Preparation of Alexa Fluor 350-conjugated nonradioactive or 3H-labeled GM1 ganglioside derivatives with different ceramides

Abstract Alexa Fluor 350 hydrazide (AF) was coupled to the aldehyde group at C-6 of terminal galactose of oxidized GM1 gangliosides containing different fatty acid residues (GM1s). The AF–GM1 hydrazones obtained were reduced with NaBH 4 or [ 3 H]NaBH 4 and purified by high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). Final yields of AF–GM1s exceeded 30%, purity was better than 97%, and radiochemical purity of 3 H-labeled AF–GM1s was more than 94.5%. Structures of AF–GM1s were confirmed by electrospray ionization–mass spectrometry (ESI–MS). When added to HL-60 cell culture media, more than 81.6 or 78.9% of the AF–[ 3 H]GM1s were taken up by cells in a bovine serum albumin- or trypsin-resistant manner, respectively. Approximately 70% of the AF–[ 3 H]GM1s were recovered in HL-60 total plasma membrane fraction.