Strategy to incorporate diazirine into gangliosides. (a) Glycosphingolipid GM1. The sialic acid is highlighted in red. (b) Enzymes in the sialic acid metabolic pathway convert ManNAc and analogs to activated CMP-sialic acids, which are added to glycoconjugates by sialyltransferases. Unnatural substituents are indicated by –R. (c) Synthesized sugar analogs Ac₄ManNAc (1), Ac₄ManNDAz (2), Ac₄GlcNDAz (3). Ac₄ManNDAz is expected to be metabolized to SiaDAz (4).

Production of diazirine-modified gangliosides. (a) Resorcinol-stained HPTLC plate reveals the profile of gangliosides produced by Jurkat cells. Ganglioside standards are shown in the first and last lanes. In untreated Jurkat cells, we observe multiple bands for each ganglioside, which we interpret as corresponding to different fatty acid chain lengths. Additional species are observed when cells are cultured with 2. (b) MALDI-TOF-MS analysis identified GM3, GM2, GM1, and GD1a gangliosides in untreated Jurkat cells; peak identity reported in supplemental information (Table S1). (c) MALDI-TOF-MS analysis identified gangliosides containing a naturally occurring sialic acid (N-acetyl neuraminic acid, NeuAc) or 4 (SiaDAz) in 2-treated cells; peak identity reported in supplemental information (Table S2). Ionization resulted in some loss of N₂ from diazirines (*). (d) GM1 gangliosides, including GM1-SiaDAz, are recognized by CTxB-Alexa488.

Photocrosslinking of GM1-SiaDAz to CTxB. All blots were probed for CTxB and β-actin. (a) A new CTxB-containing species appears in cells cultured with 2 and exposed to UV irradiation, indicative of crosslinking to GM1-SiaDAz. (b) Jurkat cells cultured with the glycosphingolipid biosynthesis inhibitor NB-DGJ demonstrate decreased production of GM1 (Fig. S6), decreased CTxB binding, and diminished levels of the crosslinked GM1-CTxB complex.