Fluorescent neoglycolipids

Abstract

A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, 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 to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (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 ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHPneoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequencespecific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Le a -and sialyl-Le x -active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.Keywords: carbohydrate recognition; fluorescence; oligosaccharide ligands; oligosaccharide probes; neoglycolipids.Neoglycolipids, derived by chemical conjugation of oligosaccharides to lipid, were introduced [1] and developed as oligosaccharide probes to address the need for a microprocedure for direct binding studies with glycans released from glycoproteins and glycolipids [2±5]. Neoglycolipids are generated by conjugation of oligosaccharide to an aminolipid such as 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) by reductive amination. As with natural glycolipids, the hydrophobic lipid enables the oligosaccharides to be coated onto matrices for solid phase binding experiments. Through clustering of the lipid moieties, the oligosaccharides are presented in an oligomeric state, which generates the avidities required for readily detectable binding. Neoglycolipids have the advantage that they contain a single lipid moiety, contrasting with the heterogeneous lipids of natural glycolipids. Following conjugation of mixtures, each oligosaccharide remains a discrete entity, rather than there being a population of oligosaccharides conjugated to a macromolecular carrier. Thus mixtures of neoglycolipids are amenable to resolution by TLC for binding experiments on chromatograms. The excellent ioni...

Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides

“…Preparation and Purification of NGL Probes-Fluorescent NGLs were prepared as described (26) by chemical conjugation of oligosaccharides to the amino phospholipid N-aminoacetyl-N-(9-anthracenyl methyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP) with the following modifications. To the lyophilized oligosaccharide fraction (typically 100 nmol) were added 5 l of water, 100 l of a solution of ADHP (10 nmol/l) in chloroform/methanol (1:3 by volume), and 20 l of a fresh solution of the reducing agent tetrabutylammonium cyanoborohydride (20 g/l in methanol).…”

“…The excess reagents in the reaction mixtures were removed (27,28) either by preparative HPTLC using as solvent chloroform/methanol/water (105:100:28 by volume) or by silica cartridge (100 mg, Strata; Phenomenex, Macclesfield, UK). Purification of NGLs by HPLC was as described (26), except that 10 mM ammonium formate was used instead of sodium chloride, and the gradient was from 70 to 30% solvent C in D at ambient temperature over 35 min. NGLs were quantified by spectrophotometry in solution and by densitometry after arraying on HPTLC plates (27,28).…”

Carbohydrate Sequence of the Prostate Cancer-associated Antigen F77 Assigned by a Mucin O-Glycome Designer Array

“…Fluorescent NGLs were prepared using N -aminoacetyl- N -(9-anthracenylmethyl)-1,2-dihexadecyl- sn -glycero-3-phosphoethanolamine (ADHP) as described (16, 21). In brief, lyophilized reducing oligosaccharides (100 nmol) were mixed with 80 μl of ADHP solution (10 nmol/μl in CHCl 3 /MeOH, 1:1, v/v) and freshly prepared 20 μl of tetrabutylammonium cyanoborohydride (20 μg/μl in MeOH).…”

“…The purified NGLs were analyzed by MALDI-MS, quantified on HPTLC by densitometry (21), and stored at −20 °C in CHCl 3 /MeOH/H 2 O, 25:25:8 (v/v).…”

10E4 Antigen of Scrapie Lesions Contains an Unusual Nonsulfated Heparan Motif

“…Preparation and Fractionation of Fluorescent Neoglycolipids-Fluorescent NGLs were prepared and resolved by high performance (HP) TLC essentially as described previously (12). In brief, the following were added to lyophilized HS fragments, typically 50 nmol Removal of ⌬UA from Tetrasaccharides-The 4,5-unsaturated hexuronic residue (⌬UA) at the nonreducing terminus of the HS-1 tetrasaccharides was removed by oxymercuration (16).…”