Synthesis of beta-D-GlcpNAc-(1-->2)-5a-carba-alpha-D-Man p-(1-->6)-beta-D- Glcp-O(CH2)7CH3: a reactive acceptor analog for N-acetylglucosaminyltransferase-V.

The branching enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) recognizes the trisaccharide beta-D-GlcpNAc-(1-->2)-alpha-D-Man p-(1-->6)-beta-D-Glcp-O(CH2)7CH3 (1) as its minimum substrate. We report here the chemical synthesis of beta-D-GlcpNAc-(1-->2)-5a- carba-alpha-D-Manp-(1-->6)-beta-D-Glcp-O(CH2)7CH3 (2), a carbocyclic analog of 1 where the ring oxygen of the alpha-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 Glc pNAc residue was the sole product as detected by 1H NMR spectroscopy and FAB mass spectrometry and was assigned the structure beta-D-GlcpNAc-(1-->2)-[beta-D-GlcpNAc-(1-->6)]-5a- carba-alpha-D-Manp-(1-->6)-beta-D-Glc p-O(CH2)7CH3 (13).