Chemoenzymatic Synthesis and Facile Purification of Gangliosides

Abstract

Gangliosides are biologically important sialic acid-containing glycolipids found commonly in human and other vertebrates. Isolation of pure gangliosides from cells or tissues is difficult, and chemical synthesis of gangliosides usually involves numerous steps with low synthetic yields. We report here a chemoenzymatic synthesis and purification protocol for two ganglioside cancer antigens, GM3 and GD3. One-pot multienzyme glycosylation reactions are used to sequentially prepare GM3 and GD3 sphingosines from chemically synthesized lactosyl sphingosine. A facile C18-cartridge purification procedure after each glycosylation reaction provides the desired pure glycosyl sphingosine product, which is readily acylated to form the target ganglioside. © 2021 Wiley Periodicals LLC. Basic Protocol: Chemoenzymatic synthesis and purification of GM3 and GD3 gangliosides Support Protocol: Monitoring reactions by thin-layer chromatography.

Keywords: carbohydrate; chemoenzymatic synthesis; ganglioside; glycolipid; glycosphingolipid; sialic acid.

Figure 1.

Structures of gangliosides GM3 and GD3. The sphingosine component of the ceramide is shown in red.

Figure 2.

Chemoenzymatic synthetic procedures described in this protocol for producing GM3 and GD3 gangliosides from lactosyl sphingosine (LacβSph).

Figure 3.

TLC analysis results of one-pot two-enzyme (OP2E) synthesis of a) GM3βSph and b) GD3βSph. Lanes: 1, LacβSph standard; 2, GM3βSph synthesis reaction mixture; 3, co-spots of GM3βSph synthesis reaction mixture and LacβSph standard; 4, GM3βSph standard; 5, GD3βSph synthesis reaction mixture; 6, co-spots of GD3βSph synthesis reaction mixture and GM3βSph standard. Developing solvent i-PrOH: H₂O: NH₄OH = 5:1:0.5 (by volume).

Figure 4.

TLC analysis results of the formation of gangliosides from a) GM3βSph and b) GD3βSph. Lanes: 1, GM3βSph standard; 2, GM3 synthesis reaction mixture; 3, Co-spots of GM3 synthesis reaction mixture and GM3βSph standard; 4, GD3βSph standard; 5, GD3 synthesis reaction mixture; 6, co-spots of GD3 synthesis reaction mixture and GD3βSph standard. Developing solvent: i-PrOH: H₂O: NH₄OH = 5:1:0.5 (by volume).