Temporary attachment of carbohydrates to cyclopeptide templates : a new strategy for single-bead analysis of multivalent neoglycopeptides

Employing a cleavable carbohydrate–peptide linker, a new strategy for single-bead analysis of multivalent cyclic neoglycopeptides based on Edman degradation is described. Edman degradation of glycopeptides is hampered by the acid lability of the glycosidic bond and potential incompatibilities of phenylthiohydantoin (PTH) derivatives of glycosylated amino acids with PTH derivatives of the proteinogenic amino acids. To overcome this problem, carbohydrates are detached from the cyclopeptide templates before single-bead analysis, allowing for micro sequencing under routine conditions. With this strategy, application of multivalent cyclic neoglycopeptides in split-mix libraries with a subsequent screening process becomes possible for the first time. Conformationally restricted cyclic peptides are frequently employed as templates for the spatially defined presentation of pharmacophores or whole peptide domains. Cyclic peptides carrying several copies of a carbohydrate ligand are of great interest as potential multivalent lectin ligands. In particular, the influence of the spatial presentation of carbohydrates on lectin mediated signal transduction processes is subject of current research in the area of glycobiology. The screening of one-bead one-compound libraries of cyclic glycopeptides obtained by the split-mix protocol, however, has not yet been described. Recently, we reported the efficient convergent synthesis of split-mix libraries of cyclic neoglycopeptides of type 1. Here we present a strategy for the direct analysis of these compounds bound to single resin beads. Together, these techniques make it possible for split-mix libraries of type 1 to be applied in a screening process. Side-chain cyclized neoglycopeptide 5 (Scheme 1) which after deacetylation is a potential trivalent lectin ligand served as a model compound for our analytical strategy. It was obtained by convergent solid-phase synthesis on aminoethyl TentaGel via intermediates 2 and 3. The carbohydrates were attached to side-chain amino groups of 3 via urethane formation by treatment with active carbonate 4 in the presence of Hünig’s base (DIEA). Quantitative derivatization was ensured by monitoring the coupling progress with bromophenol blue after having thoroughly washed the resin with N,N-dimethylformamide (DMF). Identification of active members of one-bead one-compound libraries is based on the analysis of the minute amounts of substances bound to single resin beads (typically <200 pmol). Direct analysis of synthetic products is generally advantageous over indirect coding strategies because failed/side reactions can be moni