Development of αGlcN(1↔1)αMan-Based Lipid A Mimetics as a Novel Class of Potent Toll-like Receptor 4 Agonists

Abstract

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.

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

“…The synthesis of the pentaacylated β(1→6) diglucosamine backbone commenced with the preparation of the GlcN-based donor and acceptor molecules (Scheme 3 ). To this end, the p -methoxybenzylidene acetal 8 was acylated by ( R )-3-(allyloxycarbonyloxy)tetradecanoic acid 9 in the presence of strictly equimolar amount of diisopropylcarbodiimide (DIC) and catalytic amount of 4- N , N -dimethylaminopyridine (DMAP) at 0 °C (to avoid a formation of α,β-elimination product at fatty acid),[ 44 ] which afforded 12 in nearly quantitative yield. Reductive opening of 4,6- p -methoxybenzylidene acetal with sodium cyanoborohydride and trimethylsilyl chloride in acetonitrile furnished a mixture of two isomeric products, 6-OH derivative 13 and its 4-OH regio-isomer 14 .…”

Blockade of myeloid differentiation 2 attenuates diabetic nephropathy by reducing activation of the renin‐angiotensin system in mouse kidneys

“…It still remains unclear to as how hyperglycaemia activates MD2. Apart from LPS, several other ligands have been recently reported to bind MD2 and activate the MD2/TLR4 pathway (Adanitsch et al, 2014;Aranda et al, 2014;Hussein, Liu, Skwarczynski, & Toth, 2014). Thus, an important and unsolved question remains as to the specific mechanism by which hyperglycaemia promotes MD2/TLR4 complex formation and TLR4 activation under hyperglycaemic conditions.…”

Disaccharide‐Based Anionic Amphiphiles as Potent Inhibitors of Lipopolysaccharide‐Induced Inflammation

“…The biological activity of LAMs depends on the 3D‐molecular shape of their sugar core represented by glycosidically (1↔1)‐linked disaccharides having distinct anomeric configuration. Accordingly, LAMs derived from the α,α‐1,1′‐linked nonreducing disaccharide scaffold characterized by a skewed topology of the constituting pyranose rings furnish TLR4 agonists with picomolar affinity for TLR4⋅MD‐2 . Juxtapose, rigidifying the disaccharide backbone of the Lipid A‐like TLR4 ligands in a conformation where the two GlcN rings are fixed in a “planar” orientation (achieved via β,α‐1,1′‐linkage) ensures powerful TLR4 antagonists .…”

“…Accordingly,L AMs derived from the a,a-1,1'-linked nonreducing disaccharide scaffold characterized by as kewed topology of the constituting pyranose rings furnish TLR4 agonists with picomolara ffinity for TLR4·MD-2. [66,67] Juxtapose, rigidifying the disaccharide backbone of the Lipid A-like TLR4 ligands in a conformation where the two GlcN rings are fixed in a" planar" orientation (achieved via b,a-1,1'-linkage) ensures powerful TLR4 antagonists. [65,68] Thus, tetraacylated Lipid Am imetics derived from bGlcN(1$1)aGlcN scaffold with planar 3D topology (ba-GG-LAMs, Figure 3B)d isplayed nanomolar affinity for the MD-2·TLR4 complex and efficiently inhibited LPS-induced TLR4mediated expression of pro-inflammatoryc ytokines in human and mouses ystems.…”