Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel SN1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future.

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Highly diastereoselective nucleophilic additions to cyclic nitrones derived from L-malic acid and D-arabinose have been used for the construction of enantiomerically pure polyhydroxylated pyrrolidines. The synthetic strategy adopted was based on an oxidation/reduction protocol involving hydroxylamine/nitrone pairs and demonstrates the use of reagent-and substrate-derived stereocontrol. In most cases reactions took place with total diastereoselectivity and in quantitative yield, with no purification being necessary. By this strategy, 2-(hydroxymethyl)-, 2-(aminomethyl)-, and 2-aryl-

A general method for the homologation of aldehydes to a-amino aldehydes (aminohomologation) has been developed, which employs nitrones as iminium derivatives of the aldehydes. Key operations include a) the addition of a thiazole metalated at C-2 to the N-benzylnitrone derived from the aldehyde, b) the reductive dehydroxylation of the resultant thiazolyl N-benzylhydroxylamine, and c) the unmasking of the formyl group from the thiazole ring. The homologation sequence was studied by employing nitrones derived from various chiral polyalkoxy aldehydes and dialdoses. The addition of 2-lithiothiazole to these nitrones was synselective, whereas the reaction with the same nitrones precomplexed with Lewis acids was mri-selective. Hence, from each nitrone a pair of diastereoisomeric hydroxylamines was obtained. These compounds were then converted by the above sequence into a-epimeric a-amino aldehydes. Model elaborations of some of these products afforded the amino sugars D-glucosamine, D-mannosamine, D-nOJirimycin, and advanced intermediates for the synthesis of destomic acid and lincosamine.