Synthesis of medium-sized cyclic amines by selective ring cleavage of sulfonylated bicyclic amines.

An efficient method of synthesis of functionalized medium-sized cyclic amines (eight- to ten-membered rings) by selective ring opening of sulfonylated bicyclic pyrrolizidine, indolizidine, and quinolizidine compounds is described. The key step is the selective cleavage of the central C-N bond of the bicyclic amine by means of a Julia-like desulfonylation process. Reaction: see text.

Heterologous over-expression of alpha-1,6-fucosyltransferase from Rhizobium sp.: application to the synthesis of the trisaccharide beta-D-GlcNAc(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GLcNAc, study of the acceptor specificity and evaluation of polyhydroxylated indolizidines as inhibitors.

An efficient heterologous expression system for overproduction of the enzyme alpha-1,6-Fucosyltransferase (alpha-1,6-FucT) from Rhizobium sp. has been developed. The gene codifying for the alpha-1,6-FucT was amplified by PCR using specific primers. After purification, the gene was cloned in the plasmid pKK223-3. The resulting plasmid, pKK1,6FucT, was transformed into the E. coli strain XL1-Blue MRF'. The protein was expressed both as inclusion bodies and in soluble form. Changing the induction time a five-fold increase of enzyme expressed in soluble form was obtained. In this way five units of enzyme alpha-1,6-FucT can be obtained per liter of culture. A crude preparation of the recombinant enzyme was used for the synthesis of the branched trisaccharide alpha-D-GlcNAc-(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GlcNAc (3), from chitobiose (2) and GDP-Fucose (1). After purification, the trisaccharide 3 was obtained in a 84% overall yield. In order to elucidate the structural requirements for the acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides, which are structurally related to chitobiose. The enzyme uses, among others, the disaccharide N-acetyl lactosamine as a good substrate; the monosaccharide GlcNAc is a weak acceptor. Finally, several racemic polyhydroxylated indolizidines have been tested as potential inhibitors of the enzyme. Indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10(-5) M. Interestingly, this compound turned out to be the best mimic for the structural features of the fucose moiety in the presumed transition state.