Synthetic Endotoxin mimetics as tools for studying LPS-protein interactions

Toll-like receptor 4 (TLR4) activation is mediated by the binding of lipopolysaccharide (LPS or Endotoxin) to the central hydrophobic pocket of the co-receptor protein myeloid differentiation factor 2 (MD-2), followed by the dimerisation of two ternary ligand-receptor complexes.

LPS from different bacterial species induce different degrees of receptor complex dimerisation, which regulate the subsequent induction of pro-inflammatory signaling. To elucidate the molecular basis of LPS-induced activation of innate immune responses, we design and chemically synthesise unnatural carbohydrate-based LPS mimetics that can interact with LPS-recognising proteins and are perfect tools to study the host-pathogen interaction and the structural basis of ligand recognition by the innate immune receptors.

Our recent findings, based on the immunofunctional studies of synthetic endotoxin mimetics derived from β(1↔1)α and α(1↔1)α linked disaccharide scaffolds, reveal that the 3D molecular shape and ternary structure of the protein-bound diglucosamine backbone of lipid A is one of the most critical determinants of endotoxicity.

We perform synthetic studies on the stereoselective assembly of the non-reducing α,α- β,α- and β,β-1,1'-linked orthogonally protected disaccharides under simultaneous stereocontrol at two anomeric centres. The synthetic disaccharide backbones are then chemically substituted with various functional groups to provide biologically active endotoxin mimetics as tools for immunobiology and valuable probes for studying LPS-protein interactions.


Selected contributions

Pharmaceuticals (2023), 16, 23.

Chemistry (2022), 28, e20220054  

Front. Immunol. (2021), 12, 631797    

Front. Immunol. (2021), 12, 631797

Chem. Sci. (2018), 16, 3957-3963
ChemMedChem (2018), 13, 2317-2331
J.Med.Chem. (2015), 57, 8056-8071
Innate Immun. (2015); 21, 490-503
ACS Chem.Biol. (2013), 8, 2423-2432

Synthesis of aminoarabinose / aminosugar modified lipid A

We are developing methods for the stereoselective synthesis of binary glycosyl phosphodiesters corresponding to partial structures of LPS and lipid A from Burkholderia, Pseudomonas, Francisella and Bordetella species. Modification of the phosphate groups of lipid A by positively charged appendages is part of the survival strategy of many Gram-negative bacteria. The phosphate groups of cystic fibrosis (CF)-adapted Burkholderia LPS are abundantly esterified by 4-amino-4-deoxy-β-L-arabinose (β-L-Ara4N), which confers resistance to endogenous cationic antimicrobial peptides (CAMPs) and to antibiotic treatment and contributes significantly to bacterial virulence. To study the structural basis for the unique pro-inflammatory activity of Burkholderia LPS, we synthesised lipid A modified by β-L-Ara4N at the anomeric phosphate group and its Ara4N-free counterpart. We have shown that Ara4N modification at the glycosidically-linked phosphate group of Burkholderia lipid A contributes to a significant enhancement of the induction of pro-inflammatory signaling by otherwise inactive pentaacyl lipid A. This finding is important for understanding the virulence of CF-adapted BCC species and for the development of advanced CF-related therapeutics. In addition, trisaccharide-based neoglycoconjugates containing a conserved epitope βGlcN(1→6)αGlcN(1→P←1)β-L-Ara4N characteristic of CF adapted Burkholderia cepacia/cenocepacia and Pseudomonas aeruginosa species and an epitope βGlcN(1→6)αGlcN-(1→P←1)αGalN typical of most virulent Francisella strains were synthesised. These neoglycoconjugates are useful for the generation of specific monoclonal antibodies (mAbs) to be used in diagnostic assays for rapid antigen determination in clinical samples or for screening of as yet unidentified Ara4N-producing mutants.


Selected contributions

Org. Lett. (2017), 19, 78-81
Chem. Eur. J. (2015), 21, 4102-4114
Org.Lett. (2014) 16, 3772-3775

Synthetic nucleotide activated sugars

The LPS biosynthesis pathway is a complex multi-step process involving many metabolites and small molecules. Gram-negative bacteria use ADP-β-D-heptose as a substrate for bacterial heptosyltransferases and for the autotransporter heptosyltransferase (BAHT) family, which are involved in the incorporation of a bacterial sugar, glycero-β-D-manno-heptose, into lipopolysaccharide. Recently, it has been shown that several heptose derivatives swiped from the LPS biosynthesis pathway, are also pathogen-associated molecular patterns that are directly recognised by the host's innate immune system. Our chemical synthesis of the bacterial metabolites D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP) and adenosine diphosphate L-glycero- and D-glycero-D-manno-heptopyranose enabled the discovery of novel mammalian intracellular pattern recognition receptor alpha kinase-1. ADP-L-β-D-heptose can activate NF-κB signalling in the cytosol of host immune cells by binding to alpha-kinase 1 (ALPK 1). D-β-D-heptose 1,7-diphosphate is converted to the ADP-activated sugar by host adenylyl transferases and can also bind to ALPK1, although with lower affinity.


Selected contributions

Nature (2018), 561, 122-126
EMBO Rep. (2018),  19,  e46943
Cell Rep. (2017), 20, 2384-2395
Cell Host & Microbe (2014), 16, 351-363
e-Life (2014), 10.7554/eLife.03714