Synthesis of GlcN- and GlcNAc-type inositol glycans of plant sphingolipid GIPCs for LC-MS/MS analysis.

Glycosylinositol phosphoceramides (GIPCs) are major sphingolipids in plant plasma membranes. GIPCs possess structurally diverse inositol glycan (IG) head groups. While the chemical structure of hexose-type IGs has been elucidated, the chemical structure of hexosamine (HexN)- and N-acetylhexosamine (HexNAc)-type IGs remain poorly understood. This study reports the synthesis of amino sugar-containing IGs, GlcN-α(1 → 4)-GlcA-α(1 → 2)-Ins 1 and GlcNAc-α(1 → 4)-GlcA-α(1 → 2)-Ins 2, and their application as authentic standards for LC-MS/MS analysis. GlcN- and GlcNAc-type IGs were synthesized via 4,6-O-phenylboronic ester-directed α-selective glucosylation followed by global deprotection and selective N-acetylation. Naturally occurring HexN- and HexNAc-type IGs were obtained by the enzymatic degradation of GIPCs isolated from broad bean pods and radish sprouts. Using amino sugar-containing IGs 1 and 2 as authentic standards, the chemical structures of the HexN- and HexNAc-type IGs were investigated by LC-MS/MS analysis. The retention times and MS/MS fragmentation patterns of the natural IGs were in good agreement with those of the synthetic IGs. These results demonstrate that natural HexN- and HexNAc-type IGs correspond to 1 and 2. This study provides the first direct structural confirmation of amino sugar-containing IGs in plant GIPCs and highlights the utility of authentic synthetic standards for uncovering the hidden IG structural diversity of plant GIPCs.