title: Oxidation of glycosphingolipids under basic conditions: synthesis of glycosyl "serine acids" as opposed to "ceramide acids". Precursors for neoglycoconjugates with increased ligand binding affinity.
abstract: Two types of oxidative cleavage of the double bond of glycosphingolipids (GSLs) are described. Oxidation of peracetylated GSL precursors with stoichiometric proportions of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system, gave nearly quantitative yields of the glycosyl ceramide acid, 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)oxybutyric acid [Mylvaganam, M., and Lingwood, C. A. (1999) J. Biol. Chem. 274, 20725-20732]. However, if the reaction medium was made alkaline, the hydroxyallylic function of the sphingolipid, as a whole, was oxidized and the glycosyl serine acid, 2-(N-acyl)-3-(O-glycosyl)oxypropionic acid, was obtained in good yield. This represents a new type of oxidation reaction. Optimized conditions gave glycosyl ceramide or serine acids with greater than 90% selectivity and in good yields (90%). Oxidation of dGSLs gave serine and ceramide oligosaccharides, devoid of hydrocarbon chains. An intriguing glycosyl species containing 5-hydroxy-4-oxo-3-hydroxy-2-(N-acyl)sphingosine (hydroxy-acyl intermediate) was identified via ESMS analyses. We propose that further oxidation of this intermediate is pH-dependent and will be oxidized to either serine or ceramide acids. On the basis of MS-MS analysis of specific homologues of serine and ceramide acids, two types of collision-induced dissociation (CID) patterns have been established. These CID patterns were then used in the identification of serine and ceramide acids synthesized from natural GSL samples. Also, on a qualitative basis, this oxidation protocol, in conjunction with ESMS, provides a novel method for characterizing the aglycone composition (acyl chain length, unsaturation position, dihydrosphingosine content, etc.) of natural GSLs. A novel class of neohydrocarbon conjugates were synthesized by coupling the acids to rigid hydrocarbon frames such as 2-aminoadamantane. Preliminary studies with conjugates derived from globotriaosyl ceramide (Gb3C), lactosyl ceramide (LC), and galactosyl ceramide (GalC) bound verotoxin with the expected specificity but with affinities much greater than that of the natural glycolipid. Also, the ceramide acid-based conjugates were better ligands than serine acid conjugates.
authors.affiliation: Division of Immunity, Infection, Injury and Repair, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Mylvaganam
authors.forename: L
authors.initials: L
authors.lastname: Meng
authors.forename: C A
authors.initials: CA
authors.lastname: Lingwood
mesh_terms: D000215:Acylation; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002254:Carbonates; D002417:Cattle; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D006001:Glycoconjugates / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D006028:Glycosphingolipids / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010504:Periodic Acid; D011188:Potassium; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep; D022622:Shiga Toxin 1; D016339:Spectrometry, Mass, Fast Atom Bombardment
chemical_list: D001427:Bacterial Toxins; D002254:Carbonates; D002518:Ceramides; D006001:Glycoconjugates; D006028:Glycosphingolipids; D022622:Shiga Toxin 1; D010504:Periodic Acid; D012694:Serine; C009288:metaperiodate; C037593:potassium carbonate; D011188:Potassium