Chemical Synthesis of Burkholderia Lipid A Modified with Glycosyl Phosphodiester‐Linked 4‐Amino‐4‐deoxy‐β‐L‐arabinose and Its Immunomodulatory Potential

Abstract

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.

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia

“…Here, we demonstrate a role for L-Ara4N residues in lipid A that explains why the B. cenocepacia LPS (naturally consisting of a mixture of penta-and tetra-acylated forms) acts as a strong TLR4⅐MD-2 agonist. Our results agree with a recent report by Hollaus et al (36) demonstrating that a synthetic Burkholderia lipid A substituted with L-Ara4N exclusively at the anomeric phosphate acts as a potent human TLR4⅐MD-2 agonist.…”

“…The structural features of LPS BC suggest that the molecule would be a very poor TLR4⅐MD-2 complex agonist compared with the prototypic enterobacterial LPS. In contrast, several studies show that LPS BC as well as biological and synthetic lipid A analogues is highly proinflammatory (32)(33)(34)(35)(36). Therefore, the current model for binding/activation of human TLR4⅐MD-2 complex cannot explain why LPS BC is proinflammatory.…”

Aminosugar-based immunomodulator lipid A: synthetic approaches

“…Since the PivCl-mediated H-phosphonate coupling can be often accompanied by concomitant side-reactions (formation of P-acyl byproducts [ 140 ] resulting from an over-reaction of 96 or 98 with PivCl or formation of GlcNAc-derived oxazolines in the presence of an excess of chloroanhydride) [ 141 ], phosphonium type coupling reagents were optionally explored. Accordingly, the H-phosphonate 96 was activated by 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP), which selectively reacted with the electrophilic phosphorus atom of the H-phosphonate to form a P–N activated intermediate [ 160 – 161 ]. The later was smoothly coupled to the nucleophilic component, the hemiacetal 82 .…”

“…Since the 1-phosphate group of lipid A is directly involved in the formation of the dimeric MD-2·TLR4-LPS complex [ 42 ], the appendage of β-L-Ara4N might enhance the efficiency of dimerization via ionic attraction. In order to elucidate the structural determinants responsible for the unique pro-inflammatory potential of Burkholderia lipid A, the pentaacylated Burkholderia lipid A esterified by β-L-Ara4N at the anomeric phosphate 101 and its Ara4N-free counterpart 102 corresponding to native Burkholderia LPS were chemically synthesised [ 161 ].…”

Stereoselective Synthesis of α- and β-l-Ara4N Glycosyl H-Phosphonates and a Neoglycoconjugate Comprising Glycosyl Phosphodiester Linked β-l-Ara4N

“…We have recently shown that application of the H-phosphonate approach is advantageous for such a purpose, compared to other phosphitylation methodologies. 14,15 Preparation of anomerically pure α- and β- H-phosphonate monoesters of orthogonally protected l -Ara4N in a stereoselective manner comprises a major synthetic challenge in the synthesis of Ara4N-containing phosphodiesters (Figure 2). …”