In search of specific and highly selective sugar clusters for cell receptors, such as membrane lectins, various disaccharides were coupled to small peptide cores through an amide bond. In a first step, the reducing disaccharides, i.e. lactose and three different dimannoses, were converted into glycosyl-pyroglutamyl-β-alanine derivatives. The free carboxylic group of these conjugates was then coupled to the α and ∊ amino groups of the core peptide (Lysn-Ala-Cys-NH2) with n = 1 to 5, with complete substitution leading to homogeneous glycoclusters. The thiol group of the cysteine residue was used to tag the glycosylated oligolysines upon reaction with fluorescein iodoacetamide. The affinity of these glycoclusters towards two plant lectins was assessed by surface plasmon resonance. The selectivity of their cell uptake was investigated by flow cytometry using two types of cells: a human hepatoma cell line (HepG2 cells) expressing the plasma membrane galactose-specific lectin, and monocyte-derived dendritic cells expressing the plasma membrane mannose-specific lectin. The glycoclusters containing four or five disaccharides were shown to bind plant lectins and cell surface membrane lectins with a narrow selectivity and with a high affinity.
- flow cytometry
- surface plasmon resonance
Abbreviations used: Boc, t-butoxycarbonyl; BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; ConA, concanavalin A; DC-SIGN, dendritic cell-specific ICAM-grabbing non-integrin; DIEA, N,N-di-isopropylethylamine; DMEM, Dulbecco's minimum essential medium; DMF, dimethylformamide; EDT, ethanedithiol; ESI-MS, electrospray ionization MS; FCS, fetal calf serum; Glp, pyroglutamate; H-βAla-Obzl, H-βAla-benzyl ester; H-βAla-Obzl, Tos, H-βAla-Obzl tosylate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBT, 1-hydroxybenzotriazole; HPAEC, high-performance anion-exchange chromatography; Fmoc, fluorenyl-9-methoxycarbonyl; IAF, 5-(iodoacetamido)fluorescein; Lac, lactose; NMPO, N-methylpyrrolidone; Os, saccharide; OtB, t-butyl; PBMC, peripheral blood mononuclear cells; Rca, Ricinus communis agglutinin; RU, resonance units; SPR, surface plasmon resonance; SPy, 2-thiopyridyl; TBME, t-butyl methyl ether; TCEP, tris-(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid.
- The Biochemical Society, London ©2002