Synthesis of β-d-GlcpNAc-(1 → 2)-5a-carba-α-d-Manp-(1 → 6)-β-d-Glcp-O(CH2)7CH3: a reactive acceptor analog for N-acetylglucosaminyltransferase-V

The branching enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) recognizes the trisaccharide β-d-GlcpNAc-(1 → 2)-α-d-Manp-(1 → 6)-β-d-Glcp-O(CH2)7CH3 (1) as its minimum substrate. We report here the chemical synthesis of β-d-GlcpNAc-(1 → 2)-5a-carba-α-d-Manp-(1 → 6)-β-d-Glcp-O(CH2)7CH3 (2), a carbocyclic analog of 1 where the ring oxygen of the α-d-Manp residue is replaced by a methylene group. Trisaccharide 2 was found to be fully active as an acceptor for GlcNAcT-V, both with the enzyme isolated from hamster kidney and the one cloned from rat kidney. The kinetic parameters Km and Vmax for 1 and 2 were functionally equivalent. A preparative glycosylation reaction was performed using 2 as the acceptor with the cloned rat kidney enzyme. A tetrasaccharide formed by the addition of a GlcpNAc residue was the sole product as detected by 1H NMR spectroscopy and FAB mass spectrometry and was assigned the structure β-d-GlcpNAc-(1 → 2)-[β-d-GlcpNAc-(1 → 6)]-5a-carba-α-d-Manp-(1 → 6)-β-d-Glcp-O(CH2)7CH3 (13).