Editing the C2 Position of Glycolipid Ligands Reveals Ligand-Dependent Recognition by Mincle

Macrophage-inducible C-type lectin (Mincle) induces immune responses in the presence of pathogen- and damage-associated molecular patterns, including glycolipids such as trehalose dimycolate (TDM), and is a potential target for the development of vaccine adjuvants. While previous research has extensively explored the structure-activity relationship (SAR) of the lipid component of Mincle ligands, here we focus on structural modification of the C-2 hydroxy group of trehalose in TDM. We previously reported that replacing the 2-hydroxy group of α-glucosylceramide with an exomethylene group significantly enhances its Mincle activation potential. In contrast, we found here that 2-C-substituted pseudo-TDMs, synthesized through a sequential Tsuji-Trost-type reaction, only weakly activate Mincle, suggesting a major role of TDM's C-2 hydroxy group in Mincle activation. Molecular modeling studies provided a rationale for the contrasting effects of C2-exomethylene substitution on trehalose esters and α-glucosylceramide and guided the design of a 2-exomethylene pseudo-α-GlcCer bearing a 6-behenate moiety, which showed enhanced Mincle activation. Our results suggest the existence of multiple binding modes for α-glucosylceramide-type ligands on Mincle.