Fluorescent neoglycolipids - Improved probes for oligosaccharide ligand discovery

A second generation of lipid-linked oligosaccharide probes, fluorescent neo glycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl -sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively t o generate neoglycolipids for biological and structural studies, has been m odified to incorporate a fluorescent label, anthracene. This new lipid reag ent, N-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3-pho sphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADH P, by reductive amination, can be detected and quantified by spectrophotome try and scanning densitometry, and resolved by TLC and HPLC with subpicomol e detection. Antigenicities of the ADHP-neoglycolipids are well retained, a nd picomole levels can be detected using monoclonal carbohydrate sequence-s pecific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isom eric oligosaccharide sequences, sialyl-Le(a)- and sialyl-Le(x)-active, coul d be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liqui d secondary-ion mass spectrometry. Thus the neoglycolipid technology now un iquely combines high sensitivity of immuno-detection with a comparable sens itivity of chemical detection. Principles are thus established for a stream lined technology whereby an oligosaccharide population is carried through l igand detection and ligand isolation steps, and sequence determination by m ass spectrometry, enzymatic sequencing and other state-of-the-art technolog ies for carbohydrate analysis.