SYNTHESIS OF CARBA-ALPHA-D-MANP-(1-]6)-BETA-D-GLCP-O(CH2)(7)CH3 - A REACTIVE ACCEPTOR ANALOG FOR N-ACETYLGLUCOSAMINYLTRANSFERASE-V

The branching enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) rec ognizes the trisaccharide beta-D-GlcpNAc-(1 --> 2)-alpha-D-Manp-(1 --> 6)-beta-D-Glcp-O(CH2)(7)CH3 (1) as its minimum substrate. We report h ere the chemical synthesis of beta-D-GlcpNAc-(1 --> 2)-5a-carba-alpha- D-Manp-(1 --> 6)-beta-D-Glcp-O(CH2)(7)CH3 (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 ac ceptor for GlcNAcT-V, both with the enzyme isolated from hamster kidne y and the one cloned from rat kidney. The kinetic parameters K-m and V -max for 1 and 2 were functionally equivalent. A preparative glycosyla tion reaction was performed using 2 as the acceptor with the cloned ra t kidney enzyme. A tetrasaccharide formed by the addition of a GlcpNAc residue was the sole product as detected by H-1 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)-bet a-D-Glcp-O(CH2)(7)CH3 (13).