Chemical Synthesis and Structural Determination of the Inositol Glycan Head of Plant Sphingolipid GIPC in Brassicaceae

Abstract

Glycosylinositol phosphoceramides (GIPCs), a major class of plant sphingolipids, are key components of the plasma membrane and play important roles in plants. These molecules consist of a ceramide 1-phosphate tail attached to an inositol glycan (IG) head, which typically contains an d-glucuronic acid (GlcA)-α(1→2)-myo-inositol structure. The second monosaccharide linked to the O-4 position of the GlcA residue differs depending on plant species and cell tissues. In Arabidopsis, the predominant second monosaccharide has been identified as hexose (Hex) using LC-MS/MS analysis. However, the stereochemistry and anomeric configuration of the Hex have not yet been determined using spectroscopic techniques. In this study, we chemically synthesized three different Hex-type IGs in which the GlcA residue binds to d-glucose, d-galactose, or d-mannose (Man) as the second monosaccharide. Using the synthetic standards, we determined the IG head structure of naturally occurring GIPCs isolated from cabbage by using several analytical methods. We successfully confirmed that Hex as the second monosaccharide linked to the GlcA residue is d-Man with an α-glycosidic bond.

Keywords: chemical synthesis; glycosylinositol phosphoceramide; inositol glycan; plant sphingolipid; structural determination.

Figure 1

Schematic representation of a GIPC structure in Brassicaceae and plausible chemical structures of the IG head: Glc‐type IG 1, Gal‐type IG 2, and Man‐type IG 3.

Scheme 1

Retrosynthetic analysis of IGs 1–3. BMAD: Boron‐mediated aglycon delivery; LG: Leaving group; NGP: Neighboring group participation; PG: Protecting group.

Scheme 2

Preparation of glucosyl inositol 17 as the common intermediate in the synthesis of IGs 1–3. DBTO: dibutyltin oxide; NapBr: 2‐naphthylmethyl bromide; TBAB: tetrabutylammonium bromide; NIS: N‐iodosuccinimide; TMSOTf: trimethylsilyl trifluoromethanesulfonate; MeOTf: methyl trifluoromethanesulfonate.

Scheme 3

Synthesis of IGs 1–3. *Containing trace impurities.

Figure 2

LC‐MS/MS analysis of synthetic IGs 1–3 and natural IG. A) LC‐MS/MS chromatograms: (i) 1, (ii) 2, (iii) 3, and (iv) natural IG. [Conditions] Column: ZIC‐pHILIC (2.1 mm I.D. × 150 mm); temperature: 45 °C; flow rate: 0.2 mL/min; MRM transition: m/z 517.1 > 293.1; eluent: A: 0.02 m NH₄HCO₃ (pH 9.8), B: MeCN; gradient: A/B = 25/75 (0–1 minute), 25/75 to 50/50 (1–15 minutes). B) MS/MS spectra: (i) 1, (ii) 2, (iii) 3, and (iv) natural IG. [Conditions] Collision energy: 24 V with Ar gas; N₂ gas nebulizer: 1.5 L/min; drying: 10 L/minute; temperature: 250 °C; heat block temperature: 300 °C. C) Relative signal intensity of MS² fragments from the precursor ion (m/z 517.1) obtained by the targeted MRM mode; signal intensity of the primary fragment at m/z 293.1 was set to 100%. D) Principal component analysis of the MS/MS spectra in Figure 2C.

Figure 3

Monosaccharide composition and ¹H NMR analyses of synthetic IG 3 and natural IG. A) HPLC chromatograms after acid hydrolysis followed by ABEE labeling: (i) Man, (ii) GlcA, (iii) 3, and (iv) natural IG. [Conditions]: Column: Shodex Asahipak NH2P‐50 4E (4.6 × 250 mm); temperature: 25 °C; flow rate: 0.8 mL/minute; fetection: Fluorescence (Ex. 305 nm, Em. 360 nm); eluent: A: 93% MeCN in 0.3% AcOH (pH adjusted to 7.0 with NH₄OH), B: 20% MeCN in 0.3% AcOH (pH adjusted to 7.0 with NH₄OH); gradient: A/B = 97/3 (0–5 min), 97/3 to 75/25 (5–15 min), 75/25 to 68/32 (15–30 min), 68/32 to 97/3 (30–35 min), 97/3 (35–60 min). B) ¹H NMR spectra (500 MHz) in D₂O: (i) 3 and (ii) natural IG.