHep p- (1→3)-L- gro-α-D- manHep p-(1→O-Allyl) [23] and α-D-Gal pNAc-(1→3)-L- gro-α-D- manHep p-(1→O-Allyl) [30] were synthesized via regioselective formation of a 6′,7′- O-carbonate group followed by ring opening with NH3/NH4HCO3 to give the 7- O-carbamate in high yields. Finally, glycosides of the Kdo-isosteric D- glycero-D- talo-2-octulosonic acid (Ko) occurring in Acinetobacter spp. have been prepared via intermediate orthoester formation and TMSO-triflate-catalyzed rearrangement into α-ketosides. Coupling with a Kdo bromide donor and deblocking afforded the disaccharide α-Kdo-(2→4)-α-Ko-(2→O-Allyl) [43].

Attachment of 4-amino-4-deoxy-l-arabinose to phosphates or sugar hydroxyl groups of lipopolysaccharide contributes to bacterial resistance against common antibiotics. For a detailed study of antigenic properties and binding interactions, Ara4N-containing inner core ligands related to Burkholderia and Proteus LPS have been synthesized in good yields. Glycosylation at position 8 of allyl glycosides of oct-2-ulosonic acids (Ko, Kdo) has been accomplished using an N-phenyltrifluoroacetimidate 4-azido-4-deoxy-l-arabinosyl glycosyl donor followed by azide reduction and global deprotection. The β-l-Ara4N-(1→8)-α-Kdo disaccharide was further extended into the branched β-l-Ara4N-(1→8)[α-Kdo-(2→4)]-α-Kdo trisaccharide via a regioselective glycosylation of a protected triol intermediate. Synthesis of Ara4N-modified lipid A – part structure occurring in the LPS of Burkholderia, Pseudomonas and Klebsiellla strains was accomplished using the H-phosphonate approach. The stereocontrolled assembly of the phosphodiester linkage connecting glycosidic centres of two aminosugars was elaborated employing an anomeric H-phosphonate of cyclic silyl-ether protected 4-azido-4-deoxy-β-l-arabinose which was coupled to the hemiacetal of the lipid A GlcN-disaccharide backbone. Conditions for global deprotection which warrant the integrity of “double anomeric” phosphodiester linkage were successfully developed. Introduction of thiol-terminated spacer at the synthetic ligands allows both coupling to BSA and immobilization on gold nanoparticles as well as generation of glycoarrays.

Genomic data and analytical studies of the past decade have revealed a multitude of novel structural features of the core region of the lipopolysaccharide of Gram-negative bacteria. The chapter presents an update of ongoing and recent synthetic efforts illustrating representative examples for the synthesis of complex oligosaccharides as ligands, structural analogues as well as neoglycoconjugates. A major focus has been placed on chemical and chemo-enzymatic synthesis of the higher-carbon sugars 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) as well L-glycero-D-manno-heptose followed by subsequent conversion into suitable glycosyl donors and acceptor derivatives for the assembly of inner and outer core units. Major synthetic accomplishments covering core structures from Chlamydia, Burkholderia, Proteus, Haemophilus, Nesseria, Escherichia coli and Pseudomonas aeruginosa LPS are described in this review.

Methyl 4-azido-4-deoxy-β-L-arabinopyranoside has been synthesized in five steps starting from methyl β-D-xylopyranoside in a multigram scale without chromatographic purification in 78% overall yield. The transformation relied on selective tosylation/nosylation at O-4 followed by acylation, S(N)2 displacement with sodium azide and subsequent deprotection. The methyl 4-azido-4-deoxy-arabinoside was then converted into allyl, propenyl, ω-bromohexyl and chlorethoxyethyl spacer glycosides by transglycosylation with the respective alcohols in good yields and fair anomeric selectivity. Reduction of the azido group and further transformations of the aglycon afforded ω-thiol-containing spacer derivatives. Coupling to maleimide-activated BSA provided a potent immunogen which was used to generate murine and rabbit polyclonal sera binding to LPS-core epitopes containing 4-amino-4-deoxy-arabinose residues.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

The synthetic 1,4′-bisphosphorylated penta-acyl and tetra-acyl lipid A structures representing the major molecular species of natural chlamydial lipid A were tested for their endotoxic activities as measured by interleukin-8 release from human embryonic kidney (HEK) 293 cells expressing Toll-like receptor (TLR) 2 or TLR4. Both compounds were unable to activate HEK293 cells transiently transfected with TLR2. The penta-acyl lipid A was a weak activator of HEK293 cells expressing TLR4/MD-2/CD14 whereas tetra-acyl lipid A was inactive even at high concentrations. The weak activity of the penta-acyl lipid A could be antagonized by the tetra-acyl derivative of Escherichia coli lipid A (compound 406) or the anti-CD14 monoclonal antibody MEM-18. Both, tetra- and pentaacyl lipid A were unable to antagonize the activity of synthetic E. coli-type lipid A (compound 506) or smooth lipopolysaccharide of Salmonella enterica serovar Friedenau. Tetra- and penta-acyl lipid A served as acceptors for Kdo transferases from E. coli, Chlamydia trachomatis and Chlamydophila psittaci as shown by in vitro assays and detection of the products by thin layer chromatography and immune staining with monoclonal antibody.