Aminosugar-based immunomodulator lipid A: synthetic approaches

Abstract

The immediate immune response to infection by Gram-negative bacteria depends on the structure of a lipopolysaccharide (LPS, also known as endotoxin), a complex glycolipid constituting the outer leaflet of the bacterial outer membrane. Recognition of picomolar quantities of pathogenic LPS by the germ-line encoded Toll-like Receptor 4 (TLR4) complex triggers the intracellular pro-inflammatory signaling cascade leading to the expression of cytokines, chemokines, prostaglandins and reactive oxygen species which manifest an acute inflammatory response to infection. The "endotoxic principle" of LPS resides in its amphiphilic membrane-bound fragment glycophospholipid lipid A which directly binds to the TLR4·MD-2 receptor complex. The lipid A content of LPS comprises a complex mixture of structural homologs varying in the acylation pattern, the length of the (R)-3-hydroxyacyl- and (R)-3-acyloxyacyl long-chain residues and in the phosphorylation status of the β(1→6)-linked diglucosamine backbone. The structural heterogeneity of the lipid A isolates obtained from bacterial cultures as well as possible contamination with other pro-inflammatory bacterial components makes it difficult to obtain unambiguous immunobiological data correlating specific structural features of lipid A with its endotoxic activity. Advanced understanding of the therapeutic significance of the TLR4-mediated modulation of the innate immune signaling and the central role of lipid A in the recognition of LPS by the innate immune system has led to a demand for well-defined materials for biological studies. Since effective synthetic chemistry is a prerequisite for the availability of homogeneous structurally distinct lipid A, the development of divergent and reproducible approaches for the synthesis of various types of lipid A has become a subject of considerable importance. This review focuses on recent advances in synthetic methodologies toward LPS substructures comprising lipid A and describes the synthesis and immunobiological properties of representative lipid A variants corresponding to different bacterial species. The main criteria for the choice of orthogonal protecting groups for hydroxyl and amino functions of synthetically assembled β(1→6)-linked diglucosamine backbone of lipid A which allows for a stepwise introduction of multiple functional groups into the molecule are discussed. Thorough consideration is also given to the synthesis of 1,1'-glycosyl phosphodiesters comprising partial structures of 4-amino-4-deoxy-β-L-arabinose modified Burkholderia lipid A and galactosamine-modified Francisella lipid A. Particular emphasis is put on the stereoselective construction of binary glycosyl phosphodiester fragments connecting the anomeric centers of two aminosugars as well as on the advanced P(III)-phosphorus chemistry behind the assembly of zwitterionic double glycosyl phosphodiesters.

Keywords: TLR4; glycoconjugate; glycolipids; glycosylation; immunomodulation; lipopolysaccharide.

Figure 1

(A) Gram-negative bacterial membrane with LPS as major component of the outer membrane; (B) structural constituents of LPS: lipid A, inner/outer core and O-specific chain.

Figure 2

Structures of representative TLR4 ligands: TLR4 agonists (E. coli lipid A, N. meningitidis lipid A and MPLA) and TLR4 antagonists (lipid IVa, R. sphaeroides lipid A and eritoran (E5564)); examples of post-translationally modified lipid A from Francisella, Burkholderia and Helicobacter.

Figure 3

(A) Co-crystal structure of the homodimeric E. coli Ra-LPS·hMD-2∙TLR4 complex (PDB code: 3FXI); (B) schematic representation of the E. coli lipid A induced activation of the MD-2∙TLR4 complex (C) schematic representation of the interaction of TLR4 antagonist eritoran with MD-2∙TLR4 complex. Images were generated with PyMol, ChemDraw and PowerPoint.

Figure 4

Co-crystal structures of (A) hybrid TLR4·hMD-2 with the bound antagonist eritoran (PDB: 2Z65, TLR4 is not shown); (B) homodimeric E. coli Ra-LPS·hMD-2∙TLR4 complex (PDB code: 3FXI, TLR4 is not shown, only lipid A portion is shown for clarity). Images were generated with PyMol.

Scheme 1

Synthesis of E. coli and S. typhimurium lipid A and analogues with shorter acyl chains.

Scheme 2

Synthesis of N. meningitidis Kdo-lipid A.

Scheme 3

Synthesis of fluorescently labeled E. coli lipid A.

Scheme 4

Synthesis of H. pylori lipid A and Kdo-lipid A.

Scheme 5

Synthesis of tetraacylated lipid A corresponding to P. gingivalis LPS.

Scheme 6

Synthesis of pentaacylated P. gingivalis lipid A.

Scheme 7

Synthesis of monophosphoryl lipid A (MPLA) and analogues.

Scheme 8

Synthesis of tetraacylated Rhizobium lipid A containing aminogluconate moiety.

Scheme 9

Synthesis of pentaacylated Rhizobium lipid A and its analogue containing ether chain.

Scheme 10

Synthesis of pentaacylated Rhizobium lipid A containing 27-hydroxyoctacosanoate lipid chain.

Scheme 11

Synthesis of zwitterionic 1,1′-glycosyl phosphodiester: a partial structure of GalN-modified Francisella lipid A and a neoglycoconjugate based thereof.

Scheme 12

Synthesis of a binary 1,1′-glycosyl phosphodiester: a partial structure of β-L-Ara4N-modified Burkholderia Lipid A and a neoglycoconjugate based thereof.

Scheme 13

Synthesis of Burkholderia lipid A containing binary glycosyl phosphodiester linked β-L-Ara4N.