No abstract

Research aimed at understanding the specific role of glycosylation patterns in protein function would greatly benefit from additional approaches allowing direct access to homogeneous glycoproteins. Here the development and application of an efficient approach for the synthesis of complex homogenously glycosylated peptides based on a multifunctional photocleavable auxiliary is described. The presence of a PEG polymer within the auxiliary enables sequential enzymatic glycosylation and straightforward isolation in excellent yields. The auxiliary-modified peptides can be directly used in native chemical ligations with peptide thioesters easily obtained via direct hydrazinolysis of the respective glycosylated peptidyl resins and subsequent oxidation. The ligated glycopeptides can be smoothly deprotected via UV irradiation. Here we apply this approach to the preparation of variants of the epithelial tumor marker MUC1 carrying one or more Tn, T or sialyl-T antigens.

Full control over complex post-translational modifications (PTMs), such as O-glycosylation, is a prerequisite for testing and understanding the biological role of these modifications in protein function. Despite considerable progress over the last years, high throughput and easy-to-use methods for the synthesis of complex glycosylated peptides are still missing. We present here an efficient methodology to produce homogeneous site-specifically O-glycosylated peptides. Sequential chemoenzymatic glycosylation and separation from the reaction components are achieved via the temporary attachment of a monodisperse polyethylene glycol (PEG) polymer to the N-terminus of these peptides. Subsequent proteolytic removal of the PEG moiety allows quantitative recovery of homogeneous O-glycopeptides, suitable as building blocks for glycoprotein synthesis. Here, we demonstrate the preparation of glucuronylated variants of MUC1, a well-known member of the human mucin family. Homogeneously O-glycosylated variants were synthesized and will be used to study the role of O-linked glucuronic acid epitopes within the functional environment of the human MUC1 tandem repeat.

Several members of a new family of seven-membered azasugars, which can be seen as 1-azasugar ring homologues, have been obtained by simple chemical transformations starting from a sugar-derived azidolactol. Unlike their piperidine counterparts, these molecules are chemically stable when they possess a hydroxy group at the pseudo-C-2 position. Biological assays with a range of carbohydrate-processing enzymes have revealed interesting potential for these compounds. A trihydroxymethyl-substituted azepane displayed strong competitive inhibition on almond beta-glucosidase (K(i)=2.5 microM) while a trihydroxylated carboxylic acid derivative proved to be a potent and selective L-fucosidase inhibitor (K(i)=41 nM). N-Butylation of these seven-membered 1-azasugars generated derivatives with some activity towards the Gaucher's disease-related glucosylceramide transferase (IC(50) 75 microM) that did not interact significantly with digestive glucosidases.